WO2025036963A1

WO2025036963A1 - Crebbp/p300 histone acetyltransferase inhibitors for treating cytopenias and their pathological sequalae

Info

Publication number: WO2025036963A1
Application number: PCT/EP2024/072982
Authority: WO
Inventors: Nikolai Pirmin JASCHKE; Christoph SCHEIERMANN; Tilman Daniel RACHNER; Lorenz Christian HOFBAUER
Original assignee: Technische Universitaet Dresden
Current assignee: Technische Universitaet Dresden
Priority date: 2023-08-15
Filing date: 2024-08-15
Publication date: 2025-02-20
Anticipated expiration: 2026-02-15

Abstract

The invention relates to an inhibitor of a histone acetyl transferase (HAT) activity of CREBBP(CBP)/p300 for use in the treatment of cytopenia. In embodiments, the inhibitor preferably suppresses a HAT domain of CBP/p300, more preferably by competing with acetyl CoA for binding to the HAT domain, thereby suppressing HAT enzymatic activity. In embodiments, the inhibitor induces acute and/or transient leukocytosis comprising mobilization of leukocytes from the bone marrow into the blood of a subject. The invention further relates to a pharmaceutical composition for use in the treatment of cytopenia, comprising the inhibitor according to the invention, and to a combination medication for use in the treatment of cytopenia, comprising the inhibitor according to the invention and a G-CSF or derivative thereof, preferably compound selected from G-CSF, recombinant G-CSF and/or G-CSF analogues.

Description

CREBBP/P300 HISTONE ACETYLTRANSFERASE INHIBITORS FOR TREATING CYTOPENIAS AND THEIR PATHOLOGICAL SEQUALAE

DESCRIPTION

The invention is in the field of biochemistry, hematology and medicine, particularly in the field of pharmaceutical treatment of cytopenia.

The invention relates in one aspect to inhibitors of the histone acetyl transferase (HAT) domain of cyclic adenosine monophosphate response element binding protein (CBP) and its orthologue EA1 associated protein 300 (EP300, also referred to as “p300”) for use in the treatment of cytopenia. In embodiments, the inhibitors preferably suppress the HAT domain of CBP/p300, in embodiments by competing with acetyl CoA for binding to the HAT domain. In embodiments, inhibitors induce acute and/or transient leukocytosis resulting from mobilization of leukocytes from the bone marrow into the blood of a subject. The invention further relates to a pharmaceutical composition for use in the treatment of cytopenia, comprising the inhibitor according to the invention, and further relates to a combination medication for use in the treatment of cytopenia, comprising the inhibitor according to the invention and G-CSF or a derivative thereof, such as a compound selected from G-CSF, recombinant G-CSF and/or G-CSF analogues.

BACKGROUND OF THE INVENTION

The bone marrow is the central reservoir for leukocytes, which include specialized populations such as neutrophils, monocytes and B lymphocytes. These cells arise from hematopoietic precursors and are released into the blood stream to reach distant tissue compartments¹. Leukocyte compartment sizes in the blood and tissues fluctuate physiologically within dynamic ranges²'⁴. As a function of demand, as occurs in response to infections, injury or stress, compartment sizes of leukocytes are modulated for host defense and returned to homeostasis when the demand is no longer present⁵⁶. Leukocyte numbers and their distribution are controlled at the level of production, mobilization from organ reservoirs, trafficking cues directing cells to sites of demand as well as their rate of local degradation⁷. A variety of cell-autonomous and non- cell-autonomous mechanisms that program these control mechanisms have been extensively studied (REFs³⁸'¹¹, among many others).

Top-down control of bone marrow function and leukocyte trafficking are coordinated by the central nervous system (CNS), which integrates peripheral inputs to generate adaptive leukocyte responses¹². These responses are relayed by different mechanisms including neuro-humoral circuits involving the hypothalamus-pituitary-adrenal gland (HPA)-axis and the sympathetic nervous system (SNS)⁷’¹⁰’¹³’¹⁴. Re-programming of leukocyte compartmentalization has trade-offs given the homeostatic roles that leukocytes play and less well-studied programs return leukocyte compartments to homeostatic set-points once host insults have been resolved¹⁵'¹⁷. Extreme examples of the cost of loss of homeostatic control of leukocyte compartmentalization are pathologic conditions, where normal “checkpoints” are dysregulated. This may lead to cases in which circulating leukocyte counts in the blood stream are barely detectable (e.g., acquired or genetic bone marrow failure) or excessively elevated as seen in patients with acute leukemia.

Extreme deviations from physiology, as found in rare genetic diseases, can be instructive to identify novel and druggable therapeutic targets²⁰. In severe congenital neutropenia (SCN), affected individuals display the two extreme ends of bone marrow phenotypes across their lifetime: loss of function (congenital neutropenia) to gain of function (acute myeloid leukemia)²¹. Longitudinal studies have shown that the leukemic transition of the disease is characterized by the acquisition of a distinct mutational landscape, which is not present during the cytopenic phase²². This landscape includes for example mutations in RLINX1, SLIZ12, ASXL1, CSF3R (missense mutation) as well as EP300, among others. EP300 (E1A-associated protein p300, also referred to as “p300”) loss of function in animal models is sufficient to both impair hematopoiesis (when deleted prenatally) and to induce leukocytosis or leukemia in later life²³²⁴. EP300 and its orthologue CREBBP (cyclic adenosine monophosphate response element binding protein, also known as “CBP”) share 90% sequence homology and are built from 8 functionally distinct domains, one of which confers histone acetyltransferase (HAT) activity²⁵. For example, the mutation in patients with SCN linked to leukemic transitioning is located within the HAT domainencoding genetic sequence ²², suggestive of functional relevance to leukocyte homeostasis.

Despite increasing knowledge of how leukocyte compartments are controlled, pharmacological interventions to interfere with the production, degradation, and localization of leukocytes to correct hematologic pathologies, diseases of acute and chronic inflammation, or augment normal adaptive host defenses remain limited. Therefore, the pharmacological interference with leukocyte compartmentalization is an underexplored space with an urgent need for innovation. Against this background, the inventors asked the question if the HAT domain of CBP/p300 may be targeted pharmacologically to modulate leukocyte compartment sizes (“leukocytosis on demand”). Herein the inventors demonstrate that (competitive, small molecule-mediated) inhibition of the CBP/p300 HAT domain has the capacity of inducing acute and transient leukocyte mobilization from the bone marrow, which may be relayed by a neuroendocrine loop of the HPA-axis, a process that is distinct from glucocorticoids.

The few available approaches for the modulation of leukocyte compartments include granulocyte colony stimulating factor (G-CSF) derivatives, CXC-motif chemokine receptor 4 (CXCR4) antagonists (e.g., plerixafor/AMD3100) or inhibitors of the very late antigen 4 integrin (VLA4)¹⁸’¹⁹. Among these drugs, only G-CSF derivatives confer acute neutrophil mobilization and full restoration of neutropenia, but these effects come with drawbacks including adverse events¹⁹.

Drawbacks of G-CSF and its derivatives include bone pain, allergic reactions, or pulmonary toxicity. Cytokine release syndromes are also discussed in the context of G-CSF therapeutics. In addition, there may be an increased risk for the development of secondary hematologic malignancies. Although longer-term stimulation of granulopoiesis by G-CSF is in principle desirable in certain situations (e.g., after chemotherapy), it limits the use of these therapeutics in a broader field, since in many cases only a short, pulsatile mobilization of neutrophil granulocytes into the blood is desired without modulating bone marrow homeostasis in the longer term. The latter is not possible with G-CSF-based drugs. In the prior art, different p300 inhibitors are described in the context of hematopoietic diseases. Some rather unspecific p300 inhibitors, such as (nano)curcumin have been described, e.g., in US11020372B or Mortazavi Farsani et al., 2020. However, pharmacologically curcumin is a less favorable compound due to it being a rather unspecific small molecule with a wide range of untraceable off-target effects on an array of cellular targets including NFkB, COX enzymes, iNOS, various MAPK etc ³⁶, rendering curcumin unsuitable for targeting specific molecular targets in the treatment of diseases without eliciting numerous site- and off-target effects. Also, the effects on platelets and megakaryocytes and the stimulation of myelopoiesis observed in said studies can most likely not be attributed to a selective inhibition of CBP/p300 HAT but are explained rather by off target effects because selective pharmacological inhibition of the CBP/p300 or genetic deletion of CBP from the hematopoietic system does not affect platelet homeostasis⁶⁴⁶⁵.

Effects of less specific CBP/p300 KAT inhibitors, such as C646, including apoptosis inhibition have been described, e.g., by Shunsheng et al., 2017. In this study apoptosis was inhibited by down regulation of important tumor suppressor genes in healthy bone marrow cells, but no relevant disease models were analyzed and the physiological and therapeutic relevance of these observations remained unspecified.

WO 2019/049061 A1 described the use of p300 inhibitors in the treatment of thrombocytopenia, which is medically unrelated to the proposed patent.

Other studies, such as Katavolos et al., 2020, analyzed the biological consequences of interfering with DNA binding of p300/CBP through manipulation of the protein’s bromodomain.

Hence, considering the prior art, there remains a significant need in the art to provide additional means for the treatment of cytopenia, particularly leukopenia and neutropenia.

SUMMARY OF THE INVENTION

In light of the prior art the technical problem underlying the present invention is to provide alternative and/or improved means for the treatment of cytopenia, particularly leukopenia and neutropenia, preferably by pharmacological manipulation of leukocyte compartments.

Another object of the invention was the provision of means to induce acute and/or transient leukocytosis in a subject or to restore/recover blood cells into a homeostatic range, comprising mobilization of leukocytes from the bone marrow into the blood, without eliciting longer lasting changes of bone marrow homeostasis and specifically, granulopoiesis.

The problem underlying the present invention is solved by the features of the independent claims. Preferred embodiments of the present invention are provided by the dependent claims.

The present invention addresses the problems above, in one aspect, by providing means and methods for mediating acute but transient mobilization of leukocytes, particularly neutrophil granulocytes, from the bone marrow, optionally in an additive manner to G-CSF-based therapies, to enable combination therapy as needed.

The invention therefore relates to an inhibitor of a histone acetyl transferase (HAT) activity of CREBBP (CBP) / p300 for use in the treatment of cytopenia. As discussed in more detail herein, neutropenia is a type of cytopenia (lack of blood cells), particularly of leukopenia (lack of white blood cells), e.g., characterized by a drop of (reduced) neutrophil granulocyte numbers below the lower limit of the homeostatic range. Neutropenia is associated with a high mortality and may result from genetic or acquired causes and is also a frequent complication of chemotherapy.

Pharmacological interventions to interfere with leukocyte production, degradation and localization to correct haematological conditions remain limited, despite increasing knowledge of how leukocyte compartments are controlled. Unfortunately, pharmacological interference with leukocyte compartmentalization is to date still an under-explored area with an unmet need for innovation. At present, most available therapies for the treatment of cytopenia, like neutropenia, are derivatives of the endogenous cytokine granulocyte colony stimulating factor (G-CSF).

As an example, filgrastim (recombinant methionyl human G-CSF) was first approved for this indication in 1991. Through chemical modifications, longer-acting G-CSF derivatives have been developed (e.g., pegylated filgrastim). On the one hand, G-CSF acts by mobilizing neutrophil granulocytes from the bone marrow, which can acutely restore neutropenia. At the same time, the cytokine or its derivatives act as a growth factor that stimulates the further formation ("granulopoiesis") of neutrophil granulocytes, also in the longer term.

G-CSF and its derivatives have several drawbacks, as they are associated with side effects that include bone pain, allergic reactions, or pulmonary toxicity and cytokine release syndromes. In addition, extended therapy duration or dosages may lead to an increased risk for the development of secondary hematologic malignancies. Other approved drugs that also act on the bone marrow, such as CXCR4 antagonists (e.g. AMD3100/Plerixafor) or VLA4 antagonists (e.g. Firategrast), do not achieve clinically meaningful mobilization of neutrophil granulocytes and are therefore not relevant in the treatment of neutropenia. Therefore, the inventors sought novel therapeutic approaches to treat cytopenia, specifically neutropenia, that might enhance or replace current therapies and at the same time incur fewer side effects.

Without being bound by theory, the present invention identifies means for pharmacological manipulation of leukocyte compartment sizes that may prove useful to tune host defenses, whereby none such approaches have been identified previously for the treatment of cytopenia, or more specifically leukopenia, in particular neutropenia.

The inventors have surprisingly found that small inhibitors (so-called "small molecules") of the histone acetyltransferase (HAT) domain of E1 A-associated protein (EP300, also called p300) or its ortholog cyclic adenosine monophosphate response element binding protein (“CREBBP” or “CBP”), which compete with acetyl CoA for the catalytic center of the aforementioned domain, induce a rapid onset and transient mobilization of white blood cells (leukocytes) including neutrophil granulocytes. This novel effect is entirely unexpected, since to date it has never been suggested in the art that pharmacological inhibition of the CBP/p300 HAT domain can induce rapid onset and transient bone marrow mobilization. P300 and CREB-binding protein (CREBBP/ CBP) are histone acetyltransferase orthologues with >90% sequency homology that play an important physiological role by acting as transcriptional co-activators in various cellular processes and diseases.

In the examples provided herein, the inventors further demonstrate that competitive, small molecule-mediated inhibition of the CBP/p300 HAT domain triggers acute and transient leukocyte mobilization from the bone marrow, which, without being bound by theory, may be relayed by a neuroendocrine loop of the HPA-axis, a process that is distinct from glucocorticoids. As is shown in more detail below, the inventors surprisingly found that competitive inhibition of the CBP/p300 histone acetyltransferase (HAT) domain, e.g., by the small molecule A485, triggers acute and transient mobilization of leukocytes from the bone marrow into the blood. In the examples, these effects were maintained in models of leukopenia and equally as potent, but mechanistically distinct from G-CSF.

In their experiments the inventors were surprisingly able to mobilize cells from damaged bone marrow, thereby restoring physiological numbers of these cells by treating animals with an inhibitor of a HAT activity of CBP/p300 according to the present invention. Unexpectedly this effect was able to protect animals with bone marrow injury against death when they contracted an infection (akin to the clinical scenario of neutropenic fever).

Furthermore, the inventors demonstrate that a combination therapy of competitive inhibitors of the CBP/p300 histone acetyltransferase (HAT) domain and G-CSF showed an unexpected synergistic neutrophil mobilization. The examples reveal that CBP/p300 histone acetyltransferase (HAT) domain inhibition leads to central activation of the hypothalamus-pituitary-adrenal (HPA)- axis, which relays shifts in leukocyte distribution through corticotropin-releasing hormone receptor 1 (CRHR1) and adrenocorticotropic hormone (ACTH), but independent of glucocorticoids. Further, in contrast to G-CSF, CBP/p300 HAT inhibition did not exert a persistent effect on leukocytes or neutrophil granulocytes, which may be advantageous in various clinical scenarios (e.g., infections). Moreover, as shown in the examples, the present approach activates the so- called hypothalamic-pituitary-adrenocortical (HPA)-axis, where activation of a specific receptor (corticotropin releasing hormone receptor 1) is required for bone marrow mobilization. This phenomenon is also clinically relevant in the context of stresses on the organism, such as infections. In summary, the present invention represents a novel approach for rapid expansion of the blood leukocyte compartment via a neuroendocrine loop which can be applied for the treatment of human diseases, such as cytopenia.

In embodiments, the invention relates to an inhibitor for use according to the invention wherein the inhibitor suppresses a HAT domain of CBP/p300. In embodiments the inhibitor inhibits, sterically interferes and/or blocks (reversibly or irreversibly) the HAT domain of CBP/p300.

In embodiments, the inhibitor suppresses the activity of the HAT domain of CBP/p300 by competing with acetyl CoA for binding to the HAT domain (preferably thereby suppressing HAT enzymatic activity).

Preferably the inhibitor according to the present invention has no or negligible “off target” effects, such as inhibition of other functions or functional domains of CBP/p300. In embodiments, the inhibitor of a histone acetyl transferase (HAT) activity of CREBBP (CBP) I p300 according to the invention may also be used in the prevention of and/or the treatment of a suspected cytopenia.

In embodiments, the inhibitor is a small molecule compound.

In embodiments, the inhibitor is a spirocyclic HAT inhibitor. In embodiments the spirocyclic HAT inhibitor is A-485 or salts or derivatives thereof. In embodiments the spirocyclic HAT inhibitor is IP300w or salts or derivatives thereof. In embodiments the spirocyclic HAT inhibitor is A-485 or IP300w or salts or derivatives thereof. In embodiments, the inhibitor is a non-spirocyclic HAT inhibitor. In embodiments the non-spirocyclic HAT inhibitor is C-646.

In embodiments, the inhibitor is selected from A-485, IP300w, CPI-1612 or salts or derivatives thereof.

In embodiments, the inhibitor is selected from A-485, C-646, IP300w, CPI-1612 or salts or derivatives thereof.

In embodiments, the inhibitor is selected from A-485, IP300w, CPI-1612, PU141 , B026, PU139, EML-425, Histone Acetyltransferase Inhibitor II, DS-9300, or salts or derivatives thereof.

In embodiments, the inhibitor is PU141 or salts or derivatives thereof. In embodiments, the inhibitor is B026 or salts or derivatives thereof. In embodiments, the inhibitor is PU139 or salts or derivatives thereof. In embodiments, the inhibitor is EML-425 or salts or derivatives thereof. In some embodiments, the inhibitor is Anacardic Acid or salts or derivatives thereof. In embodiments, the inhibitor is Histone Acetyltransferase Inhibitor II or salts or derivatives thereof. In embodiments, the inhibitor is DS-9300, or salts or derivatives thereof.

In some embodiments, the inhibitor is selected from A-485, IP300w, CPI-1612, PU141 , B026, PU139, EML-425, Histone Acetyltransferase Inhibitor II, DS-9300, or salts or derivatives thereof. In other some embodiments, the inhibitor is selected from A-485, C-646, IP300w, CPI-1612, PU141 , B026, PU139, EML-425, Anacardic Acid, Histone Acetyltransferase Inhibitor II, DS-9300, or salts or derivatives thereof.

In some embodiments, the inhibitor is selected from A-485, C-646, IP300w, CPI-1612, PU141 , B026, PU139, EML-425, Anacardic Acid, Histone Acetyltransferase Inhibitor II, Curcumin, DS- 9300, or salts or derivatives thereof. In embodiments, the inhibitor is A-485

A485 is the most specific CBP/p300 HAT inhibitor identified to date ⁶². A485 was validated by the provider AbbVie against a range of non-epigenetic targets without the detection of any biologically relevant off-target interactions. Likewise, also the inventors screened A485 against a panel of more than 300 human GPCRs and also did not find evidence for off-target effects. In line with biological specificity, CBP HAT inhibitors, such as A485, selectively mobilized leukocytes from the bone marrow, but did not affect platelet counts, red blood cells or hematopoietic precursor cells in the bone marrow. Conversely, more unspecific substances, such as curcumin, have been shown to inhibit p300, but also possess a wide range of side and off-target effects on an array of cellular targets including NFkB, COX enzymes, iNOS, various MAPK etc. ⁶³, limiting their therapeutic utility.

In embodiments, the inhibitor is C646

In embodiments, the inhibitor is CPI-1612

In embodiments the inhibitor is IP300w

In embodiments, the inhibitor induces acute and/or transient leukocytosis comprising mobilization of leukocytes from the bone marrow into the blood of a subject. In embodiments, the inhibitor induces acute and/or transient leukocytosis. In embodiments, the inhibitor induces the mobilization (release) of leukocytes from the bone marrow into the blood of a subject.

