WO2024246220A1

WO2024246220A1 - Methods and compositions for treating lung cancer

Info

Publication number: WO2024246220A1
Application number: PCT/EP2024/064934
Authority: WO
Inventors: Laurent YVAN-CHARVET; Marius ILIE; Paul Hofman
Original assignee: Institut National de la Sante et de la Recherche Medicale INSERM; Universite de Nice Sophia Antipolis UNSA; Centre Hospitalier Universitaire de Nice
Current assignee: Institut National de la Sante et de la Recherche Medicale INSERM; Universite de Nice Sophia Antipolis UNSA; Centre Hospitalier Universitaire de Nice
Priority date: 2023-05-31
Filing date: 2024-05-30
Publication date: 2024-12-05
Anticipated expiration: 2025-11-30

Abstract

Cholesterol efflux pathways have anti-inflammatory and anti-proliferative properties that could be exploited in tumor biology to unravel cancer vulnerabilities. Using a mouse model of lungtumor bearing KRAS^G12D mutation, the inventors identified that disruption of cholesterol efflux pathways by specific inactivation of Abca1 and Abcg1 in epithelial cancer progenitor cells and to some extent in macrophages promoted a pro-tolerogenic tumor microenvironment (TME). In particular, the inventors show that cholesterol removal therapy with cyclodextrin inhalation also reduced tumor burden in progressing tumor by suppressing the proliferation and expansion of epithelial progenitor cells of tumor-origin. The inventors' results position cholesterol removal therapy as a putative metabolic target in lung cancer progenitor cells. The present invention relates to a method for the treatment of a lung cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of cyclodextrin.

Description

METHODS AND COMPOSITIONS FOR TREATING LUNG CANCER

FIELD OF THE INVENTION:

The invention is in the field of oncology, in particular in the field of lung cancer.

BACKGROUND OF THE INVENTION:

Advances in cancer metabolism research over the last decades have extended the original observation that most tumor cells rely on glucose and glutamine catabolism to sustain the energetic and biosynthetic demands of uncontrolled proliferation^1,2. Cancer metabolic reprogramming in an in vivo setting is a complex phenomenon that depends on intrinsic tumor properties (driver mutation, tissue of origin, stage) and on constraints imposed by its microenvironment³. Recent evidence highlights a metabolic competition between tumor and immune cells that creates an immunosuppressive/tolerogenic tumor microenvironment (TME). This includes a glucose steal mechanism by tumor cells⁴, the generation of pro-tolerogenic metabolites such as lactate⁵. This global reprogramming enables tumor cells to carry outgrowth and survival instructions initiated by oncogenic mutations. Thus, gaining a better understanding of the nature of nutrient utilization by cancer cells and the metabolic routes used to sustain tumor growth is important, as it will provide insights into the mechanisms of spontaneously arising tumors.

Lung cancer is a leading cause of cancer mortality worldwide, with non-small cell lung cancer (NSCLC) representing almost 85% of all cases. It is estimated to kill 1.7 million people worldwide per year (more than 19% of the total cancer deaths)⁶. In the absence of early warning signs, patients with localized lung cancer can be cured by surgical resection. However, most of the patients are diagnosed with late-stage lung cancer with poor prognosis and clinical outcome, pressing the need to identify early biomarkers. Activating mutations of the proto-oncogene KRAS (V-KI-ras2 Kirsten rat sarcoma viral oncogene homolog) occur in -30% of NSCLC cases and cultured mutant KRAS-driven tumor cells have specific metabolic consequences in vitro¹. While increased aerobic glycolysis with glucose-to-lactate conversion (Warburg effect) is a common feature of KRAS mutant cells, preclinical studies assessing early metabolic alterations in KRAS-driven lung tumorigenesis remain sparse. While the group of DeBerardinis recently showed evidences for oxidation of multiple nutrients within and between lung tumors⁸, the group of Vander Heiden showed that KRAS-driven lung tumors in rodents are less dependent on glutamine metabolism than culture cells⁹. Nevertheless, dietary control exerts a robust anti -turn or effect in the same mouse model of KRAS-driven lung cancer, indicating that this type of tumor is sensitive to metabolic changes¹⁰.

A meta-analysis of randomized controlled trials of lipid-altering therapies with 625,000 patients and >8,000 incident cancers revealed that a 10-mg/dL increase in the plasma high-density lipoproteins (HDL)-cholesterol level was associated with a 36% lowered risk of cancer incidence¹¹. However, the role for HDL-C in cancer development remains elusive, especially the link between its cholesterol efflux capacity and the control of tumor growth¹². The ability of HDL and its major apolipoprotein A-I (apo- Al) to promote the efflux of cholesterol from cells depends in part on the ATP -binding cassette transporters ABCA1 and ABCG1, but can also occur though scavenger receptor Bl (SR-BI) or passive efflux pathways¹³. While raising HDL-C levels in syngeneic and xenogeneic mouse models of cancer metastasis limited tumor growth^14,15, increased cholesterol efflux pathways in tumor-associated macrophages (TAMs) rather generated a pro-tolerogenic TME¹⁶'¹⁸. The reason of this discrepancy remains to be clarified but could highlight the limitation of the syngeneic and xenogeneic tumor models that do not take into account the diversity of TAMs in their specific tissue environment or the lack of understanding in the metabolic dialogue between tumor and immune cells¹⁹.

To evaluate if cholesterol efflux pathways interfere with KRAS-driven lung tumorigenesis and unmask key cancer vulnerabilities, the inventors used a model of lung adenocarcinoma development induced by specific expression of oncogenic KRAS mutation in the lung²⁰. The inventors made the unexpected observation that defective cholesterol efflux pathways in macrophages had only a mild pro-tolerogenic role in a mouse model of early NSCLC. The inventors rather unravel the first in vivo evidence that defective cholesterol efflux pathways in epithelial tumor progenitor cells is a culprit of early NSCLC lesion development. Using the multiplexed capabilities of flow cytometry and single cell transcriptomics, the inventors identify that defective cholesterol efflux pathways in epithelial progenitor cells enable outgrowth and survival instructions initiated by oncogenic KRAS mutation and compromise anti-tumor immunity in vivo. Cholesterol removal strategies from the onset or in established tumors suppressed the proliferation and expansion of epithelial progenitor cells of tumor-origin. Mechanistically, the inventors identified that HDL blunted a positive feedback loop between growth factor signaling pathways and cholesterol efflux pathways that cancer cells hijack to expand. Finally, the inventors have used topology of transcriptional regulatory networks to connect perturbed cholesterol efflux pathways in human lung adenocarcinoma to systemic reduced HDL-mediated cholesterol efflux capacity. These experiments collectively indicate a novel anti-tumorigenic role of cholesterol efflux pathways in lung cancer that could be envisioned as a novel checkpoint blockade therapy on tumor progenitor cells.

SUMMARY OF THE INVENTION:

The present invention relates to a method for the treatment of a lung cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of cyclodextrin. In particular, the invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

Cholesterol efflux pathways have anti-inflammatory and anti-proliferative properties that could be exploited in tumor biology to unravel cancer vulnerabilities. Using a mouse model of lungtumor bearing KRAS^G12D mutation, the inventors identified that disruption of cholesterol efflux pathways by specific inactivation of Abcal and Abcgl in epithelial cancer progenitor cells and to some extent in macrophages promoted a pro-tolerogenic tumor microenvironment (TME) and tumor growth. Defective cholesterol efflux in epithelial cancer progenitor cells dominated tumor growth by governing their transcriptional landscape to support their expansion and creating TME heterogeneity that compromises anti-tumor immunity. Overexpression of the apolipoprotein A-I, to raise HDL levels, limited the cholesterol lung retention and protected these mice from tumor development and dire pathologic consequences. Mechanistically, HDL blunted a positive feedback loop between growth factor signaling pathways and cholesterol efflux pathways that cancer cells hijack to expand. Cholesterol removal therapy with cyclodextrin inhalation also reduced tumor burden in progressing tumor by suppressing the proliferation and expansion of epithelial progenitor cells of tumor-origin. Local and systemic perturbations of cholesterol efflux pathways was confirmed in human lung adenocarcinoma. The inventors’ results position cholesterol removal therapy as a putative metabolic target in lung cancer progenitor cells.

Method for treating luns cancer

In one embodiment, the present invention relates to a method for the treatment of a lung cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of cyclodextrin. In particular, the present invention also relates to a method for the treatment of a lung cancer in a patient in need thereof comprising administering to the lung of the patient a therapeutically effective amount of cyclodextrin

As used herein, the term “macrophage” has its general meaning in the art and refers to a type of antigen-presenting cell of the mammalian immune system that have phagocytic activities. These cells are characterized by their distinctive morphology and high levels of surface MHC- class II expression. A macrophage is a monocyte-derived phagocyte which is not a dendritic cell or a cell that derives from tissue macrophages by local proliferation. In the body these cells are tissue specific and refer to e. g. Kupffer cells in the liver, alveolar macrophages in the lung, microglia cells in the brain, osteoclasts in the bone etc. The skilled person is aware how to identify macrophage cells, how to isolate macrophage cells from the body of a human or animal, and how to characterize macrophage cells with respect to their subclass and subpopulation.

Macrophages have historically been divided into two phenotypically diverse populations, i.e. a Ml -polarized or "classically activated" population, and a macrophage M2 -polarized or "alternatively activated" population. However, it is well appreciated in the art that a continuum of phenotypes exists between the macrophage Ml- polarized and macrophage M2 -polarized populations, and in some cases macrophages assume a phenotype that does not fit well within any of these defined phenotypic groups.

Macrophages exhibiting a Ml phenotype are pro-inflammatory, and are capable of either direct (pathogen pattern recognition receptors) or indirect (Fc receptors, complement receptors) recognition of pathogens and tumor antigens (i.e. they exhibit anti -tumor activity). Ml macrophages produce reactive oxygen species and secrete pro-inflammatory cytokines and chemokines, such as, for example, but without limitation, TNFa, IL-1, IL-6, IL-15, IL-18, IL- 23, and iNOS. Ml macrophages also express high levels of MHC, costimulatory molecules, and FCyR. The Ml phenotype is triggered by GM-CSF and further stimulated by interferon-y (IFN-y), bacterial lipopolysaccharide (LPS), or tumor necrosis factor a (TNFa), and is mediated by several signal transduction pathways involving signal transducer and activator of transcription (STAT), nuclear factor kappa-light-chain-enhancer of activated B cells (NFKB), and mitogen-activated protein kinases (MAPK). These events enhance the production of agents such as the reactive oxygen species and nitric oxide (NO) and promote subsequent inflammatory immune responses by increasing antigen presentation capacity and inducing the Thl immunity through the production of cytokines such as IL-12.

In contrast, macrophages exhibiting a M2 phenotype are often characterized as being antiinflammatory and immunosuppressive as they suppress T-cell responses and are involved in the Th2-type immune response. The M2 macrophage phenotype facilitates tissue repair, wound healing, and is profibrotic. M2 macrophages often undesirably infiltrate and surround tumors, where they provide an immunosuppressive microenvironment that promotes rather than suppresses tumor progression. M2 macrophages are characterized by high surface expression of I1-4R, FcsR, Dectin- 1, CD 136, CD206, and CD209A. M2 macrophages include IL-4/IL- 13 -stimulated macrophages, IL-10-induced macrophages, and immune complex-triggered macrophages.

As used herein, the term “cholesterol” has its general meaning in the art and refers to any of a class of certain organic molecules called lipids. It is a sterol (or modified steroid), a type of lipid. Cholesterol is biosynthesized by all animal cells and is an essential structural component of animal cell membranes. When chemically isolated, it is a yellowish crystalline solid. Cholesterol also serves as a precursor for the biosynthesis of steroid hormones, bile acid[4] and vitamin D.

As used herein, the term “cholesterol efflux” or “cholesterol efflux activity” refers to the efflux of cholesterol from population of in epithelial tumor progenitor cells or from macrophages. Accordingly the term refers to the movement of cholesterol from the cell to the cell's exterior.

As used herein, the term "cancer" has its general meaning in the art and includes, but is not limited to, solid tumors and blood-borne tumors. The term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood and vessels. The term "cancer" further encompasses both primary and metastatic cancers. Examples of cancers that may be treated by methods and compositions of the invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

As used herein, the term “KRAS mutant cancer” refers to a cancer in which the initiation and/or maintenance are/is dependent, at least in part, on one or more mutations in the gene that encodes KRAS (human: UniProtKB — P01116). In certain embodiments, the one or more KRAS mutations constitutively activate KRAS and subsequently its downstream Raf/MEK/ERKl/2 and/or PI3K/PIP3/AKT survival pathways in cancer cells of the KRAS mutant cancer.

In some embodiment, the individual has a KRAS mutant cancer characterized by the presence of a solid tumor, a semi-solid tumor, a primary tumor, a metastatic tumor, a liquid tumor (e.g., a leukemia or lymphoma), and/or the like. According to some embodiments, the individual has a KRAS mutant cancer selected from breast cancer, glioblastoma, neuroblastoma, head and neck cancer, gastric cancer, ovarian cancer, skin cancer (e.g., basal cell carcinoma, melanoma, or the like), lung cancer, colorectal cancer, prostate cancer, glioma, bladder cancer, endometrial cancer, kidney cancer, leukemia (e.g., T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML), etc.), liver cancer (e.g., hepatocellular carcinoma (HCC), such as primary or recurrent HCC), a B-cell malignancy (e.g., non-Hodgkin lymphomas (NHL), chronic lymphocytic leukemia (CLL), follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, and the like), pancreatic cancer, thyroid cancer, any combinations thereof, and any sub-types thereof. In certain embodiments, the individual's KRAS mutant cancer is a human pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer, colorectal cancer, and/or biliary cancer. In a particular embodiment, the cancer is lung cancer.

