WO2025151077A1

WO2025151077A1 - A method to predict and target resistance to chemotherapy in a cancer

Info

Publication number: WO2025151077A1
Application number: PCT/SG2025/050013
Authority: WO
Inventors: Anand Devaprasath JEYASEKHARAN; Michal Marek HOPPE; Zi Yan Charmaine ONG
Original assignee: National University of Singapore
Current assignee: National University of Singapore
Priority date: 2024-01-08
Filing date: 2025-01-08
Publication date: 2025-07-17
Anticipated expiration: 2026-07-08

Abstract

This disclosure concerns methods for predicting treatment outcome to a cancer therapy (in particular combination therapy with CHOP or R-CHOP) by detecting the level of macrophage immunometabolism regulator (MACIR) expression in a subject, wherein an increased MACIR expression indicates likely non-responsiveness to the cancer therapy. Also provided are methods of treating refractory cancer using MACIR inhibitors.

Description

A Method to Predict and Target Resistance to Chemotherapy in a Cancer

Technical field

The invention relates generally to oncology biomarkers. In particular, the invention relates to a method for determining resistance to a cancer therapy by detecting levels of macrophage immunometabolism regulator (MACIR).

Background

Diffuse large B cell lymphoma (DLBCL) is the most common form of lymphoma, with a worldwide estimate of 150,000 new cases annually. This represents almost 30% of all non-Hodgkin’ s lymphoma cases. DLBCL is an aggressive neoplasm, and while many patients respond well to the standard-of-carc treatment R-CHOP (a combination of Rituximab, Cyclophosphamide, Doxorubicin, Vincristine and Prednisone), up to 40% of patients are either refractory to R-CHOP or experience relapse. Salvage treatments for these relapsed/refractory cases are effective only in a fraction of cases. It would be beneficial to be able to identify poor responders early, so that they can be treated with adjunctive therapies in addition to R-CHOP or with alternative therapies.

Accordingly, it is generally desirable to overcome or ameliorate one or more of the above-mentioned difficulties .

Summary

Disclosed herein is a method for determining the likelihood of resistance to a cancer therapy in a subject suffering from a cancer, the method comprising detecting an increased level of macrophage immunometabolism regulator (MACIR) expression as compared to a reference in a sample obtained from the subject, to thereby determine that the subject has an increased likelihood of resistance to the cancer therapy.

Disclosed herein is a method of treating a subject suffering from a cancer, the method comprising: a) determining the likelihood of resistance of the subject to a cancer therapy by detecting an increased level of macrophage immunometabolism regulator (MACIR) expression as compared to a reference in a sample obtained from the subject, wherein an increased level of MACIR expression indicates that the subject has an increased likelihood of resistance to the cancer therapy; and b) treating the subject found to have an increased likelihood of resistance to the cancer therapy.

Disclosed herein is a method for detecting a refractor}' cancer in a subject suffering from a cancer, the method comprising detecting an increased level of macrophage immunometabolism regulator (MACIR) expression as compared to a reference in a sample obtained from the subject, to thereby detecting the refractory cancer in the subject.

Disclosed herein is a method of treating a refractory cancer in a subject, the method comprising a) detecting an increased level of macrophage immunometabolism regulator (MACIR) expression as compared to a reference in a sample obtained from the subject, to thereby detect the refractory cancer in the subject; and b) treating the refractory cancer that is detected in the subject.

Disclosed herein is a method for stratifying a subject as a likely responder or nonresponder to a cancer therapy, the method comprising detecting the level of macrophage immunometabolism regulator (MACIR) expression in a sample obtained from the subject, wherein an increased level of MACIR expression indicates that the subject is likely to be non-responsive to the therapy.

Disclosed herein is a method for predicting treatment outcome of a subject to a cancer therapy, the method comprising detecting macrophage immunometabolism regulator (MACIR) expression in a cancer sample obtained from the subject, wherein an increased level of MACIR expression as compared to a reference indicates that the subject is likely to be non-responsive to the therapy or to relapse after the therapy.

Disclosed herein is a method of treating a cancer in a subject, the method comprising administering an inhibitor of macrophage immunometabolism regulator (MACIR) to the subject. Disclosed herein is a method of sensitizing a subject suffering from cancer to a cancer therapy, the method comprising administering an inhibitor of macrophage immunometabolism regulator (MACIR) to the subject.

Brief description of the drawings

Embodiments of the present invention are hereafter described, by way of non-limiting example only, with reference to the accompanying drawings in which:

Figure 1. A recursive screen identifies MACIR as a robust negative prognosticator after R-CHOP therapy in DLBCL. A, Schematic of univariate survival analysis screen conducted across all consensus genes in all datasets. B, Descriptive flow chart of the survival screen. 1mm - Immunotherapy (Rituximab); Chemotherapy (cyclophosphamide, doxorubicin, vincristine, and prednisone). C, Description of the cohorts used for the analysis. D, Summary of single gene survival associations in the discovery (left) and validation cohort (right). Survival association is expressed as a mean percentile (perc.) of hazard ratio (HR) for overall survival (OS) across all genes in all cohorts. Av. - Average. Result for MACIR is marked by a square. Result for MYC, an established poor prognosticator in DLBCL, is marked by a triangle.

Figure 2. MACIR expression is associated with poor survival in DLBCL patient cohorts: A, Cox proportional hazards model results for MACIR-High vs -Low patients (stratified using median) in all DLBCL gene expression cohorts. B, Kaplan-Meier plots of patients from each discovery cohort stratified according to median MACIR gene expression. C, Kaplan-Meier plot of the largest validation cohort, Lacy et al, stratified according to quartile (Q) MACIR gene expression. Gehan-Breslow-Wilcoxon test, pBonf - Bonferroni corrected p value. D-E, Kaplan-Meier plots of patients from other validation cohorts (excluding Lacy), stratified according to median MACIR gene expression. F, Comparison of early death rates at 12 months (mth) in MACIR-High and -Low patients (stratified according to median) across all cohorts (discovery, validation and chcmo-only inclusive). Paired t-test. G, Multivariate Cox proportional hazards model, similar to (A) but correcting for prognostically-relevant clinicop athological features - age, stage, performance status, extranodal disease, and serum lactate dehydrogenase levels in the form of 1P1 (International Prognostic Index) Risk Score, from all cohorts (discovery, validation and chcmo-only inclusive) that met the significance criteria and for which IPI scores are available.

Figure 3. Validation of antibody and establishment of immunohistochemistry staining protocol for MACIR in FFPE tissues. A, Western blot depicting HEK293T cells treated with siRNA against MACIR, probed for MACIR. B, MACIR KO SCI lymphoma cell lines created via the CRISPR method. C, Pseudo-immunohistochemistry images, obtained via multiplexed fluorescent immunohistochemistry (mflHC), of SCI wild type and MACIR KO SCI cell blocks stained with the anti-MACIR antibody. D, Grey-scale immunofluorcsccnt images of the experiment in (C). E, mflHC images from two DLBCL patient FFPE samples from the NUH cohort showing distinct patterns of MACIR staining. F, The distribution of MACIR expression (percentage extent) among patients within the NUH cohort, as detected by mflHC.