In embodiments wherein the subject is immunocompromised and/or is suffering from leukopenia, “leukocytosis” may herein generally refer to an increase of leukocytes (e.g., in the blood) in a subject, as in some of said subjects even if a large mass of leukocytes is mobilized, this may only “recover” leukocyte numbers within physiological (“healthy”, normal) ranges such that this effect would not fall under the general condition described as “leukocytosis”.

Therefore, in embodiments the leukocytosis (comprising mobilization of leukocytes from the bone marrow into the blood) induced in a subject aims to restore I recover blood cells, preferably leukocytes and/or neutrophiles, in the subject in a homeostatic range. In embodiments, when used in the medical-therapeutic field, the administration of the inhibitor according to the invention may primarily aim to treat patients with leukopenia and/or neutropenia, in whom the induced (strong) mobilization of leukocytes would not directly resemble the medical condition of ‘leukocytosis’, but rather a "balancing" of the cytopenia (namely the increase of the abundance/concentration of (white) blood cells) in the subject in a homeostatic range (recovery of normal/healthy (white) blood cell levels).

In embodiments, the inhibitor induces acute and/or transient leukocytosis comprising mobilization of leukocytes from the bone marrow into the blood of a subject, thereby restoring (the abundance/concentration of) blood cells, preferably leukocytes and/or neutrophiles, in the subject in a homeostatic range.

In embodiments, the inhibitor induces acute and/or transient leukocytosis comprising mobilization (release) of leukocytes from the bone marrow into the blood of a subject and/or the production and/or maturation of new leucocytes in the bone marrow.

In embodiments, acute leukocytosis refers to an onset, preferably a rapid onset, of leukocytosis within 10, 15, 20, 30, 40, 45, 50 or 60 minutes, or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 36, 40, 45, 48, 50, 60 or 72 hours or an a onset within less than 30, 40, 45, 50 or 60 minutes or less than 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, 36, 48, or 72 hours upon I after administration of the inhibitor according to the invention. In embodiments, acute leukocytosis refers to an onset, preferably a rapid onset, of leukocytosis within 30 minutes to 12 hours or within 15 minutes to 24 hours.

In embodiments, transient leukocytosis refers to leukocytosis that has only a short duration, or a shorter duration compared to standard G-CSF administration, preferably leukocytosis with a duration between 30 minutes and 48 hours, more preferably leukocytosis with a duration between 1 and 24 hours, or between 1 and 12 hours, or leukocytosis with a duration of 15, 20, 30, 40, 45, 50 or 60 minutes, or of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30 hours or of a duration of less than 3, 6, 12, 24, 36 or 48 hours upon I after administration of the inhibitor according to the invention.

In the context of the present invention, it may, in embodiments, be possible that the acute and/or transient leukocytosis is induced instead of or in addition to the bone marrow, from additional sources of leukocytes, such as, without limitation thereto, demargination of cells from peripheral vessels. However, herein the inventors provide strong and conclusive evidence that the cells mobilized by CBP/P300 HAT inhibition are recruited from the bone marrow, however, e.g., additional demargination phenomena may in embodiments not be ruled out.

In general, the medical condition of leukocytosis is a condition wherein white blood cells (leukocytes) are present in the blood in a concentration that is elevated (e.g., compared to a reference range of healthy individuals, or) above the upper limit of the reference range of healthy individuals. The aforementioned reference range is sex- and age-dependent and usually defined by the testing laboratory. The term “healthy” is broadly defined by the absence of disease and specifically, infection.

In preferred embodiments, the leukocytosis induced by the treatments according to the invention is a condition wherein white blood cells (leukocytes) are present in the blood at a concentration, level or amount that is elevated or increased compared to a previous concentration, level or range, namely wherein the leucocyte count in the blood of an individual is increased or elevated above a previous count (e.g., a leucocyte count during cytopenia) and preferably does not, or only shortly exceeds the upper limit of the reference range of healthy individuals and primarily restores leukocyte counts to this range. In the context of the present invention the leukocytosis induced by the present treatments preferably comprises the mobilization of leukocytes from the bone marrow into the blood of a subject and/or the production and/or maturation of new leucocytes in the bone marrow. Therefore, in embodiments the leukocytosis (comprising mobilization of leukocytes from the bone marrow into the blood) induced in a subject suffering from cytopenia (preferably leuko- and/or neutropenia) by the present inhibitors aims to restore I recover blood cells, preferably leukocytes and/or neutrophiles, in the subject in a homeostatic range.

In some embodiments, a white blood cell count of > 11 ,000 cells per pl of blood is considered leukocytosis. In some embodiments a leukocyte count above > 11 ,000 cells per pl of blood is considered an increased white blood cell count. In some embodiments an elevated white blood cell count is called a “leukemoid reaction”, comprising a count of about > 25,000-50,000 white blood cells per pL blood or of ~25, 000-50, 000 WBC/mm3, e.g., as a reaction of a healthy bone marrow to e.g., infection. These exemplary reference ranges do not serve as universal definitions of leukocytosis and may require adjustments depending on sex, age and the method used to quantify leukocytes or their subsets.

In embodiments, the inhibitor induces rapid onset and/or transient (1-24 hours) leukocytosis in a subject. In some preferred embodiments, the inhibitor induces rapid onset and transient leukocytosis in a subject. In embodiments, the inhibitor induces rapid onset of and/or transient leukocytosis in a subject, wherein the leukocytosis is induced for 1-24 hours. In embodiments, the leukocytosis is induced for 1-12 hours, for 1-6 hours, for 1-36 hours or for 1-48 hours and even longer.

At present, for patients suffering from cytopenia, the treatment of choice comprises the administration of G-CSF. However, in certain embodiments these individuals may further benefit from a combination treatment comprising G-CSF and HATi, due to the superior leukocyte mobilization and/or more rapid onset of this effect induced by a HATi-treatment. One advantage of the use of the present inhibitor in the treatment of cytopenia, especially leukopenia or neutropenia, compared to the prior art approach of G-CSF mono-administration is considered the rapid onset and/or shorter duration of the modulation of leukocyte compartments. This advantage may in embodiments especially be relevant for immunocompromised or immunocompetent subjects suffering from acute infections, or who are in danger of contracting them. Particularly in said subjects G-CSF is currently not routinely administered, in part due to the herein described drawbacks.

In further embodiments, patients who do not suffer from leukopenia, or who are leukopenic despite G-CSF treatment, and who also develop an infection may benefit from HATi monotherapy according to the invention (e.g., due to a short neutrophil mobilization for host defense). In embodiments the present inhibitor may be also particularly favorable in cases where only short bursts of neutrophil mobilization are required, such as in acute infection. In fact, unresolved monocytosis and neutrophilia may be detrimental in such scenario due to the risk of excessive inflammation, collateral tissue damage and organ failure. In other embodiments, the present combination therapy with G-CSF facilitates additional long-term hematopoietic recovery. In embodiments the present inhibitor may be applied as an alternative, a complementary and/or synergistic pharmacological strategy to G-CSF derivatives in the treatment of various conditions, e.g., where a rapid and short-term modulation of leukocyte compartments is advantageous.

In embodiments, the leukocytosis comprises B-lymphocytosis and/or neutrophilia. In general, the leukocytosis induced by the inhibitor according to the invention may comprise neutrophilia, lymphocytosis, monocytosis, eosinophilia and/or basophilia.

In embodiments, the inhibitor according to the invention induces central activation of the hypothalamus-pituitary-adrenal (HPA)-axis in a subject. In embodiments, the inhibitor according to the invention is a spirocyclic HAT inhibitor that induces central activation of the hypothalamus- pituitary-adrenal (HPA)-axis in a subject. In embodiments, the inhibitor according to the invention is A-485, which induces central activation of the hypothalamus-pituitary-adrenal (HPA)-axis in a subject.

In embodiments, the activation of the HPA-axis results in corticotropin-releasing hormone receptor 1 (CRHRI)-dependent release of ACTH and/or glucocorticoid release in a subject.

In embodiments, the cytopenia is a leukopenia or a neutropenia. In some preferred embodiments the cytopenia is a leukopenia. In some preferred embodiments the cytopenia is a neutropenia.

In embodiments, the patient has been diagnosed with cancer. In embodiments the patient has been diagnosed with cancer resulting in cytopenia. In embodiments, the patient has been diagnosed with cancer of hematological or non-hematological origin.

In embodiments the patient has been diagnosed with cancer and/or a genetic or acquired bone marrow disorder resulting in cytopenia. In embodiments the patient is experiencing/suffering from cytopenia, preferably leukopenia or neutropenia, wherein the cytopenia is associated, suspected to be caused by or caused by a cancer present in said patient.

In embodiments the patient is suffering from cytopenia caused by or associated with a cancer treatment the patient has been and/or is receiving, which may include chemotherapy, irradiation, a combination of the two or irradiation followed by bone marrow transplantation. In embodiments the patient is experiencing/suffering from cytopenia, preferably leukopenia or neutropenia, wherein the cytopenia is associated, suspected to be caused by or caused by a cancer-treatment said patient is or has been receiving.

In embodiments the patient has been or is receiving a cancer treatment comprising cytotoxic chemotherapeutics.

In embodiments of the inhibitor for use according to the invention CD34+ stem cells are mobilized, preferably by the inhibitor treatment, in a subject for autologous (donor and recipient are identical; the HATi is administered to said subject) or allogenic (donor and recipient are two independent individuals; the HATi is administered to the donor) stem cell transplantation preferably to restore hematopoiesis and/or to cure any form of bone marrow pathology. In such embodiments recipients of an allogenic graft may or may not receive a HATi (treatment) following transplantation to accelerate recovery of hematopoiesis.

In embodiments the patient exhibits one or more of the symptoms comprising swollen lymph nodes, recurrent infections (in embodiments including gingivitis), opportunistic viral, fungal and/or bacterial infections, severe infections (in embodiments including sepsis), fatigue, sore throat (pharyngitis), sweating or chills, fever (continuous, recurring or remittent), neutropenic fever (febrile neutropenia), muscle weakness, shortness of breath, severe cough, pneumonia, urinary symptoms, swelling of any part of the body, rashes and/or redness, mouth sores (ulcers) and/or diarrhea. Additional symptoms may in embodiments include any manifestation of graft vs. host disease such as colitis or skin pathology as well as polydipsia, polyuria, listlessness, weight loss, hypoglycemia or loss of appetite (the latter may be indicative of adrenal insufficiency). In embodiments the patient exhibits a cell count of ~ 1 ,000 - 1 ,500 neutrophils per microliter of blood (indicative of mild neutropenia). In embodiments the patient exhibits a cell count of between ~ 500 - 1 ,000 neutrophils per microliter of blood (indicative of moderate neutropenia). In embodiments the patient exhibits a cell count of < 500 neutrophils per microliter of blood (indicative of severe neutropenia).

In embodiments, neutropenia causes neutropenic fever (febrile neutropenia) in a subject, which is accompanied often with other signs of (severe) infection. In embodiments, neutropenic fever is associated with a patient’s body temperature of over 38.0°C. In embodiments the patient exhibits one or more of the symptoms comprising neutropenic fever (febrile neutropenia) including elevated body temperature of over 38.0°C (fever), sweating and/or chills, and signs of severe infections (in embodiments including sepsis), single or multiorgan failure, bleeding, hypotension and neurological symptoms in the presence of neutropenia.

In embodiments the inhibitor is administered once or on two or more consecutive days.

In embodiments the inhibitor is administered orally or parenterally.

In embodiments A-485 is administered via injection, e.g., intravenously, intramuscularly, subcutaneously or intrathecally. In embodiments A-485 is administered at a concentration of between 1-500 mg/kg. In embodiments A-485 is administered at 100 mg/kg.

In embodiments, the inhibitor of a histone acetyl transferase (HAT) activity of CREBBP (CBP) I p300 for use according to the present invention is administered upon diagnosis of cytopenia, preferably leukopenia or neutropenia, during the nadir of the cytopenia and/or upon occurrence of symptoms thereof. Additionally, in embodiments the inhibitor may be administered prior to administration of chemotherapeutic drugs, irradiation or any other therapeutic intervention aiming to treat an underlying disease including malignancy or bone marrow failure. In embodiments, the inhibitor according to the invention is administered prior to administration of a cancer treatment or bone marrow disease (or failure).

In embodiments, the inhibitor of a histone acetyl transferase (HAT) activity of CREBBP (CBP) I p300 is administered 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 45, 50 or 60 minutes, or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 36, 40, 45, 48, 50, 60 or 72 hours or 1 , 2, 3, 4, 5, 6, 7, 14, 21 or up to 28 days after the diagnosis of cytopenia, preferably leukopenia or neutropenia, and/or the occurrence of symptoms thereof. In embodiments, the inhibitor of a HAT activity of CBP/ p300 is administered during cancer and/or glucocorticoid and/or anti-infective therapy or after said therapy was discontinued. In embodiments, the inhibitor of a histone acetyl transferase (HAT) activity of CREBBP (CBP) I p300 is administered up to 7 days prior to therapeutic interventions, which aim at treating an underlying disease such as malignancy or bone marrow failure. In embodiments a non-limiting example of such therapeutic interventions is stem cell mobilization prior to chemotherapy for (subsequent) autologous transfer (after chemotherapy).

In embodiments of the inhibitor of a histone acetyl transferase (HAT) activity of CREBBP (CBP) I p300 is administered at least once to the subject. In embodiments the inhibitor of a HAT activity of CBP I p300 is administered at least twice, either on consecutive days, or on each other day. Accordingly, the subject may receive more than one dose of the inhibitor of a HAT activity of CBP/p300, wherein preferably not more than one dose per day is administered. In further embodiments, the subject receives at least 2, 3, 4 or 5 dosages of the inhibitor of a HAT activity CBP/p300 of the invention. In embodiments, the inhibitor of a HAT activity CBP/p300 is administered to the subject at least every 2 weeks, preferably every 1-4 weeks.

In embodiments G-CSF, recombinant G-CSF, or a G-CSF analogues (e.g., filgrastim, pegylated filgrastim and all/any of its biosimilars) is administered in combination and/or simultaneously (to the inhibitor of a HAT activity of CBP/p300) to a subject. In embodiments the G-CSF, recombinant G-CSF, or a G-CSF analogues is selected from filgrastim, pegylated filgrastim and all of its biosimilars. In embodiments G-CSF analogues are selected from filgrastim, pegylated filgrastim and optionally all of their biosimilars.

In embodiments G-CSF is administered in combination and/or simultaneously (to the inhibitor of a HAT activity of CBP/p300) to a subject. In embodiments recombinant G-CSF is administered in combination and/or simultaneously (to the inhibitor of a HAT activity of CBP/p300) to a subject. In embodiments a G-CSF analogue is administered in combination and/or simultaneously (to the inhibitor of a HAT activity of CBP/p300) to a subject.

In embodiments an anti-infectant, preferably an antibiotic, a virostatic, an antiviral and/or an antimycotic compound, is administered in combination and/or simultaneously to a subject. Said subject may be immunocompromised or not.

In another aspect the present invention relates to a pharmaceutical composition for use in the treatment of cytopenia comprising the inhibitor according to the present invention.

Surprisingly, the inventors found that a combination therapy of inhibitors of the CBP/p300 histone acetyltransferase (HAT) domain and G-CSF showed synergistic neutrophil mobilization in patients.

Therefore, in another preferred aspect the present invention relates to a combination medication for use in the treatment of cytopenia, comprising an inhibitor according to the present invention and a compound selected from the group comprising G-CSF, recombinant G-CSF and/or a G- CSF analogues.

Therefore, in embodiments the invention relates to a combination medication for use in the treatment of cytopenia comprising an inhibitor of a histone acetyl transferase (HAT) activity of CREBBP (CBP) I p300 and a compound selected from the group comprising G-CSF, recombinant G-CSF and/or a G-CSF analogue.

In embodiments the invention relates to a combination medication for use according to the invention comprising an inhibitor of a histone acetyl transferase (HAT) activity of CREBBP (CBP) I p300 that suppresses a HAT domain of CBP/p300 and a compound selected from the group comprising G-CSF, recombinant G-CSF and/or a G-CSF analogue. In embodiments the invention relates to a combination medication for use according to the invention comprising a small molecule compound inhibitor of a histone acetyl transferase (HAT) activity of CREBBP (CBP) I p300 and a compound selected from the group comprising G-CSF, recombinant G-CSF and/or a G-CSF analogue.

In embodiments the invention relates to a combination medication for use according to the invention comprising a small molecule compound inhibitor of a histone acetyl transferase (HAT) activity of CREBBP (CBP) I p300 and a compound selected from the group comprising G-CSF, recombinant G-CSF and/or a G-CSF analogue, wherein the inhibitor is a spirocyclic HAT inhibitor, such as e.g., A-485 or salts or derivatives thereof.

In embodiments the combination medication for use according to the invention comprises A-485, or salts or derivatives thereof, as inhibitor of a histone acetyl transferase (HAT) activity of CREBBP (CBP) I p300, and a compound selected from the group comprising G-CSF, recombinant G-CSF and/or a G-CSF analogues.

In embodiments the combination medication for use according to the invention comprises a small molecule compound inhibitor of a histone acetyl transferase (HAT) activity of CREBB p300 and a compound selected from the group comprising G-CSF, recombinant G-C G-CSF analogue, wherein the inhibitor is a non-spirocyclic HAT inhibitor, such as e.g salts or derivatives thereof. In embodiments the combination medication comprises C salts or derivatives thereof, as inhibitor of a histone acetyl transferase (HAT) activity

(CBP) I p300, and a compound selected from the group comprising G-CSF, recombinant G-CSF and/or a G-CSF analogues.

In embodiments the combination medication comprises an inhibitor of a histone acetyl transferase (HAT) activity of CREBBP (CBP) I p300 selected from A-485, C-646, IP300w, CPI-1612, or salts or derivatives thereof, and a compound selected from the group comprising G-CSF, recombinant G-CSF and/or a G-CSF analogues.

In embodiments the combination medication for use according to the invention is administered once to the subject.

In embodiments the combination medication may be administered to patients undergoing chemotherapy (e.g., in parallel or afterwards) and/or stem cell transplantation, or to subjects suffering from any other type of bone marrow pathology. In some embodiments the combination medication according to the invention is particularly advantageous as a rapid, but also persistent stimulation of granulopoiesis is not only desired but may be vital. In certain embodiments the synergistic effect achieved using said combination medication enables improved leukocyte and/or stem cell mobilization, reduced number of administration events and/or a reduced dosage of one or both agents of the combination medication, thereby advantageously limiting side effects of the treatment and improving disease outcomes including diminished leukopenia nadirs following chemotherapy administration, improved engraftment following allo-/autologous stem cell transplantation, infection and other embodiments previously described. Moreover, in respective embodiments the combination medication may optimize therapeutic outcomes in individuals, who respond insufficiently to G-CSF or G-CSF analogue monotherapy (“poor responders”), e.g., in the context of bone marrow mobilization for subsequent stem cell therapy.

In embodiments the combination medication for use according to the invention is administered at least twice, either at least on two consecutive days or with at least one, two, three, four, five, six, seven or even more days in between each administration.

