WO2023042209A2

WO2023042209A2 - Treatment of myeloid malignancies

Info

Publication number: WO2023042209A2
Application number: PCT/IL2022/051005
Authority: WO
Inventors: Liran Shlush; Nathali KAUSHANSKY; Naama ZIONI
Original assignee: Yeda Research And Development Co. Ltd.
Priority date: 2021-09-20
Filing date: 2022-09-20
Publication date: 2023-03-23
Also published as: WO2023042209A3

Abstract

A method of treating a myeloid malignancy in a subject comprising a mutation in DNMT3A in a genome thereof is disclosed. The method comprises: (a) analyzing in a sample of the subject for the presence of a DNMT3A mutation in the genome; and (b) administering to the subject interleukin-6 (IL-6), when the DNMT3A mutation is indicated in the genome.

Description

TREATMENT OF MYELOID MALIGNANCIES

RELATED APPLICATION/S

This application claims the benefit of priority from US Application No. 63/245,938, filed 20 September 2021, which is hereby incorporated by reference in its entirety.

SEQUENCE LISTING STATEMENT

The file, entitled 93437 Sequence Listing. xml, created on 18 September 2022, comprising 6,573 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to methods of treating myeloid malignancies and more particularly to those malignancies harboring a DNMT3A mutation.

Age related accumulation of adipocytes among humans is ubiquitous. At birth, the bone marrow (BM) contains functionally active hematopoietic tissue, known as red BM. With aging, there is a shift from red marrow to adipocyte-enriched yellow bone marrow that begins in the distal parts of the bones and expands proximally with high variability among different individuals. The initial step of adipogenesis is an enhanced lineage commitment of mesenchymal stem cells (MSCs) into pre-adipocytes followed by the expansion of preadipocytes into mature adipocytes, mainly in the cavities of trabecular bones.

BM adipocytes are located in the bone marrow cavity and accounts for 70% of adult bone marrow volume. They also accounts for approximately 10% of total fat in healthy adults above the age of 25 years Recent work has established that BMF plays an important role in energy storage, endocrine function, bone metabolism, and regulation of the growth and metastasis of tumors. Currently, fatty bone marrow is thought to be correlated with osteoporosis, aging, type 1 diabetes, Cushing’s disease, estrogen deficiency, anorexia nervosa, and bone metastasis in prostate and breast cancers (Wang et al., Frontiers in Endocrinology, 28 November 2018 doi: 10.3389/fendo.2018.00694 and US Patent Application No. 20160185851).

BM adipocytes are different from adipocytes in other parts of the body. Gene expression analysis of BM adipocytes suggested that they have a distinct immune regulatory properties and high expression of pro-inflammatory cytokines (ILIA, IL1B, IL6, IL8, IL15, IL18). Furthermore, BM adipocytes secrete IL6, IL8 and TNFa. As normal hematopoiesis and HSPCs are profoundly dependent on the interactions with the microenvironment to maintain selfrenewal capacity and normal differentiation6 it has been speculated that the inflammatory signals from fatty bone marrow (FBM) could alter hematopoiesis.

Background art includes Trikha et al., Clin Cancer Res. 2003 Oct; 9(13): 4653-4665; and US Patent Application No. 20160185851.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present invention there is provided a method of treating or preventing a myeloid malignancy in a subject comprising a mutation in DNA methyltransferase 3 alpha (DNMT3A) in a genome thereof comprising:

(a) analyzing in a sample of the subject for the presence of a DNMT3A mutation in the genome; and

(b) administering to the subject a therapeutically effective amount of an agent that down-regulates an amount and/or activity of interleukin-6 (IL-6), when the DNMT3A mutation is indicated in the genome, thereby treating or preventing the myeloid malignancy.

According to an aspect of some embodiments of the present invention there is provided a composition comprising an agent that down-regulates an amount and/or activity of IL-6 for the treatment or prevention of a myeloid malignancy in a subject, wherein the subject is selected as comprising a DNMT3A mutation in a genome thereof.

According to an aspect of some embodiments of the present invention there is provided a method of treating and/or preventing a disease associated with a fatty bone marrow in a subject comprising:

(a) analyzing cells of a bone marrow sample for the presence of adipocytes; and

(b) administering to the subject a therapeutically effective amount of an agent that down-regulates an amount and/or activity of interleukin-6 (IL-6), upon confirmation of an increased number of adipocytes as compared to an age-matched sample from a healthy subject, thereby treating and/or preventing the disease associated with a fatty bone marrow.

According to some embodiments of the invention, the DNMT3A mutation is a point mutation a deletion, a frameshift mutation, a nonsense mutation and a missense mutation.

According to some embodiments of the invention, the DNMT3A mutation is R882H.

According to some embodiments of the invention, the myeloid malignancy is selected from the group consisting of acute myeloid leukemia (AML), primary myelofibrosis, Hypereosinophilic Syndrome (HES), myelodysplastic syndrome (MDS), acute promyelocytic leukemia (APL), chronic myelomonocytic leukemia (CMML), chronic neutrophilic leukemia (CNL), acute undifferentiated leukemia (AUL), anaplastic large-cell lymphoma (ALCL), prolymphocytic leukemia (PML), juvenile myelomonocyctic leukemia (JMML), adult T-cell leukemia AML with trilineage myelodysplasia (AML/TMDS), mixed lineage leukemia (MLL), myeloproliferative disorders (MPD), chronic myeloid leukemia (CML)and myeloid (granulocytic) sarcoma, Systemic mastocytosis, mast cell neoplasm, clonal cytopenia of indetermined significance, clonal hematopoiesis, follicular lymphoma, Blastic plasmacytoid dendritic cell neoplasm and chronic neutrophilic leukemia.

According to some embodiments of the invention, the myeloid malignancy is selected from the group consisting of AML, MDS, CMML and primary myelofibrosis.

According to some embodiments of the invention, the myeloid malignancy is AML.

According to some embodiments of the invention, the sample comprises peripheral blood cells and/or bone marrow cells.

According to some embodiments of the invention, the analyzing is effected at the protein level.

According to some embodiments of the invention, the analyzing is effected at the nucleic acid level.

According to some embodiments of the invention, the disease is a metabolic disease. According to some embodiments of the invention, the subject is above 50 years old. According to some embodiments of the invention, the subject is prediabetic.

According to some embodiments of the invention, the disease is selected from the group consisting of osteoporosis, type 1 diabetes, Cushing’s disease, estrogen deficiency, and anorexia nervosa.

According to some embodiments of the invention, the analyzing is effected by MRI.

According to some embodiments of the invention, the agent is an antibody.

According to some embodiments of the invention, the agent is a polynucleotide agent that hybridizes to a nucleic acid encoding IL-6.

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

In the drawings:

FIGs. 1A-F: Models of BM adipogenesis in vivo in NSG mice: H&E staining of NSG mice tibia/femur. NSG mice were irradiated with 225 rad (X ray). A. Non-Irradiated control normal bone marrow (NBM) (n=5). B. One day following irradiation (n=5) or C. seven days (n=5) following Irradiation mice were sacrificed. Shown are the H&E staining of tibial of one experiment out of five independent experiments. D. H&E staining of NSG (n=5) mice that were administrated intraperitoneally for seven days with PPARyi (30mg/kg) before and seven days after the Irradiation. E. Adipocytes quantification by LipidTOX™ Deep Red Neutral Lipid Stain in ImageStream X Mark II Luminex imaging Flow Cytometer. * p<0.05, **p<0.005. Each dot represents a mouse. All comparisons were performed using a two-tailed, non-paired, nonparametric Wilcoxon rank sum test with 95% confidence interval and FDR correction for multiple hypothesis testing. F. Representative stacked whole-mount immunofluorescence staining of epiphyseal-metaphyseal BM femur derived from NBM, FBM and FBM & PPARyi treated NSG mice. Adipocytes are depicted by FABP4 expression and by distinctive unilocular morphology in the DIC (differential interference contrast) channel. DAPI in blue. Adipocytes are additionally marked by yellow dots. Scale Bar: 200pm; n=4 775pm x 775pm x 50pm stacked images from 2 mice and 2 bones (femur, tibia) each group.