Figure 4. MACIR detected at the protein level stratifies DLBCL patients for survival.

A, Kaplan-Meier (KM) plot depicting overall survival (OS) for patients in the NUH cohort, stratified at the 65^th percentile of MACIR expression, as determined by mflHC.

B, KM plot depicting overall survival (OS) for patients in the Taiwan CMMC cohort, stratified at the 65^th percentile of MACIR expression, as determined by mflHC. C, Multivariate analysis table demonstrating that MACIR is a still an independent prognostic factor in the NUH cohort after adjusting for International Prognostic Index (IPI) risk group, Ccll-of-origin (Hans) classification, and c-MYC translocation status.

Figure 5, MACIR accurately classifies patient survival. A, Top, Summary volcano plot denoting the positions of MACIR, MYC, BCL2 and BCL6 in terms of mean overall survival hazard ratio percentile in the context of all genes evaluated (14,667 in total). Bottom, Individual volcano plots for MACIR, MYC, BCL2 and BCL6 depicting their cohort specific overall survival hazard ratio percentile in the context of all genes evaluated. B, Receiver operating characteristic (ROC) analysis plot of MACIR (Left) and cell-of-origin (COO) classification (Right) for predicting survival. (PFS, Progression free survival; OS, Overall survival}

Detailed description The inventors performed a Bayesian bioinformatic analysis of multiple gene expression datasets to identify molecular features in diffuse large B cell lymphoma (DLBCL) which are consistently associated with poor outcomes following combination therapy with R- CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone), a standard-of-care therapy for many types of non-Hodgkin lymphomas, including DLBCL. Candidate genes were selected based on two criteria: a) poor survival outcomes in patients where the gene is overexpressed (thus providing a potential therapeutic target for overcoming resistance); and b) the gene is robustly associated with poor survival across multiple independent well-annotated cohorts of R-CHOP-treated DLBCL patients (at least 100 patients each). A transcriptomic screen of 14,575 protein-coding genes in 8 datasets (2,519 patients) showed that the gene for macrophage immunometabolism regulator (MACIR) fulfilled both criteria. MACIR was validated as a suitable resistance biomarker in 3 additional validation cohorts (1,172 patients). Overall, a robust association of MACIR with poor survival was evident in 3,944 DLBCL patients across 13 cohorts (including 2 additional cohorts where only MACIR was evaluated). Thus, the inventors have identified the MACIR gene as a biomarker for predicting the outcome of R-CHOP-based combination therapies in cancer patients.

Macrophage immunometabolism regulator (MACIR), also known as C5orf30 (Chromosome 5 open reading frame 30), is implicated in regulating immune responses in inflammatory pathways. Previous genetic studies have identified MACIR as a susceptibility locus for the autoimmune disorder rheumatoid arthritis, but the gene is not known to play a role in cancer. It is thus a surprising finding that MACIR expression is associated with treatment outcomes in lymphoma.

This disclosure concerns the use of macrophage immunometabolism regulator (MACIR) as a biomarker for detecting refractory cancers and for predicting treatment outcomes in cancer patients following therapy with CHOP, R-CHOP or a related combination therapy. Also disclosed are methods of treating refractory cancers using MACIR inhibitors.

General definitions As used herein, the term “cancer” refers to the physiological condition characterized in part by unregulated cell growth. The term “cancer” includes both non-metastatic and metastatic cancers, including early stage and late-stage cancers.

The term “refractory” or “resistant” refers to a condition where there are residual cancer cells in a subject even after a complete course of treatment with a cancer therapy.

The term “relapsed” refers to a condition where a subject who was previously in remission from cancer following one or more courses of therapy re-develops cancer. A relapse may be detected by the rc-appcarancc of cancer cells following a period of absence after completing one or more courses of treatment.

The term “sample” herein is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, including both biological and environmental sources. A “biological sample” includes within its scope a collection of similar fluids, cells, or tissues isolated from a biological source, such as a whole organism or in vitro culture. Samples include but are not limited to tissue biopsies, tissue resections, tissue aspirates, swabs (e.g., buccal swabs), whole blood, plasma, serum, urine, saliva, cerebrospinal fluid, and cell cultures, and may be obtained using any suitable method known in the art. Archival tissues, such as those having treatment or outcome history may also be used for sample extraction. The sample may be pooled from multiple aliquots. Samples include untreated, treated, diluted and concentrated samples.

A “biological fluid” herein includes, but is not limited to, intravascular fluid (e.g., blood, plasma, serum, lymph), urine, saliva, sputum, cerebrospinal fluid, pleural fluid, fluid of the respiratory, intestinal, and genitourinary tracts, synovial fluid, vaginal secretion, tear fluid, pus, breast milk, semen, fluid from ascites, cyst or tumour, amniotic fluid, or combinations thereof.

The terms “patient”, “subject”, “host” or “individual” used interchangeably herein, refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, for whom therapy or prophylaxis is desired. Suitable vertebrate animals that fall within the scope of the invention include, but are not restricted to, any member of the subphylum Chordata including primates (c.g., humans, monkeys and apes, and includes species of monkeys such from the genus Macaca (e.g., cynomologus monkeys such as Macaca fascicularis, and/or rhesus monkeys (Macaca mulatto ) and baboon (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri) and tamarins (species from the genus Saguinus), as well as species of apes such as chimpanzees (Pan troglodytes)), rodents (e.g., mice rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovincs (c.g., sheep), caprincs (c.g., goats), porcincs (c.g., pigs), equines (c.g., horses), canines (e.g., dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars etc.), marine mammals (c.g., dolphins, whales), reptiles (snakes, frogs, lizards etc.), and fish. A preferred subject is a human in need of treatment for a cancer. However, it will be understood that the aforementioned terms do not imply that symptoms are present.

As used herein, the term “antibody” includes, but is not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFv), Fab fragments, F(ab’) fragments, disulfide-linked Fvs (sdFv) (including bispecific sdFvs), anti-idiotypic (anti-Id) antibodies, singledomain antibodies including nanobodies (VHHs) and variable new antigen receptors (VNARs), diabodies, and epitope-binding fragments of any of the above. The antibodies provided herein may be monospecific, bispccific, trispccific or of greater multispecificity.

The term “pharmaceutical composition” or “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of the active ingredient(s) to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the composition or formulation would be administered. Such formulations are sterile. “Pharmaceutically acceptable” carriers and excipients (vehicles, additives) are those which can reasonably be administered to a subject mammal to provide an effective dose of the active ingredient employed.

The terms “treating”, “treatment” and the like include relieving, reducing, alleviating, ameliorating or otherwise inhibiting the effects of a disease for at least a period of time. It is also to be understood that terms “treating”, “treatment” and the like do not imply that the disease, or a symptom thereof, is permanently relieved, reduced, alleviated, ameliorated or otherwise inhibited and therefore also encompasses the temporary relief, reduction, alleviation, amelioration or otherwise inhibition of the disease, or of a symptom thereof.