In embodiments, the CBP/p300 HAT inhibitor is administered as a monotherapy to an immunocompromised or immunocompetent individual suffering from leukopenia and/or an acute infection. In embodiments, the CBP/p300 HAT inhibitor is administered as a monotherapy to an immunocompromised or immunocompetent individual suffering from an acute infection. In embodiments, the CBP/p300 HAT inhibitor is administered as a monotherapy to an immunocompromised or immunocompetent individual suffering from leukopenia. Here, the shorter effect of the inhibitor is favored over G-CSF to avoid long term stimulation of leuko- and granulopoiesis, thereby limiting pathological sequalae, such as, without limitation thereto, excessive inflammation with resultant tissue damage.

The inventors further found that the inhibitor treatment according to the present invention induces leukocytosis and/or the mobilization of leukocytes from the bone marrow into the blood of a subject faster (within a shorter time) than conventional G-CDF treatment alone. Therefore, another advantage that the present combination medication provides is the fast, stable and reliable induction of leukocytosis and/or the mobilization of leukocytes from the bone marrow into the blood, as the effect of the HAT-inhibitor according to the invention induces these effects faster than G-CSF, while the combination of both agents achieves a synergistic efficacy regarding the overall achieved induction of leukocytosis and/or the mobilization of leukocytes from the bone marrow into the blood of a subject.

In addition, the combination medication according to the invention may further comprise in embodiments an anti-infectant, preferably an antibiotic, a virostatic, an antiviral and/or an antimycotic compound, that is administered in combination and/or simultaneously to a subject.

As opportunistic and/or severe infections or sepsis are side effects of cytopenia, the inclusion of an anti-infectant in the treatment of cytopenia may be required or of advantage.

In embodiments, the combination medication comprising an inhibitor of a histone acetyl transferase (HAT) activity of CREBBP (CBP) I p300 and a compound selected from the group comprising G-CSF, recombinant G-CSF and/or a G-CSF analogues, for use according to the present invention is administered upon diagnosis of cytopenia, preferably leukopenia or neutropenia, and/or upon occurrence of symptoms thereof. Alternatively, in embodiments the combination therapy is administered prior to administration of chemotherapeutic drugs, irradiation or any other therapeutic intervention to treat an underlying disease including malignancy or bone marrow failure. In embodiments the combination therapy is administered prior to administration of a cancer treatment. In embodiments, the combination medication is administered 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 45, 50 or 60 minutes, or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 36, 40, 45, 48, 50, 60 or 72 hours or 1 , 2, 3, 4, 5, 6, 7, 14, 21 or up to 28 days after the diagnosis of cytopenia, preferably leukopenia or neutropenia, and/or the occurrence of symptoms thereof. In embodiments, the combination medication comprising an inhibitor of a histone acetyl transferase (HAT) activity of CREBBP (CBP) I p300 and a compound selected from the group comprising G-CSF, recombinant G-CSF and/or a G-CSF analogue are administered during cancer and/or glucocorticoid and/or anti-infective therapy or after said therapy was discontinued. In embodiments, the inhibitor of a histone acetyl transferase (HAT) activity of CREBBP (CBP) I p300 is administered up to 7 days prior to therapeutic interventions, which aim at treating an underlying disease, such as, without limitation thereto, a malignancy or bone marrow failure.

In embodiments of the combination medication is administered at least once to the subject. In embodiments the combination medication is administered at least twice, either on consecutive days or on each other day. Accordingly, the subject may receive more than one dose of the combination therapy, wherein preferably not more than one dose per day is administered. In further embodiments, the subject receives at least 2, 3, 4 or 5 dosages of the combination medication of the invention. In embodiments, the combination medication is administered to the subject at least every 2 weeks, preferably every 1 -4 weeks. In embodiments the inhibitor of a histone acetyl transferase (HAT) activity of CREBBP (CBP) I p300 is administered before, during and/or after the administration of the compound selected from the group comprising G-CSF, recombinant G-CSF and/or a G-CSF analogue. In embodiments inhibitor of a histone acetyl transferase (HAT) activity of CREBBP (CBP) I p300 is administered at least once, or at least on two days, and at least once per day.

The present invention also relates to a method of treating subjects suffering from the various medical conditions disclosed herein. In embodiments, the method of treatment comprises preferably the administration of a therapeutically effective amount of a compound disclosed herein to a subject in need thereof.

In embodiments, the present invention relates to a method of treating a subject, preferably suffering from cytopenia, preferably from leukopenia, more preferably from neutropenia, the method comprising administration of a therapeutically effective amount of an inhibitor of a histone acetyl transferase (HAT) activity of CREBBP(CBP)/p300 to the subject in need thereof.

In embodiments the inhibitor treatment results in/induces the mobilization of leukocytes from the bone marrow into the blood of the subject, preferably thereby restoring the abundance of blood cells, preferably of leukocytes and/or neutrophiles, in the subject in a homeostatic range.

In embodiments, the subject has been diagnosed with cancer, and/or a genetic or acquired bone marrow disorder resulting in cytopenia and/or wherein the subject is suffering from cytopenia caused by or associated with a cancer treatment the patient has been and/or is receiving.

In embodiments, the inhibitor suppresses a HAT domain of CBP/p300 by competing with acetyl CoA to bind the HAT domain. In embodiments, the compound is a small molecule compound. In embodiments, the inhibitor is a spirocyclic HAT inhibitor or a non-spirocyclic HAT inhibitor. In embodiments, the inhibitor is selected from the group comprising A-485, IP300w, or CPI-1612. . In embodiments, the inhibitor is A-485. In embodiments, the inhibitor is IP300w. In embodiments, the inhibitor is CPI-1612. In embodiments, the inhibitor induces central activation of the hypothalamus-pituitary-adrenal (HPA)-axis in the subject, preferably wherein activation of the HPA-axis results in corticotropinreleasing hormone receptor 1 (CRHRI)-dependent release of ACTH and/or glucocorticoid release in the subject.

In embodiments, the present invention relates to a method of inducing acute and/or transient leukocytosis in a subject, preferably wherein the subject suffers from cytopenia, more preferably from leukopenia, even more preferably from neutropenia, the method comprising administering a therapeutically effective amount of an inhibitor of a histone acetyl transferase (HAT) activity of CREBBP(CBP)/p300 to the subject in need thereof.

In embodiments, the induced acute and/or transient leukocytosis, comprises mobilization of leukocytes from the bone marrow into the blood of the subject. In embodiments, the inhibitor treatment restores the abundance of blood cells, preferably of leukocytes and/or neutrophiles, in the subject in a homeostatic range.

In embodiments, the inhibitor induces central activation of the hypothalamus-pituitary-adrenal (HPA)-axis in the subject, preferably wherein activation of the HPA-axis results in corticotropinreleasing hormone receptor 1 (CRHRI)-dependent release of ACTH and/or glucocorticoid release in the subject.

In embodiments, the present invention relates to a method of inducing mobilization of leukocytes from the bone marrow into the blood of the subject, the method comprising administering a therapeutically effective amount of an inhibitor of a histone acetyl transferase (HAT) activity of CREBBP(CBP)/p300 to the subject in need thereof. In embodiments, the subject suffers from cytopenia, more preferably from leukopenia, even more preferably from neutropenia. In embodiments, the inhibitor treatment restores the abundance of blood cells, preferably of leukocytes and/or neutrophiles, in the subject in a homeostatic range.

In embodiments, the present invention relates to a method of restoring the abundance of blood cells, preferably of leukocytes and/or neutrophiles, in a subject in a homeostatic range, comprising administering a therapeutically effective amount of an inhibitor of a histone acetyl transferase (HAT) activity of CREBBP(CBP)/p300 to the subject in need thereof, preferably wherein the subject suffers from cytopenia, preferably from leukopenia, more preferably from neutropenia.

In embodiments, of the afore-mentioned methods the subject has been diagnosed with cancer, and/or a genetic or acquired bone marrow disorder resulting in cytopenia and/or wherein the subject is suffering from cytopenia caused by or associated with a cancer treatment the patient has been and/or is receiving. Each optional or preferred feature of the invention that is disclosed or described in the context of one aspect of the invention is herewith also disclosed in the context of the other aspects of the invention described herein.

All features disclosed in the context of the use of a HAT-inhibitor in the treatment of cytopenia (e.g., neutropenia and/or leukopenia) in a subject according to the invention also relate to, and are herewith disclosed also in the context of the combination medications and pharmaceutical compositions for use in the treatment of cytopenia in a subject, and vice versa.

DETAILED DESCRIPTION OF THE INVENTION

All cited documents of the patent and non-patent literature are hereby incorporated by reference in their entirety.

The present invention is directed to an inhibitor of a histone acetyl transferase (HAT) activity of CREBBP(CBP)/p300 for use in the treatment of cytopenia, wherein the inhibitor preferably suppresses a HAT domain of CBP/p300, and preferably induces acute and/or transient leukocytosis comprising mobilization of leukocytes from the bone marrow into the blood of a subject.

Herein a "subject" or “patient” may be a vertebrate, preferably a mammal, more preferably a human subject or patient. In the context of the present invention, the term "subject" or “patient” includes both humans and animals, particularly mammals, more particularly humans, and other organisms.

Clinical conditions

"Cytopenia" refers to a clinical condition characterized by a deficiency of blood cells. In general, cytopenia and bone marrow insufficiency refer to a cluster of hematological disorders characterized by common features of decreased and/or ineffective blood cell production. Cytopenia and bone marrow insufficiency are accompanied by similar complications or side effects, such as an increased risk of developing leukemia, life threatening infections and the need for blood and/or platelet transfusions.

“White blood cells” (leucocytes) are part of the immune system, which are primarily produced in the bone marrow and protect the body against diseases and pathogen infection. Many homeostatic functions such as electrical conduction in the heart, metabolism, tissue repair or nutrient absorption (among many others) are also controlled, tuned or governed by leukocytes. Besides serving as an indicator of infections, elevated white blood cell counts are indicative of certain blood cancers or other bone marrow diseases.

One type of cytopenia is “leukopenia”, a clinical condition characterized by a lack of or reduced amounts of (compared to a healthy control) white blood cells. In relation to the five types of white blood cells (leukocytes) there exist five main types of leukocytosis, depending on which type of cell is affected, namely, neutrophilic leukocytosis (neutrophilia), lymphocytosis, monocytosis, eosinophilia and very rarely, basophilia. The clinical condition of “neutrophilic leukocytosis” (neutrophilia) is characterized by a high number of neutrophils (the most common type of white blood cell in humans that helps clear infections and heal damaged tissue. Although, the relative distribution of different leukocyte populations differs between species, neutrophilia is a conserved response to injury).

The clinical condition of “lymphocytosis” is characterized by a high number of lymphocytes (white blood cells that protect the lymphatic system).

The clinical condition of “monocytosis” is characterized by a high number of monocytes (white blood cells that help fight the immune system).

The clinical condition of “eosinophilia” is characterized by a high number of eosinophils (white blood cells that play a role in fighting infection and inflammation). Eosinophilia is common and is often associated with allergies, parasitic infections or autoimmune diseases.

The clinical condition of “basophilia”, which is the rarest form of leukocytosis, is characterized by an increased number of basophils (white blood cells playing a role in the response to allergic reactions, defense against parasitic infections and in the prevention of blood clotting).

“Neutropenia” refers to a clinical condition characterized by a drop (reduction, decrease) of blood neutrophil granulocyte numbers below the lower limit of the reference range of healthy individuals. Neutropenia can result from genetic or acquired causes and mostly arises from malfunctions of the bone marrow, where neutrophil granulocytes are produced. Neutropenia is also a frequent complication of medical interventions, such as chemotherapy in the context of cancer and other malignant diseases. Neutropenia is characterized by high mortality, as neutrophil granulocytes provide, among other functions, the initial defense against invading pathogens such as bacteria. Thus, methods for acute and long-term therapy of neutropenia are of high clinical relevance. Commonly, neutropenia is classified, depending on the number of neutrophils in the blood of a subject, into mild, moderate, or severe. The lower limit for adults that is considered healthy or normal is in embodiments between ~ 1 ,500 and ~1 ,800 neutrophils per microliter of blood. In embodiments mild neutropenia is diagnosed at between ~ 1 ,000 - 1 ,500 neutrophils per microliter of blood, moderate neutropenia at between ~ 500 - 1 ,000 neutrophils per microliter of blood and severe neutropenia at < 500 neutrophils per microliter of blood. One effect of neutropenia may be neutropenic fever (febrile neutropenia), which is defined as the combination of infection in the presence of neutropenia . Per definition, neutropenic fever is associated with a patient’s body temperature of over 38.0°C (i.e. fever) and may progress to fullblown sepsis with multiorgan failure. Neutropenic fever is linked to high mortality because no effective pharmacological treatments beyond supportive care exist. The latter includes antibiotics and fluid supplementation. However, in the absence of functional neutrophils, antibiotics are less effective. In embodiments herein, the treatment of neutropenia may also comprise the treatment of neutropenic fever (febrile neutropenia) and/or other related infectious complications.

Commonly leukopenia, such as neutropenia, can have various underlying causes such as genetic conditions, pharmaceuticals (drugs), cancer, autoimmune diseases, infections or even nutritional deficiencies. Genetic conditions causing leukopenia, such as neutropenia, particularly inherited leukopenia or neutropenia, may comprise, without being limited thereto, severe congenital neutropenia, benign ethnic neutropenia (BEN) and cyclic neutropenia.

Cancer and other bone marrow and/or blood malignancies may induce leukopenia, such as neutropenia, e.g., leukemias, lymphomas or other bone marrow and/or blood malignancies.

Pharmaceuticals (drugs) inducing leukopenia, such as neutropenia, may comprise, without being limited thereto, cancer treatments, e.g., chemotherapy and/or radiotherapy, or other drugs including analgesics (e.g., metamizole), antibiotics (e.g., ciprofloxacine) or anticonvulsants (e.g. valproic acid), among others. In general drug treatment, or cancer treatments may directly damage or destroy neutrophil granulocytes and/or affect or damage the bone marrow, which produces neutrophil granulocytes.

Autoimmune diseases may, e.g., through the production of (auto-)antibodies, induce destruction of healthy leukocytes, such as neutrophils. Autoimmune diseases may comprise, without being limited thereto, Crohn's disease, lupus and rheumatoid arthritis.

Also, infections, such as bacterial, viral and parasitic infections, e.g., HIV, hepatitis, sepsis, tuberculosis and Lyme disease and nutritional deficiencies, such as an insufficient intake of vitamins or minerals, e.g., of vitamin B12, folic acid or copper, may cause leukopenia, such as neutropenia.

In general, “leukocytosis” is a condition wherein white blood cells (leukocytes) are present in the blood in a concentration, level or range that is elevated or increased compared to a normal/healthy level or concentration, namely wherein the leucocyte count in the blood of an individual is increased or elevated above a level or concentration commonly considered normal or healthy (e.g., in a healthy individual, such as an individual not suffering from an acute infection). In other words, leukocytosis is a condition wherein white blood cells (leukocytes) are present in the blood in a concentration that is elevated above the upper limit of the reference range of healthy individuals. The aforementioned reference range is sex- and age-dependent and usually defined by the testing laboratory. The term “healthy” is broadly defined by the absence of disease and specifically, infection. In the context of the invention the leukocytosis (comprising mobilization of leukocytes from the bone marrow into the blood) induced in a subject preferably aims to restore I recover blood cells, preferably leukocytes and/or neutrophiles, in the subject suffering from cytopenia (preferably leuko- and/or neutropenia) into a homeostatic range. In embodiments the administration of the inhibitor according to the invention preferably aims to treat patients with cytopenia (preferably leukopenia and/or neutropenia, in whom the induced (strong) mobilization of leukocytes would not directly resemble the medical condition of ‘leukocytosis’, but rather a "balancing" of the cytopenia (namely the increase of the abundance/concentration of (white) blood cells) in the subject in a homeostatic range (recovery of normal/healthy (white) blood cell levels).

In some embodiments a white blood cell count of > 11 ,000 cells per pl of blood is considered leukocytosis. In some embodiments a leukocyte count above > 11 ,000 cells per pl of blood is considered an increased white blood cell count. In some embodiments an elevated white blood cell count is called a “leukemoid reaction”, comprising a count of about > 25,000-50,000 white blood cells per pL blood or of ~25, 000-50, 000 WBC/mm³, e.g., as a reaction of a healthy bone marrow to e.g., infection. In embodiments leukocytosis (especially neutrophilia) may be accompanied by a shift (so called "left upper shift") in the ratio of immature to mature neutrophils and macrophages. During common leukocytosis the proportion of immature leukocytes may increase in embodiments due to the proliferation and inhibition of granulocyte and monocyte precursors in the bone marrow, which are commonly stimulated, e.g., during an infection in the subject, by various inflammatory factors such as, e.g., G-CSF. In preferred embodiments herein, the leukocytosis induced by the treatments according to the invention is a condition wherein white blood cells (leukocytes) are present in the blood in a concentration, level or amount that is elevated or increased compared to a previous concentration, level or range, namely wherein the leucocyte count in the blood of an individual is increased or elevated above a previous count (e.g., a leucocyte count during cytopenia). In embodiments wherein the subject is immunocompromised and/or is suffering from leukopenia, “leukocytosis” may generally refer to an increase of leukocytes in a subject, as in some of said subjects even if a large mass of leukocytes is mobilized, this may only “recover” leukocyte numbers within physiological (“healthy”, normal) ranges such that this effect would not fall under the general condition described as “leukocytosis”.

A histone acetyltransferase (HAT) is an enzyme that acetylates lysine amino acids on histone or other proteins. The acetylation is accomplished by transferring an acetyl group from acetyl-CoA to form E-N-acetyl lysine. HATs may be divided into two classes, namely nuclear (Type A) and cytoplasmatic (Type B) HATs. Type A HATs play a role in gene regulation through acetylation of nucleosomal histones. Type A HATs, such as p300/CBP, comprise a bromodomain, which confers DNA binding. Type B HATs, e.g., Hat1 , lack a bromodomain and acetylate newly synthesized histones prior to their assembly into nucleosomes in the cytoplasm.

Common histone acetyltransferase inhibitors are A-485, C-646, IP300w, CPI-1612, PU141 , B026, PU139, EML-425, Anacardic Acid, Histone Acetyltransferase Inhibitor II, Curcumin, DS-9300, and salts or derivatives thereof.

The drug class of histone acetyltransferase inhibitors (HATi) is an established drug class commonly used in the treatment of various diseases, as described in McKenna et al., 2023 ⁶², Lasko et al., 2017 ²⁶ and others (e.g., Manzo F, Tambaro FP, Mai A, Altucci L. Histone acetyltransferase inhibitors and preclinical studies. Expert Opin Ther Pat. 2009 Jun;19(6):761-74. doi: 10.1517/13543770902895727).

Examples of further histone acetyltransferase inhibitors are described in the application WO 2016/044770 A1 from Abbvie Inc. (USA) and Lasko et al., 2017 ²⁶, which are incorporated herein by reference. In certain embodiments one or more of the compounds enlisted in said documents (WO 2016/044770 A1 ; Lasko et al, 2017²⁶) may also be used as HAT-inhibitor in the context of the present invention. Further exemplary methods of analyzing HAT activity and a characterization of certain HAT inhibitors are described in the examples herein and, for example, in the manuscript of McKenna et al., 2023 ⁶², which is incorporated by reference herein.

The skilled person is familiar with suitable assays for testing the histone acetyltransferase inhibitor activity of a compound. Exemplary assays are described, in the examples herein and, for example, by Waddell et al., 2020 ⁶¹. For example, histone acetyltransferases may be incubated with histone proteins, thereby facilitating the acetylation of specific lysine residues on the histone tails. A HATi may be added to said reaction, thereby blocking the acetylation of histones. The relative levels of site-specific histone acetylation may subsequently be measured via immunoblotting ⁶¹ or mass spectrometry.