As used herein, the term "lung cancer" includes, but is not limited to all types of lung cancers at all stages of progression like lung carcinomas metastatic lung cancer, non-small cell lung carcinomas (NSCLC) or small cell lung carcinomas (SCLC).

In some embodiments, the subject suffers from a non-small cell lung carcinomas (NSCLC).

As used herein, the term “non-small cell lung cancer” or "NSCLC" has its general meaning in the art and includes a disease in which malignant cancer cells form in the tissues of the lung. Examples of non-small cell lung cancers include, but are not limited to, squamous cell carcinoma, large cell carcinoma or lung adenocarcinoma.

In some embodiments, the subject suffers from a lung adenocarcinoma.

As used herein, the term “lung adenocarcinoma” has its general meaning in the art and refers to a subtype of non-small cell lung cancer. Lung adenocarcinoma starts in glandular cells, which secrete substances such as mucus, and tends to develop in smaller airways, such as alveoli. Lung adenocarcinoma is usually located more along the outer edges of the lungs. Lung adenocarcinoma tends to grow more slowly than other lung cancers.

In some embodiments, the KRAS mutant cancer is a KRAS mutant lung cancer. Non-limiting examples of KRAS mutant lung cancers that may be treated according to the methods of the present disclosure include KRAS mutant small cell lung cancers (SCLC) and KRAS mutant non-small cell lung cancers (NSCLC). When the individual has a KRAS mutant NSCLC, in some embodiments, the individual has a KRAS mutant lung adenocarcinoma (LU AD).

As used herein, the term “subject” denotes a mammal, such as a rodent, a feline, a canine, and a primate. Particularly, the subject according to the invention is a human. In particular, the subject has or is susceptible to have lung cancer. More particularly, the subject has or is susceptible to a non-small cell lung carcinomas (NSCLC).

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

As used herein, the expression “effective amount” means an amount of cyclodextrin or methyl- P-cyclodextrin (mpCD) used in the present invention sufficient to result in the desired therapeutic response i.e. the treatment of the lung infection. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic 20 adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day. In particular, the daily dosage of the products of the invention (i.e. cyclodextrin or methyl-P-cyclodextrin (mpCD)) may be varied over a wide range from 1 a 3 mg/kg per adult per day.

The product of the invention (e.g. cyclodextrin methyl-P-cyclodextrin (mpCD)) may be administered once, twice, three times, four times, five times per day.

As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g. cyclodextrin methyl-P-cyclodextrin (mpCD)) into the subject, such as by nasal, oral, mucosal, intradermal, intravenous, subcutaneous, intrathecal, intramuscular delivery and/or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof. In a particular embodiment, the subject is administered by aerosolization.

As used herein, the term "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of the product of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the product of the invention to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. The efficient dosages and dosage regimens for the product of the invention depend on the disease or condition to be treated and may be determined by the persons skilled in the art. A physician having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician could start doses of product of the invention employed in the pharmaceutical composition at levels lower than that required achieving the desired therapeutic effect and gradually increasing the dosage until the desired effect is achieved. In general, a suitable dose of a composition of the present invention will be that amount of the compound, which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen. Such an effective dose will generally depend upon the factors described above.

For example, a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize the progression of disease. Typically, the ability of a compound to inhibit cancer may, for example, be evaluated in an animal model system predictive of efficacy in human tumors. A therapeutically effective amount of a therapeutic compound may decrease tumor size, or otherwise ameliorate symptoms in a patient. One of ordinary skill in the art would be able to determine such amounts based on such factors as the patient's size, the severity of the patient's symptoms, and the particular composition or route of administration selected. An exemplary, non-limiting range for a therapeutically effective amount of a inhibitor of the present invention is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1- 20 mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about 1, about 3 mg/kg, about 5 mg/kg or about 8 mg/kg. An exemplary, non-limiting range for a therapeutically effective amount of a inhibitor of the present invention is 0.02-100 mg/kg, such as about 0.02-30 mg/kg, such as about 0.05-10 mg/kg or 0.1-3 mg/kg, for example about 0.5-2 mg/kg. Administration may e.g. be intravenous, intramuscular, intraperitoneal, or subcutaneous, and for instance administered proximal to the site of the target. Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some embodiments, the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time. In some embodiments, the efficacy may be monitored by visualization of the disease area, or by other diagnostic methods described further herein, e.g. by performing one or more PET-CT scans. If desired, an effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In some embodiments, the human monoclonal antibodies of the present invention are administered by slow continuous infusion over a long period, such as more than 24 hours, in order to minimize any unwanted side effects. An effective dose of a inhibitor of the present invention may also be administered using a weekly, biweekly or triweekly dosing period. The dosing period may be restricted to, e.g., 8 weeks, 12 weeks or until clinical progression has been established. As non-limiting examples, treatment according to the present invention may be provided as a daily dosage of a inhibitor of the present invention in an amount of about 0.1-100 mg/kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.

The inventors show that cholesterol removal therapy with cyclodextrin inhalation reduced tumor burden in progressing tumor by suppressing the proliferation and expansion of epithelial progenitor cells of tumor-origin.

As used herein, the term “cyclodextrin” has its general meaning in the art and refers to family of cyclic oligosaccharides, consisting of a macrocyclic ring of glucose subunits joined by a-1,4 glycosidic bonds. Cyclodextrins are typically produced from starch by enzymatic conversion. Cyclodextrins are composed of 5 or more a-D-glucopyranoside units linked l->4, as in amylose (a fragment of starch). Typical cyclodextrins contain a number of glucose monomers ranging from six to eight units in a ring, creating a cone shape. The term “a-cyclodextrin” indicates that the cyclodextrin has 6 sugar moieties in its cyclic structure, the term “P-cyclodextrin” or “P-CD” indicates that the cyclodextrin has 7 sugar moieties in its cyclic structure, and the term “y-cyclodextrin” indicates that the cyclodextrin has 8 sugar moieties in its cyclic structure.

As used herein, the term “Methyl-p-cyclodextrin” (“mpCD or “Methyl-P-CD”) or “P-methyl- cyclodextrin” refers to a macrocyclic compound that can form inclusion complexes with a number of guest molecules. It shows higher solubility in aqueous solutions and greater solubilizing and complexing power compared to the parent P-cyclodextrin. MpCD can be used to enhance the enzyme activity and enantioselectivity of subtilizing enzyme in organic solvents. Methyl-P-cyclodextrin has the following CAS Number : 128446-36-6 and the following chemical structure :

Typically, the product of the present invention is administered to the patient in the form of a pharmaceutical composition which comprises a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, di sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene- block polymers, polyethylene glycol and wool fat. For use in administration to a patient, the composition will be formulated for administration to the patient. The compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intranasal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Sterile injectable forms of the compositions of this invention may be aqueous or an oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation. The compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include, e.g., lactose. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added. Alternatively, the compositions of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols. The compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs. For topical applications, the compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyl dodecanol, benzyl alcohol and water. Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Patches may also be used. The compositions of this invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well- known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents. For example, an antibody present in a pharmaceutical composition of this invention can be supplied at a concentration of 10 mg/mL in either 100 mg (10 mL) or 500 mg (50 mL) single-use vials. The product is formulated for IV administration in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7 mg/mL polysorbate 80, and Sterile Water for Injection. The pH is adjusted to 6.5. An exemplary suitable dosage range for an antibody in a pharmaceutical composition of this invention may between about 1 mg/m² and 500 mg/m². However, it will be appreciated that these schedules are exemplary and that an optimal schedule and regimen can be adapted taking into account the affinity and tolerability of the particular antibody in the pharmaceutical composition that must be determined in clinical trials. A pharmaceutical composition of the invention for injection (e.g., intramuscular, i.v.) could be prepared to contain sterile buffered water (e.g. 1 ml for intramuscular), and between about 1 ng to about 100 mg, e.g. about 50 ng to about 30 mg or more preferably, about 5 mg to about 25 mg, of the inhibitor of the invention.

In some embodiment, the cyclodextrin is administered by aerosolization.

In some embodiment, the P-cyclodextrin (P-CD) is administered by aerosolization.

In some embodiment, the Methyl-P-cyclodextrin (mpCD) is administered by aerosolization. As used herein, the term "aerosolization" refers to a process whereby a liquid or powder or a vial formulation is converted to an aerosol. Typically, aerosolization is performed by a nebulizer.

In a particular embodiment, the cyclodextrin is administered by inhalation.

In some embodiment, the P-cyclodextrin (P-CD) is administered by inhalation.

In some embodiment, the methyl-P-cyclodextrin (mpCD) is administered by inhalation.

As used herein, the term “inhalation” refers to the absorption by the nose and the respiratory tract of a substance (air, other gas, aerosol, etc.). There are two types of administration by inhalation: administration by insufflation when the compositions are in the form of powders, and administration by nebulization when the compositions are in the form of aerosols (suspensions) or in the form of solutions, for example aqueous solutions, under pressure. The use of a nebulizer or spray will then be recommended to administer the pharmaceutical or veterinary composition.

As used herein, the term "nebulizer" or "aerosol generator" has its general meaning in the art and refers to a device that converts a liquid or powder or a vial into an aerosol of a size that can be inhaled into the respiratory tract. Pneumonic, ultrasonic, electronic nebulizers, e.g., passive electronic mesh nebulizers, active electronic mesh nebulizers and vibrating mesh nebulizers are amenable for use with the invention if the particular nebulizer emits an aerosol with the required properties, and at the required output rate. The process of pneumatically converting a bulk liquid into small droplets is called atomization. The operation of a pneumatic nebulizer requires a pressurized gas supply as the driving force for liquid atomization. Ultrasonic nebulizers use electricity introduced by a piezoelectric element in the liquid reservoir to convert a liquid into respirable droplets. Various types of nebulizers are described in Respiratory Care, Vol. 45, No. 6, pp. 609-622 (2000), the disclosure of which is incorporated herein by reference in its entirety. The terms "nebulizer" and "aerosol generator" are used interchangeably throughout the specification. "Inhalation device", "inhalation system" and "atomizer" are also used in the literature interchangeably with the terms "nebulizer" and "aerosol generator". For instance, the device can include a ventilator, optionally in combination with a mask, mouthpiece, mist inhalation apparatus, and/or a platform that guides users to inhale correctly and automatically deliver the drug (i.e. the cyclodextrin or methyl-P-cyclodextrin (mpCD)) at the right time in the breath. Representative aerosolization devices that can be used in accordance with the methods of the present invention include but are not limited to those described in U.S. Patent Nos. 6,357,671 ; 6,354,516; 6,241 ,159; 6,044,841 ; 6,041 ,776; 6,016,974; 5,823,179; 5,797,389; 5,660,166; 5,355,872; 5,284,133; and 5,277,175 and U.S. Published Patent Application Nos. 20020020412 and 20020020409.

Using a jet nebulizer, compressed gas from a compressor or hospital airline is passed through a narrow constriction known as a jet. This creates an area of low pressure, and liquid medication from a reservoir is drawn up through a feed tube and fragmented into droplets by the air stream. Only the smallest drops leave the nebulizer directly, while the majority impact on baffles and walls and are returned to the reservoir. Consequently, the time required to perform jet nebulization varies according to the volume of the composition to be nebulized, among other factors, and such time can readily be adjusted by one of skill in the art.

A metered dose inhalator (MDI) can be used to deliver a composition of the invention in a more concentrated form than typically delivered using a nebulizer. For optimal effect, MDI delivery systems require proper administration technique, which includes coordinated actuation of aerosol delivery with inhalation, a slow inhalation of about 0.5-0.75 liters per second, a deep breath approaching inspiratory capacity inhalation, and at least 4 seconds of breath holding. Pulmonary delivery using a MDI is convenient and suitable when the treatment benefits from a relatively short treatment time and low cost.

Aerosolized cyclodextrin or methyl-P-cyclodextrin (mpCD) of the invention comprise droplets that are a suitable size for efficient delivery within the lung. Preferably, the formulation is effectively delivered to lung bronchi, more preferably to bronchioles, still more preferably to alveolar ducts, and still more preferably to alveoli. Thus, aerosol droplets are typically less than about 15 pm in diameter, and preferably less than about 10 pm in diameter, more preferably less than about 5 pm in diameter, and still more preferably less than about 2 pm in diameter. For efficient delivery to alveolar bronchi of a human subject or for efficient delivery to the upper airway: Oropharynx and tracheobronchial zones of a human subject, an aerosol composition preferably comprises droplets having a diameter of about 1 pm to about 5 pm. Droplet size can be assessed using techniques known in the art, for example cascade, impaction, laser diffraction, and optical pattemation. See McLean et al. (2000) Anal Chem 72:4796-804, Fults et al. (1991 ) J Pharm Pharmacol 43:726-8, and Vecellio None et al. (2001 ) J Aerosol Med 14: 107-14.