FIGs. 2A-H: Increased engraftment of human DNMT3A mutated pre-leukemic cells under FBM conditions. A. variant allele frequency (VAF) analysis of DNMT3A and NPMlc mutation of the engrafted pre-leukemic cells by amplicon sequencing in FBM NSG mice. NSG mice were Irradiated with 225 rad, and after a week injected intra femur (IF) with CD3 depleted 1X10^A6 AML primary human cells (sample #160005) (50% /I AA 7'3 ^{RSS21 1}, 50% NPMlc). Eight weeks later mice were sacrificed, and BM was flashed from tibias and femurs and sequenced. All engrafted cells were pre-leukemic namely carrying only the DAA77'3 ^RSS211 mutation without NMPlc mutation. B. Engraftment of human pre- leukemic cells in normal bone marrow (NBM) (n=17), fatty bone marrow (FBM) (n=20) and in Irradiated NSG mice (n=10) treated with PPARyi (FBM+PPARyi). Eight weeks following 1X10^A6 AML primary human cells transplantation, BM was flashed from tibia/femur and expression of human CD45+ was measured by FACs. C. Engraftment of preleukemic cells in NBM (n=5) and in castrated (CAS) NSG mice (n=5). One month following castration, 1X10^A6 AML primary human cells were transplanted. Eight weeks following 1X10^A6 AML primary human cells transplantation, BM was flashed from tibia/femur and expression of human CD45+ was measured by FACs. * p<0.05, **p<0.005. Each dot represents a mouse. All comparisons were performed using a two-tailed, non-pared, nonparametric Wilcoxon rank sum test with 95% confidence interval and FDR multiple hypothesis correction. D-F. The same analysis was carried out for another human sample as described in A-C. (Sample #141464 CD34+ enriched) (VAF 69% of the DNMT3A^R882R in the primary sample). E. Engraftment of human pre- leukemic cells in NBM (n=8 mice), FBM (n=5 mice). F. Engraftment of preleukemic cells in NBM (n=4 mice) and in castrated (CAS) NSG mice (n=4). G. Level of chimerism in NBM (n=5 mice) or FBM NSG mice (n=5) transplanted with 50X10^A5 CD34+ WT cord blood cells, h. Transplantation of CD34+ from mobilized peripheral blood auto transplant bags to FBM and NBM NSG mice. 1.2xlO^A5 cells CD34+ from mobilized peripheral blood auto transplant bag (#141519) that have been sequenced and identified w/o ARCH mutation were transplanted intra femur to NBM (n=5 mice) and FBM NSG mice (n=5). Eight weeks following cells transplantation, mice were sacrificed, BM was flashed from tibia/femur and expression of human CD45+ was measured by FACs. Human engraftment was assessed according to presence of >0.1% human CD45+ cells. Each dot represents a mouse. All comparisons were performed using a two-tailed, non-paired, nonparametric Wilcoxon rank sum test with 95% confidence interval with FDR multiple hypothesis correction.

FIGs. 3A-E: Engraftment of DNMT3A^Mut derived BM cells in NSG mice. A. FACs analysis of young-two-month-old 6X10^A6 DNMT3A^MM (CD45.2) (red) or DNMT3A^W1 (CD45.2) (blue) BM derived cells transplanted to normal bone marrow (NBM) NSG mice (DNMT3A^Mllt to n=23 NSG mice, DNMT3A^W1 to n=24 NSG mice), and to fatty bone marrow (FBM) (DNMT3A^Mut to n=24 NSG mice, DNMT3A^W1 to n=17 NSG mice) and to Irradiated NSG mice (CD45.1) treated with PPARyi (FBM+ PPARyi) (J NMT3A^Mut to n=6 NSG mice, DNMT3A^W1 to n=4 NSG mice). Eight weeks following transplantation, BM was flashed from tibia/femur and expression of mCD45.2 was measured by FACs. Engraftment was assessed according to presence of >0.1% mCD45.2 cells. B. Self-renewal of DNMT3A^Mllt derived BM cells in FBM NSG mice. Primary transplantation of DNMT3A^{Mllt or} DAA// ^W7was performed as detailed in A. For the primary transplantation BM derived cells were transplanted to NBM mice (DNMT3A^Mut to n=4 NSG DNMT3A^W1 to n=5 NSG mice) and to FBM mice (n=4 for DNMT3A^Mllt , n=4 for DNM13A^W ). After eight weeks cells were harvested and a secondary transplantation was performed to FBM NSG mice. C. FACs analysis of one-year-old 6X10^A6 DNMT3A^Mut - (CD45.2) (red) or DNMT3A^W1 (CD45.2) (blue) BM derived cells transplanted to NBM mice (DNMT3A^Mut to n=20 NSG mice, DNMT3A^W1 to n=13 NSG mice), to FBM mice (DNMT3A^Mut to n=18 NSG mice, DNMT3A^W1 to n=12 NSG mice) and to Irradiated NSG mice (CD45.1) treated with PPARyi (FBM+ PPARyi) (DNMT3A^Mut to n=10 NSG mice, DNM73A^WT to n=8 NSG mice) performed as detailed in a. d-e. Primary and secondary transplantation of middle-aged DNMT3A^Mllt (D) (in primary: DNMT3A^Mllt derived BM was transplanted to NBM mice (n=10) and to FBM mice (n=8) and to Irradiated NSG mice treated with PPARyi (FBM+ PPARyi) (n=8). For the secondary transplantation: DNMT3A^Mut derived BM cells from NBM, FBM and FBM+ PPARyi were transplanted to FBM NSG mice (n=10, n=8, n=5 respectively) DNMT3A^W1 (E) in primary: DNMT3A^W1 derived BM cells were transplanted to NBM (n=10) and to FBM mice (n=7) and to Irradiated NSG mice treated with PPARyi (FBM+ PPARyi ) (n=8). In secondary: DNMT3A^W1 derived BM cells from NBM, FBM and FBM+ PPARyi were transplanted to FBM NSG mice (n=6, n=6, n=5 respectively). * p<0.05, **p<0.005, ***p<0.0005, ****p<0.00005. Each dot represents a mouse. All comparisons were performed using a two-tailed, non-pared, nonparametric Wilcoxon rank sum test with 95% confidence interval with FDR multiple hypothesis, n.s - not significant.

FIGs. 4A-G: DNMTSA ¹ cells exposed to FBM maintain an HSC pool characterized by an inflammatory phenotype. A. cells from DNMT3A^mut and DNMT3A^^ were injected to mice with fatty bone marrow (FBM) and normal bone marrow (NBM). Three days after injection lin- Scal+KIT+ (LSK) cells were isolated from mice bone marrow (BM) and underwent single cell RNA-Seq analysis. MetaCell algorithm was used to assign different single cells to metacells with unique gene programs and cell types³⁸. Gold, hematopoietic stem cells (HSCs); darkgreen, common myeloid progenitors (CMP); lightblue, common lymphoid progenitors, (CLP); cyan, dendritic progenitors (DcP); grey, multipotent progenitors (MPP); darkolivegreen, monocyte progenitors (MonoP); pink, megakaryocyte progenitors (MegK); red, erythroid progenitors (Ery); grey4 (Unknown). Conditions: normal bone marrow (NBM); wild type (wt); fatty bone marrow (FBM); naive cells are cells extracted directly from BM of respective mice without transplantation, ere is the ere control. B. While all injected cells show a marked reduction in HSCs after transplantation, DNMT3A^mut cells exposed to FBM maintain their HSC pool which is significantly higher that all other condition (which were transplanted). Exact fisher test was used ****p<0.0005 to compare proportions of HSCs between the groups. C. Ranked GSEA analysis on differentially expressed genes between DNMT3A^mut cells exposed to FBM cluster and other clusters exposed significant enrichment of inflammatory pathways one of them was the IL-6 JAK STAT3 response geneset. An expression score for each single cell was calculated based on the expression of each of the genes in the IL-6 gene set. All comparisons were performed using a two-tailed, non-pared, nonparametric Wilcoxon rank sum test with 95% confidence interval with FDR multiple hypothesis. * p<0.05, **p<0.005, ***p<0.0005, ****p<0.0005. D. Multiplex cytokines assay (FirePlex-96 Key Cytokines (Mouse) Immunoassay Panel (ab235656)) of 17 common cytokines analyzed by FACS based multiplex method of serum from NBM, FBM and following PPARyi administration to NSG mice, without any cell’s transplantation. E. Multiplex cytokines assay (FirePlex-96 Key Cytokines (Mouse) Immunoassay Panel (ab235656)) of 17 common cytokines analyzed by FACS based multiplex method of BM from NBM, FBM and following PPARyi administration to NSG mice, without any cell’s transplantation. F-G. FACS based multiplex method of NBM, FBM and following PPARyi administration to NSG mice transplanted with one-year-old DNMT3A^Mllt or DNMT3A^W1 BM derived cells. Each bar represents 4 to 5 mice. * p<0.05, **p<0.005, ***p<0.0005, ****p<0.00005. Analyzed by two- way ANOVA test - Sidaks multiple comparison test.

FIGs. 5A-D: Selective advantage to DNMT3A^Mllt BM derived cells under methylcellulose colony assay. A. Number of colonies in methylcellulose (MethoCult M3334) colony-forming- unit assays of DNMT3A^{Mut an}d DNMT3A^W1 BM derived cells. Mean values ± s.d. are shown, n = 3 biologically independent experiments. B. Representative photograph of the methylcellulose plating from a. All comparisons were performed using a two-tailed, non-paired, nonparametric Wilcoxon rank sum test with 95% confidence interval and FDR multiple hypothesis correction. C. Significant engraftment decrease of DNMT3A^Mut BM derived cells in NSG mice following administration of neutralizing IL-6 Ab. 6X10^A6 DNMT3A^Mllt BM derived cells were transplanted to normal bone marrow (NBM) (n=13), and fatty bone marrow (FBM) (n=l l) and FBM NSG mice (n=7) treated with neutralizing IL-6 Ab (Clone: MP5-20F3, Ultra- LEAF™ Purified anti-mouse IL-6 Antibody, BioLegend 504512). Neutralizing IL-6 Ab (50 pg /mouse) was administrated to NSG mice intraperitoneal one day before cells transplantation and during ten days after transplantation. D. Secondary transplantation of cells from A to FBM NSG mice (n=8, n=l 1, n=7 respectively). DNMT3A^Mllt BM derived cells do not perform any selfrenewal following treatment with neutralizing IL-6 Ab. * p<0.05,**p<0.005, ***p<0.0005, ****p<0.00005. Each dot represents a mouse. All comparisons were performed using a two- tailed, non-paired, nonparametric Wilcoxon rank sum test with 95% confidence interval and FDR multiple hypothesis correction. FIGs. 6A-C. FIG. 6A. Schematic presentation of different models used in this study. FIG. 6B Multilineage engraftment of AML patient-derived. FACs analysis CD3 depleted 1X10^A6 AML primary human cells (sample #160005) (50% hDNMT3A^R&&211 , 50% NPMlc). Eight weeks later mice were sacrificed, and BM was flashed from tibias and femurs. A multi-lineage engraftment is defined when a subpopulation of B cell progenitors (CD33-CD3- cells expressing CD 19+) can be identified. C. DNMT3A^Mat cells derived from one-year-old mice injected into FBM had the most significant growth advantage with a tenfold increase in comparison to NBM and PPARyi controls.