As used herein a “therapeutically effective amount” or “effective amount” is an amount that is non-toxic to the subject and sufficient to effect desired outcomes in a subject (i.c., achieve therapeutic efficacy). For purposes of this disclosure, a therapeutically effective amount of an inhibitor or composition is an amount that is sufficient to palliate, ameliorate, stabilise, reverse, prevent, slow or delay the progression of a disease state. A therapeutically effective amount can be administered in one or more administrations. The effective amount will vary depending upon the health and physical condition of the subject to be treated, the taxonomic group of subject, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

As used in this application, the singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an agent” includes a plurality of agents, including mixtures thereof.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Biomarker detection

Sequences for MACIR and expression products of MACIR may be accessed on known databases of genome, transcriptomc, or protcomc information, such as the GenBank or RefSeq databases maintained by the National Center for Biotechnology Information (NCBI), USA, or the UniProt database maintained by UniProt consortium. For example, a sequence for the human MACIR gene is provided by Gene ID 90355 on the GenBank database, and a sequence for the human MACIR protein is the provided by accession identifier Q96GV9 on the UniProtKB database.

Methods of detecting MACIR expression include methods that quantify the level of an RNA or protein expression product of the MACIR gene. Non-limiting examples of gene RNA expression products include messenger RNA (mRNA), microRNA (miRNA), small interfering RNA (siRNA) and circular RNA (circRNA). Methods of detecting expression products such as RNA and proteins are well known to persons skilled in the art. Exemplary nucleic acid detection methods include blotting techniques (e.g., Northern blots), probe hybridisation-based methods, nucleic acid amplification-based methods and nucleic acid sequencing. Exemplary protein detection methods include gel electrophoresis (e.g., 2D electrophoresis), immunoassays, protein activity assays and mass spectrometry.

In some embodiments, methods herein comprise detecting an RNA expression product of MACIR. In one embodiment, methods herein comprise detecting the level of MACIR mRNA.

RNA may be analysed directly without an amplification step, using nucleic acids that specifically hybridise with a target RNA. Alternatively, the RNA may be reverse- transcribed into complementary DNA (cDNA) before further amplification for analysis. Such reverse transcription may be performed alone or in combination with an amplification step. One example of a method combining reverse transcription and amplification steps is reverse transcription polymerase chain reaction (RT-PCR), which may be further modified to be quantitative, e.g., quantitative RT-PCR (qRT-PCR). Methods for reverse transcription with and without amplification are generally known in the art.

Nucleic acid amplification methods include, without limitation, polymerase chain reaction (PCR) and its variants such as in situ PCR and quantitative PCR, self-sustained sequence replication and its variants, and transcriptional amplification system and its variants (Kwoh et al., 1989), followed by the detection of the amplified molecules using techniques well known to those of skill in the art. Especially useful are those detection schemes designed for the detection of nucleic acid molecules if such molecules are present in very low numbers.

Complementary DNA may also be amplified using isothermal amplification, i.e., amplification of DNA that occurs at substantially the same temperature. A number of isothermal amplification methods are known in the art, including but not limited to transcription mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), signal mediated amplification of RNA technology (SMART), strand displacement amplification (SDA), nicking enzyme amplification reaction (NEAR), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), isothermal multiple displacement amplification (MDA), helicase-dependent amplification (HDA), single primer isothermal amplification (SPIA), and cross-primed amplification (CPA).

The DNA that is amplified can be detected with generic double-stranded DNA detection reagents or with sequence-specific probes. Any method of detecting amplified DNA is suitable for use in the present methods.

Methods for assessing RNA levels that do not require conversion of the RNA to cDNA are also known in the art and are suitable for use in the methods herein. For example, the nCounter™ Analysis system from NanoString Technologies uses a digital molecular barcoding technology for multiplex measurement of RNA levels. The RNA sample is mixed with pairs of capture and reporter probes, tailored to each RNA sequence of interest. After hybridisation and washing, probe-bound target nucleic acids are immobilised to a surface to detect the fluorescent barcodes of the reporter probes. This allows for up to 1000-plex measurement with high sensitivity and without amplification bias.

RNA can also be sequenced to determine gene expression. Sequencing methods can include but are not limited to RNA-seq. In some embodiments, RNA-seq comprises reverse transcribing at least one RNA molecule to produce at least one double-stranded complementary' DNA molecule (dseDNA). Methods known in the art for creating a dseDNA library' may be used. RNA-seq can further comprise appending sequencing adaptors to the at least one dseDNA molecule, followed by amplification, and finally sequencing. Methods of sequencing known in the art, including sequencing by synthesis, can be used. The various RNA-seq methods known in the art may be used, e.g., those described in Kukurba et al. (Kukurba K.R., Montgomery S.B. RNA sequencing and analysis. Cold Spring Harb Protoc. 2015; 2015(11):951-69). Base abundances obtained using RNA-seq methods can be measured as read counts and normalised using methods known in the art. Gene abundances can also be reported in Reads Per Million (RPM) or Transcripts Per Million (TPM).

“Next-generation” sequencing (NGS) or high-throughput sequencing may also performed for DNA or RNA detection. These sequencing techniques allow for the identification of nucleic acids present in low or high abundance in a sample, or which are otherwise not detected by more conventional hybridisation methods or a quantitative PGR method. NGS typically incorporates the addition of nucleotides followed by washing steps.

In some embodiments, methods herein comprise detecting a polypeptide expression product of MAC1R. In one embodiment, methods herein comprise detecting the level of MACIR protein.

The level of MACIR may be detected using antibody-based techniques such as enzyme- linked immunosorbent assay (ELISA), Luminex® assay or Western blotting. An antibody or antigen-binding fragment thereof that binds to MACIR may be used. The antibody may be further conjugated to a detectable label (such as a fluorescent, luminescent or enzyme label) to allow detection. Alternatively, the antibody may be detected using a secondary antibody that is conjugated to a label (such as a fluorescent, luminescent or enzyme label). In other embodiments, surface plasmon resonance (SPR) may be used to detect the interaction between MACIR and an antibody or antigenbinding fragment that recognises it, and used to quantify the amount of the polypeptide in a sample. Non-immunological methods may also be used to detect a polypeptide product of MACIR. For instance, labelled aptamers (e.g., a radiolabeled, chromophore-labeled, fluorophore-labeled, or enzyme-labeled aptamer) may be used for polypeptide binding and detection. An aptamer refers to a nucleic acid that has a specific binding affinity for a target molecule. A suitable aptamer can be identified using any known method, including but not limited to the SELEX process, and prepared or synthesised in accordance with any known method, including chemical synthesis methods and enzymatic synthesis methods. Other binding agents for detecting a polypeptide may include, e.g., small molecules, lectins, ligand-binding receptors, affybodies, ankyrins, alternative antibody scaffolds (e.g., diabodics), imprinted polymers, avimers, peptidomimetics, peptoids, peptide nucleic acids, threose nucleic acids, synthetic receptors, and modifications and fragments of these.