The compositions of the present invention may comprise one or more pharmaceutically acceptable excipients, in particular selected from the group of an HFC/HFA propellant, a cosolvent, a bulking agent, a non-volatile component, a buffer/pH adjusting agent, a surfactant, a preservative, a complexing agent, or combinations thereof. Suitable propellants are those which, when mixed with the solvent(s), form a homogeneous propellant system in which a therapeutically effective amount of cyclodextrin or methyl-P-cyclodextrin (mpCD) can be dissolved. The HFC/HFA propellant must be toxicologically safe and must have a vapor pressure which is suitable to enable the cyclodextrin or methyl-P-cyclodextrin (mpCD) to be administered via a pressurized MDI. According to the present invention, the HFC/HFA propellants may comprise, one or more of 1,1,1,2-tetrafluoroethane (HFA-134(a)) and 1,1, 1,2, 3, 3, 3, -heptafluoropropane (HFA-227), HFC-32 (difluoromethane), HFC-143(a) (1,1,1- trifhroroethane), HFC-134 (1,1,2,2-tetrafluoroethane), and HFC-152a (1,1 -difluoroethane) or combinations thereof and such other propellants which may be known to the person having a skill in the art.

In some embodiment, the cyclodextrin is administered by intrathecal administration, in particular by intrathecal administration via lumbar injection.

In some embodiment, the P-cyclodextrin (P-CD) is administered by intrathecal administration, in particular by intrathecal administration via lumbar injection.

In some embodiment, the Methyl-P-cyclodextrin (mpCD) is administered by intrathecal administration, in particular by intrathecal administration via lumbar injection.

As used herein, the terms “intrathecal delivery” or “intrathecal administration” refer to a route of administration for drugs via an injection into the spinal canal, more specifically into the subarachnoid space so that it reaches the cerebrospinal fluid (CSF). Intrathecal delivery may include lumbar puncture, intraventricular (including intracerebroventricular (ICV)), suboccipital/intracisternal, and/or Cl-2 puncture. For example, material may be introduced for diffusion throughout the subarachnoid space by means of lumbar puncture. In another example, injection may be into the cisterna magna.

In some embodiment, the cyclodextrin is administered by intravenous administration.

In some embodiment, the P-cyclodextrin (P-CD) is administered by intravenous administration.

In some embodiment, the Methyl-P-cyclodextrin (mpCD) is administered by intravenous administration.

As used herein, the term “intravenous (IV) administration” is a medical technique that administers fluids, medications and nutrients directly into a person's vein. The intravenous route of administration is commonly used for rehydration or to provide nutrients for those who cannot, or will not — due to reduced mental states or otherwise — consume food or water by mouth. It may also be used to administer medications or other medical therapy such as blood products or electrolytes to correct electrolyte imbalances. The intravenous route is the fastest way to deliver medications and fluid replacement throughout the body as they are introduced directly into the circulatory system and thus quickly distributed.

Combined preparation

In another embodiment, the present invention relates to i) cyclodextrin and ii) a classical treatment as a combined preparation for use in the treatment of lung cancer.

In a particular embodiment, the present invention relates to relates to i) cyclodextrin and ii) a classical treatment as a combined preparation for use in the treatment of KRAS mutant lung cancer.

In a particular embodiment, the present invention relates to relates to i) cyclodextrin and ii) a classical treatment as a combined preparation for use in the treatment of KRAS mutant nonsmall cell lung cancer (NSCLC).

In a particular embodiment, the present invention relates to i) methyl-P-cyclodextrin (mpCD). and ii) a classical treatment as a combined preparation for use in the treatment of lung cancer. In a particular embodiment, the present invention relates to relates to i) mpCD and ii) a classical treatment as a combined preparation for use in the treatment of KRAS mutant lung cancer.

In a particular embodiment, the present invention relates to relates to i) mpCD and ii) a classical treatment as a combined preparation for use in the treatment of KRAS mutant non-small cell lung cancer (NSCLC).

As used herein, the terms “combined treatment”, “combined therapy” or “therapy combination” refer to a treatment that uses more than one medication. The combined therapy may be dual therapy or bi-therapy.

Such administration may be simultaneous, separate or sequential. For simultaneous administration the product of the invention may be administered as one composition or as separate compositions, as appropriate.

As used herein, the term “administration simultaneously” refers to administration of 2 active ingredients by the same route and at the same time or at substantially the same time. The term “administration separately” refers to an administration of 2 active ingredients at the same time or at substantially the same time by different routes. The term “administration sequentially” refers to an administration of 2 active ingredients at different times, the administration route being identical or different.

As used herein, the term “classical treatment” refers to treatments well known in the art and used to lung cancer, in particular NSCLC. In the context of the invention, the classical treatment refers to radiation therapy, chemotherapy immunotherapy, HD AC inhibitor.

As used herein, the term “immunotherapy” has its general meaning in the art and refers to the treatment that consists in administering an immunogenic agent i.e. an agent capable of inducing, enhancing, suppressing or otherwise modifying an immune response. In a particular embodiment, the immunotherapy consists of use of an immune check point inhibitor as described above. As used herein, the term “chemotherapy” refers to use of chemotherapeutic agents to treat a subject. As used herein, the term "chemotherapeutic agent" refers to chemical compounds that are effective in inhibiting tumor growth.

Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaorarnide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a carnptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estrarnustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimus tine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem Inti. Ed. Engl. 33: 183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idanrbicin, marcellomycin, mitomycins, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomgrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti- adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophospharnide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defo famine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pento statin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogennanium; tenuazonic acid; triaziquone; 2, 2', 2"- trichlorotriethylarnine; trichothecenes (especially T-2 toxin, verracurin A, roridinA and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobromtol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisp latin and carbop latin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit honnone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

As used herein, the term “radiation therapy” or “radiotherapy” have their general meaning in the art and refers the treatment of cancer with ionizing radiation. Ionizing radiation deposits energy that injures or destroys cells in the area being treated (the target tissue) by damaging their genetic material, making it impossible for these cells to continue to grow. One type of radiation therapy commonly used involves photons, e.g. X-rays. Depending on the amount of energy they possess, the rays can be used to destroy cancer cells on the surface of or deeper in the body. The higher the energy of the x-ray beam, the deeper the x-rays can go into the target tissue. Linear accelerators and betatrons produce x-rays of increasingly greater energy. The use of machines to focus radiation (such as x-rays) on a cancer site is called external beam radiation therapy. Gamma rays are another form of photons used in radiation therapy. Gamma rays are produced spontaneously as certain elements (such as radium, uranium, and cobalt 60) release radiation as they decompose, or decay. In some embodiments, the radiation therapy is external radiation therapy. Examples of external radiation therapy include, but are not limited to, conventional external beam radiation therapy; three-dimensional conformal radiation therapy (3D-CRT), which delivers shaped beams to closely fit the shape of a tumor from different directions; intensity modulated radiation therapy (IMRT), e.g., helical tomotherapy, which shapes the radiation beams to closely fit the shape of a tumor and also alters the radiation dose according to the shape of the tumor; conformal proton beam radiation therapy; image-guided radiation therapy (IGRT), which combines scanning and radiation technologies to provide real time images of a tumor to guide the radiation treatment; intraoperative radiation therapy (IORT), which delivers radiation directly to a tumor during surgery; stereotactic radiosurgery, which delivers a large, precise radiation dose to a small tumor area in a single session; hyperfractionated radiation therapy, e.g., continuous hyperfractionated accelerated radiation therapy (CHART), in which more than one treatment (fraction) of radiation therapy are given to a subject per day; and hypofractionated radiation therapy, in which larger doses of radiation therapy per fraction is given but fewer fractions.

In a particular embodiment, the invention relates to i) cyclodextrin and ii) an histone deacetylase inhibitor as a combined preparation for simultaneous, separate or sequential use in the treatment of lung cancer.

In a particular embodiment, the invention relates to i) cyclodextrin and ii) an histone deacetylase inhibitor as a combined preparation for simultaneous, separate or sequential use in the treatment of KRAS mutant non-small cell lung cancer (NSCLC).

In a particular embodiment, the invention relates to i) methyl-P-cyclodextrin (mpCD) and ii) an histone deacetylase inhibitor as a combined preparation for simultaneous, separate or sequential use in the treatment of lung cancer.

In a particular embodiment, the invention relates to i) methyl-P-cyclodextrin (mpCD) and ii) an histone deacetylase inhibitor as a combined preparation for simultaneous, separate or sequential use in the treatment of KRAS mutant non-small cell lung cancer (NSCLC).

As used herein, the term histone “histone deacetylase inhibitor” called also HDACi, refers to a class of compounds that interfere with the function of histone deacetylase. Histone deacetylases (HDACs) play important roles in transcriptional regulation and pathogenesis of cancer. Typically, inhibitors of HDACs modulate transcription and induce cell growth arrest, differentiation and apoptosis. HDACis also enhance the cytotoxic effects of therapeutic agents used in cancer treatment, including radiation and chemotherapeutic drugs. In a particular embodiment, the histone deacetylase inhibitor is valproic acid (VP A). The term "valproic acid" refers to acid-2- propylpentanoic (CsHieCh), 5 which has the following CAS number and formula 99-66-1 in the art:

In a particular embodiment, the HD AC inhibitor is suberoylanilide hydroxamic acid, also called Vorinostat (N-Hydroxy-N'-phenyloctanediamide) was the first histone deacetylase inhibitor approved by the U.S. Food and Drug Administration (FDA) on 2006 (Marchion DC et al 2004; Valente et al 2014).

In a particular embodiment the HD AC inhibitor is Panobinostat (LBH-589) has received the FDA approval on 2015 and has the structure as described in Valente et al 2014.

In a particular embodiment the HD AC inhibitor is Givinostat (ITF2357) has been granted as an orphan drug in the European Union (Leoni et al 2005; Valente et al 2014).

In a particular embodiment the HDAC inhibitor is Belinostat also called Beleodaq (PXD-101) has received the FDA approval on 2014 (Ja et al 2003; Valente et al 2014).

In a particular embodiment the HDAC inhibitor is Entinostat (as SNDX-275 or MS-275). This molecule has the following chemical formula (C21H20N4O3) and has structure as described in Valente et al 2014.

In a particular embodiment the HDAC inhibitor is Mocetinostat (MGCD01030) having the following chemical formula (C23H20N6O) (Valente et al 2014). In a particular embodiment the HDAC inhibitor is Practinostat (SB939) having the following chemical formula (C20H30N4O2) and the structure as described in Diermayr et al 2012.

In a particular embodiment the HDAC inhibitor is Chidamide (CS055/HBI-8000) having the following chemical formula (C22H19FN4O2).

In a particular embodiment the HDAC inhibitor is Quisinostat (JNJ-26481585) having the following chemical formula (C21H26N6O2).

In a particular embodiment the HDAC inhibitor is Abexinostat (PCI24781) having the following chemical formula (C21H23N3O5) (Valente et al 2014).

In a particular embodiment the HDAC inhibitor is CHR-3996 having the following chemical formula (C20H19FN6O2) (Moffat D et al 2010; Banerji et al 2012).

In a particular embodiment the HDAC inhibitor is AR-42 having the following chemical formula (C18H20N2O3) (Lin et al 2012).

Pharmaceutical composition

The cyclodextrin for use according to the invention combined with classical treatment as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.

In some embodiment, the present invention relates to a pharmaceutical composition comprising cyclodextrin for use in the treatment of lung cancer.

In particular, the present invention relates to a pharmaceutical composition comprising cyclodextrin for use in the treatment of KRAS mutant non-small cell lung cancer (NSCLC).

In some embodiment, the present invention relates to a pharmaceutical composition comprising methyl-P-cyclodextrin (mpCD) for use in the treatment of lung cancer. In particular, the present invention relates to a pharmaceutical composition comprising methyl- P-cyclodextrin (mpCD) for use in the treatment of KRAS mutant non-small cell lung cancer (NSCLC).

Cyclodextrin, in particular methyl-P-cyclodextrin (mpCD) and the combined preparation as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intravitreal administration, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuumdrying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: Cholesterol removal therapy with cyclodextrin reduces tumor burden in progressing tumor in vivo. (A) Experimental outline. Six weeks after tumor initiation (TO group), tumor-bearing KrasG12D (CC-LR) mice were exposed once a week to saline and methyl-P-cyclodextrin (PCD, 4g/kg of mouse) inhalation to follow tumor regression over a 4- week period. (B) Quantification of lesion sizes (mm2) (left panel) or nodule subtypes (right panel) of CC-LR treated with saline or PCD. Tumor represents the sum of atypical adenomatous hyperplasia (AAH), adenoma and adenocarcinoma lesions. Ten weeks after tumor initiation (TO group), reporter control (CCEYFP) or tumor-bearing KrasG12D (CC-LREYFP) mice were exposed once a week to saline and methyl-P-cyclodextrin (PCD, 4g/kg of mouse) inhalation to follow tumor regression over a 4/6-week period. (C) Quantification of tumor nodules (number per cm2) throughout the study period. (D) Quantification of C45-CD31-EpCAM+EYFP+ epithelial progenitors determined by flow cytometry in the lung of CC-LREYFP treated with saline or PCD. All values are mean ± SEM and are representative of at least one experiment (n=4-5 independent animals). *P<0.05 compared to TO CC-LR. §P<0.05 compared to CC-LR control mice or CC-LREYFP control mice.

EXAMPLE:

Human samples.

Human samples were provided by the Laboratory of Clinical and Experimental Pathology (LPCE) and the Hospital -related biobank (BB-0033-00025), which are accredited according to the ISO 15189 since 2012 for clinical and molecular biology and certified with the S96-900 since 2010 for management of human bioresource. This biobank is IBiSA certified since 2014 and belongs to the BBMRI consortium in Europe. Informed signed consent was obtained from all patients.

Human blood samples (n=32) were divided in 4 groups: 5 normal patients, 5 patients with chronic non-cancerous lung disease (chronic obstructive pulmonary disease, COPD), 14 patients with early-stage lung adenocarcinoma (LU AD) (stage I/II) and 8 with late-stage LU AD (stage IIEIV).