FIGs. 7A-F: Engraftment of DNMT3A^hapl° derived BM cells in FBM NSG mice. A. FACs analysis of young-two-month-old 6X10^A6 DNMT3A^hapl° (CD45.2) BM derived cells transplanted to normal bone marrow (NBM) (n=8) and fatty bone marrow (FBM) (NSG mice are CD45.1) (n=7). Eight weeks cells following transplantation, BM was flashed from tibia/femur and expression of mCD45.2 was measured by FACs. Engraftment was assessed according to presence of >0.1% mCD45.2 cells. B. Self-renewal of DNMT3A^hapl° derived BM cells in FBM NSG mice. Primary transplantation of DNMT3A^hapl° was performed as detailed in a. Then, a secondary transplantation was performed to FBM NSG mice (n=6, n=7 respectively). C. FACs analysis of one-year-old DNMT3A^hapl° , 6X10^A6 BM derived cells transplanted to control (n=15), to a week following Irradiation (n=14) and to Irradiated NSG mice (CD45.1) treated with PPARyi (n=6) performed as detailed in a. D. Primary transplantation of one-year DNMT3A^hapl° BM derived cells to NBM mice (n=l l), and to FBM mice (n=l 1) and to Irradiated NSG mice treated with PPARyi (FBM+ PPARyi ) (n=6). E. Secondary transplantation of cells from d. to FBM NSG mice (n=8, n=9, n=6 respectively). F. Differences (FBM-NBM) between engraftment of middle-aged DNMT3A^Mllt~ and DNMT3A^hapl° BM derived cells when transplanted to FBM. * p<0.05, ****p<0.00005. Each dot represents a mouse. All comparisons were performed using a two-tailed, non-paired, nonparametric Wilcoxon rank sum test with 95% confidence interval and FDR for multiple hypothesis correction, n.s - not significant.

FIGs. 8A-B: Engraftment analysis of SRSF2^Mllt or control S/ SF2 ^WT BM derived cells in FBM. A. FACs analysis of young-two-month-old 6X10^A6 SRSF2^Mllt (purple) or control SRSF2^WT (pink) (CD45.2) BM derived cells transplanted to normal bone marrow mice (NBM) (SRSF2^W1 to n=15 NSG mice, SRSF2^Mllt to n=9 NSG mice) and to fatty bone marrow (FBM) NSG mice (CD45.1) (SRSF2^WT to n=12 NSG mice, SRSF2^Mut to n=13 NSG mice). Eight weeks following transplantation, BM was flashed from tibia/femur and expression of mCD45.2 was measured by FACs. Engraftment was assessed according to presence of >0.1% mCD45.2 cells. B. Primary transplantation of middle-aged SRSF2^Mllt (purple) (NBM, n= 4; FBM, n=5) or control SRSF2^WT ⁽pink) (NBM, n=9; FBM, n=9) was performed as detailed in A. Then, a secondary transplantation of middle-aged SRSF2^Mut (purple) BM derived cells was performed to FBM NSG mice (n=3, n=5 respectively), n.s- not significant.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

Age-related clonal hematopoiesis is not only due to the random accumulation of mutations, but also how phenotypes are selected by the aging environment. While fatty bone marrow (FBM), is one of the hallmarks of bone marrow ageing, it is unknown whether FBM can modify the evolution of the early stages of leukemia and clonal hematopoiesis (CH). To address this question, the present inventors established different mouse models to recapitulate the high percentage of adipocytes that are found in humans during aging (Figures 6A-C). They transplanted both human and mice preleukemic hematopoietic stem cells (PreL-HSCs) carrying DNMT3A mutations into mice with FBM. A significant increase in self-renewal was found when DNMT 3A^Mut-preL-HSPCs were exposed to FBM (Figures 3A-E). A ten fold increase in DNMT 3A^Mut-preL-HSCs was observed under FBM conditions in comparison to other conditions in which myeloid differentiation occurred (Figures 4A-C).

The present inventors further demonstrated that mice PreL-HSPCs exposed to FBM exhibited an activated inflammatory signaling (IL-6 and IFNy). Cytokine analysis of BM fluid demonstrated increased IL-6 levels under FBM conditions, which significantly decreased after treatment with a PPARy inhibitor (Figures 4D-G).

Whilst reducing the present invention to practice, the present inventors showed that administration of anti-IL-6 neutralizing antibodies significantly reduced the selective advantage of DNMT 3A^Mut-preL-HSPCs exposed to FBM (Figure 5C).

Overall, the data shows that age related paracrine FBM inflammatory signals promote D/VAfTJA-driven clonal hematopoiesis, which can be inhibited by blocking the IL-6 receptor. Accordingly, the present inventors propose treatment of myeloid malignancies which harbor a DNAT3A mutation using agents which block the activity of IL-6. Furthermore, since the amount of IL-6 in the bone marrow was shown to correlate with the fattiness of the bone marrow, the present inventors propose prevention of metabolic and age-related diseases by blocking the activity of IL-6.

Thus, according to an aspect of the invention there is provided a method of treating or preventing a myeloid malignancy in a subject comprising a mutation in DNA methyltransferase 3 alpha (DNMT3A) in a genome thereof comprising:

As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms the myeloid malignancy or substantially preventing the appearance of clinical or aesthetical symptoms of the condition (also referred to as a disease or disorder).

As used herein, the term “subject” or “subject in need thereof’ refers to mammals, preferably human beings, male or female, who are diagnosed with, or are at risk of developing a myeloid malignancy.

According to a specific embodiment, the subject is an infant, a child, an adolescent or an adult as defined by the classification tables of the Food and Drug Administration (FDA).

According to one embodiment, the subject is under 70 years old, under 65 years old, under 60 years old, under 55 years old, under 50 years old, under 45 years old, under 40 years old, under 35 years old, under 30 years old, under 25 years old or under 20 years old.

According to an embodiment of the invention the subject is 18-75 years old, or between 50- 80 years old.

According to an embodiment of the invention the subject is up to 18 years old.

According to an embodiment of the invention the subject is 3-18 years old.

According to an embodiment of the invention the subject is 0-3 years old.

According to an embodiment, the subject is diagnosed with cancer but has not been subject to anti-cancer therapy (e.g., chemotherapy, radiation, radiotherapy or immunotherapy). In such case the treatment described herein may be the first line treatment.

According to one embodiment, the subject is undergoing a routine well-being check-up.

The subject may be diagnosed as having a pre-myeloid malignancy. As used herein “a pre-myeloid malignancy” refers to medical conditions in which asymptomatic subjects for a myeloid malignant disease, at times also referred to as healthy subjects, display (also referred to as “positive for”) a somatic mutation in the DNMT3A gene in the DNA of the peripheral blood (e.g., peripheral blood cells).

According to a particular embodiment, the pre-myeloid malignancy is an acute or chronic leukemia.

The term “leukemia” refers to a disease of the blood forming tissues characterized by an abnormal increase in the number of leukocytes in the tissues of the body with or without a corresponding increase of those in the circulating blood. Leukemia of the present invention includes lymphocytic (lymphoblastic) leukemia and myelogenous (myeloid or nonlymphocytic) leukemia.

The term "acute leukemia" means a disease that is characterized by a rapid increase in the numbers of immature blood cells that transform into malignant cells, rapid progression and accumulation of the malignant cells, which spill into the bloodstream and spread to other organs of the body.

The term "chronic leukemia" means a disease that is characterized by the excessive build up of relatively mature, but abnormal, white blood cells.

Myeloid malignant diseases comprise chronic (including, but not limited to, myelodysplastic syndromes, myeloproliferative neoplasms) or acute (such as acute myeloid leukemia) stages. They are clonal diseases arising in hematopoietic stem or progenitor cells.

Examples of particular myeloid malignancies associated with DNMT3A mutations include, but are not limited to:

Acute myeloid leukemia (AML), primary myelofibrosis, Hypereosinophilic Syndrome (HES), myelodysplastic syndrome (MDS), acute promyelocytic leukemia (APL), chronic myelomonocytic leukemia (CMML), chronic neutrophilic leukemia (CNL), acute undifferentiated leukemia (AUL), anaplastic large-cell lymphoma (ALCL), prolymphocytic leukemia (PML), juvenile myelomonocyctic leukemia (JMML), adult T-cell leukemia AML with trilineage myelodysplasia (AML/TMDS), mixed lineage leukemia (MLL), myeloproliferative disorders (MPD), chronic myeloid leukemia (CML)and myeloid (granulocytic) sarcoma, Systemic mastocytosis, mast cell neoplasm, clonal cytopenia of indetermined significance, clonal hematopoiesis, follicular lymphoma, Blastic plasmacytoid dendritic cell neoplasm and chronic neutrophilic leukemia. According to a specific embodiment, the myeloid malignancy is acute myeloid leukemia (AML), myelodysplastic syndromes, acute myeloid leukemia with myelodysplasia-related changes, chronic myelomonocytic leukemia or myeloid plastic syndrome.