MACIR protein may also be quantified, via its proteotypic peptides, by known mass spectrometry techniques, non-limiting examples of which include: electrospray ionization mass spectrometry (ESI-MS), ESI-MS/MS, ESI-MS/(MS)n, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDT- TOF-MS), desorption/ionization on silicon (DIOS), secondary ion mass spectrometry (SIMS), quadrupole time-of-flight (Q-TOF), tandem time-of-flight (TOF/TOF) technology, atmospheric pressure chemical ionization mass spectrometry (APCI-MS), APCI-MS/MS, APCI-(MS)n, atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS/MS, and APPI-(MS)n, quadrupole mass spectrometry, Fourier transform mass spectrometry (FTMS), quantitative mass spectrometry, and ion trap mass spectrometry. Any of these techniques may be combined with selected reaction monitoring (SRM) to produce more targeted quantitative measurements for a polypeptide product.

Kits

Provided herein arc kits for use in the methods herein to detect MACIR expression. The kits may comprise nucleic acids, such as oligonucleotide probes or primers, for detecting an RNA expression product (e.g., an mRNA) of the MACIR gene. Alternatively or additionally, the kits may comprise antibodies or antigen-binding fragments thereof for detecting a polypeptide expression product (c.g., a protein or fragment thereof) of MACIR.

Disclosed herein is a kit for determining the likelihood of resistance to a cancer therapy in a subject with cancer, the kit comprising a reagent for detecting macrophage immunometabolism regulator (MACIR) expression in a sample obtained from the subject.

Disclosed herein is the use of a macrophage immunometabolism regulator (MACIR) detection reagent in the manufacture of a kit for determining the likelihood of resistance to a cancer therapy in a subject with cancer.

In some embodiments, the kit comprises a nucleic acid for detecting MACIR mRNA or cDNA. The nucleic acid may be, for example, an oligonucleotide probe or primer capable of hybridising to MACIR mRNA or cDNA. The design of hybridisation probes and primers are well-known in the art.

In some embodiments, the nucleic acid is an oligonucleotide probe for detecting MACIR mRNA. Oligonucleotide probes herein are single-stranded DNA or RNA molecules designed to be substantially complementary to specific target nucleic acids (e.g., RNA), such that hybridisation of the target sequence and the probes occur. This complementarity need not be perfect, so long as the probe is able to hybridise with its target sequence under suitable hybridisation conditions. For example, an oligonucleotide probe can comprise a sequence having a complementarity to a corresponding target sequence of at least about 70%, at least about 75%>, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 99%, or 100%. In general, hybridisation will be influenced by the length of the probe, the pH, the temperature, the concentration of mono- and divalent cations, the proportion of G and C nucleotides in the region of hybridisation, and the possible presence of denaturants. Such variables also influence the time required for hybridisation. The preferred conditions will therefore depend upon the particular application. Such empirical conditions, however, can be routinely determined without undue experimentation. In some embodiments, the nucleic acid in the kit is a primer for reverse transcription of MACIR RNA to eDNA and/or for amplifying the RNA or eDNA product (e.g., primers for PCR amplification or isothermal amplification).

In certain embodiments, more than one oligonucleotide probe or primer is used per target sequence. The probes or primers may target overlapping sections or separate sections of the target sequence. That is, two, three, four or more probes or primers may be used to build in a redundancy for a particular target.

In some embodiments, the kit comprises an antigen-binding molecule for detecting MACIR protein. The antigen-binding molecule may be, for example, an antibody or antigen-binding fragment thereof which binds specifically to MACIR. The antibody may be a polyclonal or monoclonal antibody. The kit may comprise a plurality of different antigen-binding molecules targeting MACIR.

The detection reagent in the kit may comprise a detectable label. Detectable labels include, for example, chromogens, fluorophores, near-infrared dyes, chemiluminescent molecules, bioluminescent molecules, colloidal metal particles (such as gold or silver particles), lanthanide ions (e.g., Eu³⁺), semiconductor nanocrystals (e.g., quantum dots), radioisotopes, epitopes, enzymes, and colored beads (such as glass or plastic beads). Detectable labels also include barcodes such as molecular- barcodes and fluorescent barcodes.

The detection reagent in the kit may comprise an affinity tag for separating MACIR mRNA or protein that is bound to the detection reagent. As used herein, the term “affinity tag” refers to a component of a multi-component complex, wherein the components of the multi-component complex specifically interact with or bind to each other. For example, an affinity tag can include biotin that can bind streptavidin. Other examples of multiple-component affinity tag complexes include ligands and their receptors; binding proteins/peptides, including maltose/maltose binding protein (MBP), calcium/calcium binding protcin/pcptidc (CBP); antigen-antibody, including epitope tags, such as c-MYC, HA, VSV-G, HSV, V5, and FLAG Tag™, and their corresponding anti-epitope antibodies; haptens, for example, dinitrophenyl and digoxigenin, and their corresponding antibodies; aptamers and their corresponding targets; fluorophorcs and anti-fluorophore antibodies; and the like.

The detection reagent may be immobilised on a solid substrate to form an array. Suitable substrates include plastics (e.g., polytetrafluoroethylene, polystyrene, acrylic polymers), ceramics, silicon, glass, ferro- or paramagnetic materials, titanium dioxide, latex, crosslinked dextrans such as Sepharose, and cellulose. Signal detection from an array may be performed using a chip or plate reader. Alternatively, when the array is made using a mixture of individually addressable kinds of labelled particles, the reaction may be detected using flow cytometry.

Kits herein may comprise reagents for generating a detectable signal upon binding of the detection reagent to its target. For example, for detection reagents containing an enzyme, the kit may contain reagents and substrates for the enzyme to generate a detectable signal. The kit may also comprise double-stranded DNA detection reagents, such as ethidium bromide, Picogreen™ (Life Technologies) and SYBR™ Green (Life Technologies), among others.

Where the detection reagents are primers, the kit may comprise an enzyme for performing the reverse transcription (e.g., a reverse transciptase) and/or DNA- dependent DNA polymerisation (e.g., a DNA polymerase). The reagents may also comprise dcoxynuclcotidc triphosphates (dNTPs), such as dcoxyadcnosinc triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxythymidine triphosphate (dTTP), deoxyuridine triphosphate (dUTP), deoxycytidine triphosphate (dCTP), deoxyinosine triphosphate (dTTP), and deoxyxanthosine triphosphate (dXTP). The enzyme and/or deoxynucleotide triphosphates are preferably present in a combination and an amount sufficient to generate a DNA copy of the RNA template and to amplify the DNA copy into a detectable amount.

Some or all of the reagents in the kit may be provided together as a mix in dried form (e.g., a powder) and arc reconstituted with a liquid prior to use. The reagents may be dried through lyophilisation or other methods known in the art. The kit may further comprise an aqueous buffer for reconstituting the dried components. Samples

Samples herein may be tissue samples, biological fluids, or cultures derived from tissue or fluid samples.