“EP300” (E1A-associated protein p300, also referred to as “p300”) and its orthologue “CREBBP” (cyclic adenosine monophosphate response element binding protein, also known as “CBP”) share 90% sequence homology and are built from 8 functionally distinct domains, one of which confers histone acetyltransferase (HAT) activity²⁵. EP300 loss of function of p300 in animal models is sufficient to both impair hematopoiesis (when deleted prenatally) and to induce leukocytosis or leukemia in later life²³²⁴. P300/CBP comprise a bromodomain and acetylate histones H2A, H2B, H3 and H4, but also acetylate numerous non-histone substrates.

“A-485” (C25H24F4N4O5) is a potent, selective catalytic small molecule inhibitor of the catalytic active site of p300 and CBP where it competes with acetyl coenzyme A (acetyl-CoA) for binding (acetyl-CoA-competitive p300/CBP catalytic inhibitor) ²⁶ (Fig. 7 A, B). A-485 is selective over BET bromodomain proteins and was shown to inhibit the activity of the p300-BHC (bromodomain HAT- C/H3) domain ²⁶. A-485 is considered to only inhibit H3K27AC and H3K18AC. Herein, A-485 is considered a spirocyclic compound or may also be termed an “indane spiro-oxazolidinedione”.

In general, spirocyclic compounds are compounds comprising at least two molecular rings with only one shared atom (e.g., with two rings connected through a single common atom).

“C646” (C24H19N3O6) is a reversible histone acetyltransferase (HAT) inhibitor, e.g., for p300. Herein, C646 is considered a non-spirocyclic HAT inhibitor. C646 treatment was shown to reduce histone H3 and H4 acetylation levels.

Herein, an “inhibitor according to the invention” is an inhibitor of a histone acetyl transferase (HAT) activity of CREBBP (CBP) I p300.

In embodiments an example of an indane spiro-oxazolidinedione (spirocyclic compound) may be A-485, an example of a spiro-hydantoin may be iP300w, and an example of an aminopyridine may be CPI-1612.

The present invention encompasses both treatment and prophylactic treatment of a subject. A "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology. The present invention relates further to pharmaceutically acceptable salts of the compounds described herein. The term "pharmaceutically acceptable salt " refers to salts or esters prepared by conventional means that include basic salts of inorganic and organic acids, including but not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, maleic acid, oxalic acid, tartaric acid, citric acid, malic acid, acetic acid, lactic acid, fumaric acid, succinic acid, salicylic acid, benzoic acid, phenylacetic acid, mandelic acid and the like. Any chemical compound recited in this specification may alternatively be administered as a pharmaceutically acceptable salt thereof.

"Pharmaceutically acceptable salts" are also inclusive of the free acid, base, and zwitterionic forms. Descriptions of suitable pharmaceutically acceptable salts can be found in Handbook of Pharmaceutical Salts, Properties, Selection and Use, Wiley VCH (2002). For therapeutic use, salts of the compounds are those wherein the counter-ion is pharmaceutically acceptable. However, salts of acids and bases which are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.

A dotted line in the position of a double bond represents an optional double bond, which may be present or absent.

Protected derivatives of the disclosed compound also are contemplated. A variety of suitable protecting groups for use with the disclosed compounds are disclosed in Greene and Wuts Protective Groups in Organic Synthesis; 3rd Ed.; John Wiley & Sons, New York, 1999. In general, protecting groups are removed under conditions which will not affect the remaining portion of the molecule. These methods are well known in the art and include acid hydrolysis, hydrogenolysis and the like.

It is understood that substituents and substitution patterns of the compounds described herein can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art and further by the methods set forth in this disclosure.

Small molecule drugs/compounds (for example, with a relatively low molecular weight of ~ less than 1000 daltons or 800 g/mol) are significantly smaller than antibodies, other biological molecules (biopharmaceuticals) and larger drug molecules. Therefore, small molecule compounds may in embodiments be administered orally because they may not by degraded within and may be absorbed from the gastrointestinal tract, and/or they are small enough to easily enter cells and/or cross the blood-brain barrier (BBB). Also, small molecule compounds may be synthesized chemically in large amounts at moderate cost, as they are relatively simple chemical compounds.

In certain embodiments, the inhibitors disclosed herein may be used for treating cytopenia in a subject.

Another aspect of the disclosure includes “pharmaceutical compositions” prepared for administration to a subject and which include a “therapeutically effective amount” of one or more of the compounds disclosed herein. In certain embodiments, the pharmaceutical compositions are useful for treating cytopenia, particularly leukopenia or neutropenia. The therapeutically effective amount of a disclosed compound will depend on the route of administration, the species of subject and the physical characteristics of the subject being treated. Specific factors that can be taken into account include disease severity and stage, weight, diet and concurrent medications. The relationship of these factors to determining a therapeutically effective amount of the disclosed compounds is understood by those of skill in the art.

Pharmaceutical compositions for administration to a subject can include at least one further pharmaceutically acceptable additive such as carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions can also include one or more additional active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like. The pharmaceutically acceptable carriers useful for these formulations are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 19th Edition (1995), describes compositions and formulations suitable for pharmaceutical delivery of the compounds herein disclosed.

Combined administration

According to the present invention, the term “combined administration”, otherwise known as joint treatment or co-administration, encompasses in embodiments the administration of separate formulations of the compounds described herein, whereby treatment may occur within minutes of each other, in the same hour, on the same day, in the same week or in the same month as one another. Alternating administration of two agents is considered as one embodiment of combined administration. Staggered administration is encompassed by the term combined administration, whereby one agent may be administered, followed by the later administration of a second agent, optionally followed by administration of the first agent, again, and so forth. Simultaneous administration of multiple agents is considered as one embodiment of combined administration. Simultaneous administration encompasses in some embodiments, for example the taking of multiple compositions comprising the multiple agents at the same time, e.g., orally by ingesting separate tablets simultaneously. A combination medicament, such as a single formulation comprising multiple agents disclosed herein, and optionally additional anti-infective, may also be used in order to co-administer the various components in a single administration or dosage.

A combined therapy or combined administration of one agent may precede or follow treatment with the other agent to be combined, by intervals ranging from minutes to weeks. In embodiments where the second agent and the first agent are administered separately, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the first and second agents would still be able to exert an advantageously combined synergistic effect on a treatment site. In such instances, it is contemplated that one would contact the subject with both modalities within about 1-96 hours of each other and, in embodiments preferably, within about 6-48 hours of each other. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) lapse between the respective administrations. According to the present invention, a “pharmaceutical combination” or “combination medicine” is the combined presence of an HAT-inhibitor according to the invention and a G-CSF or analogue thereof according to the invention, in proximity to one another. In one embodiment, the combination is suitable for combined administration.

In one embodiment, the pharmaceutical combination or combination medicine as described herein is characterized in that HAT-inhibitor according to the invention is in a pharmaceutical composition in admixture with a pharmaceutically acceptable carrier, and the G-CSF or analogue thereof is in a separate pharmaceutical composition in admixture with a pharmaceutically acceptable carrier, and - optionally - the anti-infective is in a separate pharmaceutical composition in admixture with a pharmaceutically acceptable carrier. The pharmaceutical combination of the present invention can therefore in some embodiments relate to the presence of two or three separate compositions or dosage forms in proximity to each other. The agents in combination are not required to be present in a single composition.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually contain injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

The pharmaceutical compositions can be administered by intramuscular, intravenous, subcutaneous, intra-arterial, intra-articular, intraperitoneal, intrathecal, intracerebroventricular, or parenteral routes. Optionally, compositions can be administered to subjects by a variety of administration modes, including oral delivery, or by topical delivery to other bodily surfaces.

The compositions of the disclosure can alternatively contain as pharmaceutically acceptable carrier substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate. For solid compositions, conventional nontoxic pharmaceutically acceptable vehicles can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.

In accordance with the various treatment methods of the disclosure, the compound can be delivered to a subject in a manner consistent with conventional methodologies associated with management of the disorder for which treatment or prevention is sought. In accordance with the disclosure herein, a prophylactically or therapeutically effective amount of the compound and/or other biologically active agent is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent, inhibit, and/or ameliorate a selected disease or condition or one or more symptom(s) thereof.

"Administration of and "administering a" compound should be understood to mean providing a compound, a prodrug of a compound, or a pharmaceutical composition as described herein. The compound or composition can be administered by another person to the subject (e.g., intravenously) or it can be self-administered by the subject (e.g., tablets).

Dosage can be varied by the attending clinician to maintain a desired concentration at a target site (for example, systemic circulation or the bone marrow). Higher or lower concentrations can be selected based on the mode of delivery, for example, oral delivery versus intravenous delivery. Dosage can also be adjusted based on the release rate of the administered formulation, for example, of a sustained release oral versus injected particulate delivery formulations, and so forth.

The present invention also relates to a method of treatment of subjects suffering from the various medical conditions disclosed herein. The method of treatment comprises preferably the administration of a therapeutically effective amount of a compound disclosed herein to a subject in need thereof.

A "therapeutically effective amount" refers to a quantity of a specified agent sufficient to achieve a desired effect in a subject being treated with that agent. For example, this may be the amount of a compound disclosed herein useful in treating a disease of cytopenia, particularly leukopenia or neutropenia, in a subject. The therapeutically effective amount or diagnostically effective amount of an agent will be dependent on the subject being treated, the severity of the affliction, and the manner of administration of the therapeutic composition. Dosage regimens can be adjusted to provide an optimum prophylactic or therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental side effects of the compound and/or other biologically active agent is outweighed in clinical terms by therapeutically beneficial effects.

As used herein, the term “subject that is or has been receiving (e.g., cancer) therapy” includes subjects that are currently under ongoing (e.g., cancer) therapy, or subjects that have received a (e.g., cancer) therapy that was either discontinued, for example after cytopenia, leukopenia or neutropenia and/or symptoms thereof occurred, or completed.

A non-limiting range for a dosage of an inhibitor according to the invention or a therapeutically effective amount thereof, or of another compound according to the present disclosure is about 0.001 mg/kg body weight to 500 mg/kg body weight, 0.01 mg/kg body weight to about 100 mg/kg body weight, such as about 0.05 mg kg to about 5 mg/kg body weight, or about 0.5 mg/kg to about 10 mg/kg body weight.

As used herein, the term “approximately” or “about” (“~”) is used to describe and account for small variations. For example, the term may refer to less than or equal to 10, such as less than or equal down to 1 , when appropriate, also the term may refer to more than or equal to 10, such as more than or equal up to 100 or more, when appropriate. It is to be understood that range format is used for the sake of simplicity and brevity and is to be flexibly understood to include numeric values expressly stated as boundaries of a range, encompassing each numeric value and subranges.

The instant disclosure also includes kits, packages and multi-container units containing the herein described pharmaceutical compositions, active ingredients, and/or means for administering the same for use in the prevention and treatment of diseases and other conditions in mammalian subjects.

FIGURES

The invention is further described by the following figures. These are not intended to limit the scope of the invention but represent preferred embodiments of aspects of the invention provided for greater illustration of the invention described herein.

Brief description of the figures:

Figure 1. CBP/p300 HAT inhibition triggers transient leukocytosis.

Figure 2. A485-induced leukocytosis augments host defenses.

Figure 3. A485 mobilizes leukocytes from the bone marrow.

Figure 4. Distinct effector pathways drive changes in leukocyte subsets in response to A485.

Figure 5. The HPA axis is activated by A485.

Figure 6. HPA-axis activity relays the A485 leukocyte response independently of glucocorticoids.

Figure 7. CBP/p300 HAT or bromodomain inhibition, related to Figure 1.

Figure 8. Pharmacokinetics of A485 and toxicity screen, related to Figure 1.

Figure 9. Comparing A485 to other drugs, related to Figure 1 .

Figure 10. Tissue origins of A485-induced leukocytosis and intravascular labeling, related to Figure 3.

Figure 11. A485, Cxcl12 and integrins, related to Figure 4.

Figure 12. Cytokines and the SNS, related to Figure 4.

Figure 13. Activation of the HPA-axis, related to Figure 5.

Figure 14. GC, CRHR1 and leukocytes, related to Figure 6.

Figure 15. Mc2r expression in the murine bone marrow, related to Figure 6.

Figure 16. Gating strategy for flow cytometry.

Figure 17. Gating strategy for flow cytometry: intravascular labeling, related to STAR Methods.

Figure 18. Gating strategy for flow cytometry. Figure 19. Gating strategy for flow cytometry.

Detailed description of the figures:

Figure 1. CBP/p300 HAT inhibition triggers transient leukocytosis. (A) Blood leukocytes in C57BL/6J wild-type (WT) mice 2 h following A485 (100 mg/kg) or vehicle injection (n = 4- 5/group). (B and C) Neutrophil, monocyte, and B and T lymphocyte counts in the same mice assessed by flow cytometry. (D) Blood leukocytes and neutrophils in response to increasing concentrations of A485 or vehicle solution (“0”) (n = 3/group). (E) Dynamic changes in blood leukocyte and neutrophil numbers at different time points following A485 injection (n = 5-9/time point). (F) Neutrophil kinetics in the blood over time in response to treatment with recombinant murine G-CSF i.v., A485 i.p., or the combination of the two drugs (n = 6-8/group). Quantification of the neutrophil area under the curve (AUC) (0-12 h) from the same animals is shown on the right. (G) Schematic illustration of the percentage of individuals in a cohort of patients with Rubinstein-Taybi syndrome (RSTS) exhibiting leukocyte counts above (elevated) or below (“normal”) the age-adjusted upper reference limit of normal (n = 46 total). (H) Percentage of patients with or without elevated leukocyte counts as stratified by mutational profile (HAT domain likely functionally affected: HATmut or not affected: HATwt). Data are shown as mean ± SEM or frequency of total *p < 0.05, **p < 0.01 , ***p < 0.001 , ****p < 0.0001 . (A-C) Unpaired, two-tailed Student’s t test (D-F) one-way ANOVA with Holm Sidak’s post hoc test.

Figure 2. A485-induced leukocytosis augments host defenses. (A) Leukocyte and neutrophil blood counts in 12-week-old female Vav1-NUP98-HOXD13 mice, a model for myelodysplastic syndrome (MDS), orwild-type littermate controls (WT) (n = 11- 12/group). (B) Blood leukocyte and neutrophils in MDS mice 2 h following A485 or vehicle treatment (n = 3/group). (C) Experimental design. (D) Leukocyte and neutrophil counts in naive (PBS injected) or 5-flurouracil (5FU)-treated C57BL/6J WT mice exposed to vehicle solution (white) or A485 (gray) on day 10 of the experiment (refers to experimental design in (C) (n = 3-5/group). Blood was collected 5 h post-injection. (E) Experimental design of the bone marrow injury and bacterial sepsis model. (F) Blood analysis of mice on day 6 following 5FU (150 mg/kg) exposure. Results are expressed as percentage of vehicle controls (n = 3/group). (G) Relative change in body weight from baseline in mice infected with 7.5 3 104 CFUs Listeria monocytogenes on day 6 after 5FU exposure, followed by treatment with a single bolus of A485 or vehicle 1 h post-infection, (n = 2-10/group; low sample size beyond day 3 due to mortality. Cross symbol indicates 100% mortality.) (H) Survival curves. 5FU-treated mice were challenged with 7.5 3 104 CFUs, while PBS controls received 1 3 105 CFUs. The latter group was sacrificed on day 9 without any signs of sickness. The dashed line corresponds to predicted survival of immunocompetent (PBS-treated) mice (n = 9-10 for 5FU groups, n = 3 for PBS group). (I and J) Number of colony-forming units (CFUs) of Listeria monocytogenes recovered from liver and spleen lysates on day 2 of the infection. Results were normalized according to tissue weight (n = 5/group). Data are shown as mean ± SEM. *p < 0.05, **p < 0.01 , ***p < 0.001 , ****p < 0.0001 . (A, B, and F) Unpaired, two-tailed Student’s t test (D) one-way ANOVA with Holm Sidak’s post hoc test. (G) Two-way ANOVA with Fisher’s least square difference (LSD), (H) Mantel-Cox test, and (I) Mann-Whitney U test. Figure 3. A485 mobilizes leukocytes from the bone marrow. (A) Experimental design. (B) Exemplary flow cytometry plot of adoptively transferred CD45.1 neutrophils in the blood and bone marrow 8 h post i.v. injection of cells. (C) Quantification of endogenous CD45.2 and transferred CD45.1 neutrophils in different organs 5 h after A485 injection expressed as percentage of the mean neutrophil number in the respective organ of vehicle-treated mice (n = 4/group). (D) Estimated loss of transferred CD45.1 neutrophils in the bone marrow vs. gain in the blood. (E) Experimental design of intravascular labeling of leukocytes using fluorophore conjugated anti- CD45 antibodies (left) with the corresponding gating strategy (right). Exemplary plots are shown. (F) Extravascular (referred to as “single CD45+ ”) and intravascular (“double CD45+ ”) neutrophils in lung tissue 5 h after A485 or vehicle injection. Results were normalized according to tissue weight (n = 5/group). (G) Intra- or extravascular neutrophils in the bone marrow of the same animals. (H) Quantification of leukocytes in the bone marrow and leukocytes and neutrophils in the blood 3 days after sub-lethal irradiation followed by injection of A485 or vehicle (n = 3-4/group for bone marrow and n = 7/group for blood). Data are shown as mean ± SEM. *p < 0.05, **p < 0.01 , ***p < 0.001 , ****p < 0.0001 . (C, F, and G) Unpaired, two-tailed Student’s t test (H) one-way ANOVA with Holm-Sidak’s post hoc test.