Biospies from human LU AD (stage I/II) and adjacent normal tissues (n=68) were collected from patients undergoing surgical resection of primary tumors and transcriptomic analyses were performed at the genomics platform of the Institute Molecular and Cellular Pharmacology (IPMC) as previously described⁴⁵. Briefly, data were normalized using the quantile method. A linear modeling approach was used to calculate log ratios, moderated Lstatistics, and -values for each comparison of interest. -values were adjusted for multiple testing using the Benjamini-Hochberg method that controls the false discovery rate. RNA sequencing has been deposited in the GEO database: GSE117049.

Mice.

LSL-Kras^G12D/+ (B6.129S4- ^mV ), R26R-EYFP (B6.129Xl-Gt(ROSA)26So^{tml(EYFP Cos}/J Tg Hu apoA-I (C57BL/6-Tg(APOAl)lRub/J), CCSP-CreER™^/+ (B6N.129S6(Cg)- Scgblal^{tml(cre/ERT)Blh}A hypoxia-inducible factor-la (HIFla^fl/fl; B6.129-HIFla^tmlKats/J), hypoxia-inducible factor-2 (HIF2^fl/fl; B6.129-Epasl^tmlMcs) and Abcal ¹¹ ^bcg l ^{11 11} (B6.Cg- Abcal^tmlJp Abcgl^tmlTall/y) were obtained from the Jackson Laboratory. Kras^LSL'^G12D/+ mice carry a Lox-Stop-Lox (LSL) termination sequence with the K-ras G12D point mutation. The stop codon can be excised by Cre-mediated recombination. First, Kras^LSL'^G12D/+ mice were crossed to Rosa26^LSL'^EYFP reporter mice (LR^EYFP). Kras^LSL'^G12D/+ mice were also crossed to apoAI transgenic mice (LR/apoAI-Tg). We then used mice containing an inductible Cre recombinase inserted into the Clara cell secretory protein (CCSP) locus, CCSP-CreER™^/+ mice, and crossed them to Kras^LSL'^G12D/+ mice, to generate the CC-LR mice. CC-LR mice were then crossed with Abcal; Abcgl floxed mice (CC-LR^DKO). For each experiment, co-housed littermate controls were used. Animal protocols were approved by the Institutional Animal Care and Use Committee of the French Ministry of Higher Education and Research and the Mediterranean Center of Molecular Medicine (Inserm U1065) and were undertaken in accordance with the European Guidelines for Care and Use of Experimental Animals. Mouse survival was closely monitored during the entire experimental period. Animals had free access to food and water and were housed in a controlled environment with a 12h light-dark cycle and constant room temperature (22°C).

Integrated network analyses.

Network-based integration of gene expression datasets was conducted using Shiny Gam as previously described⁴⁶. In brief, common up and down regulated metabolic genes were selected from the LPCE gene expression datasets and the publicly available gene expression datasets of the cancer genome atlas (TCGA) cohort containing 442 samples (Stage I and II LU AD). These dysregulated transcripts were mapped into models maintaining all essential KEGG pathway attributes. For cholesterol efflux pathway mapping, a precompiled Gene Ontology (GO) terms list, annotated with the term ‘cholesterol’, was added to the network. We also manually mapped nodes representing reactions that are connected to cholesterol homeostasis to generate a global combined network resulting in a topological description of cholesterol homeostasis pathway.

Laboratory measurements.

Blood specimens were analyzed for total cholesterol, HDL-chole sterol and HDL- phosphatidylcoline levels. Other plasma parameters were determined using commercial kits (ApoA-I, Fibrinogen, Ang II and TNFa from Abeam and SP-B and SP-D from Abbexa).

Plasma HDL preparation.

ApoB-containing particles were precipitated from serum by adding 100 pL of serum to 40 pL of 20% polyethylene glycol (Sigma P-2139 in 200 mmol/L glycine, pHlO) solution. This mixture was incubated at room temperature for 15 minutes then was centrifuged at 4000 rpm for 20 minutes. The supernatant, containing HDL fractions, was removed and used for experiments.

Isotopic cholesterol efflux assay.

The functionality of the HDL to mediate cholesterol efflux was assessed on macrophages. THP- 1 monocytes were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) at 37 °C in 5% CO2. Cells were treated with lOOnM PMA (Phorbol myristate acetate) for 24 h to facilitate differentiation into macrophages. Then, adherent macrophages were incubated in complete media with 2pCi/mL of [l,2-3H(N)]-cholesterol for 24 hours before cholesterol efflux studies. Cholesterol efflux was performed for 6h in 0.2% BSA RPMI containing different concentrations or volumes ofHDL as acceptors. The cholesterol efflux was expressed as the percentage of the radioactivity in cells plus medium.

Adenovirus inhalation.

Lightly anesthetized mice were infected intranasally with adenoviruses containing gene for ere recombinase (Genecust, Luxembourg) at 5xl0⁸ PFU per mouse to induce Kras-driven lung tumorigenesis.

Tamoxifen induction.

Mice were i.p. injected with 200pl com oil Tamoxifen solution [10 mg/ml] (Sigma Aldrich) at 6-8 weeks of age to induce ra -driven lung tumorigenesis.

Bone marrow (BM) transplantation.

Recipient CC-LR or CC-LR^DKO mice were lethally irradiated the day before transplantation as previously described⁵⁶. Femurs and tibias of donor LyzM-Cre Abcal^fl/flAbcgl^fl/fl mice, LyzM- Cre CD36^fl/fl mice and LyzM-Cre SR-BI^fl/flmice were kindly provided by Pr. Marit Westerterp, Pr. Ira Goldberg and Pr. Thierry Huby, respectively. Briefly, femurs and tibia were flushed with ice-cold RPMI 1640 and centrifugated at 1,400 rpm for 5 minutes to extract BM cells. Red blood cells were lysed, and each recipient mouse was injected with 5xl0⁶ BM cells through the vein. After 4 weeks of reconstitution, mice were i.p injected with tamoxifen to induce lung cancer development.

Spirometry.

Lung capacity was measured using a spirometer (Adinstrument, Oxford, UK). Inspiratory and expiratory volumes and debit were measured and plot using LabChart (ADinstrument). Briefly, mice were lightly anesthetized with 6,3 mg/kg xylasine and 125 mg/kg ketamine (Virbac, Carros, France). Spirometer mask was attached to mice head and lung capacity was measured during 5 min. Debit by volume was plotted to represent lung capacity.

In vivo treatment studies.

Tumor-bearing Kras^G12D mice (6weeks after tamoxifen administration) were treated by inhalation with either 50pl of PBS or the LXR agonist T0901317 (Cayman Chemical) (50mg/kg of mouse) twice a week for 4weeks. In independent set of experiments, mice were treated by inhalation with 50 pl of PBS or the P-methyl-cyclodextrin (Sigma) (4g/kg of mouse) once a week for 4weeks. For the anti-PD-1 treatment, mice were i.p injected with either the IgG2a isotype control antibody (Clone 2A3, BioXCell) (200pg/mouse) or with the anti-mouse PD-1 antibody (Clone RPM1-14, BioXCell) (200pg/mouse) twice a week. Animals were sacrificed at the indicated time point after treatments.

In vivo )1 ,2-Al ( ) /-Cholesterol tracing experiment.

We analyzed the retention and distribution of [1,2-³H(N)] -Cholesterol in the lung and peripheral tissues, respectively. In brief, tumor-bearing Kras^G12D mice (6weeks after tamoxifen administration) received 4pCi of [1,2-³H(N)] -Cholesterol (PerkinElmer) by inhalation and organs were collected 24h later. The amount of [1,2-³H(N)] -cholesterol was determined by standard procedures using a liquid scintillation counter. The relative cholesterol retention in the lung or distribution in peripheral tissues was calculated by dividing the amount of deposited dose and expressed as percentage. Imaging for [1,2-³H(N)]-Cholesterol was performed on TyphoonTM Biomolecular Imager (Amersham). For in vivo reverse cholesterol transport (RCT) assay³³, tumor-bearing Kras^G12D mice (6weeks after tumor induction) were i.p injected with [1,2-³H(N)]- cholesterol -labeled bone marrow-derived macrophages (5x10⁶ cells were radiolabeled with 5 pCi/ml [³H]-cholesterol for 24 hours before injection). Plasma was collected at 24 hours and 48 hours and was used for liquid scintillation counting. Plasma radioactivity is expressed as percent of total injected [³H]-cholesterol. Feces and liver were collected at 48 hours prior lipid extraction and data are expressed as a percent of total [³H]-cholesterol-injected.

White blood cell counts.

Leukocytes, differential blood counts, platelets and red blood cells were quantified from whole blood using a hematology cell counter (HEMAVET 950).

Blood parameters.

Plasma angiotensin II, G-CSF and IL-17 levels were determined using commercial kits (all from Raybioteck, Inc).

Bronchoalveolar lavage (BAL).

After sacrifice, the trachea was exposed with pincers and a catheter 24G x 0.75po. (BD Biosciences) was inserted. The catheter was stabilized with cotton thread and we injected gently 750mL of sterile ice-cold PBS. Fluid was centrifuged at 400g for 10 minutes to separate cells from supernatant.

ELISA and colorimetric measurements.

Cholesterol, SP-D and phosphatidylcholine from plasma, BAL and lung homogenates were measured using colorimetric kits LabAssay™ total cholesterol (Wako Chemicals), free cholesterol (Clinisciences), Mouse SP-D Elisa (R&D) and Phosphatidylcholine Assay Kit (Sigma Aldrich) respectively, according to the manufacturer’s instructions.

Adipose tissue cellularity.

Cellularity of epididymal adipose tissue was determined from images of isolated adipocytes. The measurement of ~400 cell diameters was performed using Image J software, allowing calculation of a mean fat cell weight.

Histopathology and tumors quantification.

Mice were euthanized and tissues were harvested and fixed in 4% paraformaldehyde. Lung was serially paraffin sectioned (6-pm sections) using a Microm HM340E microtome (Microm Microtech, Francheville France) and stained with H&E for morphological analysis. Images were captured with an Olympus BX53 (Olympus Life Science, Germany). Routine anatomopathological examination of the slides was made by a senior pathologist (M.I., LPCE, Nice, France) to determine and quantify lesion type (i.e, histological nodule subtypes), reflecting the stage of lung cancer, according to the recommendations of the mouse models of human cancers consortium. The number of the lesions were manually measured by the pathologist on the cellSens Imaging Software (Olympus Life Science). Tumor represents the sum of atypical adenomatous hyperplasia (AAH), adenoma and adenocarcinoma lesions. Hyperplasia lesions were scored independently. Quantification of the lesion size was performed in a blind fashion by two independent research scientists using FIJI software and all nodule subtypes were normalized per cm² of lung tissue.

Immunostainings.

Paraffin sections were deparaffinized and antigen retrieval was carried out in citrate buffer. Sections were further permeabilized in 0.3% Triton X-100 for 10 minutes and then blocked with in 10% BSA for 1 hour. The following primary antibodies were used for incubation during 2 hours at RT: anti-E-cadherin (clone 24E10, Cell Signaling), anti-fibronection (BDBiosciences), anti-vimentin (polyclonal, Novus Biologicals), anti-CCSP (clone S-20, Santa Cruz), anti-SP-C (polyclonal, Abeam), anti -Ki-67 (Clone Sol Al 5, ThermoFisher), anti- LC3A/B (Clone D3U4C, Cell Signaling). Alexa Fluor 594 donkey anti-chicken (703-586-155), Alexa Fluor 594 donkey anti-mouse (715-586-150), Alexa Fluor 594 donkey anti-rat (712-586- 150) antibodies from Jackson ImmunoResearch and Alexa Fluor 594 chicken anti -goat (A21468), Alexa Fluor 488 donkey anti-rabbit (A21206) antibodies from Thermo Fisher were used as secondary antibodies for 2 hours at RT. Images were captured with a Leica video-TIRF epifluorescence microscope.

Tissue clearing and 3D-recontrusction microscopy.

After sacrifice, lungs were fixed with 4% paraformaldehyde and then, permeabilized for several days with 0.2% Triton X-100. For staining procedures, lungs were incubated with anti-CCSP (clone S-20, Santa Cruz) overnight at 4°C. Alexa Fluor 594 chicken anti-goat (A21468) antibody from Thermo Fisher was used as secondary antibody for 2 hours at RT. Lung were then dehydrated with different ethanol baths (from 70% to 100%) and directly immersed in Methyl Salicylate for 15 minutes at RT. Z-stack images of cleared tissue were captured with a Nikon confocal microscope.

Single-cell RNA-seq data analysis.