In one embodiment, the sample is a bodily fluid or a liquid biopsy. Bodily fluids include but are not limited to peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, bronchoalveolar lavage fluid, semen (including prostatic fluid), Cowper’s fluid or prc-cjaculatory fluid, female ejaculate, sweat, fecal matter, tears, cyst fluid, pleural fluid, peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates and other lavage fluids. In one embodiment, the sample is a blood, serum or plasma sample.

In some embodiments, the sample is a cancer sample. As used herein, a “cancer sample” refers to a sample derived from a cancerous cell or tissue. Cancer samples would typically contain cancer cells, circulating tumor cells (CTCs), and/or cellular material (e.g., cellular vesicles, secretions or debris) derived from cancer cells or CTCs.

Samples may be treated prior to MACIR detection. For example, cellular fractions, nucleic acid fractions or protein fractions may be extracted for analysis. Extraction of nucleic acids and proteins may be achieved with various methods known in the art. For example, RNA extraction may be achieved using protein precipitation according to standard procedures and techniques known in the art. Such methods may utilise a nucleic acid-binding column to capture nucleic acids contained within the extracellular vesicles. Once bound, the nucleic acids can then be eluted using a buffer or solution suitable to disrupt the interaction between the nucleic acids and the binding column, thereby eluting the nucleic acids. Alternatively, commercially available kits for purifying proteins and RNA may be used.

This disclosure also provides a composition comprising a sample obtained from a subject with cancer and a reagent for detecting macrophage immunometabolism regulator (MACIR) expression in the sample. The detection reagent may be as provided herein, such as a nucleic acid that hybridises to MACIR mRNA or eDNA, or antigenbinding molecule that binds to MACIR protein or a fragment thereof.

Cancers

Methods herein may be used to predict treatment outcome in subjects with any type of cancer for which combination therapies based on R-CHOP or component agents thereof are suitable. In certain preferred embodiments, the cancer is a lymphoma.

As used herein, “lymphoma” refers to a group of blood cell cancers that develop from lymphocytes. Lymphomas include, but are not limited to, Hodgkin lymphoma, and nonHodgkin lymphoma, e.g., B cell lymphoma (e.g., diffuse large B cell lymphoma (DLBCL), follicular' lymphoma, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphomas, Burkitt’s lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, primary central nervous system lymphoma and the like), T cell lymphoma (e.g., precursor T-lymphoblastic lymphoma/leukemia, peripheral T-cell lymphomas and the like) and NK cell lymphoma. In some embodiments, the cancer is a non-Hodgkin lymphoma or a B cell lymphoma. In one embodiment, the cancer is diffuse large B cell lymphoma (DLBCL).

Cancer therapies

Methods herein may be used to predict treatment outcome for various combination therapies based on R-CHOP, i.e., therapies based on two or more of rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone, or therapies incorporating two or more therapeutic agents that are equivalent to rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone.

An “equivalent” therapeutic agent refers to a drug that achieves substantially the same therapeutic effect as the reference drug, and may be substituted for the reference drug for treating a particular indication. For example, the equivalent drug may be a generic version of the reference drug, a drug with the same molecular target or the same targeted pathway as the reference drug, or a multivalent drug incorporating the reference drug.

In some embodiments, the combination therapy comprises rituximab or an equivalent therapeutic agent. The equivalent therapeutic agent may be an inhibitor of CD20, including but not limited to antibodies, antigen-binding fragments thereof, and other antigen-binding molecules capable of binding and inhibiting CD20. Equivalents of rituximab include, for example, the CD20-targeting monoclonal antibodies ofatumumab, obinutuzumab and ublituximab, the bispccific antibodies mosunctuzumab and glofitamab targeting both CD20 and CD3, the antibody-drug conjugate loncastuximab tcsirinc (Zynlonta), and antigen-binding fragments thereof which bind CD20.

In some embodiments, the combination therapy comprises one or more of the chemotherapeutic agents cyclophosphamide, doxorubicin and vincristine, or equivalent compounds.

Therapeutic agents equivalent to cyclophosphamide include but are not limited to other DNA alkylating agents, such as ifosfamide, melphalan, chlorambucil andbendamustine. Therapeutic agents equivalent to doxorubicin include but are not limited to other anthracycline compounds (e.g., daunorubicin, epirubicin, idarubicin and valrubicin), anthracenediones (e.g., mitoxantrone and pixantrone), and other topoisomerase II inhibitors (e.g., etoposide and teniposide). Therapeutic agents equivalent to vincristine include but are not limited to other vinca alkaloids (e.g., vinblastine, vinorelbine and vindesine), taxanes (e.g., paclitaxel, docetaxel, cabazitaxel), and other microtubuletargeting and anti-mitotic agents (e.g., ixabepilone and eribulin).

In some embodiments, the combination therapy comprises prednisone or an equivalent therapeutic agent. The equivalent therapeutic agent may be a glucocorticoid or corticosteroid, including but not limited to prednisolone, methylprednisolone, hydrocortisone (cortisol), dexamethasone, betamethasone, and triamcinolone. Alternatively, the equivalent therapeutic agent may be a non-corticostcroid immunosuppressant, such as azathioprine or methotrexate.

In some embodiments, the cancer therapy is a combination therapy comprising two or more of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone, and additionally comprises etoposide and/or polatuzumab vedotin. Polatuzumab vedotin is an antibody-drug conjugate consisting of an antibody specifically targeting CD79b (polatuzumab) linked to the anti-mitotic agent monomethyl auristatin E (MMAE).

In one embodiment, the cancer therapy is CHOP (i.e., a combination of cyclophosphamide, doxorubicin, vincristine and prednisone) or R-CHOP (i.e., a combination of rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone). In one embodiment, the cancer therapy is a combination of rituximab, etoposide, prednisone, vincristine, cyclophosphamide and doxorubicin (i.e., R-EPOCH). In one embodiment, the cancer therapy is a combination of polatuzumab vedotin, rituximab, cyclophosphamide, doxorubicin and prednisone (i.e., pola-R-CHP).

Methods of detection and prediction

The expression level of MACIR may be used to identify cancer patients who are not likely to respond to R-CHOP-based combination therapies, or who are likely to relapse following R-CHOP-based combination therapies. In particular, increased expression of MACIR is predictive of refractory cancers and poor response to R-CHOP-based combination therapies.

Disclosed herein is a method for predicting treatment outcome of a subject to a cancer therapy, the method comprising detecting macrophage immunometabolism regulator (MACIR) expression in a sample obtained from the subject, wherein an increased level of MACIR as compared to a reference indicates that the subject has an increased likelihood of resistance to the therapy or relapse after the therapy.

Disclosed herein is a method for detecting a refractor}' cancer in a subject suffering from a cancer, the method comprising detecting an increased level of macrophage immunometabolism regulator (MACIR) expression as compared to a reference in a sample obtained from the subject, to thereby detect the refractory cancer in the subject.