Figure 4. Distinct effector pathways drive changes in leukocyte subsets in response to A485. (A) Cell surface expression of the VLA44 subunit CD49d on B lymphocytes and neutrophils assessed by flow cytometry 5 h following A485 or vehicle injection (n = 5-6/group). (B) Quantification of Itga4 (encoding for Cd49d) mRNA levels in CD31+ and CD31- bone marrow cells 1 h following A485 or vehicle injection assessed by qPCR (n = 4/group). (C) Cell surface expression of Cd49d on CD45+ cells in bone marrow cultures following exposure to 5 mM A485 for 30 or 60 min at 37 or 4 deg C ex vivo. Results are expressed as fold change compared with vehicle-treated controls (n = 3/time point). (D) Itga4 mRNA levels in bone marrow cultures treated with vehicle or A485 (5 mM) for 30 min assessed by qPCR. (E) Dynamic changes in blood leukocytes and neutrophils upon VLA4 inhibition (by firategrast) or vehicle treatment expressed as percentage of the mean cell count at baseline (n = 4/group). A485 is overlayed for reference in light gray. (F) Blood neutrophils in mice with global genetic Myd88 deletion (Myd88 KO) following vehicle or A485 administration. Blood was collected at 2 h post-injection (n = 4-7/group). (G) Circulating G-CSF levels in vehicle and A485-treated mice 12 h post exposure to the respective substance (n = 8/group). (H) Peak blood neutrophil levels (5 h post injection) in A485- or vehicle- exposed mice following pre-treatment with an anti-G-CSF antibody or isotype control (IgG) (n = 4/group). (I) Immunoblot of acetylated lysine 18 of histone H3 (H3K18ac) in bone marrow cultures treated with recombinant murine G-CSF (rG-CSF) ex vivo. Stat3 phosphorylation was used as a positive control for confirmation of successful induction of CSF3R signaling. (J) Blood leukocytes and neutrophils in A485- or vehicle-treated mice following chemical sympathectomy by 6-OHDA (+) or vehicle (PBS) injection (-) (n = 4-7/group). Blood was collected 5 h after drug exposure. (K) Leukocytes and neutrophils in the blood 2 h following A485 or vehicle treatment with (+) or without (-) concomitant muscarinic receptor blockade by scopolamine (n = 4/group). Data are shown mean ± SEM. *p < 0.05, **p < 0.01 , ***p < 0.001 , ****p < 0.0001 . (A, B, D, F, and G) Unpaired, two-tailed Student’s t test. (C) Two-way ANOVA with Holm-Sidak’s post hoc test. (H, J, and K) One-way ANOVA with Holm-Sidak’s post hoc test. Figure 5. The HPA axis is activated by A485. (A) Circulating CORT levels over time assessed by ELISA in response to vehicle or A485 treatment. Baseline levels were determined in 4 randomly sampled mice (orange dot) prior to drug injection (n = 3-6/time point). (B) Design of the HPA-axis. Neurons of the paraventricular nucleus (PVN) secrete corticotropin-releasing hormone (CRH) into the hypophyseal portal system, which triggers adrenocorticotropic hormone (ACTH) release from the anterior pituitary via CRH receptor 1 (CRHR1). ACTH promotes glucocorticoid (GC) secretion from the adrenal cortex through melanocortin receptor 2 (Mc2r). GCs bind to the glucocorticoid receptor (Nr3c1) and exert negative feedback on HPAaxis activity. (C) CORT concentration in cell culture supernatants of adrenal cortical Y1 cells exposed to vehicle (DMSO), increasing concentrations of A485 or the cAMP inductor forskolin (5 mM) (n = 4/group). (D) cFos (immediate-early gene) transcript levels in hypothalamus cell lysates of A485- vs. vehicle-treated mice 1 h post-injection (n = 8/group). (E) Representative immunostaining of cFos in the PVN. “3V” denotes third ventricle. (F) Circulating ACTH concentration 1 h after A485 or vehicle injection (n = 8/group). (G) A485 abundance in hypothalamus (“hypo”) lysates or lysates of the brain excluding the hypothalamus (“brain”) measured by LC-MS/MS and normalized according to tissue weight (n = 4/group). (H) Glucose levels 45 min following A485 or vehicle administration (n = 3/group). (I) Rectal body temperature across the three indicated conditions 2 h after injection (n = 3-4/group). (J) CORT levels in the same mice. (K) CORT serum concentration in response to A485 or vehicle challenge in mice with (RTX) or without (DMSO) ablation of TRPV1+ sensory neurons measured by ELISA. Blood was collected 2 h post challenge (n = 3-4/group). (L) Circulating CORT levels in A485- or vehicle-treated mice 2 h following exposure to the respective substances. A subset of mice (+) received ketamine/xylazine (ket/xyl) anesthesia prior to the challenge (n = 3-4/group). (M) Serum concentration of CORT in mice injected i.v. with kappa bungarotoxin (kBTX) or PBS 15 min prior to A485 or vehicle challenge. Blood was collected at 2 h post-injection (n = 4-5/group). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01 , ***p < 0.001 , ****p < 0.0001 . (A and K) Two-way ANOVA with Holm-Sidak’s post hoc test (C, I, J, L, and M) one-way ANOVA with Holm-Sidak’s post hoc test. (D, F, and H) unpaired, two-tailed Student’s t test.

Figure 6. HPA-axis activity relays the A485 leukocyte response independently of glucocorticoids. (A) Quantification of leukocytes and neutrophils in the blood of A485- or vehicle-treated mice with (Nr3c1 Rosa26CreERT2 , referred to as Nr3c1 KO ) or without (Nr3c1 fl/fl) inducible global GC receptor (encoded by Nr3c1) deletion (n = 3-4/group). (B) Leukocytes and neutrophils in A485- or vehicle-treated mice with (+) or without (-) pharmacologically induced GC deficiency by metyrapone (n = 5/group). (C) Leukocytes and neutrophils in the blood of ADX- or sham-operated animals treated with A485 for 5 h. Results are expressed as percentage of naive controls (n = 5/group). (D) Blood leukocytes and neutrophils in mice exposed to the indicated treatments. CRHRI i denotes a corticotropin-releasing hormone receptor 1 inhibitor (Dmp696). Results are expressed as percentage of vehicle controls (n = 7-8/group). (E) Validation of the result from (D) using a second CRHRI i (antalarmin) (n = 8/group). Blood was collected 2 h following drug injection. (F and G) (F) Experimental design (G) Neutrophils in vehicle-, A485-, or A485+CRHR1 i-treated mice supplemented with (+) or without (-) recombinant ACTH (n = 3- 4/group). (H) Number of neutrophils in the blood of WT (C57BL/6J), Nr3c1 fl/fl, or Nr3c1 KO mice 2 h following injection of rACTH (+) or vehicle (-) (experimental design akin to F) (n = 4-5/group). (I and J) Blood neutrophils and total lymphocytes in mice treated with rACTH (+) or vehicle (-) in the presence (blue) or absence (white) of pharmacologically induced GC deficiency by metyrapone (n = 5-10/group) Data are presented as mean ± SEM. *p < 0.05, **p < 0.01 , ***p < 0.001 , ****p < 0.0001 . (A) Two-way ANOVA with Holm-Sidak’s post hoc test. (B, D-G, I, and J) One-way ANOVA with Holm-Sidak’s post hoc test (C and H) unpaired, two-tailed Student’s t test.

Figure 7. CBP/p300 HAT or bromodomain inhibition, related to Figure 1. (A) Chemical structure of the spirocyclic drug A485. The characteristic dispirane structure of the spiro compound is highlighted in blue (B) Mechanism of action of A485 (competitive inhibition) (C) Acetyl CoA levels in murine bone marrow derived macrophages assessed by LC/MS-MS at various time points following A485 or vehicle (DMSO) treatment. Results are expressed as fold change from baseline (n=3-4/time point) (D) Total number of lymphocytes in the blood of A485- or vehicle-treated mice 2h post injection (n=5/group) (E) Red blood cell and platelet counts of the same animals (F) Hematopoietic stem and progenitor cells (defined as Sca1+cKitCD150+CD48-) in the blood 2h following A485 or vehicle injection (G) Validation of A485-induced leukocytosis and neutrophilia in a second animal facility (Yale University, Y.U.) (n=6-8/group) (H) Blood leukocytes in male and female mice 2h after being challenged with A485 or vehicle (n=3-7/group) (I) Neutrophils in female mice treated with A485 or vehicle (n=5-7/group) (J) Leukocyte and neutrophil counts 2h following injection of A485 at 100 or 200 mg/kg vs. vehicle (denoted as “0”) (n=4-8/group) (K) Neutrophils in the blood after 3 injections of A485 or vehicle on 3 consecutive days expressed as percentage of vehicle treated controls (n=7-8/group). The left panel schematically illustrates the experimental design (L) Leukocyte and neutrophil counts in the blood of mice treated with vehicle solution or a non-spirocyclic CBP/p300 HAT domain inhibitor (C646) (n=3-6/group) (M) Blood leukocytes and neutrophils in mice injected with vehicle or a CBP/p300 bromodomain inhibitor (SGCCBP30) (n=7/group) (N) Leukocytes, lymphocytes and neutrophils in the blood of mice injected with a Tip60 inhibitor (NU9056) or vehicle solution expressed as percentage of vehicle. Data is shown as mean ± s.e.m. *p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001 . (C,H) Two-way ANOVA with Holm Sidak’s post hoc test (D-G, I, L-M) two-tailed, unpaired student’s t-test (J) one-way ANOVA with Holm-Sidak’s post hoc test.

Figure 8. Pharmacokinetics of A485 and toxicity screen, related to Figure 1. (A) Serum kinetics of A485 following a single injection of the compound assessed by LC/MS (n=3/time point) (B) Pharmacokinetics of A485 across different tissues determined by LC/MS. Results were normalized according to tissue weight (n=3-6/time point) (C) Early changes in leukocytes in response to A485 exposure (n=3-4/time-point) (D) A485-induced changes of lympho- and monocytes in the blood over time (n=4-10/time-point) (E) Numbers of leuko-, lympho-, monocytes and neutrophils in the blood 1 week after a single injection of A485 or vehicle solution. Results are expressed as percentage of vehicle controls (n=6/group) (F) Weight change from baseline in the same animals (G) Serum levels of various markers of organ damage 24h following vehicle or A485 injection expressed as fold change compared to vehicle controls (n=3-4/group) (H) LDH levels in cell culture supernatants of bone marrow derived macrophages treated with vehicle (DMSO) orA485 at 5pM (n=4/time-point). Data is shown as mean ± s.e.m. *p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001 . (C,D) one-way ANOVA with Holm-Sidak’s post hoc test. Figure 9. Comparing A485 to other drugs, related to Figure 1 (A) Changes in neutrophil numbers in the blood overtime in response to A485 (injected i.p. or i.v.) or recombinant murine G-csf (rG-csf, i.p. or i.v.) (n=5-8/group and time-point) (B) Neutrophils at 1 h post r-Gcsf orA485 injection (i.p.) (n=5-8/group) (C) Number of leukocytes, neutrophils and monocytes in the blood 24h after a single injection of A485 or rG-csf (n=6-8/group) (D) Blood leukocyte and neutrophils 1 and 2h after injection of the CXCR4 antagonist AMD3100 (5mg/kg, i.p.) or A485 (i.v.) (n=4- 7/group and time point) (E) Absolute leukocyte counts in patients with RSTS stratified according to their mutational profile (HAT function likely affected by mutation: “HATmut “ vs. not affected: “HATwt”).Data is shown as mean ± s.e.m. *p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001 . (B,C,E) two-tailed, unpaired student’s t-test.

Figure 10. Tissue origins of A485-induced leukocytosis and intravascular labeling, related to Figure 3. (A) Number of progenitor populations (hematopoietic stem cells: HSCs, multipotent progenitor population 2: MPP2, multipotent progenitor population 3/4: MPP3/4, common myeloid progenitors: CMP) in the bone marrow 2h following A485 or vehicle injection, expressed as percentage of vehicle controls (n=5-6/group) (B) Leukocytes and lymphocytes in the blood of A485- or vehicle-treated mice with (+) or without (-) concomitant sphingosine-1 -phosphate receptor inhibition by fingolimod to prevent lymphocyte egress from lymph nodes (n=4/group) (C) Extrapolated concentration of transferred CD45.1 leukocyte and neutrophils per ml of tissue in the bone marrow and blood 8h post adoptive transfer (D) Quantification of endogenous CD45.2 and transferred CD45.1 leukocytes in different organs 5h following A485 injection expressed as percentage of the mean leukocyte number in the respective organ of vehicle treated mice (n=4/group) (E) Extravascular (referred to as single CD45+) and intravascular (double CD45+) leukocytes in lung tissue 5h after A485 or vehicle injection. Results were normalized according to tissue weight (n=5/group) (F,G) Extra- and intravascular neutrophils, leukocytes and Blymphocytes in the spleen and bone marrow of the same animals (n=5/group). Data is shown as mean ± s.e.m. *p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001. (B) One-way ANOVA with Holm- Sidak’s post hoc test (C-G) two-tailed, unpaired student’s t-test.

Figure 11. A485, Cxcl12 and integrins, related to Figure 4. (A) Vcaml cell surface expression on CD31+Sca1 + bone marrow endothelial cells at 2h post challenge (n=6/group) (B) Cxcl12 serum levels 2h post injection (n=4-6/group) (C) Protein levels of Cxcl12 in bone marrow lysates (n=6-9/group) (D) Protein levels of acetylated lysine 18 of histone 3 (H3K18ac), a marker of CBP/p300 HAT activity, in bone marrow lysates 1 h following vehicle or A485 injection. Quantified band intensities are shown in the right panel (n=4/group) (E) Quantification of Cxcl12 mRNA levels in CD31 + and CD31- bone marrow cells 1 h post injection (n=4/group) (F) Cxcl12 protein levels in bone marrow lysates at the same time point (n=4/group) (G) Cxcr4 mRNA levels 1 h post injection of A485 or vehicle in CD31 + and CD31- bone marrow cells (n=4/group) (H) Cxcl12 protein levels in cell culture supernatants of bone marrow cultures treated with A485 (5pM) or vehicle for 2h (n=3-4/group) (I) Dynamic changes in blood lymphocytes upon VLA4 inhibition (by firategrast) or vehicle treatment expressed as percentage of the mean cell count at baseline (n=4/group). A485 is overlayed for reference in light grey (J) Quantification of murine Bcr-Abl ALL cells migrated into the bottom chamber of a two-chamber setup by flow cytometry. Bottom chambers contained recombinant Cxcl12 (100ng/ml), A485 (1 pM), the combination of the two treatments or medium without any supplements (n=6/group). Data is shown as mean ± s.e.m. *p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001. (A-H) two-tailed, unpaired student’s t-test (J) Oneway ANOVA with Holm-Sidak’s post hoc test.

Figure 12. Cytokines and the SNS, related to Figure 4. (A) Tumor necrosis factor alpha (Tnfa) quantification in the serum of A485 or vehicle treated mice over time. Tnfa remained undetectable throughout the 12h (n=4/group) (B) Undetectable levels of Interleukin 1 beta in the circulation (n=4/group) (C-E) Blood leukocyte and/or neutrophil numbers in mice with Myd88-, interferon alpha/beta receptor alpha chain (Ifnarl)- or ST2/TSLPR/IL25 (“alarmins”) knock-out (KO) challenged with A485 or vehicle (n=4-7/group) (F) Leukocytes in mice pre-treated with an IL6 receptor-neutralizing antibody (alL6R) or isotype control (IgG), followed by A485 or vehicle exposure (n=3-4/group). In panels (C-F), blood was analyzed 2h post challenge (G) Blood leukocyte and lymphocyte numbers over time in mice pre-treated with anti-G-csf antibodies or an isotype control (IgG) followed by A485 or vehicle injection (n=4/group) (H) Blood lymphocytes in A485- or vehicle-treated mice following chemical sympathectomy by 6-OHDA (+) or vehicle injection (-) (n=4-7/group) (I) ) Total leukocytes and subsets in mice with genetic deficiency of adrenoceptor beta 1 and beta 2 (Adrb1/b2 KO) and C57BL6/J wildtype mice (B6) treated with A485 or vehicle. Blood was collected at 2h post injection (n=3-5/group) (J) Circulating G-Csf levels in mice supplemented with rG-Csf (250 pg/kg) with or without A485 over time measured by ELISA (n=4/time point). Data is shown as mean ± s.e.m. *p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001 . (C-E) two-tailed, unpaired student’s t-test (F,H) One-way ANOVA with Holm- Sidak’s post hoc test (G , I) Two-way ANOVA with Holm-Sidak’s post hoc test.

Figure 13. Activation of the HPA-axis, related to Figure 5. (A) Quantification of transcript levels of Cyp11a1 (encoding for cholesterol side-change cleavage enzyme, P450scc) and Cyp11 b1 (encoding 11 -betahydroxylase) in adrenocortical Y1 cells in response to various concentrations of A485, the cAMP inductor forskolin (5pM) or vehicle (DMSO) (B) cFos mRNA expression in hypothalamic explants following ex vivo exposure to A485 (5pM) or vehicle (DMSO) for 3h assessed by qPCR (n=3/group) (C) Corticosterone levels in mice pre-treated with an IL6 receptor-neutralizing antibody (alL6R) or isotype control (IgG) followed by injection with A485 or vehicle. Blood was collected at 2h post drug/vehicle challenge (n=3-4/group) (D) Leukocytes and neutrophils in the blood of mice subjected to chemical ablation of TRPV1 + sensory neurons by resiniferatoxin (RTX) or vehicle treatment (DMSO), followed by A485 or vehicle challenge. Blood was analyzed at 2h post injection (n=3-4/group) (E) Blood leukocytes and neutrophils in A485- or vehicle challenged mice with (+) or without (-) prior induction of anesthesia using ketamine/xylazine (n=3-4/group) Data is shown as mean ± s.e.m. *p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001 . (A,C,E) One-way ANOVA with Holm-Sidak’s post hoc test (D): Two-way ANOVA with Holm-Sidak’s post hoc test.

Figure 14. GC, CRHR1 and leukocytes, related to Figure 6. (A) Leukocytes in A485 or vehicle treated animals with (+) or without (-) concomitant GC receptor inhibition by mifepristone (RU486) at 5h post injection (n=5/group) (B) Transcript levels of the GR (encoded by Nr3c1) in bone marrow lysates of mice with GR knockout (Nr3c1 Rosa26CreERT2, referred to as Nr3c1 KO ) or wildtype littermate controls (Nr3c1 fl/fl) expressed as fold and assessed by qPCR (n=3/group) (C) Lymphocytes in mice of the same genotypes upon A485 or vehicle injection (n=3-4/group) (D) Corticosterone levels in mice treated with A485 or vehicle in the presence (+) or absence (-) of pharmacologically-induced GC deficiency by metyrapone (n=5/group) (E,F) Corticosterone and Acth levels in the circulation of ADX or shamoperated mice 5h post A485 injection expressed as percentage of naive controls (no surgical intervention) (n=5/group) (G) Lymphocytes in the blood of the same animals (H) Crhrl mRNA levels in bone marrow, bone and brain lysates (n=4/group). “n.d.” denotes not detected (I) Blood lymphocytes 2h following injection of A485 with or without concomitant Crhrl blockade by dmp696. Results are expressed as percentage of vehicle-treated controls. CRHRI i denotes corticotropin-releasing hormone receptor 1 inhibitor (n=7-8/group) (J) Leukocytes and lymphocytes in response to the indicated treatments 2h post injection (n=3/group) (K) Circulating corticosterone levels in mice treated with recombinant Acth (rActh) (+) or vehicle (-) for 2h in the presence (blue) or absence (white) of pharmacological GC synthesis inhibition (n=4-5/group). Data is shown as mean ± s.e.m. *p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001 . (A,D, l-K) One-way ANOVA with Holm-Sidak’s post hoc test (B,EG) two-tailed, unpaired student’s t-test (C) Two-way ANOVA with Holm-Sidak’s post hoc test.

Figure 15. Mc2r expression in the murine bone marrow, related to Figure 6. (A) Transcript levels of melanocortin receptors 1-5 (Mc1-5r) in murine bone marrow cell lysates assessed by qPCR. The right panel shows relative Mc2r mRNA abundance in CD31 + and CD31- bone marrow cells (n=3-4/group) (B) Relative mRNA levels of Mc2r in CD31 + bone marrow cells vs. whole adipose tissue assessed by qPCR (n=4/group) (C) Absence of Mc2r in hematopoietic cells (Tabula Muris Dataset) (D) Expression (color bar, TP10K) of Mc2r at single cell resolution in the murine bone marrow stroma. t-Distributed stochastic neighbor embedding (t-SNE) of non- hematopoietic cells annotated post hoc and colored by clustering, bone or bone marrow location is shown in the right panel. The relevant populations are numbered (1-4) (E) Cxcl12 protein levels in bone marrow lysates of A485-exposed mice treated with (light blue) or without (grey) a CRHR1 antagonist (CRHR1 i, Dmp696) (n=4/group). Data is shown as mean ± s.e.m. **p<0.01 . (E) two- tailed, unpaired student’s t-test.

Figure 16. Gating strategy for flow cytometry: bone marrow progenitors, related to STAR Methods.

Figure 17. Gating strategy for flow cytometry: intravascular labeling, related to STAR Methods. The figure shows an exemplary gating of a blood sample.

