METHODS AND COMPOSITIONS FOR DIAGNOSING AND TREATING CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML)

Abstract

In the present invention, inventors have used high throughput sequencing to identify novel mutations in ABCA1 in CM ML patient samples. Further studies in a mouse model of myelomonocytic leukemia driven by hematopoietic Tet2 deficiency have shown that these somatic mutations abrogate the tumor suppressor function of WT ABCA1, resulting in the failure to suppress canonical IL3-receptor beta signaling-driven myelopoiesis. The loss of the myelo-suppressive function of ABCA1 mutants can be overcome by raising HDL levels through overexpression of the human apolipoprotein A-1 (apoA-1) transgene. Inventors have also shown that both IL-3Rbeta blocking antibody and cyclodextrin prevented the proliferation of ABCA1 mutant-transduced Tet2 deficient BM cells similar to the effect of ABCA1-WT overexpression. Accordingly, the invention relates to a method for predicting the survival time of a subject NI suffering from CM ML comprising the step identifying at least one ABCA1 and to a method for treating said subject with HDL/ABCA recombinant (ApoA-1); cylodextrin and/or anti-IL-3Rbeta antibody.

Claims

1. A method for diagnosing a chronic myelomonocytic leukemia (CMML) in a subject, wherein said method comprising a step of detecting a at least one mutation in a ATP-binding cassette A1 (ABCA1) gene, RNA or protein in a biological sample obtained from said subject, wherein the presence of a mutation is indicative of a CMML.

2. A method for predicting the survival time of a subject suffering from chronic myelomonocytic leukemia (CMML) comprising the steps of i) identifying at least one mutation in ATP-binding cassette A1 (ABCA1) at gene, RNA or protein in a biological sample obtained from the subject; and ii) concluding that the subject will have a short survival time when at least one mutation in ABCA1 is identified or concluding that the subject will have a long survival time when any mutation is not identified in ABCA1.

3. The method according to claim 1, wherein the at least one mutation is ABCA1-P711L, ABCA1-A1291T, ABCA1-G1421R, ABCA1-P1423S and/or ABCA1-A2011T.

4. The method according to claim 1, wherein multiple mutations are identified simultaneously, separately or sequentially.

5. The method according to claim 1, wherein the biological sample is a tissue biopsy.

6. The method according to claim 1, wherein the biological sample is a bone marrow sample.

7. The method according to claim 1, wherein the biological sample is a blood sample.

8. A method for treating a subject suffering from chronic myelomonocytic leukemia (CMML) comprising a step of administering to the subject a therapeutically effective amount of HDL/ABCA recombinant (ApoA-1); cylodextrin and/or anti-IL-3Rbeta antibody.

9. The method according to claim 8 wherein said subject is i) diagnosed as having CMML by detecting, in a biological sample obtained from said subject, at least one mutation in an ATP-binding cassette A1 (ABCA1) gene, RNA or protein, wherein the presence of a mutation is indicative of CMML, and/or ii) identified as having a short survival time by identifying at least one mutation in an ATP-binding cassette A1 (ABCA1) at gene, RNA or protein in the biological sample.

10. A kit for performing the methods of the present invention, wherein said kit comprises means for measuring at least one mutation in ABCA1 protein and/or detecting ABCA1 SNP that is indicative of the risk of having a short survival time in a subject.

11. The method according to claim 8, wherein the at least one mutation is ABCA1-P711L, ABCA1-A1291T, ABCA1-G1421R, ABCA1-P1423S and/or ABCA1-A2011T.

12. The method according to claim 8, wherein multiple mutations are identified simultaneously, separately or sequentially.