According to a particular embodiment, the myeloid malignancy is AML.

The subject may also harbor additional mutations for these diseases in genes whose encoded proteins include NPM1 , IDH1/2, and FLT3.

As mentioned, the method comprises analyzing in a sample of the subject for the presence of a DNMT3A mutation.

In one embodiment, the sample is a fluid sample, including, but not limited to whole blood, plasma and serum. According to a particular embodiment, the sample is a peripheral blood sample.

In another embodiment, the sample is a tissue sample (e.g. a tissue biopsy).

In still another embodiment, the sample is a bone marrow sample.

As used herein, the term "DNMT3A", or " DNA (cytosine-5-1-methyltransferase 3 alpha" refers to the wild-type (non-mutated) human DNMT3A amino acid sequence, which encodes the protein annotated under NCBI Genbank accession numbers NP_072046.2, and is further reproduced in SEQ ID NO: 5.

The genomic sequence of DNMT3A is set forth in NG_029465.2.

The DNMT3A protein is encoded on human chromosome 2, and serves as a DNA methyltransferase that is believed to function in de novo methylation, rather than maintenance methylation. DNMT3A localizes to the cytoplasm and nucleus and its expression is developmentally regulated.

Non-limiting examples of DNMT3A alterations include a missense mutation, i.e., a mutation which changes an amino acid residue in the protein with another amino acid residue and thereby abolishes the enzymatic activity of the protein; a nonsense mutation, i.e., a mutation which introduces a stop codon in a protein, e.g., an early stop codon which results in a shorter protein devoid of the enzymatic activity; a frame-shift mutation, i.e., a mutation, usually, deletion or insertion of nucleic acid(s) which changes the reading frame of the protein, and may result in an early termination by introducing a stop codon into a reading frame (e.g., a truncated protein, devoid of the enzymatic activity), or in a longer amino acid sequence (e.g., a readthrough protein) which affects the secondary or tertiary structure of the protein and results in a non-functional protein, devoid of the enzymatic activity of the non-mutated polypeptide; a readthrough mutation due to a frame-shift mutation or a modified stop codon mutation (i.e., when the stop codon is mutated into an amino acid codon), with an abolished enzymatic activity; a promoter mutation, i.e., a mutation in a promoter sequence, usually 5' to the transcription start site of a gene, which results in downregulation of a specific gene product; a regulatory mutation, i.e., a mutation in a region upstream or downstream, or within a gene, which affects the expression of the gene product; a deletion mutation, i.e., a mutation which deletes coding nucleic acids in a gene sequence and which may result in a frame-shift mutation or an in-frame mutation (within the coding sequence, deletion of one or more amino acid codons); an insertion mutation, i.e., a mutation which inserts coding or non-coding nucleic acids into a gene sequence, and which may result in a frame- shift mutation or an in-frame insertion of one or more amino acid codons; an inversion, i.e., a mutation which results in an inverted coding or non-coding sequence; a splice mutation i.e., a mutation which results in abnormal splicing or poor splicing; and a duplication mutation, i.e., a mutation which results in a duplicated coding or non-coding sequence, which can be in-frame or can cause a frame-shift.

According to specific embodiments, the mutation of DNMT3A is comprises in at least one allele of the gene.

The term "allele" as used herein, refers to any of one or more alternative forms of a gene locus, all of which alleles relate to a trait or characteristic. In a diploid cell or organism, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes.

Examples of DNMT3A mutations include the missense mutations at position R882 - for example R882H, R882C, R882P, and R882S. Other missense mutations include E30A P904L and A368D. Nonsense mutations may be at position R598 and L805. Frameshift insertions may be at E733. Frameshift deletion may be at F827.

Methods of analyzing for the presence of DNMT3A mutations are known in the art and are detailed herein below.

1. Chromosomal and DNA staining methods:

Exemplary methods include FISH: High-resolution multicolor banding (MCB) on interphase chromosomes and Quantitative FISH (Q-FISH).

2. Analysis of sequence alterations at the DNA level:

To determine sequence alterations in the DNMT3A gene, DNA is first obtained from a biological sample (as described herein above) of the tested subject.

Once the sample is obtained, DNA is extracted using methods which are well known in the art, involving tissue mincing, cell lysis, protein extraction and DNA precipitation using 2 to 3 volumes of 100% ethanol, rinsing in 70% ethanol, pelleting, drying and resuspension in water or any other suitable buffer (e.g., Tris-EDTA). Preferably, following such procedure, DNA concentration is determined such as by measuring the optical density (OD) of the sample at 260 nm (wherein 1 unit OD=50 qg/ml DNA). To determine the presence of proteins in the DNA solution, the OD 260/OD 280 ratio is determined. Preferably, only DNA preparations having an OD 260/OD 280 ratio between 1.8 and 2 are used in the following procedures described hereinbelow.

The sequence alteration (or SNP) of some embodiments of the invention can be identified using a variety of methods. One option is to determine the entire gene sequence of a PCR reaction product (see sequence analysis, hereinbelow). Alternatively, a given segment of nucleic acid may be characterized on several other levels. At the lowest resolution, the size of the molecule can be determined by electrophoresis by comparison to a known standard run on the same gel. A more detailed picture of the molecule may be achieved by cleavage with combinations of restriction enzymes prior to electrophoresis, to allow construction of an ordered map. The presence of specific sequences within the fragment can be detected by hybridization of a labeled probe, or the precise nucleotide sequence can be determined by partial chemical degradation or by primer extension in the presence of chain-terminating nucleotide analogs.

Exemplary techniques include restriction fragment length polymorphism (RFLP): sequencing analysis, micro sequencing analysis, mismatch detection assays based on polymerases and ligases, Ligase/Polymerase-mediated Genetic Bit Analysis™, hybridization Assay Methods, single-strand conformation polymorphism (SSCP), Dideoxy fingerprinting (ddF), pyrosequencing™ analysis (Pyrosequencing, Inc. Westborough, MA, USA), Acycloprime™ analysis (Perkin Elmer, Boston, Massachusetts, USA) and reverse dot blot.

3. Analysis of sequence alteration at the RNA level:

Alteration in the sequence of RNA can be determined using methods known in the arts.

Exemplary techniques include Northern Blot analysis, RT-PCR analysis, RNA in situ hybridization stain, in situ RT-PCR stain and DNA microarrays/DNA chips.

4. Analysis of equence alterations at the protein level:

Sequence alterations can also be determined at the protein level. While chromatography and electrophoretic methods are preferably used to detect large variations in molecular weight, such as detection of the truncated ETS protein, immunodetection assays such as ELISA and Western blot analysis, immunohistochemistry and the like, which may be effected using antibodies specific to smaller sequence alterations are preferably used to detect point mutations and subtle changes in molecular weight.

Thus, the invention according to some embodiments thereof also envisages the use of serum immunoglobulins, polyclonal antibodies or fragments thereof, (i.e., immunoreactive derivatives thereof), or monoclonal antibodies or fragments thereof. Monoclonal antibodies or purified fragments of the monoclonal antibodies having at least a portion of an antigen-binding region, including the fragments described hereinbelow, chimeric or humanized antibodies and complementarity determining regions (CDR).

Exemplary methods for analyzing protein alterations include Western blot, Fluorescence activated cell sorting (FACS), Immunohistochemical analysis. Once the subject has been shown to harbour the DNMT3A mutation, it is advisable to treat the subject with an IE-6 inhibitor or an agent which can down-regulate the activity and/or amount of IL-6. Such agents are further described herein below.

As mentioned, the present inventors also found that the amount of fat cells in the bone marrow corresponded with the amount of interleukin-6 signaling. Accordingly, the present inventors propose prevention of metabolic and age-related diseases by blocking the activity of IL- 6.

Thus, according to another aspect of the present invention, there is provided a method of preventing and/or treating a disease associated with a fatty bone marrow in a subject comprising:

(a) analyzing cells of a bone marrow sample for the presence of adipocytes; and

As used herein, the term “preventing” refers to preventing at least one clinical symptom of a disease from occuring in a subject. The subject may be at risk for the disease, but has not yet been diagnosed as having the disease. Thus, for example the subject may be pre-diabetic and may be at risk for having diabetes. The subject may show high levels of cholesterol and/or triglycerides and/or show markers for being at risk for stroke or a cardiac event.

In one embodiment, the subject is older than 40 years old, older than 50 years old, older than 60 years old or even older than 70 years old.

Exemplary diseases which may be prevented include metabolic disease (e.g. Diabetes type I or type II), obesity, and age-related diseases (e.g. atherosclerosis, cardiovascular disease, arthritis, cataracts, osteoporosis, type 2 diabetes, hypertension and Alzheimer’s disease).

Other exemplary diseases include osteoporosis, type 1 diabetes, Cushing’s disease, estrogen deficiency, and anorexia nervosa.