Figure 18. Gating strategy for flow cytometry: Hematopoietic stem and progenitor cells in the blood, related to STAR Methods.

Figure 19. Gating strategy for flow cytometry: adoptive CD45.1 transfer, related to STAR Methods. The figure shows an exemplary gating of a blood sample.

EXAMPLES

The invention is further described by the following examples. These are not intended to limit the scope of the invention but represent preferred embodiments of aspects of the invention provided for greater illustration of the invention described herein.

EXPERIMENTAL MODEL AND STUDY PARTICIPANTS DETAILS: Mice: C57BL/6J wildtype mice were obtained from Janvier Laboratories (Le Genest-Saint-lsle, France) at the age of 6-8 weeks or bred inhouse at Yale University. Mice from external sources were allowed to acclimatize to local conditions for a minimum of 7-10 days before being subjected to experimental procedures. Animals were housed in groups of up to 5 mice/cage at the animal facility of the Technical University (TU) of Dresden or Yale University and kept under a 12h light:dark cycle with ad libitum access to water and food. Nr3c1 fl/fl mice were kindly provided by Jan Tuckermann (University of Ulm, Germany) and crossed with Rsa26-CreERT2 mice to obtain Nr3c1fl/fl;Rosa26-CreERT2 animals. Mice from both sexes were used, which were heterozygous for the Cre allele, whereas Cre-negative littermates served as controls. DNA was isolated from ear clips and genotyping was performed according to standardized protocols. Both genotypes were injected with tamoxifen dissolved in sunflower oil (100ml, 10g/L) at the age of 6-7 weeks and used for in vivo experiments 2 weeks later. B6.S6JL-Ptrprca Pepcb /BoyJ (B6 CD45.1) and C57BL/6-Tg(Vav1-NUP98/HOXD13) G2Apla/J (MDS model) mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and bred at TU Dresden. Adrb1/b2 double knock-out (KO), Myd88 KO, and IFNAR1 KO mice were all obtained from the Jackson Laboratory and bred at Yale University. YRS ST2/TSLPR/IL25 TKO were kindly provided by Dr. Richard Locksley (University of San Francisco). Animals were used between the age of 6-12 weeks for experimental procedures. Adrenalectomized (ADX) animals were also purchased commercially. Following surgical removal of the two adrenal glands, animals were closely monitored for 2 weeks at the local facility. Sham operated animals served as controls. All mice received 0.9% saline solution instead of regular drinking water until further experimental procedures, which were conducted 3 weeks post-surgery. After experimental interventions (see next sections) were terminated, mice were anaesthetized using ketamine/xylazine anesthesia and sacrificed by cervical dislocation. Blood was obtained by cardiac puncture or retroorbital bleeding. Organs were either collected in PBS or formaldehyde for further processing or immediately snap frozen in liquid nitrogen. For small volume blood sampling, the retroorbital plexus was punctured using thin capillaries. Blood was collected in EDTA- or heparin-coated tubes. All mouse experiments were initiated between ZT23 (05:00 AM) and ZT2 (08:00 AM) if not otherwise stated. Animal experiments were approved by local authorities (TUD, protocol TW 22/2022 and Yale University’s Institutional Animal Care and Use Committee) and performed according to institutional guidelines.

Human study participants: Patients from both sexes were included in our study if the following inclusion criteria were met (1) diagnosis of Rubinstein Taybi syndrome (RSTS) by genetic testing (either pathogenic or likely pathogenic mutation in CREBBP or EP300) (2) at least one determination of white blood cell count (3) written informed consent for collection, analysis, and publication of relevant data obtained from the patient or legal guardians. Exclusion criteria were: (1) insufficient data available, (2) refusal to participate in the study. The PID-GENMET and FISIOPAT-PID studies were approved by the institutional review boards/ethic committee of Comitato Etico Brianza (Monza, Italy) and conformed to the Declaration of Helsinki. Data was collected between 2007 and 2019. All laboratory results were analyzed with reference to age- matched normal ranges. Leukocytosis was defined as leukocyte counts exceeding the age- matched upper reference limit of normal. Bone marrow cultures and murine bone marrow derived macrophage (mBMDM) differentiation: Animals were sacrificed to obtain femora and tibias. Bones were crushed in FACS buffer (PBS, 2% FCS and 5 mM EDTA) using a mortar. Residual skeletal tissue was removed and cell suspensions were filtered through 70mm cell strainers to yield single cells. Suspensions were centrifuged at 1800 RPM for 5 min, followed by erythrocyte lysis using ACK buffer (Thermo Fisher Sci, Waltham, MA). After another centrifugation step, the pellet was dissolved in RPMI medium supplemented with 10% FCS, 1 % penicillin/streptomycin (P/S), 2mM glutamine and 1x non- essential amino acids (NEAAs) (Gibco, Thermo Fisher Sci, Waltham, MA). Cells were seeded into 6-well plates at a density of 5x106 cells/well and used for in vitro studies on the same day. Following treatment, cell cultures were washed, pelleted by centrifugation and snap-frozen in liquid nitrogen for downstream analysis. Each bone marrow culture corresponds to a biologically independent replicate (mouse). For experiments with flow cytometry read-outs, cells were directly seeded into FACS tubes, centrifuged and resuspended in media containing the indicated treatments. Cells were then incubated at 37 or 4 deg C, followed by downstream processing for flow cytometry as described elsewhere in this section. To obtain macrophages, bone marrow cultures were exposed to differentiation medium for 7 days consisting of RPMI supplemented with 30% L929 conditioned medium as a source for G-csf, 10%FCS, 1 % P/S, 2mM glutamine and 1x NEAAs. After confirmation of successful differentiation by microscopy, cells were subjected to experimental treatments.

Y1 cell culture: Immortalized murine adrenocortical cells (Y1) were kindly provided by the Bornstein Lab (TU Dresden, Germany). Cells were grown in 75mm2 flasks and F-12 K Medium supplemented with 2.5% horse serum, 15% FCS, 1 % P%S. Cultures were maintained under a humidified atmosphere at 37 deg C and 5% CO2. One day before being challenged with different reagents, cells were seeded in 6-well plates at a density of 150 000 cells/well. Treatment conditions are specified in the figure legends.

METHOD DETAILS:

Drugs and treatments: A485 was injected i.p. or i.v. (10 or 5ml/g body weight) at 100 mg/kg as previously reported. For selected experiments, lower or higher doses were chosen as denoted in the respective figures. A485 stock solutions were prepared by dissolving the drug in prewarmed DMSO at 200 mg/ml, followed by steady shaking for 5-10 min. For in vivo experiments, the stock solution was diluted in a mix of PEG300 (Sigma Aldrich, St.Louis, MO), Tween80 (Serva, Heidelberg, Germany) and sterile water (30/5/60%, respectively). The resulting liquid was mixed rigorously and ultrasonication was applied if necessary. Aliquots were prepared after all components were successfully dissolved. Controls received an equivalent amount of vehicle solution containing 5% pure DMSO instead of A485 in DMSO. Aliquots were stored at -80 deg C and used within 4-6 weeks. All other drugs used in this study were likewise dissolved in DMSO/PEG300/Tween80/water or PBS as appropriate. Recombinant proteins were freshly dissolved in sterile PBS immediately before use and injected i.v. (100ml/mouse) under short isoflurane anesthesia. The following drug doses were used: 20 mg/kg C646, 20 mg/kg SGC- CBP30, 150 mg/kg 5-FU, 0.2 mg/kg fingolimod, 5 mg/kg NU9056 (Tip60 inhibitor), 5 mg/kg AMD3100, 50 mg/kg firategrast, 25 mg/kg dmp696, 20 mg/kg antalarmin, 20 mg/kg mifepristone (RU486), 50 mg/kg metyrapone, 1 mg/kg scopolamine, 1 mg/kg kappa bungarotoxin, 250 mg/kg rG-csf, 10mg rActh/mouse, 20mg anti-G-csf or isotype control (IgG)Zmouse, 8 mg/kg anti-IL6R or rat IgG isotype control. Metyrapone and rActh were administered twice (90 and 45 min between injections, respectively) during the corresponding experiments due to their short half-lives. Neutralizing antibodies and isotype controls were applied 16-18h prior to the experimental challenge. The RTX model is described elsewhere in this section. Loss of function of peripheral sympathetic nervous system neurons was induced by i.p. injection of 6-OHDA (100mg/kg) twice (d1 and d3) into 8 to 10-week-old wildtype mice. Experiments were conducted two days after the second injection (d5).

Listeria monocytogenes infection and quantification of bacterial burden: Toxic bone marrow injury was induced by a single i.p. injection of 5-FU (150 mg/kg). Controls received an equivalent volume of PBS. Listeria monocytogenes (L. monocytogenes) strain 10403s was originally obtained from the laboratory of Dr. Daniel Portnoy and kindly provided by Dr. Ruslan Medzhitov. L. monocytogenes was grown to log-phase in brain heart infusion (BHI) broth, washed once with PBS, and stored as stock solutions at -80 deg C (2x1010 CFUs/ml). For infection of mice, stock solutions were diluted in PBS and mice were injected retro-orbitally with 7.5x104 CFUs of L. monocytogenes (in 100ml PBS). Mice were infected on the sixth day after 5FU exposure around ZT21 (4:30 AM), followed by i.p. treatment with A485 (100 mg/kg) or an equivalent volume of vehicle solution 1 h later (5:30 AM). For L. monocytogenes infection in immunocompetent hosts (PBS- instead of 5FU-injected), a higher dose (1x105 CFUs) was used, but no mortality was noted. Survival was monitored twice daily for a minimum of 9 (immunocompetent mice) or 21 (immunocompromised mice) days. Immunocompetent mice were sacrificed on day 9 of the experiment without any signs of sickness. Quantification of bacterial burden was achieved by pushing 50-100 mg of liver or spleen tissue though 70mm cell strainers using the plunger of a 5ml syringe, followed by flushing with plain RPMI medium. The resulting homogenates were mixed with 1% TritonX O (diluted in H2O) and subjected to serial dilutions, which were plated on BHI agar plates (Hardy Diagnostics) using sterile plastic loops. Bacteria were allowed to grow at 37 deg C for 24 hours. CFU’s were counted manually, and results were expressed as number of CFUs/g of tissue.

Intravascular leukocyte labeling: Labeling of the intravascular leukocyte compartment was achieved by retroorbital injection of 2mg of fluorophore labeled CD45 antibodies (CD45 APC) dissolved in 100ml PBS 2-3 min before sacrifice, followed by immediate organ collection. Blood and tissues were subjected to preparation for flow cytometry as described elsewhere in this section. Intravascular leukocytes were defined as “double CD45 positive”, if they were labeled with both the intravascularly applied CD45 antibody as well as the second CD45 antibody (CD45 PE-Cy7), which was added to cell preparations during flow cytometry staining. “Single CD45 positive” cells stained positive for CD45 PE-Cy7 only (extravascular).

Cell tracking: In vivo tracking of CD45+ cells was achieved as previously described. The bone marrow of B6-.S6JL-Ptrprca Pepcb /BoyJ (referred to as B6 CD45.1) mice was obtained as described elsewhere in this section; 10-20x106 bone marrow cells (diluted in 200 ml PBS) were adoptively transferred into C57BL/6J wildtype mice through retroorbital injection at ZT16. After 8 to 9h (ZT0/1), mice were either sacrificed for confirmation of successful bone marrow homing or treated with A485 or vehicle for 5h, followed by organ collection and flow cytometry analysis. Conventional and automated flow cytometry analysis: Blood was collected in EDTA-coated tubes and erythrocyte lysis was performed using ACK lysis buffer (Thermo Fisher Sci . , Waltham, MA). Tissues (lung, liver, muscle) were collected in PBS, weighed and minced in a mixture of DMEM supplemented with collagenase (1.2 mg/mL) and dispase II (25 U/mL) (both from Sigma-Aldrich, St.Louis. MO), followed by digestion at 37 deg C under steady shaking at 800 rpm for 45 minutes. Homogenates were then flushed through 40 or 100mm cell strainers (Sigma Aldrich, St.Louis, MO) to yield single cell suspensions. Spleens were immediately pressed through cell strainers without prior digestion. Bone marrow homogenates were obtained by crushing tibias and femora in FACS buffer (PBS, 2% FCS and 5 mM EDTA) using a mortar. Residual skeletal tissue was removed and homogenates were passed through 40mm cell strainers. For both the spleen and bone marrow, erythrocyte lysis was performed as described above. Single cell suspensions were stained in FACS buffer containing fluorophore-conjugated antibodies at a concentration of 1 :400 at 4 degrees for 30 min. Prior to adding primary antibodies, unspecific bindings sites were blocked using anti-CD16/32 antibodies. Live and dead cells were differentiated by DAPI or 7-AAD staining (both diluted 1 :1000) as shown in the respective gating examples. For quantification of progenitor populations in the bone marrow, single cell suspensions were stained by DAPI (0.1 mg/ml) and DAPI-negative cells were counted by MACSQuant Analyzer (Miltenyi Biotec, Cologne, Germany), followed by stained with a c-Kit bio antibody. Anti-Biotin MicroBeads were added to enrich for c-Kit+ cells using LS columns.

Cells were identified as: 1.) HSCs: Lin- (negative for B220, CD3E, CD19, NK1 .1 , Gr1 , Teri 19, and CD11 b) Sca1+ c-Kit+ (LSK) CD48-CD150+ , 2.) MPP2: LSK CD48+ CD150+ , 3.) MPP3/4: LSK CD48+ CD150, 3.) MPP3/4: LSK CD48+ CD150- , 4.) CMP: LK CD16/32-CD41- CD105- CD150- , 5.) Leukocytes: single CD45+, 6.) Neutrophils: CD45+,Cd11 b+,Gr1 +, 7.) B lymphocytes: CD45+, Cd11 b-, CD3-, CD19+, 8.) T lymphocytes: CD45+, Cd11 b-, Cd3+, CD19-.

All flow cytometry experiments were performed using counting beads (Countbright, Thermo Fisher Sci., Waltham MA) if not otherwise stated. Data were acquired on a BD LSR Fortessa (BD Biosciences), BD LSR II, BD FACSAria II, BD LSRFortessa X-20, or a BD FACSCanto II (all from BD Bioscience) and analyzed using FlowJo v10 software (Tree Star Inc.). The abundance of cells within tissues was normalized according to the respective weight and expressed as number of cells per mg of tissue. Exemplary gating strategies are shown in Figures 16-19. Automated fluorescence-flow cytometry analysis was performed using XN-1000 (Sysmex) or Hemavet 950 (Drew Scientific) devices, allowing for complete blood count and white blood count differential measurements. Murine blood (2-3 drops) was collected in EDTA- or heparin-coated tubes, followed by analysis on the same or subsequent day.

Hypothalamic explants: Following sacrifice, brains were extracted from WT mice and hypothalami were explanted as previously described under sterile conditions. Whole tissue explants were cultivated in DMEM F-12 (Gibco, Thermo Fisher Sci, Waltham, MA) supplemented with 10% FCS, 2 mM glutamine, 1x non-essential amino acids and 1 % pencilline/streptomycine. A485 (5mM) or vehicle (DMSO) were immediately added, and tissue was harvested 3h later for downstream processing. A485 measurements: A rapid and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of A485 in serum and tissue. Frozen serum samples (-20 deg C) were thawed at room temperature, mixed and centrifuged. Volumes of 50 ml serum were diluted with 100 ml acetonitrile and mixed and centrifuged for 10 minutes (14.000 U/min). Volumes of 50 ml cell lysates were diluted with 100 ml solvent AB (a mixture of acetonitrile, 2 mM ammonium acetate solution and formic acid; 50/50/0.05, v/v/v); 20 ml of the clear supernatant was injected into the API 4000 LC-MS/MS system. Measurements were performed using an Ultimate 3000 HPLC system from Thermo Scientific (Waltham, MA, USA) The flow rate was 0.5 ml/min. A485 was determined using a Synergi 4m Fusion-RP 80A, 150 x 2.0 mm column (Phenomenex, Torrance, CA, USA) with a mobile phase gradient. The flow rate was 0.5 ml/min. The retention time of A485 was 3.77 min. The HPLC system was coupled to an API 4000 tandem mass spectrometer (AB Sciex, Framingham, MA, USA) with an electrospray interface. The detection was performed in multiple reaction monitoring (MRM) mode, using the three most intensive transitions. The product ion transition with the highest intensity was used for quantification (quantifier) and the other transitions were used for confirmation (qualifier).

In preliminary tests, we found that the expected concentration range was large. Accordingly, the upper standard was set at 1 ,000 ng/mL. Two-fold serial dilutions were performed to yield a standard curve with the lowest standard set at 3.91 ng/mL. Finally, the standard samples were completed with a blank serum. Samples above the calibration range were diluted 1 :10 or 1 :100 with solvent AB and measured again.

Ablation of TRPV1 + sensory neurons: Mice received increasing doses (30, 70 and 100mg/kg) of resiniferatoxin (referred to as “RTX”, Alomone Labs, Israel) on three consecutive days via subcutaneous injections at the age of 4 weeks as previously described. The RTX stock solution was dissolved in DMSO and diluted in PBS to the desired concentration. Controls received an equivalent volume of DMSO/PBS solution. Successful targeting of TRPV1 sensory neurons was confirmed by progressive greying of fur79 and loss of heat sensitivity. Mice were subjected to further experimental procedures 4 weeks after the last injection.

Acetyl CoA quantification: Murine bone marrow derived macrophages were differentiated from bone marrow precursors as described elsewhere in this section. Following treatment with DMSO or A485, cells were harvested at different time points as indicated in the respective figure, washed, centrifuged to yield a cell pellet and immediately snap-frozen in liquid nitrogen. The pellet was dissolved in 200 ml 30% methanol in Acetonitrile with 0.1 mM ammonium acetate, and 0.01 % NH4OH. As an internal standard, 5 mM Acetyl-1 ,2-13C2-Coenzym A lithium salt (Sigma Aldrich, St.Louis, MO) was used. After adding 1/3 volume of 0.5 mm metal beads, samples were homogenized for 10 min at 4 deg C and 300g in a TissueLyser II (Qiagen, Hilden, Germany). Next, 20 mL of homogenate solution was isolated for protein quantification by the BCA Protein Quantification Kit (Thermo Sci., Waltham, MA). The resulting mixture was centrifuged at 13,000g for 30 min and the supernatant was transferred to a new tube, followed by LC-MS/MS. Chloropropamide (100nM) was used as an additional internal standard. Results were normalized according to the internal standard and total protein content of the respective sample.

The LC-MS/MS analysis was performed on high performance liquid chromatography (HPLC) system (1200 Agilent) coupled online to G2-S QTof (Waters). For normal phase chromatography, the Bridge Amide 3.5ul (2.1x100mm) column from Waters was used. For the normal phase, the mobile phase composed of eluent A (95% acetonitrile, 0.1 mM ammonium acetate, and 0.01 % NH4OH) and eluent B (40% acetonitrile, 0.1 mM ammonium acetate, and 0.01% NH4OH) was applied with the following gradient program: Eluent B, from 0% to 100% within 18 min; 100% from 18 to 21 min; 0% from 21 to 26 min. The flow rate was set at 0.3 ml/min. The spray voltage was set at 3.0 kV and the source temperature was set at 120 deg C. Nitrogen was used as both cone gas (50 L/h) and desolvation gas (800 L/h), and argon as the collision gas. MSE mode was used in negative ionization polarity. Mass chromatograms and mass spectral data were acquired and processed by MassLynx software (Waters).