Whole lungs were dissociated with fine scissors and then proteolytic digestion was performed with DMEM containing 2,5mg/mL collagenase D (Roche) at 37°C for 30min. Single-cell suspension was submitted to red blood cell lysis. Single-cells were encapsulated in droplets using 10X Genomics GemCode Technology and processed following manufacturer’s specifications. Briefly, every cell and every transcript are uniquely barcoded using a unique molecular identifier (UMI) and cDNA ready for sequencing on Illumina platforms is generated using the Single Cell 3’ Reagent Kits v2 (10X Genomics). Libraries were sequenced across a Nextseq 500 (Illumina) in paired-end to reach approximately 50,000 reads per single-cell. Alignment, barcode assignment and UMI counting with Cell Ranger v4.0.0 was used to perform sample demultiplexing, barcode processing and single-cell 3' counting. Cell Ranger’s mkfastq function was used to demultiplex raw base call files from the HiSeq4000 sequencer into sample specific FASTQ files. Barcodes in both samples that were considered to represent noise and low-quality cells were filtered out using knee-inflection strategy available in DropletUtils package (version 1.4.3). For analysis, Seurat package (version 3.1.0) was used, genes which express in less than 2 cells and cells which have non-zero counts in less than 200 genes were additionally filtered from both barcode expression matrices, and the result matrices were used as analysis inputs. Low quality cells, doublets and potentially dead cells were removed according the percentage of mitochondrial genes and number of genes and UMIs expressed in each cell. The fraction of mitochondrial genes was calculated for every cell, and cells with a mitochondrial fraction >2% were filtered out. After all filtering procedures, 3,193 cells were left in the scRNA-seq data of control sample, 1,785 cells were left in scRNA-seq of CC-LR sample and 2,269 cells were left in scRNA-seq of CC-LR^DKO sample. All samples were normalized using SCTransform function with mitochondrial percentage as variable to regress out in a second non-regularized linear regression. For integration purpose, variable features across the samples were selected by SelectlntegrationFeatures function with the number of features equal to 2000. Then the object was prepared for integration (PrepSCTIntegration function), the anchors were found (FindlntegrationAnchors function) and the samples were integrated into the whole object (fntegrateData function). The dimensionality of the object was reduced by principal component analysis (PCA), and the first 20 principal components (PCs) were used further to generate uniform manifold approximation and projection (UMAP) dimensionality reduction by RunUMAP function. Graph-based clustering was run using FindNeighbors and FindClusters with a resolution of 1.0 and the first 20 PCs as input, and the 18 clusters were identified. In order to exclude the technical bias across the samples, both the counts slot from SCTransform assay and the data slot from integrated assay were used as input for trajectory inference. For visualization purposes, the custom labels were assigned to several clusters by merging multiple clusters for simplification. Violin plots were drawn using the data slot of SCT assay. To generate pathway enrichment plots we took expression of genes from the pathway from the data slot of SCT counts assay, used standard normalization (z-score) for these vectors and then calculated the average vector. The gene signature heatmap was drawn using the scaled data slot of the integrated assay.

In vivo flow cytometry analysis.

Cells were collected from peripheral blood or tissues, lysed to remove red blood cells and filtered before use. Whole lungs were dissociated with fine scissors and then proteolytic digestion was performed with DMEM containing 2,5mg/mL collagenase D (Roche) at 37°C for 30min. Splenocytes were extracted by pressing spleens through a stainless-steel grid and after filtration (Cell Strainer lOOpM), cells were centrifugated at 1,500 rpm for 5 minutes. Freshly isolated cells were stained with the appropriate antibodies for 30min on ice protected from light. Cells were analyzed by flow cytometry using BDFACSCanto (BD Biosciences). Data were analyzed with FlowJo software (Tree Star).

Cholera Toxin staining.

Cells were stained 15min at 37°C, in 1 pg/ml working solution of Cholera Toxin Subunit B, Alexa Fluor 594 conjugate (Invitrogen, C34777). Cells were then stained with Ing/ml working solution of DAPI (4^Z ,6-diamidino-2-phenylindole) and washed in PBS IX. Immunostaining of cells was read on a Nikon Confocal AIR microscope.

Antibodies.

For lung epithelial cells, the following antibodies were used: CD45 APC/Cy7 conjugated (clone 30-F11, BD Biosciences), CD31 PerCP/Cy5.5 conjugated (Clone 390, BioLegend), EpCAM PE conjugated (Clone G8.8, BioLegend), CD36 PE/Cy7 conjugated (Clone HM36, BioLegend), CD24 PB conjugated (Clone MI/69, BioLegend) and Siglec-F BV510 conjugated (Clone E50-2440, BD Biosciences) were used to quantify epithelial progenitor cells. Cellular cholesterol content was quantified using the Bodipy-cholesterol probe (Life Technologies). For DNA content analysis, cells were fixed in 1% paraformaldehyde in PBS, washed, and stained with 5pg/mL Hoechst 33342 (Molecular Probes).

For lung myeloid cells, the following antibodies were used: CD45 APC/Cy7 conjugated (clone 30-F11, BD Biosciences), Ly6C APC conjugated (Clone HK1.4), CD206 PerCP/Cy5.5 conjugated (clone C068C2, BioLegend), Siglec-F PE conjugated (Clone E50-2440, BD Biosciences), CDl lc PE/Cy7 conjugated (Clone N418, BioLegend), CD64 BV421 conjugated (Clone X54-5/7.1, BD Biosciences), CDl lb BV510 conjugated (Clone MI/70, BioLegend). Cellular cholesterol content was quantified using the Bodipy-cholesterol probe (Life Technologies).

For lung T-cells, the following antibodies were used: CD45 APC/Cy7 conjugated (clone 30- Fl l, BD Biosciences), CD62L APC conjugated (Clone MEL-14, eBioscience), CTLA-4 CD152 PerCP/Cy5.5 conjugated (Clone UC10-4B6, BioLegend), PD-1 PE conjugated (Clone RPM1-30, BioLegend), CD44 PE/Cy7 conjugated (Clone IM7, BioLegend), TCRp PB conjugated (Clone H57-597, BioLegend), CD8a BV510 conjugated (Clone 53-6.7, BioLegend). For peripheral blood leukocytes analysis, the following antibodies were used: CD45 APC/Cy7 conjugated (clone 30-F11, BD Biosciences), CD62L APC conjugated (Clone MEL-14, eBioscience), Gr-1 PerCP/Cy5.5 conjugated (Clone RB6-8C5, BD Biosciences), B220 Fite conjugated (Clone RA3-6B2, eBioscience), CD115 PE conjugated (Clone AFS98, eBioscience), CD44 PE/Cy7 conjugated (Clone IM7, BioLegend), TCRb PB conjugated (Clone H57-597, BioLegend), CD8a BV510 conjugated (Clone 53-6.7, BioLegend).

For splenocytes, the following antibodies were used: CD45 APC/Cy7 conjugated (clone 30- Fl l, BD Biosciences), CD62L APC conjugated (Clone MEL-14, eBioscience), CTLA-4 CD152 PerCP/Cy5.5 conjugated (Clone UC10-4B6, BioLegend), PD-1 PE conjugated (Clone RPM1-30, BioLegend), TCRb PB conjugated (Clone H57-597, BioLegend), CD8a BV510 conjugated (Clone 53-6.7, BioLegend).

Colony-Forming Assay.

Whole lungs were dissociated with fine scissors and then proteolytic digestion was performed with DMEM containing 2,5mg/mL collagenase D (Roche) at 37°C for 30min. Single-cell suspension was submitted to red blood cell lysis and were plated in a precoated-Matrigel™ matrix (Corning Life-Science). Cells (lxl0⁴/well) were grown in DMEM with (10% FBS) or without serum (replaced by 0,2% BSA) containing IX Gibco™ Insuline-Transferrine- Selenium (ThermoFisher Scientific) in presence or absence of different treatments. Briefly, treatments included 3pM of the LXR agonist T0901317 (Cayman Chemical), 5mM of methyl- P-cyclodextrin (Sigma), 5, 10 or 25pg/mL ofHDL, lOng/mL EGF or IGFl (PrepoTech), 5, 25, 50nM of EGFR inhibitor (erlotinib) and IGF1R inhibitor (linsitinib) (Abeam). The number of epithelial colony forming unit (eCFU) per dish was scored after 8 days of culture using the ImageJ analysis software.

IncuCyte proliferation assay.

Kras-driven tumor-derived cell line generated from Kras^G12D/+ ;p53'^/_ lung tumors were kindly provided by Pr. Eileen White and cultured in RPMI 1640 medium supplemented with sodium pyruvate (ImM), penicillin (100 U/ml)/streptomycin (100 mg/ml) and 10% fetal bovine serum (FBS) at 37 °C in 5% CO2. KP cells were incubated at the density of 8.10⁴ for 24 hours into a 48-well plate in complete medium, then medium was replaced with RPMI 1640 medium supplemented with sodium pyruvate, penicillin/streptomycin containing 2% of FBS in presence or absence of lOpg/pl of HDL. Proliferation was monitored by analysis of the occupied area (% confluence) of cell images over a 72-hours period of time. The graphs from the phase of cell confluence area were recorded every 2 hours according to the IncuCyte (Essen BioScience) manufacturer’s instructions.

Prot comic analysis.

Mass spectrometry analyses were performed at the Taplin Biological Mass Spectrometry Facility of the University of Harvard Medical School. Briefly, Kras-driven tumor-derived cell line was cultured for 6 hours in presence or absence of 25 g/mL HDL prior enrichment of plasma membrane fraction by sucrose gradient. Proteins were extracted on Coomassie stained gel according to facility’s instructions before analysis by mass spectrometry. Functional enrichment analysis of protein-protein interaction networks was performed with STRING.

Bone marrow derived macrophages (HMD Ms).

BM cells were collected from mouse femur and tibia and differentiated in the presence of recombinant mouse M-CSF (20 ng/ml; Miltenyi) in complete RPMI 1640 medium (Corning) containing 10 mM glucose, 2 mM 1-glutamine, 100 U/ml of penicillin/streptomycin and 10% FBS for 7 d at 37 °C and 5% CO2. In some experiments macrophages were cultured for the last 24 hours with conditioned media obtained from colony-forming assays.

Real Time qPCR.

Total RNA was isolated using the RNeasy Plus Mini Kit (QIAGEN) and quantified using a Nanodrop (Ozyme). cDNA was prepared using 10 ng/pl total RNA by a RT-PCR using a high- capacity cDNA reverse transcription kit according to the manufacturer’s instructions (Applied Biosystems). Real-time qPCR was performed on cDNA using SYBR Green. qPCRs were performed on the StepOne device (Applied Biosystem). Results were normalized on GAPDH gene expression. All percentage changes are expressed normalized to the untreated control. Primers are listed in the table.

Quantification and statistical analysis

Data are shown as mean ± SEM. Statistical significance was performed using two-tailed parametric Student’s t test or by one-way analysis of variance (ANOVA, 4-group comparisons) with Tukey’s post-test analysis according to the dataset (GraphPad software, San Diego, CA). Results were considered as statistically significant when p < 0.05. All the statistical details of experiment can be found in the figure legends. Results:

Reduced cholesterol efflux pathways is part of the global metabolic rewiring of tumors from KRAS-mutant mice of non-small cell lung cancer.

To examinate the phenotypic, transcriptomic and metabolic diversity of lung tumor cells, we generated single cell transcriptomics (scRNAseq) from tumor-bearing Kras^G12D (LR) mice bred with the epithelial-specific cell secretory protein (CCSP)-CreER™^/+ mouse model (CC-LR) (Data not shown)²⁰. In some experiments, CC-LR mice were also crossed with Rosa26^LSL'^EYFP reporter mice. Whole lung tissue from control and CC-LR mice 6 weeks after initiating lung cancer development was enzymatically digested and scRNA-seq libraries were prepared. After sequencing, we detected up to 1,638 different genes with approximately 60,000 sequencing reads per cell (data not shown) and applied unbiased clustering on 7,247 cells in the merged dataset. To group cells with similar gene expression, we applied an unsupervised cluster detection algorithm (SEURAT3) and detected 17 distinct cell clusters (Data not shown). Gene expression data from cells extracted from all conditions were aligned and projected in a 2- dimensional space through t-stochastic neighbor embedding (t-SNE) to allow identification of isolated lung cell populations (Data not shown). Gene expression patterns allowed us to align putative biological identities to each cluster (Data not shown). We observed both immune and nonimmune cell clusters based on established canonical markers of leukocytes (Cd45), endothelial cells (Cd31) and pulmonary epithelial cells (Wfdc2) (Data not shown).

The immune cell cluster included 9 populations (macrophages, two distinct monocytes, DCs, neutrophil/eosinophils, mast cells, NK-cells, B-cells and T-cells) (Data not shown). Macrophages characterized by canonical MertK and Mrcl expression were the dominant immune cell type (Data not shown). The use of Trem2 and Lipa markers confirmed the presence of a distinct metabolically active macrophage subset²¹, that expended in the lung of CC-LR mice (Data not shown). Consistently, gene set enrichment analysis (GSEA) of the differentially expressed genes between control and CC-LR macrophages revealed an upregulation of several genes involved in metabolism (i.e, ‘mitochondria and lipid metabolism’) along with genes related to ‘intracellular trafficking’ and downregulation of genes involved in anti -tumoral signatures such as ‘response to oxygen or cytokine’ or ‘cell differentiation and motility’ (Data not shown).