13. The method according to claim 8, wherein the biological sample is a tissue biopsy.

14. The method according to claim 8, wherein the biological sample is a bone marrow sample.

15. The method according to claim 8, wherein the biological sample is a blood sample.

Description

FIGURES

[0108] FIG. 1. Identification of loss of function ABCA1 mutants in CMML. (A) [3H]-thymidine proliferation assay (pulsed for 2 h) were performed in HEK293 cells transiently transfected with plasmid constructs expressing ABCA1-WT, ABCA1 mutants or empty vector. (B) Abca1 transcripts expressed as arbitrary unit (a.u.) in several leukemic cell lines. THP-1 cells are monocytic leukemia cells that express relatively high amount of Abca1 transcripts compared to myeloblastic MV411 and HL60 cells, acute myeloid leukemia (AML) HEL, OCIAML3, MOLM13 and KG1 cells, chronic myeloid leukemia (CML) MOLM6 and K562 cells or lymphoma U237 and OCILY3 cells (C) or in THP-1 monocytic leukemia cells transduced for 72 h with lentiviral particles expressing ABCA1-WT, ABCA1 mutants or empty vector. (D) THP-1 cells transduced for 72 h with ABCA1 mutants exhibit growth advantage over a 7-day period compared with ABCA1-WT. (E) Expression of phosphoSTAT5. (F) Cholesterol efflux from the aforementioned THP-1 macrophages was quantified after overnight [.sup.3H]-cholesterol loading and determined 6 hours after incubation with apoA-I (15 μg/mL) and BODIPY staining (G) determined by flow cytometry in these cells.

[0109] FIG. 2. Loss of functional ABCA1 reduces tumor suppression in myelomonocytic leukemia-induced by Tet2 loss. (A) Modulation of Abca1 mRNA expression levels in the BM of the aforementioned mouse models. (B) Quantification of the percentage of peripheral blood myeloid cells determined by hematology cell counter over the course of 12 weeks after Poly:IC injection in recipients mice transplanted with empty, ABCA1-WT or ABCA1 mutants expressing Mx1-Cre.sup.+Tet2.sup.fl/fl BM. (C) Peripheral blood myeloid subsets (CD115.sup.+Ly6C.sup.hi and CD115.sup.+Ly6C.sup.lo monocytes and CD115.sup.−Ly6C.sup.hi neutrophils) were also quantified in these mice at the indicated time point. (D) Original magnification, ×200. Arrows indicate extensive cellular infiltrate. Quantification of spleen weight of these mice. (E) Representative quantification of CD11b.sup.+ GrI.sup.+ myeloid cells determined by flow cytometry in the spleen of recipient mice transplanted with control or Mx1-Cre.sup.+Tet2.sup.fl/fl BM expressing empty, ABCA1-WT or ABCA1 mutants. The results are means ±SEM of 5-9 animals per group. ND, not detectable. * P<0.05 versus empty control on a Tet2 deficient background. § P<0.05 versus ABCA1-WT #P<0.05 and ##P<0.001 versus Mx1-Cre.sup.+ controls.

[0110] FIG. 3. ABCA1 mutants supports Tet2-deficient HSPC expansion and myeloid lineage commitment and spreads CMML-like disease in serial BM transplantation. Quantification of hematopoietic stem (A) and progenitor (B) cells in the BM of recipient mice transplanted with control or Mx1-Cre.sup.+ Tet2.sup.fl/fl BM expressing empty, ABCA1-WT or ABCA1 mutants. Lineage(Lin).sup.− Sca1.sup.−c-Kit.sup.+ LSK cells are hematopoietic stem and progenitor cells; Lin.sup.−Sca1.sup.−c-Kit.sup.+CD34.sup.hiFcγR.sup.hi GMPs are granulocyte-monocyte progenitors and Lin.sup.−Sca1.sup.−c-Kit.sup.+CD34.sup.hiFcγR.sup.low CMPs are common myeloid progenitors. Results are mean±SEM of 5-9 animals per group. (C) Quantification of hematopoietic progenitors and (D) myeloid cells in BM cultures isolated from Mx1-Cre.sup.+Tet2.sup.fl/fl BM expressing empty, ABCA1-WT or ABCA1 mutants and grown ex vivo for 72 h in liquid culture in presence or absence of 6 ng/ml IL-3 and 2 ng/mL GM-CSF. (E) Spleen weight or (F) cholesterol content of recipient mice serially transplanted with control or Mx1-Cre.sup.+Tet2.sup.fl/fl BM expressing empty, ABCA1-WT or ABCA1 mutants. Results are means±SEM of 5-9 animals per group. * P<0.05 versus empty control on a Tet2 deficient background. § P<0.05 versus ABCA1-WT #P<0.05 versus Mx1-Cre.sup.+ controls.