Methods of determining the amount of fat cells in a bone marrow sample are known in the art and include imaging techniques such as magnetic resonance imaging (MRI) and computed tomography (CT) and histological methods. The amount of adipocytes (or size thereof) is measured and compared to control age- matched samples derived from healthy subjects.

When the amount of adipocytes (or size thereof) is above (a statistically significant increase, e.g. at least 1.5 fold higher, at least 2 fold-higher or even at least 3 fold higher) the amount present in the control sample, it is indicative that an agent which down-regulates an amount and/or activity of interleukin-6 (IL-6) is useful in preventing and/or treating the disease.

For any aspect of the invention, “down regulation”, “inhibition” or “decrease” in the context of the present invention means that expression or activity of the interleukin-6 is reduced, such as by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in the presence of the inhibitor as compared to the level of expression or activity in the absence of the inhibitor (i.e., control). Complete inhibition means that there is no detectable expression or activity of the target gene such as qualified at the RNA or protein level or appropriate activity assay.

It will be appreciated that the “inhibitor” can also be referred to collectively as an “agent”.

Non-limiting examples of inhibitors of interleukin-6 are described in details hereinbelow.

According to one embodiment, the IL-6 inhibitor directly downregulates an activity or expression of IL-6. The term “directly” means that the inhibitor directly interacts with IL-6 nucleic acid sequence or protein and not on a co-factor, an upstream activator or downstream effector of a component of a IL-6 pathway. Such an agent may block the IL-6 activity in the cell.

According to a specific embodiment the inhibitor refers to a specific inhibitor having a specific activity for IL-6 and not for an interleukin other than IL-6.

According to a specific embodiment the inhibitor refers to a non-specific interleukin inhibitor having a non-specific activity on a number of interleukins.

In addition to the agents discussed above, IL-6 inhibitors include molecules which binds to and/or cleave the protein. Such molecules can be small molecules, antagonists, or inhibitory peptides.

It will be appreciated that a non-functional analogue of at least a catalytic or binding portion of IL-6 can be also used as an agent.

Additional agents capable of inhibiting IL-6 include antibodies, antibody fragments, and aptamers.

Antibodies include IL-6 antibodies, IL-6R antibodies and gpl30 antibodies. These antibodies bind to IL-6, IL-6R or gpl30 to inhibit binding between IL-6 and IL-6R, or IL-6R and gpl30.

Preferably, the antibody specifically binds at least one epitope of the IL-6. As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.

Exemplary commercial IL-6 antibodies include siltuximab, olokizumab (CDP6038), elsilimomab, BMS-945429 (ALD518), MH-166, and sirukumab (CNTO 136).

Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are well known in the art (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference).

Another agent which can be used along with some embodiments of the invention to downregulate IL-6 is an aptamer.

As used herein, the term “aptamer” refers to double stranded or single stranded RNA molecule that binds to specific molecular target, such as a protein. Various methods are known in the art which can be used to design protein specific aptamers. The skilled artisan can employ SELEX (Systematic Evolution of Ligands by Exponential Enrichment) for efficient selection as described in Stoltenburg R, Reinemann C, and Strehlitz B (Biomolecular engineering (2007) 24(4):381-403).

Down-regulation at the nucleic acid level is typically effected using a nucleic acid agent, having a nucleic acid backbone, DNA, RNA, mimetics thereof or a combination of same. The nucleic acid agent may be encoded from a DNA molecule or provided to the cell per se.

Thus, downregulation of IL-6 can be achieved by RNA silencing. As used herein, the phrase "RNA silencing" refers to a group of regulatory mechanisms [e.g. RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co-suppression, and translational repression] mediated by RNA molecules which result in the inhibition or "silencing" of the expression of a corresponding protein-coding gene. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.

As used herein, the term "RNA silencing agent" refers to an RNA which is capable of specifically inhibiting or "silencing" the expression of a target gene. In certain embodiments, the RNA silencing agent is capable of preventing complete processing (e.g, the full translation and/or expression) of an mRNA molecule through a post-transcriptional silencing mechanism. RNA silencing agents include non-coding RNA molecules, for example RNA duplexes comprising paired strands, as well as precursor RNAs from which such small non-coding RNAs can be generated. Exemplary RNA silencing agents include dsRNAs such as siRNAs, miRNAs and shRNAs. The IL-6 inhibitor can be provided to the subject per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.

As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

Herein the term "active ingredient" refers to the IL-6 inhibitor accountable for the biological effect.

Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in “Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, inrtaperitoneal, intranasal, or intraocular injections.

Conventional approaches for drug delivery to the central nervous system (CNS) include: neurosurgical strategies (e.g., intracerebral injection or intracerebroventricular infusion); molecular manipulation of the agent (e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB) in an attempt to exploit one of the endogenous transport pathways of the BBB; pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers); and the transitory disruption of the integrity of the BBB by hyperosmotic disruption (resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide). However, each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.

Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.

The pharmaceutical composition of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

Pharmaceutical compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (multispecific antibody) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., cancer) or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 P-l).

Dosage amount and interval may be adjusted individually to provide blood levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

Compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.

It is expected that during the life of a patent maturing from this application many relevant antagonistic IE-6 antibodies will be developed and the scope of the term anti CD40 antibody is intended to include all such new technologies a priori.

As used herein the term “about” refers to ± 10 %.

The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

The term “consisting of’ means “including and limited to”.

The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure. As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides. Itisappreciated thatcertainfeaturesoftheinvention,which are,forclarity,describedin the context of separate embodiments, may also be provided in combination in a single embodiment.Conversely,variousfeaturesoftheinvention,which are,forbrevity,described in the context of a single embodiment,may also be provided separately or in any suitable subcombination or as suitable in any other described embodimentof the invention.Certain features described in the contextofvarious embodiments are notto be considered essential featuresofthoseembodiments,unlesstheembodimentisinoperativewithoutthoseelements.

Variousembodimentsand aspectsofthepresentinvention asdelineatedhereinaboveand asclaimedintheclaimssectionbelow findexperimentalsupportinthefollowingexamples.

EXAMPLES

Reference is now made to the following examples,which together with the above descriptionsillustratesomeembodimentsoftheinventioninanonlimitingfashion.

MATERIALS AND METHODS

Mice:Male immune-deficientNSG (NOD/SCID/IL-2Rgc-null)mice:NSG (Stock No: 005557) (The Jackson Laboratory, Bar-Harbor, ME, USA). NOD.Cg- Prkdc^scid//2rg^tm1j Tg(PGKl-KITLG*220)441Daw/SzJmice(stock No:017830)(TheJackson Laboratory, Bar-Harbor, ME, USA). NOD.Cg-Prkdc''"¹Il2rg^tmlWjl Tg(CMV- IL3,CSF2,KITLG)lEav/MloySzJ mice (Stock No:013062)NSG-SGM3(The Jackson Laboratory,Bar-Harbor,ME,USA)DNMT3A^R88H KImice,constitutively expressthehuman DNMT3A mutation. SRSF2^P95Hfloxed micepossessloxP sitesflanking theendogenouscoding region of the serine/arginine-rich splicing factor 2 (SRSF2)gene (Stock No:028376)(The Jackson Laboratory,Bar-Harbor,ME,USA).DNMT3A^R882H orSRSF2^F95Hwere crossed with VAV Cre (Stock No: 008610) (The Jackson Laboratory, Bar-Harbor, ME, USA).All experimentswereperformedinaccordancewithinstitutionalguidelines.

CD3 depletion:CD3 cellswereisolated from thawed human samples(peripheralblood AML sample,mobilized peripheralblood mononuclearcells(PBMCs)and cord blood)using magnetic beads according to manufacturer’s protocol (EasySepTM Human CD3 Positive SelectionKitII,StemCellTechnologies,Vancouver,Canada)

Xenotransplantation assays: PBMCs from AML patients were CD3 depleted as describedabove.MobilizedPBMCsorcordbloodwereenrichedforCD34⁺ cellsusingmagnetic beadsaccording to manufacturer’sprotocol(CD34 MicroBead Kit,MiltenyiBiotec,Bergisch Gladbach,Germany).CD3 depletion and CD34 enrichmentwere validated by flow cytometry unless specified otherwise, l-2.5xl0⁶ CD3 depleted mononuclear cells were injected intrafemoral (right femur) into 8 to 12-week-old male mice.

Flow cytometry: Primary human samples and cells extracted from mice bone marrows were stained with the antibodies shown in Table 1. Table 1: Antibodies and viability staining used for flow cytometry

Primary mouse samples and cells extracted from mice bone marrows were stained with the antibodies shown in Table 2.

Table 2: Antibodies and viability staining used for flow cytometry

All flow cytometry analyses were performed on FirePlex (Beckman Coulter, Brea, CA,

USA), using CytExpert software v 2.4.0.28 (Beckman Coulter, Brea, CA, USA). BM transplantation: Freshly dissected femora and tibiae were isolated from two months old or one-year mice DNMT3A^Mut, DNMT3A^hapl° , DNMT3A^W1- SRSF2^Mut or control SRSF2^WT mice CD45.2. BM was flushed with a lee (23G) into IMDM (Iscove's Modified Dulbecco's Medium). The BM was spun at 0.3 g by centrifugation and RBCs were lysed in ammonium chloride-potassium bicarbonate lysis buffer for 1 min. After centrifugation, cells were resuspended in PBS, passed through a cell strainer, and counted. Then 6X10^A6 cells were injected intra femur into NSG (CD45.1) mice that were Irradiated (FBM) seven days before with low dose Irradiation (225 rad) or to non-irradiated (control) NSG mice. Eight weeks following cells transfer, mice were sacrificed. Right femur and the other bones (left femur and tibias) were cut and BM cells were flushed with IMDM (Iscove's Modified Dulbecco's Medium) and analyzed by FACS. Engraftment was defined by the presence of mCD45.2. Engraftment was assessed according to presence of >0.1% mCD45.2 cells. Ab’s that were used: APC anti mouse CD45.2 (Biolegend, clone 104), PE anti mouse CD45.1 (Biolegend clone A20).