The reference may be MACIR expression level in a sample from a subject of the same species without cancer, or an average expression level in samples from a population of subjects of the same species (e.g., of varying ages, ethnic backgrounds and genders) without cancer. The reference may be MACIR expression level in a non-cancerous tissue sample from the same subject. Alternatively, the reference may be the expression level in a sample from the same subject before the suspected onset of a cancer. Reference values may be MACIR mRNA or protein levels. The reference values can be a predetermined value, and can be stored in a database and used as a reference in subsequent analyses. In one embodiment, methods herein comprise the step of comparing the level of MACIR expression in the sample to the reference.

The measured expression level of a gene may first be normalised before comparison with a reference. Normalisation is typically used to control for unwanted biological variation. In a non-limiting example, biological variation can result from some feature of the subject or the sample collection that is not relevant to the methods of the present disclosure, such as variations created by collecting samples at different times of the day and variations due to the age or gender of the subject.

Normalisation can be performed using methods known in the art. In a non-limiting example, normalisation is performed by dividing the measured expression level of MACIR by the expression level of a reference or housekeeping gene. Useful reference or housekeeping genes are genes that show a low variation in their expression level across a variety of different samples and subjects. For example, a useful reference gene will show the same expression level in samples derived from subjects who have cancer and in samples derived from subjects who do not have cancer. Such reference or housekeeping genes are typically genes which are crucial for fundamental cellular processes such as metabolism and cell structure maintenance.

As used herein, the term “increase” or “increased” with reference to a biomarker refers to a statistically significant and measurable increase in the biomarker as compared to a reference. The increase may be an increase of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%>.

As used herein, the term “decrease” or “decreased” with reference to a biomarker refers to a statistically significant and measurable decrease in the biomarker as compared to a reference. The decrease may be a decrease of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%.

In one embodiment, an increase in the level of MACIR as compared to a reference is an increase of 1 .1 fold, 1 .2 fold, 1 .3 fold, 1 .4 fold, 1 .5 fold, 1 .6 fold, 1 .7 fold, 1 .8 fold, 1 .9 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold, 16 fold, 17 fold, 18 fold, 19 fold, 20 fold, 21 fold, 22 fold, 23 fold, 24 fold, 25 fold, 26 fold, 27 fold, 28 fold, 29 fold, 30 fold, 31 fold, 32 fold, 33 fold, 34 fold, 35 fold, 36 fold, 37 fold, 38 fold, 39 fold, 40 fold, 41 fold, 42 fold, 43 fold, 44 fold, 45 fold, 46 fold, 47 fold, 48 fold, 49 fold, 50 fold, 51 fold, 52 fold, 53 fold, 54 fold, 55 fold, 56 fold, 57 fold, 58 fold, 59 fold, 60 fold, 61 fold, 62 fold, 63 fold, 64 fold, 65 fold, 66 fold, 67 fold, 68 fold, 69 fold, 70 fold, 71 fold, 72 fold, 73 fold, 74 fold, 75 fold, 76 fold, 77 fold, 78 fold, 79 fold, 80 fold, 81 fold, 82 fold, 83 fold, 84 fold, 85 fold, 86 fold, 87 fold, 88 fold, 89 fold, 90 fold, 91 fold, 92 fold, 93 fold, 94 fold, 95 fold, 96 fold, 97 fold, 98 fold, 99 fold or 100 fold increase, or anywhere in between.

Methods herein may be combined with other biomarkers or other clinical methods to provide a more comprehensive prediction of treatment outcome. Such biomarkers may include non-genetic factors such as demographic or clinical variables, non-limiting examples of which include gender, age, lifestyle (e.g., diet, frequency of physical activity, alcohol intake, tobacco use), physiological parameters (e.g., body mass index, blood pressure), family history of disease, clinical history of cancer and/or other comorbid diseases or conditions, and other diagnostic indications of cancer (e.g., levels of other biomarkers, medical imaging). Clinical methods may include imaging methods and histopathological analyses of biopsy samples.

Methods herein may be performed prior to, during or after a cancer therapy (such as R- CHOP therapy or similar combination therapies), and may be performed on multiple occasions over a period of time.

Methods of treatment

MACIR expression may guide or assist a physician in deciding a treatment path, for example, whether to administer CHOP or R-CHOP, adjust therapeutic doses or dosages for CHOP or R-CHOP therapy, or implement adjunct or alternative treatments.

Disclosed herein is a method of treating a subject suffering from a cancer, the method comprising: a) determining the likelihood of resistance of the subject to a cancer therapy by detecting an increased level of macrophage immunometabolism regulator (MACTR) expression as compared to a reference in a sample obtained from the subject, wherein an increased level of MACIR expression indicates that the subject has an increased likelihood of resistance to the cancer therapy; and b) treating the subject found to have an increased likelihood of resistance to the cancer therapy.

The cancer therapy may be a combination therapy as disclosed herein, i.e., a combination of two or more of rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone.

In some embodiments, the cancer is a lymphoma, such as a non-Hodgkin lymphoma or a B cell lymphoma. In one embodiment, the cancer is diffuse large B cell lymphoma (DLBCL).

In some embodiments, treating the subject or refractory cancer comprises administering to the subject an adjusted regimen of CHOP or R-CHOP. The adjustment may include, for example, a change in dose or dosage of one or more therapeutic agents in CHOP or R-CHOP, or a change in the schedule of CHOP or R-CHOP therapy. In other embodiments, CHOP or R-CHOP may be administered in combination with other therapeutic agents to subjects who are likely to be non-responsive to CHOP or R-CHOP therapy. For example, CHOP or R-CHOP may be administered in combination with an MACIR inhibitor.

In some embodiments, treating the subject comprises administering to the subject a therapy other than CHOP or R-CHOP. The therapy may be an equivalent combination therapy. For example, the therapy may comprise a variant of CHOP or R-CHOP where one or more components are substituted with an equivalent therapeutic agent, as provided in this disclosure. Alternatively, the therapy may comprise therapeutic agents in addition to the components of CHOP or R-CHOP, such as other therapeutic agents indicated for the cancer.

In embodiments where the cancer is a lymphoma, the therapy may be, for example, R- EPOCH (rituximab, etoposide, prednisone, vincristine, cyclophosphamide and doxorubicin) or pola-R-CHP (polatuzumab vedotin, rituximab, cyclophosphamide, doxorubicin and prednisone). Alternatively, one or more components of CHOP or R- CHOP may be substituted with an equivalent therapeutic agent, as provided in this disclosure.