CXCL12 migration assay: Murine Bcr-Abl lymphoma cells (kindly provided by Joao Pereira, Yale University) were grown in petri dishes in DMEM supplemented with 20% FCS, 1 % penicillin/streptomycin, HEPES and 2-Mercaptoethanol. Cells were collected, centrifuged and pellets were dissolved in migration buffer (DMEM with 0.5% fatty acid free BSA, HEPES and penicillin/streptomycin). Cells were then transferred into FACS tubes and incubated at 37 deg C for 30 min before being subjected to experimental procedures. Next, 50 000 cells (in 100ml migration buffer) were added to migration chambers (Corning, NY), in which the bottom chamber contained A485 (1 mM), vehicle (DMSO), recombinant Cxcl12 (100 ng/ml) or medium without any supplements as indicated in the corresponding figure. Cells were allowed to migrate for 3h at 37 deg C before being collected from the bottom chamber. Quantification of migrated cells was achieved by flow cytometry following live/dead staining with DAPI.

Magnetic-assisted cell sorting (MACS): CD45- and CD31 -positive cells were sorted from the blood and bone marrow using microbeads (Miltenyi Biotec, Cologne, Germany) and magnetic columns according to the manufacturer’s protocol by positive selection. Following preparation of single cell suspensions, cells were counted, centrifuged and the supernatant was removed, followed by resuspension in MACS buffer and addition of microbeads, both of which were adjusted according to the total number of cells. Suspensions were then incubated at 4 deg C for 15-30 min, followed by addition of the staining antibody, washing steps and centrifugation. Cells were resuspended in MACS buffer and separated using magnetic columns. The flow-through was collected and labelled as “negative”, whereas the bound fraction corresponded to “positive” cells (i.e. cell population of interest). Both fractions were subjected to downstream analysis. Successful enrichment of the population of interest was confirmed by qPCR analysis.

Quantitative polymerase chain reaction (qPCR): RNA isolation was performed using TRIzol reagent (Thermo Fisher Sci., Waltham MA) or the Reliaprep kit (Promega, Fitchburg, MA) according to the manufacturer’s instructions. RNA pellets were diluted in RNAse-free water and its quality was assessed by Nanodrop2000 (Thermo Fisher Sci., Waltham, MA); 250-500 ng of RNA were subjected to cDNA synthesis using random primers (Thermo Fisher Scientific, Waltham, MA), dNTPs (Carl Roth GmbH, Karlsruhe, Germany), M-MLV RT, and RNAsin (both from Promega Corp., Fitchburg, Wl). Quantitative polymerase chain reaction was performed using GoTaq Mastermix (Promega Corp., Fitchburg, Wl) and forward and reverse primer pairs. Primer sets were validated by melting curve analysis. All qPCRs were run on a StepOnePlusTM cycler (Applied Biosystems, Carlsbad, CA). Relative mRNA expression of selected targets was calculated using the DCT or DDCT method as appropriate. Beta actin (Actb) served as the housekeeping gene for normalization.

Protein isolation and immunoblotting: Proteins were isolated from cells and tissues using RIPA or T-PER buffer (Thermo Fisher Sci, Waltham, MA), supplemented with protease and phosphatase inhibitor cocktail (Thermo Fisher Sci, Waltham, MA). To obtain lysates, T-PER was added to tissue according to its weight, followed by homogenization using metal beads. Lysates were rested on ice for 10 min before undergoing centrifugation (10 000g, 5 min, 4 deg C). Total protein concentration in supernatants was assessed by BCA assay (Thermo Fisher Sci, Waltham, MA) and lysates were subjected to downstream analysis. Immunoblotting was performed as previously described65 using SDS page gel electrophoresis, followed by transfer of separated proteins onto 0.2 mm nitrocellulose membranes, which were blocked with 5% BSA. Primary antibodies were applied at 1 :500-1 :1000 dilution and incubated at 4 deg C overnight under steady shaking.

Signals were visualized using HRP conjugated secondary antibodies and ECL substrate (Thermo Fisher Sci, Waltham, MA). All antibodies used are listed elsewhere in this section.

Enzyme-linked sorbent assays (ELISAs): Serum was collected from whole blood following centrifugation at 5000 RPM and 4 deg C for 15 min and used for further assays. The following commercially available ELISAs were used: corticosterone and Acth ELISA (both from abeam, Camebridge, MA), Cxcl12-, interleukin 1 beta-, tumor necrosis factor alpha- and G-CSF-ELISA (all from R&D, Minneapolis, MN). Dilutions were chosen depending on the experimental conditions (undiluted to 1 :250). Cxcl12 levels in bone marrow lysates were normalized according to total protein content of the respective samples. For the Acth ELISA, blood was immediately cooled on ice, centrifuged and subjected to the downstream assay within 1 h.

Sublethal Irradiation: For sublethal irradiation, mice were exposed to 4 gray and successful induction of bone marrow injury was confirmed by flow cytometry 3 days later.

Immunofluorescence analysis: Following sacrifice, mice were perfused with PBS and 4% PFA for immediate fixation of organs. Brains were extracted and placed in 4% PFA for 8 h, followed by 3 x 10-min washes in 1xPBS. Tissues were then placed in 30% sucrose (diluted in 0.1 M phosphate buffer) at 4 deg C for 48h. After another wash, brains were embedded in OCT and stored at -80 deg C until further processing. Sectioning of brains in 40mm slices was achieved using a cryostat. Free-floating sections were washed in PBS-T (0.1 % Tween 20 in PBS) 3x for 10 min each under steady shaking at room temperature (RT). Sections were blocked with 10% BSA in PBS-T for 2h under steady shaking at RT, followed by incubation with anti-cFos antibodies (Sigma-Aldrich, 1 :2000 diluted in PBS-T) at 4 deg C for 48h. Free-floating sections were washed again with PBS- T (3 x 10 min, under steady shaking at RT) and finally incubated with an Alexa Fluor 594- conjugated goat anti-rabbit IgG (1 : 1000) at RT for 2 h. After 3 x 10 min washes with PBS-T, sections were mounted using Fluoroshield with DAPI (Sigma-Aldrich, F6057) and coverslipped. Fluorescence imaging was performed on a Nikon ECLIPSE Ti2 inverted microscope (Nikon Instruments Inc.) using a triple bandpass filter cube (TRITC, FITC, DAPI) from Nikon, with a Cool LED pE-300 and excitation wavelengths at 370nm for DAPI and 550nm for Alexa Fluor 594. Images were obtained with an Andor Sona Camera with a 20x objective and consistent settings including exposure time (DAPI: 300ms, Texas Red: 500ms). The images were further processed on the software Fiji (is just Imaged ), where the set display rate was kept consistent across images for comparison.

LDH release assay: Cell toxicity was estimated by measuring LDH activity in cell culture supernatants over time using the LDH-Glo assay (Promega, Fitchburg, MA) according to the manufacturer’s instructions.

Organ toxicity screen: Markers of organ damage including alanine and aspartate aminotransferase (ALT and AST, respectively), blood urea nitrogen (BUN), creatinine kinase (CK) and troponin T (TnT) were determined in serum samples 24h post A485 injection by routine measurements at the Department of Clinical Chemistry of the Technical University of Dresden.

Quantification and statistical analysis: Experimental data is shown as mean ± s.e.m. if not otherwise stated. Each dataset comprises a minimum of 3 biologically independent replicates. Key experiments were repeated at least twice and confirmed by independent scientists where applicable. Sample size calculations were based on expected effect sizes and previously published literature. Baseline measures of selected variables in kinetic experiments with genetically identical organisms were obtained from 3-6 randomly selected animals. Comparisons between groups of two were assessed by student’s unpaired t-test for parametric and Mann Whitney U test for non-parametric data. Groups of three or more were compared by ordinary oneway ANOVA with Holm Sidak’s post hoc test adjusted for multiple comparisons or Fisher’s LSD as stated in the figure legends. Groups stratified according to two independent variables (e.g. genotype and treatment) were compared by two-way ANOVA with Holm-Sidak’s post-hoc test. Frequencies of categorical variables were analyzed by chi square (c2 ) test. Probability of survival was assessed by Mantel Cox Test. All statistical tests were two-tailed. Where applicable, datasets were screened for outliers using Grubb’s test, which allowed for exclusion of a single data point per group and dataset (alpha=0.05). Statistical significance was assumed at P-values <0.05. Data analysis and visualizations were performed using Prism V9 (Graphpad Inc, LaJolla, CA).

RESULTS:

CBP/p300 HAT inhibition triggers transient leukocytosis:

We used the spirocyclic, small-molecule CBP/p300 HAT inhibitor A485, which reversibly competes with acetyl coenzyme A (CoA) for binding to the HAT domain’s catalytic center and exhibits high selectivity for CBP and p300 compared with other HATs (Figures 7A and 7B). 26 The crystal structure of the HAT-binding pocket in complex with A485 has previously been published. 26 We predicted that this mechanism of action should result in a rapid increase in cellular acetyl CoA levels and confirmed this hypothesis in primary murine cells (bone marrow- derived macrophages) (Figure 7C). Wild-type (WT) mice injected intraperitoneally (i.p.) with A485 developed substantial leukocytosis within 2 h compared with vehicle-treated controls (Figure 1 A), which resulted from increased numbers of most leukocyte populations in the blood, including neutrophils, B lymphocytes, and monocytes, whereas T lymphocytes, red blood cell, and platelet counts remained unchanged (Figures 1 B, 1 C, 7D, and 7E). Moreover, hematopoietic stem and progenitor cells (Lin-Sca1+ cKit+ Cd150+ CD48-) were rare in the circulation, irrespective of vehicle or A485-exposure (Figure 7F), suggesting that the leukocyte response induced by A485 was specific. The efficacy of the compound to expand blood leukocytes was validated in a second animal facility, both sexes and independent of handlers (Figures 1 and 7G-7I). Leukocytosis occurred in a dose-dependent fashion with a ceiling effect, was not subject to relevant tachyphylaxis, and could be recapitulated with an independent, non-spirocyclic CBP/p300 HAT inhibitor (C646) 27 (Figures 1 D and 7J-7L). By contrast, inhibition of the protein’s bromodomain 28 - which confers DNA binding - or the HAT domain of another major mammalian HAT (Tip60) did not induce leukocytosis (Figures 7M and 7N).

To understand the pharmacokinetics of A485, we profiled the distribution of the drug in the circulation and across different tissues using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We found that the serum concentration of A485 peaked rapidly following i.p. injection and dropped to barely detectable limits by 12 h and non-detectable amounts by 24 h (Figure 8A). We noted accumulation of A485 in most tissues analyzed including the bone marrow, adipose tissue (AT), liver, spleen, aorta, and the kidney (Figure 8B). In line with previous reports, 26 we failed to detect relevant levels in whole brain lysates, arguing against blood-brain barrier penetration (Figure 8B).

The dynamics of leukocytes in the blood closely paralleled these kinetics: their numbers started to rise within 1 h upon A485 exposure, peaked between 4 and 6 h, and returned to baseline by 12 h post-injection (Figures 1 E, 8C, and 8D). This pattern was identical for neutrophils, lymphocytes, and monocytes (Figures 1 E and 8D). One week after a single bolus of A485, leukocyte counts were indistinguishable from controls, arguing against a lasting effect on these cells (Figure 8E). We found no evidence for toxicity as mice appeared and behaved normal, did not lose weight, and markers of organ damage were similar between groups (Figures 8F and 8G). We also did not detect signs of toxicity in murine bone-marrow-derived macrophages in vitro (Figure 8H).

We next compared the efficacy of A485 to expand blood leukocytes with established pharmaceuticals and first focused on G-CSF, a potent endogenous regulator of neutrophil compartments and the treatment of choice for neutropenia in humans. 32 We injected recombinant murine G-CSF (rG-CSF, 250 mg/kg as previously reported33 ) i.v. or i.p. in mice and compared the resulting neutrophil kinetics with A485. We found that dynamic changes in circulating neutrophils were comparable between A485 and G-CSF, irrespective of the route of administration, although neutrophilia onset appeared slightly faster with A485 (Figures 1 F, 9A, and 9B). Importantly, the combination of the two drugs resulted in superior neutrophil mobilization compared with treatment with either agent alone (Figure 1 F). We further noted that, after 24 h, leukocytes, neutrophils, and monocytes were substantially more abundant in the circulation of G- CSF-, compared with A485-treated mice (Figure 9C), suggesting that the A485 neutrophil response is shorter and distinct from G-CSF. Inhibition of CXCR4 is another clinically used approach to mobilize leukocytes and hematopoietic stem cells. 66 We observed that acute neutrophil and leukocyte kinetics were similar between the CXCR4 antagonist AMD3100 and A485 (Figure 9D).

To study potential analogies between our observations in mice and human biology, we obtained data from a previously characterized cohort of patients with Rubinstein-Taybi syndrome (RSTS), a rare developmental disorder with an estimated prevalence of 1 :125,000 among live-born infants. RSTS is characterized by a distinct phenotype, which includes a short stature, learning difficulties, and facial features. The inheritance of the disease follows an autosomal dominant pattern. 42 Heterozygous mutations in CREBBP (CBP) cause RSTS type 1 , while those in EP300 (p300) result in RSTS type 2. The two types of RSTS account for 55% and 8% of cases, respectively. 43 We included 46 individuals (21 females and 25 males) of whom full genetic data and leukocyte counts were available (Figure 1G). The median age of patients at diagnosis was 11 years (range: 1-43 years). Consistent with the literature, RSTS type 1 was much more common than RSTS type 2 in our cohort (40 vs. 6 cases). The majority individuals (65%) displayed leukocyte counts above the age adjusted upper limit of normal (“elevated”) (Figure 1G). We calculated that this distribution was skewed compared with expected frequencies in the general population 45, 46 (p < 0.0001 , c2 test, two-tailed). Patients were then stratified according to their mutational profile: the first group comprised mutations that were predicted to involve the HAT domain (missense within/ frameshift before or within/deletion before or including the HAT domain) of CREBBP or EP300 (n = 35), while the second group included all other mutations (n = 11) (Figure 1 H). HAT-domain-affecting mutations (referred to as “HATmut”) were linked to leukocytosis in 71% (25/35) of cases, while those that spared it (“HATwt”) were less likely associated with this phenotype (45%) (p = 0.11 , c2 test, two-tailed) (Figure 1 H). The absolute number of leukocytes did not differ between the two groups (Figure 9E). Altogether, these data support a function of the CBP/p300 HAT domain in controlling leukocyte compartment sizes in mice and humans.

A485-induced leukocytosis augments host defenses:

We then aimed to explore the therapeutic utility of our findings and first used Vav1-Nup98- Hoxd13 mice, an experimental model of myelodysplastic syndrome (referred to as “MDS”) (Figure 2A). We found that the expansion of the blood leukocyte compartment in response to A485 was maintained in MDS animals (Figure 2B). Because neutropenia is the clinically most relevant leukopenia, but neutrophils were only mildly reduced in these mice (Figure 2A), we next moved to a model of chemotherapy-associated bone marrow injury, which we induced by 5- fluorouracil (5FU) administration (Figure 2C). As expected, 5FU treatment resulted in significant leuko- and neutropenia, which could be acutely recovered by A485 treatment (Figure 2D). Accordingly, we investigated whether these effects would protect immunocompromised hosts against infections, which are major drivers of mortality in such individuals. 67, 68 We modeled Listeria monocytogenes sepsis, where neutrophils are indispensable for host defense, 69 and introduced the pathogen in the context of overt 5FU-induced pancytopenia (day 6 post 5FU) (Figures 2E and 2F). To directly test the functional effects of A485-induced leukocyte mobilization, we did not administer antibiotics. A485 was injected in a therapeutic fashion after infection had been established (Figure 2E). Although we did not note mortality in mice without bone marrow injury during the observational period, leukopenic hosts treated with vehicle solution lost weight, became moribund, and succumbed to disease (Figures 2G and 2H). By contrast, a single bolus of A485 was sufficient to ameliorate disease trajectories as reflected by reduced weight loss and improved survival (Figures 2G and 2H). This protective effect was linked to enhanced pathogen clearance as indicated by diminished recovery of viable bacteria from liver tissue lysates of A485-treated animals (Figure 2I). Less pronounced changes were noted in the spleen (Figure 2J). Taken together, our data imply a therapeutic potential of A485 for the acute recovery of leukocyte counts and augmentation of host defenses in the presence of bone marrow injury.

A485 mobilizes leukocytes from the bone marrow:

Having established that A485 induced a functional leukocytosis, we next sought to understand its mechanism of action. First, we reasoned that a prolonged half-life could not explain leukocytosis due to its rapid onset. Second, we found no evidence for induction of “emergency hematopoiesis” as numbers of progenitor populations in the bone marrow were unchanged by A485 (Figure 10A). Third, blocking lymphocyte egress from lymph nodes by sphingosine-1 -phosphate receptor antagonism also did not impair the ability of A485 to increase leukocyte and specifically, lymphocyte counts (Figure 10B). Therefore, we focused on bone marrow mobilization as a potential mechanism of A485-induced leukocytosis. To test whether A485 mobilized cells from the bone marrow, we used a previously described cell tracking approach, 13 in which bone marrow cells from CD45.1 mice were adoptively transferred into CD45.2 mice by intravenous injection (Figure 3A). By 8 h post transplantation, transferred cells (CD45.1 + ) had efficiently migrated from the blood into the bone marrow (Figures 3B and 10C) and could be mobilized from this compartment by A485: both CD45.1- and CD45.2-positive cells increased in the circulation and peripheral tissues following injection of the drug, while their numbers decreased significantly only in the bone marrow (Figures 3C and 10D). Consistent with previous reports, 13 we found that the relative drop in leukocytes in the bone marrow was modest (Figure 3C) but sufficient to explain the changes in the periphery due to the large number of cells stored in this compartment throughout the body. This was supported by estimate calculations based on our cell tracking data (Figure 3D) as well as published analyses of the numbers and distribution of leukocytes across tissues. 70 Using fluorophore-conjugated antibodies, we found that the distribution of cells within tissues in response to A485 was mostly intravascular, while some cells had also emigrated (Figures 3E, 3F, 10E, and 10F). Likewise, cells recruited from the bone marrow included those from intra- (B lymphocytes) and extravascular (neutrophils) pools (Figures 3G and 10G). Of note, A485 could no longer increase leukocyte counts in sublethally irradiated mice with acute hematopoietic failure (day 3 post irradiation), where bone marrow leukocytes were strongly diminished (Figure 3H). In summary, these results indicate that small-molecule-mediated CBP/p300 HAT inhibition rapidly and transiently expands the blood leukocyte compartment primarily by mobilizing cells from the bone marrow. However, we cannot formally rule out that demargination phenomena may also contribute to leukocytosis. Distinct effector pathways drive changes in leukocyte subsets in response to A485:

We then set out to explore the molecular mechanism underlying A485-induced bone marrow mobilization. The speed by which leukocytosis occurred in response to A485 led us to speculate that a tonic mechanism that retains cells in the bone marrow might be disrupted. CXCR4 signaling is induced by CXCL12 and regulates the cell surface expression of adhesion molecules, including VLA4, the latter of which is built from CD49d (encoded by Itga4 in mice) and CD29 (Itgbl). 29, 30 When CXCL12 levels decline, Cd49d is downregulated. 31 Peak leukocytosis in response to A485 was paralleled by reduced CD49d cell surface expression on B lymphocytes and neutrophils together with a concomitant decrease of the VLA4-binding partner vascular cell adhesion molecule 1 (VCAM1) on bone marrow endothelial cells as assessed by flow cytometry (Figures 4A and 11 A). We further detected reduced CXCL12 protein levels in the circulation and bone marrow of A485-treated mice (Figures 11 B and 11C). To understand which of these changes occurred first, we sampled mice 1 h post-injection, a time point prior to the onset of apparent leukocytosis. Here, HAT activity was already strongly suppressed as shown by reduced protein levels of acetylated lysine 18 residues of histone H3 (H3K18ac) in bone marrow cell lysates of A485-treated mice (Figure 11 D). Isolation of CD31+ and CD31- bone marrow cells demonstrated that Cxcl12 transcript levels were indifferent between groups at this early time point, and Cxcl12 protein abundance was likewise unchanged (Figures 11 E and 11 F). By contrast, Cxcr4 and Itga4 expression was reduced in both CD31- and CD31 + cells (Figures 4B and 11G). Using ex vivo cultures, we found that the downregulation of VLA4 involved transcriptional repression as well as internalization events, confirming a direct effect of A485 on the bone marrow (Figures 4C and 4D). We did not note changes in Cxcl12 protein secretion in the same system (Figure 11 H), indicating that A485 modulates the niche via direct and indirect pathways. Pharmacological inhibition of VLA4 (VLA4i) by firategrast resembled A485-induced leuko- and lymphocytosis, but with lower amplitude and different dynamics, while eliciting much milder neutrophilia (Figures 4E and 111). We further found that A485 did not inhibit leukocyte migration toward CXCL12 in vitro (Figure 11 J), suggesting that the compound does not act as a CXCR4 antagonist upstream of VLA4. Together, these results indicate that, while lymphocytosis may result from disruption of VLA4-VCAM1 -interactions, neutrophilia could not be explained by this mechanism. This led us to study other candidate pathways that may drive this response.