BADGE administration: NSG mice were treated intraperitoneally with the PPARyi (which is a critical transcription factor in adipogenesis) inhibitor, bisphenol ADiGlycidyl Ether (BADGE) (30 mg/kg) (Sigma, Cat 15138) for seven days, irradiated and treated for seven more days following cell transplantation.

IL-6 neutralization in-vivo. NSG mice were administrated intraperitoneally with IL-6 (50 pg /mouse) neutralizing Ab (BioLegend 504512) for two days. Then mice were irradiated with low dose (225 rad), and treated intraperitoneally with anti IL-6 neutralizing Ab for seven days, followed by DNMT3A^MM or DNMTSA^ ^BM derived cell transplantation.

Histological analysis and adipocytes quantification: Mice were sacrificed and autopsied, and dissected tissue samples were fixed for 24 h in 4 % paraformaldehyde, dehydrated, and embedded in paraffin. Paraffin blocks were sectioned at 4 mm and stained with H&E. Images were scanned by Pannoramic SCAN II (3DHISTECH, Hungary). BM adipocytes were quantified by intracellular staining of the FBM lipid with LipidTox (fluorescent dye that stains neutral lipids; Life technologies) and analyzed using ImageStream X Mark I, Luminex.

Image Stream analysis: To quantify the number of adipocytes in mice, three mice per sample were sacrificed. All bones (femur and tibia) were cut and BM cells were flushed with IMDM (Iscove's Modified Dulbecco's Medium). Furthermore, the bones were crushed in order to obtain the adipocytes attached to the bones. The BM was filtered through 30 pM mesh, centrifuged and 1ml PBSxl and 1ml 8 % PFA were added. The sample was vortexed and PBSxl was added. Next, the sample was centrifuged and fixed with 200 pL designated fixative (00- 5223-56 and 00-5123-43, eBioscience) and incubated for 30 min at 4 °C (in the dark). Next the cells were washed with 1 ml designated permeabilization buffer (00-8333-56, eBioscience), centrifuged, and stained with 1:100 AB (PE anti-mouse CD45 Antibody BLG-103106) overnight spinning in the fridge. Next day, the sample was centrifuged at max speed 30 sec and washed with PBSxl twice and stained with 1ml DAPI (dilute 1:1000 in PBS) 7 min in the ice. To quantify the number of adipocytes, HCS LipidTOX™ Deep Red neutral lipid stain was used (H34477, Thermofisher). The LipidTOX™ neutral lipid stain has an extremely high affinity for neutral lipid droplets and can be detected by fluorescence microscopy or an HCS reader. The sample was centrifuged, then 40 pl PBSxl were added, and 1:50 LipidTox was added to each sample. The samples quantified by ImageStream X Mark II ,Luminex, and analyzed by ImageStream software.

Whole-mount immunofluorescence staining: The procedure was performed as described previously in Sacma et al⁶⁰. The following antibodies were used: Goat anti Mouse/Rat FABP4/A-FABP Antibody, R&D Systems, Cat#AF1443-SP; RRID: AB„2102444 and Alexa Fluor® 647 AffiniPure Donkey Anti-Goat IgG (H+L), Jackson ImmunoResearch, Cat#705-605- 003; RRID: AB_2340436. For evaluation, fluorescently labelled bone tissues were placed onto a 4 well-p- slide and covered in antifade or PBS to prevent tissue desiccation. The preparations were examined using a Leica TCS SP8 confocal microscope and analyzed with the image analysis software Volocity (v6.2, Perkin Elmer) and ImageJ. In addition, bone matrix and adipocytes were detected using the DIC (TLD ) mode of the microscope.

Z-stacked confocal images are generated from whole-mounts of bisected mouse bones in which the structural and cellular integrity is highly preserved, and they show epiphyseal/metaphyseal BM regions. FABP4 is also expressed by endothelial cells, however adipocytes have a higher expression. The DIC (differential interference contrast) channel was additionally used for the detection of the typical unilocular morphology of the adipocytes and other structures. Indeed, in BM sections, big sinusoidal vessels are often collapsing, however protective whole-mounts were used and the “empty” spaces shown are trabecular bone structures (without surrounding FABP4 cells, see Figure 6C) which are very frequently present in epiphyseal/metaphyseal BM. The sinusoidal vessels, with specific morphology, are clearly visible by lower FABP4 expression, surrounded by FABP4 low endothelial cells and in the DIC channel bone structures and sinusoidal vessels are reflected differently (Figure 6B-C).

Colony forming unit (CFU): DNMT3A^{Mu t} or DNMT3A^W1 mice were sacrificed, all bones (femur and tibia) were cut and BM cells were flushed with IMDM (Iscove's Modified Dulbecco's Medium), the cells where counted and seeded at a density of 2x10⁴ cells per replicate into cytokine- supplemented methylcellulose medium (MethoCult M3434, Stemcell Technologies). After 10-14 days, the colonies propagated and were scored. The remaining cells were resuspended and counted, and a portion was taken for replating (2xl0⁴ cells per replicate) with human (GenScript Z03034-50) or mouse (GenScript Z02767-10) IL-6 or w/o.

Amplicon sequencing: An amplicon-based approach was used to sequence DNMT3A and NPMlc from human samples after and before engraftment.

DNMT3A Fw primer for amplicon sequencing:

CTACACGACGCTCTTCCGATCTttgtttgtttgtttaactttgtg (SEQ ID NO: 1)

DNMT3A Rev for amplicon sequencing

CAGACGTGTGCTCTTCCGATCTcactatactgacgtctccaacat (SEQ ID NO: 2)

NMP1 Fw for amplicon sequencing

CTACACGACGCTCTTCCGATCTgttgaactatgcaaagagacatt (SEQ ID NO: 3)

NMP1 Rev for amplicon sequencing primer

CAGACGTGTGCTCTTCCGATCTagaaatgaaataagacggaaaat (SEQ ID NO: 4)

Single RNA seq: Cells from two-month-old and one-year-old DNMT3A^Mut or DNMTSA^⁷ were injected to FBM and normal mice. The quantity of LSK was calibrated at three time points: one day, three and five days post injection. The results demonstrated that following three days of injection, the highest number of LSK cells from all conditions (data not shown). Subsequently, three days post injection, CD45.2 LSK cells were isolated. Cells were also isolated from the same donor mice before they were injected. These were termed naive cells. From all the above conditions, LSK cells were isolated and single cell sorted. For sorting, the following antibodies were used: CD45.2-APC, anti-mouse (BLG), Ly-6G/Ly-6C (Gr-1) FITC anti-mouse (BLG), CDl lb FITC anti-mouse/human (BLG), CD45R/B220 FITC anti- mouse/human (BLG), CD4 FITC anti mouse (BLG), TER-119 FITC anti-mouse (BLG), CD8a FITC anti mouse (BLG), Sca-1-PE-Vio770 anti-mouse (Miltenyi), CD117 (c-kit)-BV605 anti mouse (BLG). Cells were sorted to 384 low binding plates and sequenced according to MARS- seq protocol (Keren Shaul et al., Nature Protocols 14, 1841-1862 (2019)) A total of 3923 cells were available for analysis after mapping. To filter empty cells and doublets, single cells were selected with more than 450 UMIs per cells, and all cells in the 3% upper percentile of UMI counts were excluded. After filtering cells, a total of 2198 cell were available for further analysis. The Metacell library was used for noise reduction, clustering and cell type annotation 3838383838. Removal of lateral effects (cell-cycle, stress) and batch effects was performed using gene module analysis to filter genes used for metacells grouping. The metacell analysis partitioned 1999 cells to 32 metacells, while filtering 199 cells as outliers. Metacells’ cell types were annotated based on known genes defining cell populations (Nestorowa, S. et al (2016) doi:10.1182/blood-2016-05). The following genes were used: HSCs (Procr); MLP (Dntt); CMP (Mpo), MegK (Pf4); ERY (Hba-a2); MonP (Irf8), DC (Cd74), MPP (Fgd5 and no other conditions).

Another method was used for single cell clustering reduction of dimension and clustering and differential expression analysis based on the UMAP algorithm. After filtration of cells (as described above for the Metacell analysis), no batch effects could be noticed in the ERCC counts between the different conditions. Clustering of the UMAP data was used to perform differential expression (DE) analysis on the different clusters.

GSEA analysis: The DE genes of cluster 1 were ranked based on fold change and analyzed using the GSEA software version 4.1.06868686868. Significant genes set had FDR q- val<0.2. The Hallmark genes sets were used for the analysis.

Gene sets scores (IL-6, TNFa, IFNa and IFNy): To generate scores for the different gene sets across cells, the present inventors down-sampled the original UMI matrix to 750 UMIs, and calculated the score per cell as the sum over all genes in the respective gene sets. These scores per cell were used to generate the plots per experimental condition in Figure 4C.