[0111] FIG. 4. ABCA1 invalidation propagates myelopoiesis and accelerates extramedullary hematopoiesis on a Tet2 deficient background. (A) Modulation of Abca1 and Tet2 mRNA expression levels in the BM of the aforementioned mouse models. (B) Quantification of the percentage of peripheral blood myeloid cells determined by hematology cell counter over the course of 20 weeks after Poly:IC injection in recipients mice transplanted with the BM from Mx1-Cre.sup.+, Mx1-Cre.sup.+Abca1.sup.fl/fl, MX1-Cre.sup.+Tet2.sup.fl/fl and Mx1-Cre.sup.+Tet2.sup.fl/flAbca1.sup.fl/fl mice. (C) Peripheral blood myeloid subsets (CD115.sup.+Ly6C.sup.hi and CD115.sup.+Ly6C.sup.lo monocytes and CD115.sup.−Ly6C.sup.hi neutrophils) were also quantified in these mice at the indicated time point. (D) Original magnification, ×200. Arrows indicate extensive cellular infiltrate. Quantification of myeloid subsets (oesinophils, neutrophils, monocytes and red pulp macrophages (RPMs)) in the spleen. (E) Quantification of hematopoietic stem and (F, G) progenitor cells in the BM of these mice. Lineage(Lin).sup.−Sca1.sup.−c-Kit.sup.+ LSK cells are hematopoietic stem and progenitor cells; Lin.sup.−Sca1.sup.−cKit.sup.+CD34.sup.hiFcγR.sup.low MEPs are megakaryocyte-erythrocyte progenitors; Lin.sup.−Sca1.sup.−c-Kit.sup.+CD34.sup.hiFcγR.sup.hi GMPs are granulocyte-monocyte progenitors and Lin.sup.−Sca1.sup.−c-Kit.sup.+CD34.sup.hiFcγR.sup.low CMPs are common myeloid progenitors. Results are mean±SEM of 5-7 animals per group. ND, not detectable. #P<0.05 and ##P<0.001 versus Mx1-Cre.sup.+ controls.

[0112] FIG. 5. Cholesterol accumulation couples ABCA1 invalidation and Tet2 deficiency to IL-3 receptor β signaling hypersensitivity. (A-D) Quantification of BODIPY staining by flow cytometry expressed as mean fluorescence intensity (MFI) as a surrogate of cellular cholesterol neutral lipid per cell in BM hematopoietic stem (A and C) and progenitor (B and D) cells (i.e, LSKs, MEPs, CMPs and GMPs) of recipient mice transplanted with Mx1-Cre.sup.+, Mx1-Cre.sup.+ Abca1.sup.fl/fl, MX1-Cre.sup.+Tet2.sup.fl/fl and Mx1-Cre.sup.+Tet2.sup.fl/flAbca1.sup.fl/fl BM (A and B) or control and Mx1-Cre.sup.+Tet2.sup.fl/fl BM expressing empty, ABCA1-WT or ABCA1 mutants (C and D). (E) Expression of mRNA was normalized to m36B4. mRNA levels were expressed as percentage over WT whole BM cells. Proliferation rates were determined after 2 h [.sup.3H]-thymidine pulse labeling in BM cells from empty, ABCA1-WT and ABCA1 mutants-transduced animals on a Tet2 deficient background that were grown for 72 h in liquid culture in the presence or absence of 6 ng/mL IL-3 and 2 ng/mL GM-CSF and the indicated chemical compounds.

[0113] FIG. 6. HDL overcome loss-of-function ABCA1 mutants and limit the myeloproliferative disorder induced by these mutants in Tet2 deficient mice. (A) Proliferation rates were determined after 2 h [.sup.3H]-thymidine pulse labeling in BM cells from empty, ABCA1-WT and ABCA1 mutants-transduced animals on a Tet2 deficient background that were grown for 72 h in liquid culture in the presence or absence of 50 μg/mL PEG-HDL. (B) Quantification of BODIPY staining was determined by flow cytometry in these cells and expressed as the mean fluorescence intensity (MFI). (C) Results are means±SEM of cultures from at least three independent mice. (E) Levels of hapoA-I (D) and plasma HDL-cholesterol levels were determined in WT or apoA-I transgenic recipient mice transplanted with Mx1-Cre.sup.+Tet2.sup.fl/fl BM transduced with lentiviral particles expressing ABCA1-WT, ABCA1 mutants or empty vector. (F) Peripheral blood CD11b.sup.+GrI.sup.+ myeloid subsets and spleen weight were quantified at the end of the study period (i.e, 7 weeks post Poly:IC injection that followed a 5 week recovery period post BMT) in WT or apoA-I transgenic recipient mice transplanted with Mx1-Cre.sup.+Tet2.sup.fl/flBM transduced with lentiviral particles expressing ABCA1-WT, ABCA/mutants or empty vector. Results are means±SEM of 4-5 animals per group. * P<0.05 versus empty control transduced animals on a Tet2 deficient background. § P<0.05 versus ABCA1-WT #P<0.05 versus non-HDL treated conditions or transduced animals on a non-ApoAI.sup.Tg background.