Among non-hematopoietic cells, we retrieved the two major subsets of general and aerocyte capillary cells (Data not shown). We also identified two clusters of epithelial cells (Data not shown) expressing multiciliated markers (FoxJl and Cd24a) and bronchiolo-alveolar type II (AT2) markers, respectively (Data not shown). Enzymatic tissue dissociation limits isolation of large adhesive epithelial cell populations and one of the epithelial cluster co-expressed the secretoglobin family 1 A member 1 (Scgblal, also known as CCSP) and the surfactant protein C (Sftpc, referred as SP-C) previously identified as the pulmonary stem cell population and termed bronchioalveolar stem cells (BASCs)²². This suggested an enrichment of epithelial progenitors in our dataset that are thought to be at the origin of lung adenocarcinoma development in mice bearing the KRAS^G12D oncogene^23,24. By using Rosa26^LSL'^EYFP reporter mice, we first confirmed by flow cytometry that expression of the EYFP expanded over lung tumor development in CD3 FCD45 EpCAM⁺ epithelial lung cells (Data not shown), indicating that these cells are most likely stem/progenitor cells at the origin of the tumor²⁵. Consistent with earlier studies^26,27, we confirmed the presence of CD24^int and CD24^hl epithelial progenitors in the CD3 FCD45 EpCAM⁺EYFP⁺ population (Data not shown). Quantification of these cells indicated an expansion of CD24^hl progenitors at 1- and 6-weeks post-tumor induction (Data not shown) arguing that this epithelial subset is enriched in tumor propagating cells (TPCs). Coincidently, half of the animals used for the scRNAseq carried the EYFP reporter, which allowed us to identify its specific expression in the two CD24^int and CD24^hl epithelial clusters (Data not shown). By comparing the transcriptional profile of these EYFP⁺ cells, we confirmed that the CD24^int epithelial cluster exhibited a stem cell signature associated with features of translation, autophagy and Myc signaling (Data not shown). In contrast, the CD24^hl epithelial cluster exhibited a more aggressive carcinoma signature associated with several migratory features (Data not shown). Consistently, we found that the previously identified BASC signature^28,29 was enriched in the CD24^int epithelial cluster (Data not shown). High-plasticity cell state (HPCS) signature occurring in late adenocarcinoma stage³⁰ was not observed in our model (Data not shown). However, the transition phase signature that occurs earlier during cellular transformation³⁰ was enriched in the CD24^hl epithelial cluster (Data not shown). An enrichment of the previously identified ‘epithelial cancer cell origin’ and ‘distant epithelial cancer cell’ signatures³¹ was also observed in the CD24^int and CD24^hl epithelial clusters, respectively (Data not shown). Finally, colony-forming assays from primary sorted CD24^int and CD24^hl epithelial progenitors confirmed epithelial colony -forming unit (eCFU) potential of both cell fractions with a highest eCFU potential of CD24^hl epithelial progenitors from the lung of mice bearing the KRAS^G12D oncogene (Data not shown). Altogether these findings identify two epithelial progenitor clusters enriched in BASC and TPC features. We next sought to unravel the phenotype of these BASC-rich (epi^BASC) and TPC-rich (epi^TPC) epithelial clusters by analyzing the specific expression patterns differentiating control and CC- LR mice. GSEA of the differentially expressed genes identified ‘survival and metabolic processes’ and ‘adaptation and metabolic processes’ as GO terms as the most enriched terms for genes that were upregulated in epi^BASCs and epi^TPCs from the lung of CC-LR mice, respectively (Data not shown). Cell dynamics (i.e, ‘Adhesion, extracellular matrix and morphogenesis’) and cell plasticity were enriched for downregulated genes in epi^BASCs and epi^TPCs, respectively (Data not shown). The metabolic diversity highlighted by the GSEA analysis of macrophage and epithelial progenitor clusters was also illustrated by their metabolic enrichment signature (Data not shown). Deeper analysis highlighted an inverse enrichment of genes involved in carbohydrate metabolism between macrophages and epithelial progenitors in the lung of CC-LR mice compared to normal lungs while a general decrease in lipid and cholesterol metabolism KEGG pathways was observed in these cell populations (Data not shown).

To explore the predictive reduced cholesterol homeostasis observed in lung tumor cell types, we next investigated how lung tumor handles cholesterol 24 hours after [³H] cholesterol inhalation in CC-LR mice (Data not shown). We found higher [³H] cholesterol accumulation and retention in the lung of these animals 6 weeks after initiating lung cancer development as compared to tumor-free controls (Data not shown). Phosphoimaging indicated that [³H] cholesterol accumulated within tumors (Data not shown). This local cholesterol retention was associated with an accumulation of both free and esterified cholesterol (Data not shown) and a systemic perturbation of cholesterol homeostasis as evidenced by the progressive decrease in plasma HDL-chole sterol (HDL-C) levels in tumor-bearing Kras^G12D mice at 4 and 9 weeks after lung cancer initiation (Data not shown), indicative of impaired cholesterol efflux pathways³². To test the relevance of these observations, we performed an in vivo reverse cholesterol transport (RCT) assay by i.p injecting mice with [³H]-labeled cholesterol bone-marrow derived macrophages and we followed the fate of macrophage-derived [³H]cholesterol³³. Plasma [³H]cholesterol was slightly lower at both 24 hours and 48 hours in CC-LR mice compared to controls with significantly higher [³H] tracer in the liver and the feces of these mice at 48 hours (Data not shown). These effects were associated with increased mRNA expression of Scarbl (i.e, SR-BI) in the liver (Data not shown), known to participate to RCT³³, and reduced mRNA expression of two cholesterol efflux transporters Abcal and Abcgl in the lung of these mice (Data not shown). Thus, tumor-bearing Kras^G12D mice exhibit distal and local cholesterol metabolism rewiring.

To pinpoint the cellular origin of pulmonary cholesterol accumulation, BODIPY (bore- dipyrromethene)-neutral lipid staining was quantified by flow cytometry in alveolar macrophages (AMs) and interstitial macrophages (IMs) or in CD24^int epi^BASCs and CD24^hl epi^TPCs from CC-LR mice (Data not shown). Low neutral lipid staining was observed in IMs with no difference between genotypes (Data not shown). In contrast, higher neutral lipid accumulation was observed in AMs and both epithelial progenitors from CC-LR mice (Data not shown). Thus, we next took advantage of the scRNAseq to investigate the specific expression of cholesterol efflux (i.e, ABCA1 and ABCG1) or influx transporters (i.e, SR-BI and CD36)¹³. We observed that both macrophages and epithelial progenitors from the lung of CC-LR mice exhibited lower Abcal mA A beg 1 expression but similar Scarbl and higher Cd36 expression (Data not shown). Thus, both macrophages and epithelial progenitors showed transcriptional perturbation of cholesterol efflux pathways in lung-tumor bearing Kras^G12D mice. Altogether, these findings reveal that impaired HDL-mediated cholesterol efflux pathways are linked to the development of adenocarcinoma in Kras-driven mice lung tumors.

Defective ABCA1 and ABCG1 but not SR-BI dependent cholesterol efflux pathways in macrophages promotes a mild pro-tolerogenic tumor microenvironment in KRAS-driven non-small cell lung cancer.

Several studies have suggested a pro-tumoral role of cholesterol efflux pathways in tumor- associated macrophages (TAMs) from orthotopic or ectopic mouse models of cancer¹⁶'¹⁸. Thus, to delineate the role of macrophage cholesterol efflux pathways in the non-metastatic mouse models of lung-tumor bearing KRAS^G12D mutation, we first investigated the role of SR-BI by carrying out bone marrow (BM) transplantation from mice with SR-BI deficiency (LyzM-Cre x Scarb 1^ mice, referred as MO^SRBI) (Data not shown)³⁴. To interpret the potential role of SR- BLmediated bidirectional cholesterol flux, we compared MO^SRBI mice to mice with defective macrophage CD36-mediated unidirectional lipid influx pathway (LyzM-Cre x Cd3^ mice, referred as MO^CD36)³⁵. After BM transplantation from these mice into irradiated CC-LR recipient mice and a recovery period of four weeks, tumor initiation was induced by CreER™ tamoxifen induction (Data not shown). Peripheral hematologic parameters and plasma HDL- C levels were not affected in lung-tumor bearing Kras^GI2D mice transplanted with M ^SRB1 BM compared to control or MO^CD36 BM transplanted mice (Data not shown). In contrast to CD36 deficiency, knockdown of SR-BI did not alter neutral lipid accumulation in AMs or IMs (Data not shown) Consistent with the lack of effect of SR-BI deficiency on lung macrophage cholesterol homeostasis, we also did not observe significant changes in lung or bronchoalveolar lavage (BAL) cholesterol levels nor in surfactant composition as measured by similar amount of surfactant protein D (SP-D) or phosphatidylcholine (PC) levels in lung-tumor bearing Kras^G12D mice transplanted with MO^SRBI BM compared to control or M ^CD36BM transplanted mice (Data not shown). In contrast to CD36 deficiency, there was also no significant perturbation in the number of AMs in MO^SRBIBM transplanted animals or in different myeloid populations (Data not shown). Consequently, the tumor analysis revealed that while macrophage CD36 deficiency limited tumor development (Data not shown), SR-BI did not (Data not shown). These findings suggest that in contrast to CD36, SR-BI-mediated bidirectional macrophage cholesterol flux is dispensable to modulate the tumor microenvironment (TME).

We next carried out BM transplantation from mice with combined deficiencies of cholesterol efflux transporters ABCA1 and ABCG1 in myeloid cells (LyzM-Cre Abcal^Abcgl^¹ mice, referred as MO^DKO)³⁶ into irradiated CC-LR recipient mice (Data not shown). After a recovery period of four weeks, tumor development was initiated by CreER™ tamoxifen induction (Data not shown). Peripheral hematologic parameters were overall unaffected in CC-LR mice transplanted with MO^DKOBM and similar plasma HDL-C and SP-D levels were observed (Data not shown). In contrast, impaired Abcal and Abcgl mRNA expression in pulmonary macrophages was associated with higher neutral lipid accumulation in AMs from CC-LR mice transplanted with MO^DKOBM (Data not shown). This cholesterol loading was ultimately linked to the disappearance of AMs in the lung of these mice (Data not shown). Thus, we investigated whether this could influence lung cholesterol homeostasis, surfactant clearance or lung inflammation. We did not observe significant changes in lung or BAL cholesterol levels nor in SP-D or PC levels in CC-LR mice transplanted with MO^DKO BM (Data not shown). However, we observed a strong pro-inflammatory environment in the lung of these animals as characterized by an increased number of CD206'SiglecF⁺ eosinophils, CDl lb¹¹¹ PMNs and Ly6C^hl monocytes (Data not shown). Consequently, the tumor analysis showed that despite similar size and amount of tumor nodules, there was an abnormal hyperplasia in CC-LR mice with defective macrophage cholesterol efflux pathways (Data not shown). Higher E-cadherin (CDH1) over vimentin immunostaining in CC-LR mice transplanted with MO^DKO BM (Data not shown) also suggested more differentiated epithelial cells independently of mesenchymal transition. Thus, defective macrophage cholesterol efflux pathways shape a mild pro- tolerogenic TME in the KRAS^G12D-driven lung tumor progression phase.

Defective ABCA1 and ABCG1 cholesterol efflux pathways in epithelial tumor progenitor cells exacerbates tumor growth in KRAS-driven non-small cell lung cancer.

Accumulation of cholesterol in the lung of a mouse model of whole body cholesterol homeostasis perturbation induced by liver X receptor (LXR) deficiency had been previously associated to spontaneous lung cancer development³⁷. Thus, we first generated epithelial- specific ABCA1/ABCG1 knockouts by breeding CCSP-CreER™ mice with Abcal^Abcgl^ mice (referred as CC and CC^DKO, respectively). We did not observe spontaneous lesion development (Data not shown) or inflammatory infiltrate (Data not shown) at 7 months of age. Then, to test whether defective cholesterol efflux pathways increased tumorigenesis in Kras- driven cancers, CCSP-CreER™Xra5^L‘^{SL G72I)} mice were crossed to Abcal^Abcgl^ mice, generating a conditional epithelial tumor-specific ABCA1/ABCG1 deficiency (referred as CC- LR and CC-LR^DKO, respectively) (Data not shown). Quantification of BODIPY staining revealed that deficiency of these transporters exacerbated the neutral lipid accumulation observed in epi^BASCs and epi^TPCs from lung-tumor bearing Kras^GI2D mice (Data not shown). In contrast, similar BODIPY staining was observed between macrophages from CC-LR and CC- LRDKO _mjce (j)_at_{a no}t shown). We next further characterized the tumor growth in absence of cholesterol efflux pathways in epithelial tumor progenitor cells. Although similar amount of tumor nodules or hyperplasia was observed in CC-LR^DKO mice compared to CC-LR mice, the size of the lesion was significantly increased (Data not shown). CCSP and SP-C co-staining indicated an expansion of BASCs at the bronchoalveolar duct junction in the lung of CC-LR^DKO compared to CC-LR mice (Data not shown). Consistently, scRNAseq performed on whole lung tissue from CC-LR^DKO mice (Data not shown) confirmed an increased number of epithelial progenitor cells expressing Sftpc (Data not shown). Quantification of CD45' nonimmune cell clusters confirmed that defective cholesterol efflux promoted an increase in the percentage of epi^BAscs _{at t}h_{e ex}p_{ense o}f endothelial cells (Data not shown). A trend towards higher epithelial progenitors was also observed by flow cytometry in the lung of CC-LR^DKO mice compared to CC-LR mice (Data not shown). A higher and more diffuse CDH1 immunostaining in CC- LRDKO _mjce f_urther supported an expansion of epithelial cells (Data not shown). GSEA of the differentially expressed genes revealed that the deficiency of cholesterol efflux pathways in CC-LR mice induced higher expression in various genes assigned to epi^BASCs ‘cell signaling’ and ‘energy metabolism’, which could partly explain the expansion of these cells (Data not shown). Notably, these cells showed a gene signature that was higher for epidermal growth factor (EGF) and insulin-like growth factor (IGF) signaling pathways but not for pre-metastatic markers (Data not shown). Epi^TPCs from CC-LR^DKO also showed enhanced expression of genes involved in ‘translation’ and ‘tumor invasiveness’ (Data not shown). Additionally, lower expression of genes involved in ‘cellular dynamics’ and ‘response to stress’ was observed in epi^BASCs _anc| _epi^TPCs from CC-LR^DKO mice, respectively (Data not shown), suggesting tumor cells could perturb the TME. Thus, defective cholesterol efflux pathways in tumor progenitor cells promote their expansion and modulate their transcriptomic signature.