Neutrophil mobilization typically involves cytokine signaling. 5 However, inflammatory cytokines linked to emergency myelopoiesis and neutrophilia (tumor necrosis factor alpha, interleukin [IL]-1 beta) remained undetectable in the circulation of both A485 and vehicle-treated mice (Figures 12A and 12B). Consistently, disruption of IL-1-/Toll-like receptor family signaling by global genetic Myd88 deletion did not interfere with A485-induced neutrophilia or leukocytosis (Figures 4F and 12C). We also found no evidence for type 1 interferon, alarmin, or IL-6 dependency of the drug’s effects on leukocytes (Figures 12D-12F). Therefore, we next explored contributions of G-CSF signaling. We first measured the concentration of G-CSF in the circulation and detected increased levels in response to A485 treatment (Figure 4G). Administration of anti-G-CSF- antibodies diminished A485-induced neutrophilia while leaving total leukocyte and lymphocyte counts unchanged (Figures 4H and 12G). The lack of effect of G-CSF neutralization on lymphocytes was expected as these cells are not mobilized by G-CSF. 34 We did not detect changes in H3K18ac protein levels in bone marrow cultures treated with G-CSF, ruling out that CBP/p300 HAT activity resides downstream of the G-CSF receptor (CSF3R) (Figure 4I). We thus asked whether A485 and G-CSF converge onto a common effector cascade. We chose to manipulate the SNS as well as cholinergic signaling, both of which are required for G- CSFinduced bone marrow mobilization. 7,10 SNS outflow was dispensable for A485-triggered leukocytosis as chemical sympathectomy using 6-hydroxydopamine (6-OHDA) left neutrophilia, leuko-, and lymphocytosis intact (Figures 4J and 12H). These results were recapitulated in adrenoceptor beta 1/2 double-deficient mice (Adrb1/b2 KO) (Figure S6I), which lack the receptor repertoire engaged by the SNS to modulate leukocyte compartments. 71 Likewise, the bloodbrain barrier penetrating muscarinic receptor antagonist scopolamine did not interfere with neutrophilia or leukocytosis in response to A485 (Figure 4K). Lastly, combining A485 and G-CSF did not result in meaningful increases in total (sum of endo- and exogenous) G-CSF levels (Figure 12J), suggesting that the additive effects of the two compounds on neutrophils that we observed (Figure 1 F) cannot be explained by augmented G-CSF activity. We conclude that A485 requires both G-CSF-dependent and -independent pathways to elicit neutrophilia.

The HPA axis is activated by A485:

Our findings led us to hypothesize that the A485 leukocyte response was fueled by neurohumoral pathways. Therapeutic doses of glucocorticoids (GCs) trigger rapid-onset leukocytosis in humans, and GCs control leukocyte trafficking under homeostatic and stressful conditions. 35, 36 Circulating levels of corticosterone (referred to as CORT) - the main endogenous GC in mice - were strongly increased in response to A485 (Figure 5A), indicative of HPA-axis activation. The HPA axis operates according to control loop principles 37 and is built from 3 main components (Figure 5B). Unlike forskolin - which promotes cyclic adenosine monophosphate (cAMP) production 38 - increasing concentrations of A485 had no effect on the transcription of key steroidogenic enzymes and CORT release of adrenal cortical cells (Y1) in vitro (Figures 5C and 13A), arguing against a direct effect of the drug on the adrenal gland. We thus asked whether A485 induces HPA-axis activity at the level of the CNS. Consistent with this idea, the mRNA abundance of cFos - an immediate-early gene indicative of neuronal activity - was nearly twice as high in hypothalamus cell lysates of A485-treated animals compared with controls 1 h postinjection (Figure 5D). We further observed that cFos protein was elevated in the paraventricular nucleus (PVN) of the hypothalamus, where corticotropin-releasing hormone (CRH)-producing neurons reside (Figure 5E). These changes translated into augmented systemic adrenocorticotropic hormone (ACTH) release (Figure 5F). We thus screened several mechanisms by which A485 may engage the HPA axis at the level of the hypothalamus.

Some hypothalamic neuronal populations are surrounded by permeable capillary networks, which allow for exposure to systemic cues. 39 However, we found that levels of A485 in the hypothalamus were as low as in other parts of the brain, and hypothalamic explants did not upregulate cFos when exposed to the drug ex vivo (Figures 5G and 13B), suggesting that A485 acts on the PVN via an indirect route. Systemic hypoglycemia was not the underlying trigger as blood glucose levels increased, rather than decreased upon A485 treatment (Figure 5H). Body temperature declined in response to A485 exposure, but warmth supplementation did not prevent CORT release, demonstrating that core body temperature is not correlated with HPA-axis activation (Figures 5I and 5J). We also found no evidence for contributions of IL-6 signaling (Figure 13C), which is physiologically induced in response to stress and regulates the HPA axis. 72, 73 We further noted that HPA-axis activation did not require nociception or consciousness because mice subjected to ablation of TRPV1 + sensory neurons using resiniferatoxin (RTX) or ketamine/xylazine (ket/xyl) anesthesia still showed strongly elevated levels of CORT and leukocytes in response to the drug (Figures 5K, 5L, 13D, and 13E). We thus wondered whether sensory nerve fibers that are spared by RTX (i.e., TRPV1-) may convey peripheral A485 distribution to the CNS. We hypothesized that such fibers are cholinergic and likely carried by the vagus nerve because most viscera receive sensory innervation via this route. 74 Vagal ganglia mainly express nicotinic acetylcholine receptors containing the a3-subunit (nAchRa3), which are sensitive to inhibition by kappa bungarotoxin (kBTX), a snake venom. 75, 76 kBTX has high affinity for a3, but not other nAchRs. 77 We found that nAchRa3 blockade by kBTX also did not prevent A485-induced CORT release (Figure 5M). We conclude that A485 elicits central activation of the HPA axis via an indirect pathway that remains unknown.

HPA-axis activity relays the A485 leukocyte response independently of glucocorticoids:

GCs are the main effectors of the HPA axis. To study the contributions of GC to A485-induced bone marrow mobilization, we used several orthogonal tools. First, we blocked the GC receptor pharmacologically (mifepristone/RU486) and genetically (Nr3c1 fl/fl Rosa26CreERT2, referred to as Nr3c1 KO ), both of which did not affect leukocyte populations in response to A485 (Figures 6A and 14A-14C). Second, induction of GC deficiency failed to prevent increases in leukocytes (Figures 6B and 14D). Third, we subjected mice to adrenalectomy (ADX) or sham surgery, which successfully resulted in adrenal insufficiency as indicated by diminished systemic CORT abundance and a counterregulatory surge in ACTH levels (Figures 14E and 14F). ADX did not abrogate leuko- and lymphocytosis or neutrophilia (Figures 6C and 14G), demonstrating that GC, mineralocorticoids, and systemically released catecholamines are dispensable for the effects of A485 on leukocytes. By contrast, ADX mice exhibited even higher leukocyte counts than shamcontrols (Figures 6C and 14G), implying that loss of HPA feedback supports the A485 leukocyte response. Therefore, we reasoned that the signal mediating the drug’s effect on leukocytes derives from the CNS and is induced upon loss of HPA feedback. We hypothesized that this signal is ACTH because, unlike CRH, it is secreted into the systemic circulation, 40 where it can reach peripheral tissues such as the bone marrow.

Pharmacological inhibition of corticotropin-releasing hormone receptor 1 (CRHR1), which is upstream of ACTH (Figure 5B) and highly expressed in the brain but not in the bone or bone marrow (Figure 14H), by two independent small molecules (dmp696 and antalarmin) prevented A485-induced leukocytosis and neutrophilia (Figures 6D, 6E, 141, and 14J). When we supplemented mice with recombinant ACTH (rACTH) in the presence of CRHR1 inhibition, neutrophils increased again (Figures 6F and 6G). We further noted that WT mice treated with rACTH developed neutrophilia, which was maintained upon disrupted GC signaling (Figures 6H, 6J, and 14K). By contrast, lymphopenia was lost when GCs were blocked (Figure 6J), demonstrating that neutrophils and lymphocytes are controlled by divergent effector mechanisms of the HPA axis. In the murine bone marrow, transcriptional expression of the high-affinity ACTH receptor melanocortin receptor 2 (Mc2r) together with other melanocortin receptors was detected (Figure 15A). Both CD31 + and CD31- bone marrow cells expressed Mc2r (Figure 15A), although at lower levels than the AT (Figure 15B), for which direct ACTH effects have previously been described. 78 To narrow down the bone marrow cell type that may potentially be targeted by ACTH, we screened two publicly available single-cell RNA sequencing datasets. We found that Mc2r was expressed by the stroma but not hematopoietic precursor cells or mature leukocytes (Figures 15C and 15D). Within the bone marrow stroma, Mc2r transcripts were confined to the leptin-receptor- positive mesenchymal stromal cell population (LepR+ MSCs) (Figure 15D), the main endogenous source of CXCL12.62 Consistent with these observations, blockade of CRHR1 was sufficient to increase bone marrow CXCL12 protein levels in A485-treated mice (Figure 15E). These data imply that the bone marrow is ACTH responsive. In summary, our results show that central activation of the HPA axis by A485 relays leukocyte mobilization via CRHR1 -regulated signals including ACTH, but independently of GCs.

DISCUSSION

With this study, we introduce the inhibition of the CBP/p300 HAT domain by the small-molecule A485 as a mechanism to elicit acute and transient bone marrow mobilization, which is equally effective as G-CSF in mice. Although we found that A485-induced neutrophil mobilization depended on endogenous G-CSF activity, the pharmacodynamic effects of A485 and G-CSF treatment are distinct: first, neutrophilia onset is faster with A485 compared with G-CSF. Second, A485 neutrophilia requires CRHR1 but not GC, muscarinic receptors, or nociceptive nerves, all of which are involved in the G-CSF-mediated bone marrow response. 10 Third, B lymphocytes are mobilized by A485 but not G-CSF. Fourth, the SNS is not involved in relaying bone marrow mobilization by A485, while it is an important component of the G-CSF effector cascade, 7 and fifth, our results collectively suggest that CBP/p300 HAT activity is upstream, rather than downstream of CSF3R. Consistent with these observations, combining A485 and G-CSF resulted in superior neutrophil mobilization compared with treatment with either agent alone. We conclude that A485 requires endogenous G-CSF to evoke neutrophilia but engages additional effector pathways that are G-CSF independent.

We also noted that the effects of A485 on the bone marrow were shorter than those of G-CSF, which manifested as lower cell counts by 24 h post-injection. This difference between the two drugs has two important implications: On the one hand, A485 may be favored in cases where only short bursts of neutrophil mobilization are required such as in acute infection. In fact, unresolved monocytosis and neutrophilia can be detrimental in this context due to the risk of excessive inflammation and collateral tissue damage. 13, 36 On the other hand, G-CSF could be superior to A485 in promoting long-term hematopoietic recovery. In our study, we did not find evidence for an acute effect of A485 on HSCs. However, this does not preclude the possibility, that repeated injections of A485 alone or in combination with G-CSF elicit mobilization of these cells. These avenues deserve to be explored in the future.

We infer that A485 might be introduced as a complementary pharmacological strategy to G-CSF derivatives in the clinics with potential benefits for rapid and short-term modulation of leukocyte compartments. Specifically, individuals suffering from acute neutropenic fever, in whom G-CSF supplementation does not confer clear clinical benefits, 79 may be a candidate population for A485 treatment. However, the therapeutic window of the drug needs to be better defined, as we only focused on a single time point post-infection. Accordingly, we do not know whether A485 treatment also confers benefits at later stages of the infection. This aspect is critical because patients typically present at variable stages of sepsis in the clinics, corresponding to a much more heterogeneous population than rodents, in which the onset of infection is clear to the investigator. Further, we only explored the effects of A485 in experimental listeriosis, a standard murine sepsis model, but whether the drug exerts protective effects in other bacterial and/or viral infections remains to be assessed. Another important consideration of A485 therapy is the trade-off between augmentation of host defenses and immunopathology, 60 which could be dose limiting and needs to be rigorously characterized in preclinical models. From a broader perspective, the established anti-tumor effects of A485 26, 80, 81 together with the herein reported immune modulation render the use of the compound attractive in the adjuvant setting of cancer therapy, where combating infectious complications and interference with (residual) malignant growth are both desirable. Vice versa, it is unknown whether inhibition of the CBP/p300 HAT domain can also exert pro-tumorigenic effects under certain conditions, but the bulk of available experimental data 26, 80, 81 argues against this notion.

The shifts in leukocyte populations induced by A485 reported in this study are unique because other types of challenges that promote HPA-axis activation (e.g., psychological stress) as well as the delivery of exogenous GC drive opposing movements of leukocyte subsets characterized by increases in blood neutrophils and concurrent lymphocyte migration into the bone marrow, which culminates in lymphopenia. 13 We observed similar changes when ACTH was injected into GC- competent animals, and lymphopenia but not neutrophilia was lost when GCs were blocked. Lymphocyte homing into the bone marrow is strongly shaped by CXCR4, the expression of which is typically augmented when GC levels rise. 51 By contrast, A485 triggers HPA-axis activity while simultaneously suppressing CXCR4 transcription, inhibiting the production of the receptor’s ligand (CXCL12) and downregulating a key downstream effector molecule, VLA4. Our data suggest that some of these changes result from a direct effect of the drug on the bone marrow, which could explain lymphocyte egress into the blood despite high levels of GC.

ACTH is sufficient to evoke neutrophilia in patients suffering from adrenal insufficiency, who have a diminished GC response. 52 In rodents, ADX, but not sham surgery, is linked to neutrophilia. Likewise, high ACTH levels in subclinical Cushing’s syndrome can be associated with substantial neutrophilia, even if cortisol levels are only minimally elevated. 53 Consistent with these observations, we found that ACTH promoted increases in neutrophils in the absence of functional GC signaling. These observations support a role of ACTH in controlling neutrophil compartments and raise the question which other homeostatic functions classically attributed to GC are partly or fully mediated by its upstream regulator ACTH. Such GC-independent functions of ACTH are reminiscent of those of other pituitary hormones, which can directly affect organ physiology independent of the gland-derived endocrine mediators that they control. 54, 55 The relatively low expression of MC2R in the murine bone marrow suggests that ACTH signaling may not occur in the steady state in this tissue. Instead, a certain threshold of HPA-axis activation and ACTH release may be required to engage bone marrow MC2Rs. This could imply that ACTH modulates leukocyte compartments only upon stress, injury, or other types of insults, where bone marrow mobilization is a physiological response. Lastly, although G-CSF activates the HPA axis, 10 the reciprocal pathway in which ACTH regulates G-CSF biology should be further investigated.

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Claims

1 . An inhibitor of a histone acetyl transferase (HAT) activity of CREBBP(CBP)/p300 for use in the treatment of cytopenia, wherein the inhibitor induces acute and/or transient leukocytosis, comprising mobilization of leukocytes from the bone marrow into the blood of a subject.

2. The inhibitor for use according to the preceding claim, wherein the inhibitor suppresses a HAT domain of CBP/p300 by competing with acetyl CoA to bind the HAT domain.

3. The inhibitor for use according to any one of the preceding claims, wherein the compound is a small molecule compound.

4. The inhibitor for use according to any one of the preceding claims, wherein the inhibitor is a spirocyclic HAT inhibitor or a non-spirocyclic HAT inhibitor.

5. The inhibitor for use according to any one of the preceding claims, wherein the inhibitor is selected from the group comprising A-485, IP300w, or CPI-1612.

6. The inhibitor for use according to any one of the preceding claims, wherein the inhibitor is A- 485

7. The inhibitor for use according to the preceding claims 1-4, wherein the inhibitor is C646

8. The inhibitor for use according to any one of the preceding claims, wherein the inhibitor induces acute and/or transient leukocytosis, thereby restoring the abundance of blood cells, preferably of leukocytes and/or neutrophiles, in the subject in a homeostatic range.

9. The inhibitor for use according to any one of the preceding claims, wherein the inhibitor induces central activation of the hypothalamus-pituitary-adrenal (HPA)-axis in a subject, preferably wherein activation of the HPA-axis results in corticotropin-releasing hormone receptor 1 (CRHRI)-dependent release of ACTH and/or glucocorticoid release in a subject.

10. The inhibitor for use according to any one of the preceding claims, wherein the cytopenia is a leukopenia.

11 . The inhibitor for use according to any one of the preceding claims, wherein the cytopenia is a neutropenia.

12. The inhibitor for use according to any one of the preceding claims, wherein the patient has been diagnosed with cancer, and/or a genetic or acquired bone marrow disorder resulting in cytopenia and/or wherein the patient is suffering from cytopenia caused by or associated with a cancer treatment the patient has been and/or is receiving.

13. The inhibitor for use according to any one of the preceding claims, wherein the patient exhibits one or more of recurrent infections (including gingivitis), opportunistic viral, fungal and/or bacterial infections, severe infections (including sepsis), severe fatigue, muscle weakness, pneumonia, fever, swelling and redness, mouth sores, severe cough, shortness of breath and/or diarrhea.

14. The inhibitor for use according to any one of the preceding claims, wherein G-CSF or derivative thereof, such as recombinant G-CSF or a G-CSF analogue, is administered in combination and/or simultaneously to a subject.

15. The inhibitor for use according to any one of the preceding claims, wherein an anti-infectant, preferably an antibiotic, a virostatic, an antiviral and/or an antimycotic compound, is administered in combination and/or simultaneously to a subject.

16. A pharmaceutical composition for use in the treatment of cytopenia comprising the inhibitor according to any one of claims 1-8.

17. A combination medication for use in the treatment of cytopenia, comprising an inhibitor according to any one of claims 1-8 and a compound selected from the group comprising G- CSF, recombinant G-CSF and/or a G-CSF analogues.