Statistical analysis: In all figures and tables comparison between medians was performed using the Mann-Whitney U test with FDR correction for multiple hypothesis testing. Calculations were performed using the compare_means function of the R library ggpubr. The comparison between cytokine levels were analyzed by two-way ANOVA test with Sidaks multiple comparison correction.

RESULTS

Establishment of fatty bone marrow models in NSG mice

Murine BM does not recapitulate the dramatic age-related increase in FBM which can be observed in humans. Accordingly, in order to be able to test the effect of FBM on primary human HSPCs, FBM was induced in NSG mice by external stress. Previous reports documented the accumulation of FBM few days to weeks after total body irradiation. Nevertheless, total body irradiation causes cytokine storm and dramatic remodeling of all components of the BM microenvironment including osteoblasts, megakaryocytes, and vasculature. To control all these off targets effects of irradiation and other external stresses, a control group of mice was used that were irradiated and treated with a PPARyi, bisphenol ADiGlycidyl Ether (BADGE). Previous studies have shown that PPARyi treatment inhibits adipogenic differentiation in vitro.

To better characterize this sub-lethal irradiation model (225 rad) NSG mice were irradiated. An enhanced adipocyte presence in the BM was noted a week following irradiation. High FBM was maintained even two months after irradiation. These changes did not appear 24- 48 hours following irradiation (which is the usual time frame in which human HSPCs are injected to NSG mice) (Figure 1A-C). Indeed, PPARyi treatment seven days prior to and post irradiation resulted in reduced FBM accumulation (Figure ID). Quantifying lipid levels in the BM by LipidTOX™ Deep Red Neutral Lipid Stain demonstrated significantly reduced FBM (30 folds) in the irradiated mice treated with PPARyi compared to irradiation without PPARyi treatment (Figure IE). These results were validated by staining irradiated BM with the adipocyte marker fatty acid binding protein 4 (FABP4) (Figure IF).

Altogether, these results provide evidence that low dose irradiation of NSG mice causes more than just a hypocellular marrow, but also an active accumulation of BM adipocytes. A castration model was also examined as it is known to recapitulate the age related decline in testosterone among males. It was found that a month after castration (CAS), male mice developed FBM, (data not shown). A similar effect could be achieved by treating NSG mice with the PPAR-y activator (rosiglitazone maleate) for three weeks (data not shown). Interestingly, analyzes of tibia bones derived from one-year-old NSG-SGM3 mice (which express human IL-3 (hIL-3), hSCF and hGM-CSF) demonstrated high FBM levels compared to one-year-old NSG or NSG-hSCF mice (data not shown).

FBM provides selective advantage to human preL-HSPCs carrying the DNMT3A R882H mutation

Following establishment of the different FBM models in NSG mice, the present inventors studied the interaction between FBM and primary preL-HSPCs carrying the DNMT3A R882H mutation. To this end, an AML sample was selected carrying both the DNMT3A mutation (VAF of 50%) and the NPMlc mutation (VAF of 50%). However, when injected into FBM NSG mice the engrafting cells following amplicon sequencing were only carrying the DNMT3A mutation, suggesting that only the preL-HSPCs with DNMT3A mutation could engraft in NSG mice at this specific sample (sample #160005) (Figure. 2A). Engraftment of sample #160005 cells was much higher under FBM conditions compared to normal BM (NBM) mice and the PPARyi -treated control, in which no adipocytes were accumulated (Figure 2B). The graft had a multilineage differentiation capacity. Furthermore, NBM samples were samples, but due to the limited engraftment, no human cells after sorting were obtained and no human reads were available after sequencing. This experiment was repeated on the castration (CAS) FBM model and again a significantly higher engraftment of sample #160005 cells was detected under FBM conditions (Figure 2C). To further validate these results, HSPCs collected for auto-transplantation (Sample# 141464) were used. This sample had a high variant allele frequency (VAF) of the DNMT3A R882H mutation, and had DNMT3A R882H mutation following transplantation to FBM as determined by amplicon sequencing (Figure 2D). Again, a significantly higher engraftment was observed in both the irradiation and castration FBM models (Figures 2E-F).

To study the interaction between FBM and normal hematopoiesis, wild type (WT) CD34+ cells from pooled cord blood samples were transplanted and also from aged matched healthy donor without clonal hematopoiesis. In contrats to the DNMT3A R882 mutated cells, the CD34+ cells from both sources had no increased engraftment when exposed to FBM (Figures 2G, H). These results suggest a role for an adipocyte-rich environment in enhancing engraftment of human preL-HSPCs, but not for normal HSPCs. To validate these results and better understand the mechanisms behind the increased engraftment of human preL-HSPCs under FBM conditions, a rodent model of mutant DNMT3A (DNMTSA ^¹) was used.

FBM support DNMT3A^Mut preL-HSPCs derived from a genetic rodent model

For the next set of experiments, the human DNMT3A R882H knock- in mice model, were crossed with mice carrying a Cre recombinase allele under the VAV promotor which is expressed only in the hematopoietic system to create hematopoietic specific DNMT3A mutant ( AAfT3A^Mut) mice. C57BlxVAV-cre mice were used as a control group (denoted DNMT3A^WT).

The injection of 2 months BM derived DNMT3A^Mat cells (CD45.2) intra femorally (IF) to NSG (CD45.1) mice with FBM resulted in significantly elevated engraftment of DNMT3A^Mat cells in comparison to control mice with NBM or mice who were irradiated and treated with the PPARy inhibitor (PPARyi) (Figure 3A). This increased engraftment under FBM conditions could not be observed when DNMT3A^WT cells were injected (Figure 3A). To test whether the increased engraftment of DNMT3A^Mat preL-HSPCs under FBM conditions was due to increased selfrenewal, the experiment was repeated, and the cells exposed to FBM and NBM were injected into secondary recipients that had FBM. Engraftment analysis demonstrated that the highest increase in self-renewal was present in DNMT3A^Mat cells exposed to FBM (Figure 3B).

Next, it was hypothesized that as DNMT3A R882 mutations are known to cause hypomethylation over time and can cause human leukemia after a long latency period, the effects of FBM could be even more pronounced if older preL-HSPCs were to be studied. Indeed, significant increase in engraftment was detected following transplantation of old DNMT3A^Mat cells derived from one-year-old mice injected into NBM, FBM and PPARyi controls compare to two months DNMT3A^Mat derived BM (Figure 6C). DNMT3A^Mat cells derived from one-year-old mice injected into FBM had the most significant growth advantage in comparison to NBM and PPARyi controls. When DNMT3A^W cells were injected, this effect of FBM could not be observed (Figure 3C, Figure 6C). Interestingly, the administration of PPARyi to FBM mice transplanted with one-year-old DNMT3A^W cells resulted in a significant increase of engraftment (Figure 3C, Figure 6C). To test whether the increased engraftment of one-year-old cells was due to increased self-renewal, secondary engraftment was performed. In agreement with the results derived from two-month-old mice, preL-HSPCs carrying the DNMT3A^Mat and exposed to FBM had significantly higher secondary engraftment compared to controls (Figures 3D-E). These results suggest that FBM provides higher selective advantage to older DNMT3A^Mat cells through increased self-renewal.

To extend the results beyond DNMT3A R882 mutations, the effects of FBM were tested on DNMT3A haplo-insufficient mice ( AA7 3A^hapl°) as frameshift mutations causing such genotype are common among humans³⁵. Significantly increased engraftment of DNMT3A^hapio was noted when cells from two-month-old mice were exposed to FBM both in primary and secondary recipients (Figures 7A-C). Similar results were obtained when one-year-old cells were transplanted (Figures 7C-E). No significant differences were observed between engraftment of one-year-old DNMT3A^hapio and DNMT3A^Mat mice when transplanted to FBM (Figure 7F). This result suggests that different DNMT3A mutations (and not just R882) have a selective advantage under FBM conditions.

FBM does not support preL-HSPCs carrying SRSF2 P95H mutations derived from a genetic rodent model

In order to understand whether the same effect holds for other pre-Leukemic mutations (pLMs), a SRSF2 P95H knock in model was used. BM cells derived from two-month-old mice were injected IF into FBM or NBM NSG mice. In contrast to the findings with DNMT3A^Mat cells, no significantly higher engraftment of SRSF2^Mat cells under FBM conditions was observed (Figure 8A). Furthermore, no differences in engraftment were observed in NBM or FBM mice following transplantation of one-year-old SRSF2^Mat BM-derived cells in both primary and secondary transplantation (Figure 8B). Altogether, these results so far indicate that FBM provides a selective advantage to preL-HSPCs carrying DNMT3A^Mat.

PreL-HSPCs carrying DNMT3A^Mut exposed to FBM maintain their stem cell pool

To further study why preL-HSPCs carrying DNMTSA^¹ have increased self-renewal upon exposure to FBM, we injected one-year-old DNMT3A''^A'^M and DNMT3A^ HSPCs to either FBM or NBM mice. Three days following injection, (10 days after low dose irradiation), single LIN-KIT+ (LK) cells were isolated and single cell RNA-seq (scRNA-seq) was performed using the MARS-Seq technology. After filtering outlier cells, 1999 LK cells were identified from the different conditions.