The therapy may comprise any drug, pharmaceutical composition, combination therapy, or clinical intervention effective to treat the cancer of the subject. A non-exhaustive list of anti-cancer therapies include surgery, chemotherapy, radiation therapy, photodynamic therapy, radionuclides, metal-containing compounds (c.g., platinum compounds), arsenic compounds, cytotoxic antibiotics, anti-metabolites, anti-mitotic agents, anti-angiogenic agents, alkylating agents, DNA topoisomerase inhibitors, signal transduction pathway inhibitors, cell cycle signalling inhibitors, inhibitors of LDH-A, inhibitors of fatty acid biosynthesis, HDAC inhibitors, protcasomc inhibitors, pro- apoptotic compounds, taxanes, nucleoside analogues, plant alkaloids, toxins, cytokines, hormones and hormonal analogues, proteolysis targeting chimeras (PROTACs), gene therapy (e.g., antisense polynucleotides, ribozymes, RNA interference molecules, triple helix polynucleotides, gene editing complexes, etc.), cell therapy (e.g., CAR-T therapy), immunotherapy (e.g., monoclonal antibodies, antibody-drug conjugates, bispecific antibodies including bispecific T-cell engagers), radioimmunotherapy and synthetic derivatives thereof. The anti-cancer therapy may also be combinations of the aforementioned therapies. The anti-cancer therapy may be administered in one or more doses, procedures or administrations. A skilled person can select a suitable therapy based on, for example, the type of cancer to be treated, the assessment of the clinical history and clinical condition of the subject, and prevailing medical guidance.

In some embodiments, treating the subject comprises administering to the subject an inhibitor of MACIR. The MACIR inhibitor may be administered in combination with CHOP, R-CHOP or an equivalent therapy.

Disclosed herein is a method of sensitizing a subject suffering from cancer to a cancer therapy, the method comprising administering an inhibitor of macrophage immunometabolism regulator (MACIR) to the subject. The MACIR inhibitor may be administered in combination with CHOP, R-CHOP or an equivalent therapy.

The cancer therapy may be a combination therapy comprising two or more of rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone. In some embodiments, the cancer therapy is CHOP or R-CHOP therapy.

Disclosed herein is a pharmaceutical composition comprising an inhibitor of macrophage immunometabolism regulator (MACIR).

Pharmaceutical compositions herein can further comprise a pharmaceutically acceptable carrier and/or a pharmaceutically acceptable salt. Suitable carriers include isotonic saline solutions, for example phosphate-buffered saline. Suitable diluents and excipients also include, for example, water, saline, dextrose, glycerol, and the like, and combinations thereof. In addition, if desired, substances such as wetting, solubilising or emulsifying agents, stabilising or pH buffering agents, viscosity controlling agents. preservatives, antioxidants, emollients, odour controlling or fragrance compounds may also be present.

The term “pharmaceutically acceptable carrier” refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which can be administered without undue toxicity. Suitable carriers can be large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, and amino acid copolymers.

Pharmaceutically acceptable salts can also be present, e.g., mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.

Pharmaceutical compositions herein may be administered locally or systemically, such as via an oral, intra-adiposal, intra-arterial, intra-articular, intracranial, intradermal, intra-lesional, intramuscular, intranasal, intraocular, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrarectal, intrathecal, intratracheal, intra-tumoral, intra- umbilical, intravaginal, intravenous, intravesicular, intravitreal, liposomal, local, mucosal, parenteral, subconjunctival, cutaneous, subcutaneous, sublingual, topical, transbuccal or transdermal route, or combinations thereof. The pharmaceutical composition may be administered via a catheter, via a lavage, via continuous infusion, via infusion, via inhalation, via injection, via local deliver}', via an implant, via a dressing, or via any combination thereof.

Depending on the intended route of administration, the pharmaceutical compositions herein may, for example, take the form of tablets, caplets, capsules, hard capsules, soft capsules, gelatin capsules, cachets, troches, lozenges, dispersions, suppositories, ointments, creams, gels, hydrogels, foams, poultices, pastes, powders, dressings, plasters, solutions, patches, aerosols, nasal sprays, inhalers, salves, suspensions, aqueous liquid suspensions, non-aqueous liquid suspensions, oil-in-water emulsions, water-in-oil emulsions, solutions, sterile solids, crystalline solids, amorphous solids, solids for reconstitution, delayed release formulations, sustained release formulations, or combinations thereof. A skilled person may select an appropriate formulation and dosage depending on, for example, the type of cancer and the desired route of administration.

Disclosed herein is a method of treating a cancer in a subject, the method comprising administering an inhibitor of macrophage immunometabolism regulator (MACIR) to the subject.

Disclosed herein is an inhibitor of macrophage immunometabolism regulator (MACIR), for use in treating a cancer in a subject.

Disclosed herein the use of an inhibitor of macrophage immunometabolism regulator (MACIR) in the manufacture of a medicament for treating a cancer in a subject.

In some embodiments, the subject is one who has an increased level of macrophage immunometabolism regulator (MACIR) expression in a sample as compared to a reference.

The MACIR inhibitor may be an inhibitory nucleic acid molecule, a site-specific nuclease (SSN) system, a peptide, a polypeptide, a proteolysis targeting chimera (PROTAC), or a small molecule capable of disrupting expression or activity of MACIR. Methods herein may comprise administering a vector encoding the MACIR inhibitor to the subject, for inhibitors that comprise a nucleic acid, SSN system, peptide or polypeptide.

In one embodiment, the MACIR inhibitor is a site-specific nuclease (SSN) system capable of modifying a gene encoding MACIR, or an RNA product of the gene, to reduce MACIR expression. SSN systems generally comprise a sequence-specific nuclease that recognises a target nucleic acid sequence. The sequence-specific nuclease may work in conjunction with one or more guide nucleic acids that target the nuclease to the target nucleic acid sequence. The sequence- specific nuclease may be a wild-type, engineered or chimeric nuclease. The SSN system may further comprise a donor template nucleic acid molecule for introducing specific sequence modifications (such as an insertion, deletion or substitution of one or more nucleotides) at or adjacent to the target nucleic acid sequence. SSNs capable of being engineered to generate target nucleic acid scqucncc-spccific modifications include zine-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced palindromic repeats/CRISPR-associated nuclease (CRISPR/Cas) systems. In some embodiments, the MACTR inhibitor is a CRTSPR/Cas SSN system, such as a CRISPR/Cas9 system, CRISPR/Casl2 system or a CRISPR/Casl3 system.

In some embodiments, the MACIR inhibitor is an inhibitory nucleic acid molecule capable of hybridising with a MACIR mRNA molecule, and the inhibitory nucleic acid molecule is selected from an antisense nucleic acid molecule, a small interfering RNA (siRNA), and a short hairpin RNA (shRNA).

In some embodiments, the MACIR inhibitor is an antigen-binding molecule that specifically binds to MACIR and inhibits MACIR activity. Suitable antigen-binding molecules include but are not limited to aptamers, monobodies, antibodies, anticalins, Kunitz domains, avimers, knottins, fynomers, atrimers, DARPins, affibodies, nanobodies (i.e., single-domain antibodies (sdAbs)), affilins, armadillo repeat proteins (ArmRPs), OBodies.

Methods herein may comprise administering the MACIR inhibitor in combination with CHOP or R-CHOP therapy. The MACIR inhibitor may be administered simultaneously or sequentially with the CHOP or R-CHOP therapy.