Quantification of CD45⁺ immune cell clusters in the scRNAseq analysis revealed an imbalance in the percentage between the macrophage and the myeloid cell, especially monocyte, clusters (Data not shown). We confirmed that deficiency of cholesterol efflux pathway in tumor progenitor cells limited the expansion of infiltrated myeloid cells (i.e, eosinophils, Mos and PMNs) by flow cytometry (Data not shown), which most likely reflect a pro-tolerogenic TME. Interestingly, defective cholesterol efflux pathways in epithelial tumor progenitor cells strongly affected the transcriptomic signature of macrophages with an upregulation of genes involved in ‘intracellular trafficking’ and ‘translation initiation’ and a downregulation of genes related to apoptotic process’ and ‘cytokine response and secretion’ (Data not shown). Of note, transplantation of MO^DKO BM into CC-LR^DKO mice (Data not shown), to test the synergy of defective cholesterol efflux pathways in these cell types, showed that despite the expected loss of AMs and higher myeloid cell infiltrates (Data not shown), there was no additive effect in tumor progression (Data not shown). Thus, defective cholesterol efflux pathways in epithelial tumor progenitor cells dominates tumor growth in lung-tumor bearing Kras^GI2D mice. Inversely, reactivation of anti -turn or activity in CC-LR^DKO mice with PD-1 blockade antibody (Data not shown) prevented the exacerbated tumor growth of these mice (Data not shown), supporting the concept that impaired cancer cell cholesterol efflux is part of the tumor strategy to escape immune surveillance. Altogether, these findings highlight the mechanism by which defective cholesterol efflux pathways in epithelial tumor progenitor cells promote a progression of the lesion size associated with an expansion of epithelial cancer cells that diverts the immune TME.

ApoA-I overexpression protects mice from mutant KRAS-driven non-small cell lung cancer by limiting the expansion of epithelial progenitor cells of tumor-origin. Since defective cholesterol efflux participates to tumor growth, we next explored the effect of human apoA-I overexpression to increase HDL levels in lung-tumor bearing Kras^GI2D mice (Data not shown . We first observed that apoA-I transgenic animals (LR/apoAI-Tg) survived longer compared to lung-tumor bearing Kras^GI2D mice (LR) (Data not shown). This was accompanied by prevention of metabolic alterations induced by lung tumor development including body weight loss (Data not shown) and epididymal fat mass atrophy (Data not shown). However, in absence of metastasis in lung-tumor bearing Kras^G12D mice, we did not observe significant changes in systemic inflammatory or hematological parameters³⁸ in presence or absence of the apoA-I transgene (Data not shown), indicating these mechanisms were unlikely involved in the global beneficial effects of the apoA-I transgene. Pneumotachographs generated by spirometry rather indicated a direct link between increased apoA-I and HDL-C levels and the breathing rate (i.e, amount of volume and debit of air that can inhaled and exhaled) of lung-tumor bearing Kras^GI2D mice (Data not shown). Histological analysis with Hematoxylin and Eosin (H&E) stain and 3-dimensional reconstruction microscopy with CCSP and DAPI stains revealed that tumor progression was indeed significantly blunted by the apoA-I transgene 8 weeks after tumor induction (Data not shown) with a strong reduction in all histological subtypes (i.e, adenocarcinoma, adenoma and atypical adenomatous hyperplasia (AAH)) (Data not shown). The underlying mechanism most likely involved the removal of excess tumor cholesterol as accumulation of cholesterol in the lung of tumor-bearing Kras^G12D mice was prevented by the raise in plasma HDL-C levels induced by overexpression of the human apoA-I transgene (Data not shown). Altogether, these findings reveal that HDL-mediated cholesterol efflux plays a preventing role in lung tumor development and long-term pathologic consequences.

We further investigated the relation of epithelial tumor progenitor cell expansion to HDL- mediated cholesterol efflux. Quantification of epi^BASCs and epi^TPCs by flow cytometry revealed reduced numbers of these cells in the lung of LR-ApoAI^Tg compared to LR mice (Data not shown). Consistently, overexpression of the human apoA-I transgene in lung-tumor bearing Kras^G12D mice suppressed epithelial progenitor expansion as shown by CCSP and SP-C costaining (Data not shown). Colony-forming assays confirmed that the expansion of epithelial colony-forming units (eCFUs) from the lung of mice bearing the KRAS^G12D oncogene was almost blunted by the in vivo overexpression of the human apoA-I transgene (Data not shown). Both small dense saccular colonies and large airway-like lobular cystic colonies were reduced (Data not shown), indicating a complementary effect of cholesterol efflux pathways on differentiation of progenies into alveolar and airway epithelial cells (i.e, reduced differentiation and tumorigenic potentials)²⁵. Using Rosa26^LSL'^EYFP reporter mice crossed with LR and LR- ApoAI^Tg mice, we also observed reduced expansion of CD3 l'CD45 EpCAM⁺EYFP⁺ epithelial progenitor cells of tumor origin (Data not shown). The prevention of epithelial tumor progenitor cell expansion paralleled a significant decrease in the SG2M cycling fraction in CD3 l'CD45 EpCAM⁺ epithelial progenitors from LR-ApoAI^Tg compared to LR mice (Data not shown) along with reduced levels of pErkl/2 (Data not shown). We also observed a significant decrease in Ki67-positive proliferative cells in the lung of these mice that paralleled a decrease in the LC3 autophagy marker (Data not shown), known to facilitate protein recycling and the survival of cancer cells in tumors³⁹. Exogenous addition of different concentrations of HDL or methyl-0-cyclodextrin (m0CD), which mediates cholesterol efflux from plasma membrane⁴⁰, recapitulated the reduced number of eCFUs (i.e, both small and large colonies) from controls or mice bearing the KRAS^G12D oncogene (Data not shown). Altogether, these findings indicate that HDL-mediated cholesterol efflux cause an anti-tumoral effect by preventing the cell intrinsic expansion of epithelial progenitor cells of tumor-origin.

Cholesterol efflux pathways are intimately linked to growth factor signaling pathways.

To apprehend the mechanistic interconnection between cholesterol efflux pathways and tumorigenesis, we next evaluated the expression of key transcriptional regulators oiAbcal and Abcgl, namely liver X receptor (LXR), microRNAs (miRNAs) and hypoxia inducible factor (HIF). The expression of LXRa and 0 (i.e, Nrlh2 and Nrlh3) and their post-transcriptional regulators (i.e, Sult2bl and Mylip) were barely detectable in the lung epithelial clusters of CC- LR mice compared to macrophages (Data not shown). This could explain the lack of cell intrinsinc effect of LXR on epithelial progenitor expansion isolated from CC-LR mice (Data not shown). Animals were then exposed to liver X receptor (LXR) activator (T0901217; 50mg/kg twice a week) (Data not shown)¹⁴. After 4 weeks of inhalation exposure, microscopic examination of the lung (Data not shown), revealed that LXR activator treatment had no effect on tumor development (Data not shown) despite limiting myeloid cell infiltrates (Data not shown). We also did not find significant changes in the expression of miRlOb, miR26, miR33 and miR183 in the lung of CC-LR mice compared to controls (data not shown). In contrast, we observed an increased expression of HIFla and HIF2 (i.e, Hifla and Epasl) in in lung epithelial clusters of CC-LR mice (Data not shown). Thus, we generated epithelial-specific HIF la or HIF2 knockouts by breeding mice bearing the KRA P^I2D oncogene with Hifl and Epasf mice named LR^HlfaKO and LR^EpaslKO, respectively. However, deficiency of these master regulators of hypoxic tumor did not significantly impact tumor growth (only a trend with HIF2 deficiency) o Abcal nd Abcgl expression in the lung of these animals (Data not shown). We next considered the regulation of cholesterol efflux transporters by growth factor signaling pathways¹³, especially IGF and EGF signaling pathways that were upregulated in CC-LR^DKO mice (Data not shown). Stimulation of eCFU with these growth factors significantly reduced the expression of Abcal and Abcgl (Data not shown). There is evidence that IGF and EGF in the TME could promote a non-classical macrophage polarization^41,42. We now observed that inhibition of IGF1R and EGFR with linsitinib or erlotinib prevented the downregulation of Abcal and Abcgl expression induced by eCFU conditioned media in macrophages (Data not shown). Thus, growth factor signaling pathways are the culprit of reduced cholesterol efflux pathways in tumors and TME.

We next reasoned that reduced cholesterol efflux pathways induced by growth factors could provide cholesterol build up to support cancer cell proliferation. The expression of cholesterol efflux transporters is heterogenous among lung cancer lines (Data not shown) but a tumor- derived cell line from Kras^G12D mice has been previously generated harboring a strong sensitivity to metabolic changes recapitulating the in vivo setting³⁹. Treatment of these cells with HDL reduced BODIPY neutral lipid staining and levels of pErkl/2 and prevented their clonogenic survival in nutrient-poor or -rich growth media (Data not shown). This led us to investigate the molecular basis of the HDL effect in these cells by investigating the proteomic composition of the plasma membrane (PM) in presence or absence of HDL treatment. Functional enrichment analysis of protein-protein interaction networks after mass spectrometry of enriched PM fraction revealed that HDL treatment reduced the expression of proteins involved in four main pathways that were interconnected namely ‘membrane ordering’, ‘IGF1 signaling’ along with ‘retrograde trafficking and PM recycling’ pathways, known to support ‘translation and protein life cycle’ during proliferation (Data not shown). Consistently, reduced cellular BODIPY cholesterol content was associated with reduced cholera toxin subunit B (CTx-B) staining in HDL treated cancer cells (Data not shown), suggesting reduced formation of cholesterol -rich lipid rafts. To further validate these findings, we showed that HDL treatment reversed the increased CTx-B staining observed in cell-sorted epi^BASCs and epi^TPCs from CC-LR mice (Data not shown) as well as free cholesterol accumulation determined by fl lipin staining (Data not shown). Consistently, we observed that HDL also reversed the KRAS^G12D-dependent EGFR and IGFR1 autocrine loop^43,44 in eCFU from cell sorted CD45 EYFP⁺ and CD31 CD45' EpCAM⁺ (Data not shown). Inversely, dual EGFR and IGF1R inhibition prevented the eCFU expansion from the lung of CC-LR^DKO mice (Data not shown). Finally, by comparing single or dual growth factor inhibition (Data not shown) with HDL treatment in eCFU expansion from the lung of CC-LR mice, we have unrevealed that HDL act similarly to a dual inhibitors of receptor tyrosine kinase (Data not shown).

Cholesterol removal prevents progressing tumor in KRAS-driven non-small cell lung cancer. We next investigated whether therapeutic modulation of cholesterol efflux pathways could be beneficial in established tumors induced for 6 weeks in CC-LR mice. We exposed mice with established tumors to m0CD (4g/kg once a week) (Figure 1A). After 4 weeks of inhalation exposure, microscopic examination of the lung (Data not shown), revealed that despite similar lesion size and hyperplasia of tumor nodules, m0CD treatment limited the number of nodules in progressing tumors (Figure IB). Activation of antitumor activity with PD-1 blockade antibody in mpCD-treated CC-LR mice (Data not shown) repressed hyperplasia but had not significant benefit on lesion size and tumor number (Data not shown) most likely because cholesterol removal therapy directly impacted the expansion of epithelial progenitor cells of tumor-origin. Thus, to delineate whether mpCD treatment directly limited the expansion of epithelial progenitor cells of tumor-origin, we repeated a similar experiment taking advantage of CC-LR Rosa26^LSL'^EYFP reporter mice. We first confirmed that mpCD treatment limited the progression of tumor nodules at 4 and 6 weeks after inhalation exposure (Data not shown and Figure 1C). These observations paralleled reduced percentages of epi^BASCs and epi^TPCs (Data not shown) and CD31 CD45 EpCAM⁺EYFP⁺ epithelial progenitors (Figure ID and Data not shown) in mpCD-treated CC-LR mice compared to saline nailing down that mpCD treatment prevented the expansion of epithelial progenitor cells of tumor-origin. Altogether, our findings indicate that cholesterol removal therapy reduces tumor burden in progressing lung tumor from mice bearing the Kras^G12D oncogene.

Systemic and local perturbations of cholesterol efflux pathways in human lung adenocarcinoma.

To investigate the translational value of our findings, we first evaluated the systemic modulation of cholesterol efflux pathways by measuring plasma HDL-C levels in twenty-two patients with different stage of lung adenocarcinoma from the LPCE biobank compared to ten patients without lung adenocarcinoma (healthy volunteers and patients with chronic obstructive pulmonary disease, COPD) (Data not shown). Plasma concentrations of HDL-C were lower by approximately 25% compared to controls (Data not shown). The ability of these HDL-C to mediate cholesterol efflux was also decrease by approximately 20% in patients with lung adenocarcinoma (Data not shown), correlating with plasma HDL-C levels (Data not shown). These HDL were not dysfunctional as similar cholesterol efflux was observed when HDL levels were matched by concentration (Data not shown). In lung adenocarcinoma patients, plasma HDL-PC and apoA-I levels were also reduced by -25% and 50%, respectively, confirming reduced number of HDL particles (Data not shown). Finally, we evaluated whether this decrease could be linked to other lung cancer biomarkers such as surfactant molecules, fibrinogen or a chronic inflammatory status. In this cohort, SP-B, SP-D, fibrinogen and angiotensin II (Angll) levels were similar within patient with or without lung adenocarcinoma (Data not shown). Although plasma tumor necrosis factor a (TNFa) levels were statistically increased in patients with lung adenocarcinoma (Data not shown), there was no correlation with plasma HDL-C levels (Data not shown). This indicates that HDL is not merely a classical inflammatory biomarker.