All the experiments involving injection of cells to either NBM or FBM mice, showed a marked reduction in HSCs. The only exception to this rule were the DNMTSA^ cells exposed to FBM who maintained their HSC pool and clustered together with naive cells (LSK cells extracted directly from BM with no injection)(Figures4A-B).DNMT3A^Mat cellsexposed to FBM had 10foldmoreHSCscomparedtoDNMT3A^ cellsexposedtoFBM (p=2.3e-9),and a significantincreaseintheirHSC poolcomparedtoDNMT3A^Mat cellsexposedtoNBM (p=0.03) (Figure4C).ThemaintenanceofHSCsamongDNMT3A^Mat cellsexposedtoFBM wasfollowed by expansion ofmyeloid progenitorsasopposed to enrichmentoflymphoid progenitorsin the naiveLSK cells(Figures4A-B).LSK cellsfrom otherconditionsaftertransplantationlosesome oftheirHSCswhichdifferentiatemainlyintothemyeloidlineage(Figures4A-B).Altogetherthe scRNA-seqdataconfirmedthehypothesisthatDNMT3A^Mat cellsexposedtoFBM undergo selfrenewal,whileallotherconditionsundergo(mostlymyeloid)differentiation.

PreL-HSPCscarryingDNMT3A^Mut exposedtoFBM activateinflammatorypathways.

To betterunderstand why DNMT3A^Mut HSCsexposed to FBM undergo increased selfrenewalandnotdifferentiation,adifferentialexpression analysisfrom thescRNA-seq datawas performed.Altogether,theanalysisofthescRNA-seq suggestedthatDNMT3A^Mat andtoalesser extentDNMTSA^ exposedtoFBM activatetheIFNy,andIL-6pathwaysandpossiblytheIFNa andTNFasignalingpathways(datanotshown).

IL-6and TNFaaresecretedbyFBM

As itwasdiscoveredthatDN3TSA^ cellsexposed toFBM demonstrated an activation ofseveralinflammatorypathways,thepresentinventorsnexthypothesizedthatadipocytesmight secrete pro-inflammatory cytokineswhich willinduce such a response.A multiplex cytokine assay (FirePlex,Abeam)wasused to measurecytokinelevelsin theBM fluid from micewith and withoutFBM.Cytokinesecretion wasanalyzed in 1yearold NBM donormicecomparing Mutto WT (no cellsinjected). No significantdifferencesweredetected in cytokinessecretion between donorMutand WT micewith NBM (datanotshown). They also examined cytokine secretion in NSG micewith eitherNBM orFBM priorto donorinjection.NSG FBM micehad higherlevelsofIL-6,IL-2,TNFa,MIPla,MIPlb and IL-ip secretion (Figure4D).NSG FBM mice following castration (FBM+CAS) also demonstrated an increase of IL-6 secretion comparedtoNBM (Datanotshown).Importantly,followingadministrationofPPARyitoFBM NSG mice,priorto donorinjection,thelevelsofIL-6 and MIPlb weresignificantlydecreased, suggesting correlation to FBM condition.To further validate thatthe cytokine secretion is specifictotheFBM,cytokinelevelsin themiceserum weremeasuredinparallel.A significant differenceswerenoted only in MCP1between FBM and NBM (Figure4E).To learn whether cytokine levels under FBM conditions remain increased following cell transplantation,the presentinventorsfirsttransplanted two monthsDNMT3A^Mat andDNMT3A^ BM cellsto FBM and NBM mice. A significant increase in IL-6 secretion was noted regardless of the genotype of injected cells (data not shown). Nonetheless, transplantation of one-year-old DNMT3A^Mat and DNMT3A^W[ BM cells to FBM also resulted in an increase in IL-6 secretion compare to NBM, regardless of the genotype of injected cells and decreased following administration of PPARyi (Figures 4F-G). These results suggest that the increased IL-6 score (IL-6 signaling activation) observed in DNMT3A^Mat cells might be the result of IL-6 secretion from FBM regardless of which cells are transplanted. Furthermore, following transplantation of one-year-old DNMTSA^⁷ or DNMT3A^Mat BM derived cells to FBM NSG mice, a significant increase in the secretion of TNFa was observed (Figures 4F-G). Altogether it may be concluded that IL-6 levels in BM fluid were increased under all conditions and were independent of cells injected. TNFa showed a similar but a less consistent pattern.

IL-6 provides selective advantage to preL-HSPCs carrying DNMT3A^Mut in a methylcellulose colony forming assay

The interaction between adipocytes and DNMT3A^Mat BM-derived cells via IL-6 was validated in vitro by serial replating of preL-HSPCs carrying the DNMT3A^Mat in methylcellulose. DNMT3A^Mut or DNMT3A^WT BM-derived cells were used for the Colony Forming Cell (CFC) assay with and without IL-6. DNMT3A^Mat cells in the presence of IL-6 demonstrated increased self-renewal over the three control groups (DNMT3A^WT cells with IL-6, DNMT3A^WT without IL- 6, and DNMT3A^Mat without IL-6). The control groups did not survive the third replete compared to the DNMT3A^Mat BM derived cells with IL-6 who survived two more cycles of replating than the controls (Figures 5A-B).

In vivo treatment with neutralizing IL-6 antibodies (Ab) results in decreased self- renewal of DNMT3A^Mut cells under FBM conditions

To study the effects of IL-6 on DNMT3A preL-HSPCs in vivo the present inventors transplanted one-year-old DNMT3A^Mat BM-derived cells into NBM and FBM NSG mice that had been treated with neutralizing IL-6 antibodies (Ab) two days before and seven days after transplantation. The administration of neutralizing IL-6 Ab resulted in a significant decrease in engraftment of DNMT3A^Mat BM derived cells (Figure 5C). For the secondary transplantations cells were harvested from primary mice and injected to NBM NSG mice with no further treatment. Secondary transplantation experiments provided evidence that anti IL-6 neutralizing Abs can reduced the self-renewal advantage harbored by DNMT3A^Mat preL-HSPCs under FBM conditions (Figure 5D).

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is/are hereby incorporated herein by reference in its/their entirety.

Claims

WHAT IS CLAIMED IS:

1. A method of treating or preventing a myeloid malignancy in a subject comprising a mutation in DNA methyltransferase 3 alpha (DNMT3A) in a genome thereof comprising:

(b) administering to the subject a therapeutically effective amount of an agent that down-regulates an amount and/or activity of interleukin-6 (IL-6), when said DNMT3A mutation is indicated in the genome, thereby treating or preventing the myeloid malignancy.

2. A composition comprising an agent that down-regulates an amount and/or activity of IL-6 for the treatment or prevention of a myeloid malignancy in a subject, wherein the subject is selected as comprising a DNMT3A mutation in a genome thereof.

3. The method or composition of claims 1 or 2, wherein said DNMT3A mutation is a point mutation a deletion, a frameshift mutation, a nonsense mutation and a missense mutation.

4. The method or composition of any one of claims 1-2, wherein said DNMT3A mutation is R882H.

5. The method or composition of any one of claims 1-4, wherein said myeloid malignancy is selected from the group consisting of acute myeloid leukemia (AML), primary myelofibrosis, Hypereosinophilic Syndrome (HES), myelodysplastic syndrome (MDS), acute promyelocytic leukemia (APL), chronic myelomonocytic leukemia (CMML), chronic neutrophilic leukemia (CNL), acute undifferentiated leukemia (AUL), anaplastic large-cell lymphoma (ALCL), prolymphocytic leukemia (PML), juvenile myelomonocyctic leukemia (JMML), adult T-cell leukemia AML with trilineage myelodysplasia (AML/TMDS), mixed lineage leukemia (MLL), myeloproliferative disorders (MPD), chronic myeloid leukemia (CML)and myeloid (granulocytic) sarcoma, Systemic mastocytosis, mast cell neoplasm, clonal cytopenia of indetermined significance, clonal hematopoiesis, follicular lymphoma, Blastic plasmacytoid dendritic cell neoplasm and chronic neutrophilic leukemia.

6. The method or composition of any one of claims 1-4, wherein said myeloid malignancy is selected from the group consisting of AML, MDS, CMML and primary myelofibrosis.

7. The method or composition of any one of claims 1-4, wherein said myeloid malignancy is AML.

8. The method or composition of any one of claims 1-7, wherein the sample comprises peripheral blood cells and/or bone marrow cells.

9. The method or composition of any one of claims 1-8, wherein the analyzing is effected at the protein level.

10. The method or composition of any one of claims 1-9, wherein the analyzing is effected at the nucleic acid level.

11. A method of treating and/or preventing a disease associated with a fatty bone marrow in a subject comprising:

(a) analyzing cells of a bone marrow sample for the presence of adipocytes; and

12. The method of claim 11, wherein said disease is a metabolic disease.

13. The method of claim 11, wherein the subject is above 50 years old.

14. The method of claim 11, wherein the subject is prediabetic.

15. The method of claim 11, wherein said disease is selected from the group consisting of osteoporosis, type 1 diabetes, Cushing’s disease, estrogen deficiency, and anorexia nervosa.

16. The method of claim 11, wherein said analyzing is effected by MRI.

17. The method or composition of any one of claims 1-12, wherein the agent is an antibody.

18. The method or composition of any one of claims 1-16, wherein the agent is a polynucleotide agent that hybridizes to a nucleic acid encoding IL-6.