The terms “a combination” and “in combination with” are not intended to imply that the therapy or the therapeutic agents must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope described herein. The therapeutic agents in the combination can be administered concurrently with, prior to, or subsequent to, one or more other additional therapies or therapeutic agents. The therapeutic agents or therapeutic protocol can be administered in any order. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent. It will further be appreciated that the additional therapeutic agent utilised in the combination may be administered together or separately in different compositions. In general, it is expected that therapeutic agents utilised in combination be utilised at levels that do not exceed the levels at which they are utilised individually. In some embodiments, the levels utilised in combination are lower than those utilised individually.

As used herein, “simultaneously” is used to mean that two or more therapeutic agents are administered concurrently or in a substantially concurrent manner (such as immedi a tely after each other). “Sequentially” refers to the administration of two or more therapeutic agents at different times. In sequential administration, the MAC1R inhibitor may be administered before or after the CHOP or R-CHOP therapy. A time delay may exist between sequential administration of the therapeutic agents. The time interval may be any pre-determined time interval, but is preferably one that provides for a cooperative effect of the MACIR inhibitor and the CHOP or R-CHOP therapy. The therapeutic agents may be administered simultaneously or sequentially via the same route or via different routes.

Those skilled in the art will appreciate that the invention described herein in susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

EXAMPLES

The expression of MACIR has been identified to be robustly associated with poor survival in multiple R-CHOP treated DLBCL datasets. This gene has only previously been studied in the setting of rheumatoid arthritis, where it was determined to be a negative regulator of tissue damage. An unbiased overall survival screen in R-CHOP- treated DLBCL cohorts was conducted in which genes were investigated independently for their association with patient survival (Figure 1A-C). In both the discovery and validation screens (11 cohorts in total), overexpression of MACIR was consistently associated with poor survival (Figure ID). This trend was further confirmed in two chemotherapy-only validation cohorts (Lenz and Hummel, Figure 2A) and observed in all cohorts after orthogonal survival analyses (Figures 2B-F). A multivariate analysis correcting for multiple prognostically relevant clinicopathological features confirmed MACIR as an independent prognosticator in majority of the cohorts (Figure 2G). A commercially available rabbit polyclonal antibody targeting the first 100 amino acids of human MACIR (Abeam, abl50906) was first validated for the appropriate detection via Western blot and multiplexed fluorescent immunohistochemistry (mflHC). HEK293T cells were treated with siRNA targeting MACIR (sense: 5’- CUUCACGACUGGCGAGGAAdTdT-3’ [SEQ ID NO: 1], antisense: 5’- UUCCUCGCCAGUCGUGAAGdTdT-3’ [SEQ ID NO: 2J) and the knockdown was validated through Western blot (Figure 3A). Additionally, MACIR knockout cell lines were generated with CRISPR (gRNA: 5’-

CCTGACACGGTCCTCCGCATGTTTTAGAGCTATGCT-3’ [SEQ ID NO: 3]) and the knockout was validated through Western blot and mflHC with the same antibody (Figures 3B-D). Interrogating patient samples with this antibody, it was found that MACIR expression patterns was present but variable across patient samples (Figure 3E- F). As expected, MACIR detected at the protein level was able to stratify DLBCL patients for survival (Figures 4A and B), the association with poor survival holding true after a multivariate analysis, confirming MACIR as an independent prognosticator (Figure 4A-C).

The prognostic significance of MACIR was also compared to known DLBCL prognostic markers MYC, BCL2 and BCL6. As shown in Figure 5 A, MACIR is more consistently and more strongly associated with poor survival when highly expressed than MYC, BCL2 or BCL6. Additionally, receiver operating characteristic (ROC) analysis indicated that MACIR accurately classifies patient survival in the NUH cohort, outperforming the cell-of-origin classification (Figure 5B).

In summary, MACIR is potentially a multifactorial biomarker that can predict for the likelihood of R-CHOP resistance, itself being a potential therapeutic target to improve the outcome of DLBCL.

Claims

1. A method for determining the likelihood of resistance to a cancer therapy in a subject suffering from a cancer, the method comprising detecting an increased level of macrophage immunometabolism regulator (MACIR) expression as compared to a reference in a sample obtained from the subject, to thereby determine that the subject has an increased likelihood of resistance to the cancer therapy.

2. The method of claim 1, wherein the sample is a cancer sample.

3. The method of claim 1 or 2, wherein the method comprises detecting an increased level of MACIR mRNA or protein expression.

4. The method of any one of claims 1 to 3, wherein the method comprises detecting MACIR protein with an antibody or antigen- binding fragment thereof.

5. The method of any one of claims 1 to 4, wherein the subject is suffering from a lymphoma.

6. The method of claim 5, wherein the lymphoma is a non-Hodgkin lymphoma or B cell lymphoma.

7. The method of claim 6, wherein the lymphoma is diffuse large B cell lymphoma (DLBCL).

8. The method of any one of claims 1 to 7, wherein the cancer therapy is a combination therapy comprising two or more of rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone.

9. The method of claim 8, wherein the cancer therapy is CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) or R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone).

10. A method of treating a subject suffering from a cancer, the method comprising: a) determining the likelihood of resistance of the subject to a cancer therapy by detecting an increased level of macrophage immunometabolism regulator (MACIR) expression as compared to a reference in a sample obtained from the subject, wherein an increased level of MACIR expression indicates that the subject has an increased likelihood of resistance to the cancer therapy; and b) treating the subject found to have an increased likelihood of resistance to the cancer therapy.

11. A method for detecting a refractory cancer in a subject suffering from a cancer, the method comprising detecting an increased level of macrophage immunometabolism regulator (MACIR) expression as compared to a reference in a sample obtained from the subject, to thereby detect the refractory cancer in the subject.

12. A method of treating a refractory cancer in a subject, the method comprising a) detecting an increased level of macrophage immunometabolism regulator (MACIR) expression as compared to a reference in a sample obtained from the subject, to thereby detect the refractory cancer in the subject; and b) treating the refractory cancer that is detected in the subject.

13. A method for stratifying a subject as a likely responder or non-responder to a cancer therapy, the method comprising detecting the level of macrophage immunometabolism regulator (MACIR) expression in a sample obtained from the subject, wherein an increased level of MACIR expression indicates that the subject is likely to be non-responsive to the therapy.

14. A method for predicting treatment outcome of a subject to a cancer therapy, the method comprising detecting macrophage immunometabolism regulator (MACIR) expression in a cancer sample obtained from the subject, wherein an increased level of MACIR expression as compared to a reference indicates that the subject is likely to be non-responsive to the therapy or to relapse after the therapy.

15. A method of treating a cancer in a subject, the method comprising administering an inhibitor of macrophage immunometabolism regulator (MACIR) to the subject.

16. A method of sensitizing a subject suffering from cancer to a cancer therapy, the method comprising administering an inhibitor of macrophage immunometabolism regulator (MACIR) to the subject.

17. The method of claim 16, wherein the cancer therapy is a combination therapy comprising two or more of rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone.

18. The method of any one of claims 15 to 17, wherein the subject is one who has increased level of macrophage immunometabolism regulator (MACIR) expression in a sample as compared to a reference.