To predict local perturbations in cholesterol efflux pathways, we performed a metabolic pathway enrichment analysis from RNAseq performed on fifty-seven lung adenocarcinoma specimens and eleven normal lung tissues⁴⁵. We first showed that 32% of metabolic transcripts were significantly modulated (i.e., 286 transcripts were increased and 286 were decreased) in stage I/II lung adenocarcinoma (Data not shown). We next validated our approach by showing an enrichment of upregulated transcripts in carbohydrate metabolism, TCA cycle, oxidative phosphorylation (OXPHOS) and amino acid (AA) metabolism from KEGG pathways (Data not shown), consistent with the Warburg effect of tumors^1,2. In contrast, lung adenocarcinoma metabolic signature showed down-regulation of transcripts involved in phospholipid (PL), sphingolipid (SM) and lipid metabolism with cholesterol pathway showing the greatest number of disrupted transcripts (Data not shown). To validate these findings in a larger cohort (i.e., 399 patients), we interrogated the publicly available cancer genome atlas (TCGA) gene expression dataset (Data not shown). The transcriptomic analysis confirmed that up- and down- regulated transcripts recapitulated the same metabolic pathway enrichment signature (Data not shown). Venn diagram highlighted a core signature of cholesterol metabolism genes that was commonly regulated between the two independent cohorts (Data not shown). Thus, cholesterol-associated transcripts were the most down-regulated in lung adenocarcinoma. Topological analyses⁴⁶ further illustrated how the cholesterol metabolism module was connected to the global metabolic transcriptome signature (Data not shown). Mapping regulation of cholesterol-associated transcripts at the cellular level highlighted an enrichment of down-regulated transcripts involved in cellular cholesterol trafficking that ultimately support cholesterol efflux pathway towards apolipoprotein A-I and HDL (Data not shown). Therefore, cholesterol efflux pathways are systemically and locally perturbed in patients with lung adenocarcinoma highlighting the translational value of our preclinical findings.

Discussion

Our data support the requirement of cholesterol efflux pathways and HDL raising therapies as a potential metabolic liability of KRAS-driven lung tumor growth that might be exploited for NSCLC therapy. Given that diet-induced obesity and dyslipidemia are linked to tumor growth^47,48, there has been a long interest in the relationship between cholesterol and cancer. A role of cholesterol for tumor growth was first suggested a century ago following cholesterol injection into xenografts⁴⁹. However, this concept has been challenged by the lack of consistency in cell-culture studies aimed at understanding the metabolic phenotype of cancer cells, the diversity of cholesterol-related metabolic signature between different types of tumors and the limitation of syngeneic and xenogeneic mouse models of cancer⁵⁰. These observations highlight the need to better understand the cholesterol routes that most likely depend on intrinsic tumor properties to enable tumor growth initiated by oncogenic mutations. Due to the multifaceted protective behavior of HDL-mediated cholesterol efflux in various inflammatory diseases¹³, its association with cancer has regained a strong interest by the scientific community¹². In syngeneic and xenogeneic mouse models of melanoma and ovarian cancer, a protective effect of HDL has been observed on tumor metastasis^14,15. However, most of the in vivo studies highlight a role of cholesterol efflux pathways in TAMs¹⁶'¹⁸. Thus, the in vivo relevance of cholesterol efflux pathways to lung epithelial cancer cells has not yet been addressed to our knowledge. The metabolic fingerprint of mouse and human lung adenocarcinoma, defined in this study, demonstrates a strong perturbation in cholesterol efflux pathways, compared with nonmalignant lung. This was associated with systemic perturbations in apoA-I and HDL-C levels. Our tritium cholesterol labeling experiments in a mouse model of KRAS-driven lung tumors supports a model in which lung tumors locally and distally rewire HDL cholesterol metabolism to retain cholesterol. Consistently, raising HDL levels through overexpression of the human apo-AI transgene or cholesterol removal with m0CD resulted in robust inhibition of tumor growth in this biologically relevant model of NSCLC. These results emphasize that lung adenocarcinoma has elevated cholesterol requirements that is highly dependent on HDL-mediated cholesterol efflux.

Tissue-resident macrophages provide a pro-tumorigenic niche to NSCLC cells⁵¹ and enhanced production of myeloid cells through extramedullary myelopoiesis has also been shown to enhance cancer growth in mouse models of lung cancer metastasis³⁸. Thus, we originally hypothesized that the anti-tumorigenic effects of cholesterol efflux pathways could possibly be due to their roles in preventing peripheral extramedullary myelopoiesis or modulating local tissue inflammation¹³. However, non-metastatic lung-tumor bearing Kras^G12D mice did not present any sign of enhanced myeloid cells in the periphery or in the spleen, excluding the involvement of extramedullary myelopoiesis. We next tested the contribution of macrophage cholesterol efflux pathways to limit tumor growth. Deficiency of macrophage ABCA1 and ABCG1 did specifically increase alveolar macrophage cholesterol content, which was associated with a remodeling of the myeloid landscape leading to an abnormal hyperplasia in lung-tumor bearing Kras^G12D mice but no major impact on tumor growth. These findings contrasted with prior studies showing that defective cholesterol efflux pathways in TAMs converted tumor promoting into anti-tumor macrophages¹⁶'¹⁸. However, these previous studies were performed in syngeneic and xenogeneic mouse models of cancer metastasis, which do not take into account the diversity of tissue macrophages in their specific tissue environment that could be multifaceted in lung cancer with pro- and anti-tumoral functions¹⁹. For instance, AMs have a specific metabolic signature to shape lipid and surfactant homeostasis and maintain lung homeostasis^52,53. Although, we did not find modulation of surfactant composition in lung-tumor bearing Kras^G12D mice with defective cholesterol efflux pathways in macrophages, we and others previously found that ABCA1 and ABCG1 deficiency created a pro-oxidative milieu in the lung, which could explain the loss of AMs in the present study and the expansion of inflamed myeloid cells⁵⁴'⁵⁶. While beyond the scope of the present study, future studies should help to clarify how cholesterol efflux in AMs could limit lung hyperplasia in NSCLC mouse model and whether this may be related to the foamy TREM2⁺ macrophage subset that has been previously shown to promote chronic lung diseases²².

In the present study, we found that the tumor-driven immune signature⁵⁷ was directly impacted by defective cholesterol efflux pathways in epithelial tumor progenitor cells. Indeed, defective cholesterol efflux in epithelial tumor progenitor cells limited the inflammatory myeloid cell infiltrate favoring tumor to escape from immune surveillance in lung-tumor bearing Kras^GI2D mice. A cholesterol steal mechanism by ovarian cancer cells through secretion of hyaluronic acid has recently been proposed¹⁸. However, cholesterol accumulated in both epithelial cells and macrophages isolated from the lung of mice bearing the KRAS^G12D oncogene. The unlikeliness of this scenario in NSCLC mouse model was also illustrated by the barely detectable expression of hyaluronan synthase 2 (Has2 the rate-limiting enzyme in hyaluronic acid synthesis, in epithelial cells from our single cell dataset and the absence of changes in pulmonary hyaluronic acid levels between the different genotypes (data not shown). One explanation could be the diversity of tumor origin, oncogenic mutations and tissue context that can differently affect tumor metabolic reprogramming and cellular cholesterol routes^1,2. Here, we identified that growth factors such as EGF and IGF1 could reduce expression of cholesterol efflux transporters at distance in macrophages, most likely as part of a global metabolic reprogramming that drives non-classical macrophage polarization associated to a pro- tolerogenic TME^41,42. Indeed, single-cell transcriptomic analysis indicated an upregulation of genes involved in generation of energy, carbohydrates and OXPHOS in epithelial tumor progenitor cells at the expense of macrophages that could reflect a glucose steal mechanism also participating to divert the immune response⁴. Consistently, the inflammatory transcriptomic signature of AMs was downregulated in both CC-LR and CC-LR^DKO mice.

The classical hallmarks of cancer are intimately intertwined with an assortment of metabolic processes that a tumor cell effectively hijacks to facilitate malignant transformation^1,2. Oncogenic transformation in mutant KRAS cells increases the demand for cholesterol most likely to provide the essential building blocks required to maintain their aberrant survival and growth or to amplify growth-factor receptor signaling pathways^43,44. However, most of these observations are made from in vitro studies using lung cancer lines that are heterogenous. In the present study, multiplexed flow cytometry and single-cell transcriptomic analyses identified a role of cholesterol efflux pathways in the previously identified epithelial progenitors termed BASCs and TPCs^{22,26,26,27,29}. Although epi^TPCs were preferentially expanded in lung adenocarcinoma, single-cell transcriptomic analysis identified in an unbiased manner cholesterol efflux-dependent gene signatures of epi^BASCs that included an upregulation of genes involved in ‘intracellular protein dynamic’ including receptor tyrosine kinase signaling. Defective cholesterol efflux pathways also promoted a more aggressive transcriptional signature of Epi^TPCs, resembling the transition phase signature of tumor progenitors or the aggressive signature of distant tumor progenitors^30,31. Despite these signatures and the enhanced tumor growth in CC-LR^DKO mice, we did not observe significant modulation of transcription factors Nkx2.1 or Runx2 or target genes associated with ECM components suggesting that NSCLC mouse model with defective cholesterol efflux did not transit to a pre-metastatic state^30,58. Additionally, we unraveled that overexpression of apoA-I in vivo reversed the expansion of both epi^BASCs and epi^TPCs and their cell cycling. We also show a cell intrinsic role of membrane cholesterol removal in limiting the proliferation and expansion of epithelial progenitor cells of tumor origin, in part by overcoming KRAS^G12D-dependent EGFR and IGF 1R autocrine loop along with limiting phospho-ERK activation, a culprit in epithelial tumor cell proliferation. This could be of therapeutic interest since EGFR and IGF1R act as mutual compensation pathways and high co-expression of these receptor tyrosine kinases correlated with worse disease-free survival in NSCLC⁵⁹.

Our findings aimed at identifying the best therapeutic option to modulate cholesterol efflux pathways are surprising, as they do not support an intuitive model whereby transcriptional activation of cholesterol efflux pathways with a LXR agonist limits the progression of established tumors⁶⁰. Notably, LXR activation had no effect on the expansion of epithelial tumor progenitor cells despite having a direct role in reducing myeloid infiltrate. Future studies will be required to evaluate whether the lack of anti -proliferative effect of LXR in lung tumor cells could be attributed to its repression depending on the disease context⁶⁰. In strong contrast, removal of plasma membrane cholesterol after mpCD inhalation reduced tumor growth after 4 weeks treatment in post-tumor-onset CC-LR mice and revealed some signs of regression after 6 weeks. This effect was dominated by a decrease in the expansion of epithelial progenitor cells of tumor-origin. Our pharmacological data using mpCD treatment indicated that cholesterol removal therapy may be a strong candidate in preventing progression of established tumor in the mouse model of NSCLC. Our data warrant thorough clinical assessment as results on checkpoint blockage therapy in lung cancer patients with KRAS mutation has been questioned⁶¹.

In the clinic, conventional chemotherapy remains the major option to treat patients with KRAS- mutant NSCLC, although chemotherapy plus immune checkpoint blockade or novel oncogenic KRAS inhibition⁶² has been recently approved as the first-line regimen for NSCLC^63,64. The metabolic fingerprint of mouse and human lung adenocarcinoma now indicates that defective cholesterol efflux is exquisitely intertwined to the metabolic rewiring of epithelial tumor progenitor cells to meet the unique cholesterol requirements for proliferation and expansion. Our preclinical findings also unveil that plasma membrane cholesterol removal with HDL- raising therapies or mpCD can overcome this defect to limit tumor growth. Because mpCD treatment in humans is safe and can also be nebulized, it may therefore be used clinically as a novel mechanism-based therapy beyond alternative horizons for treating KRAS-mutant lung cancer such KRAS, MEK or dual inhibitors of receptor tyrosine kinase.

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Claims

CLAIMS:

1. A method for the treatment of a lung cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of cyclodextrin.

2. The method according to claim 1 wherein the lung cancer is a KRAS mutant lung cancer.

3. The method according to claim 1 wherein the KRAS mutant lung cancer is a KRAS mutant non-small cell lung cancer (NSCLC).

4. The method according to claim 1 wherein the KRAS mutant NSCLC is a KRAS mutant lung adenocarcinoma (LU AD).

5. The method according to claim 1 wherein the cyclodextrin is methyl-P-cyclodextrin (mpCD).

6. The method according to claim 1 wherein the cyclodextrin is administered by aerosolization or by inhalation or by intrathecal administration or by intravenous administration.

7. Cyclodextrin and ii) a classical treatment, as a combined preparation for use in the treatment of lung cancer.

8. The combined preparation according to claim 7 wherein the lung cancer is KRAS mutant lung cancer.

9. The combined preparation according to claim 7 wherein the lung cancer is KRAS mutant non-small cell lung cancer (NSCLC).

10. The combined preparation according to claims 7 to 9 wherein the cyclodextrin is methyl-P-cyclodextrin (mpCD).

11. A pharmaceutical composition comprising cyclodextrin for use in the treatment of lung cancer.

12. The pharmaceutical composition according to claim 11 comprising i) cyclodextrin and ii) a classical treatment, as a combined preparation for use in the treatment of lung cancer.

13. The pharmaceutical composition according to claims 11 to 12 wherein the cyclodextrin is methyl-P-cyclodextrin (mpCD).

14. The pharmaceutical composition according to claims 11 to 12 wherein the lung cancer is KRAS mutant lung cancer.

15. The pharmaceutical composition according to claims 11 to 12 wherein the KRAS mutant non-small cell lung cancer (NSCLC).