EXAMPLE

Material & Methods

[0114] Genetic Analysis of Primary Patient Samples.

[0115] Peripheral blood and/or bone marrow samples were collected from 26 patients with CMML; informed consent was obtained from all patients included in this study. Matched normal tissue in the form of a buccal swab was available for all patients. Genomic DNA was extracted from viably frozen peripheral blood granulocytes and buccal swabs. High-throughput DNA sequence analysis was used to screen for mutations in ABCA1, ABCG1, NR1H2, and NR1H3. All DNA samples were whole genome amplified using ∅29 polymerase to ensure sufficient material was available for sequence analysis. M13-appended gene-specific primers were designed to amplify and sequence all coding exons of all isoforms of the above mentioned genes. Primer sequences and the genomic coordinates of all amplicons sequenced are included in Supplemental Table 1. Bidirectional sequence traces were analyzed for missense and nonsense mutations using Mutation Surveyor (Softgenetics, Inc., State College, Pa.), and all traces were reviewed manually and with Mutation Surveyor for the presence of frameshift mutations. Mutations were annotated according to the predicted effects on coding sequence using NM_005502.2, NM_004915.3, NM_007121.4, and NM_001130101.1 as the reference sequence for ABCA1, ABCG1, NR1H2, and NR1H3 respectively. Non-synonymous mutations were first compared to published SNP data (dbSNP, http://www.ncbi.nlm.nih.gov/projects/SNP) such that previously annotated SNPs were not considered pathogenic mutations. Missense mutations not in the published SNP database were annotated as somatic mutations based on either on reported data demonstrating these are somatic mutations or sequence analysis of that demonstrated these mutations were present in tumor and not in matched normal DNA. All somatic mutations were validated by resequencing non-amplified source DNA for the particular amplicon where the mutation was noted. Genomic DNA from paired samples was verified to belong to the same patient by genotyping of the specimens for 42 highly polymorphic single-nucleotide polymorphisms using mass-spectrometry based genotyping as described previously. In order to determine whether the genes in which non-synonymous mutations were identified were mutated at a rate higher than expected by chance alone, we first calculated the rate of non-synonymous mutations in the sequenced genes. We then performed binomial test in R (http://www.r-project.org/) to compare the rate of non-synonmous mutations in the genes identified in this study with the expected background rate of 0.22-2.5×10.sup.−6 synonymous mutations identified in several prior large-scale sequencing studies.sup.2-10 as well as the expected ratio of silent:non-silent mutations (0.31-0.41) from the same studies.

[0116] Mice and Treatments.

[0117] WT, Mx1-Cre.sup.+ (B6. Cg-Tg(Mx1-cre).sup.1CgnJ), Tet2.sup.fl/fl (B6; 129 S-Tet2.sup.tm1.1Iaai/J) Abca1.sup.fl/fl mice (B6. 129S6-Abca1.sup.tm1IJp/J) and human apoA-1 transgenic (B6.Tg(ApoA1).sup.1Rub/J), were obtained from the Jackson Laboratory. Human apoA-1 transgenic mice were selected based on the human apoA-1 levels in the range of 150-300 mg/dL (ELISA do not detect mouse apoA-1) as previously described (Rubin et al., 1991; Yvan-Charvet et al., 2010). Mx1-Cre.sup.+ Tet2.sup.fl/fl mice, Mx1-Cre.sup.+ Abca1.sup.fl/fl mice and Mx1-Cre.sup.+ Tet2.sup.fl/flAbca1.sup.fl/fl littermates mice were used for this study. Bone marrow (BM) transplantation into lethally irradiated WT recipients and serial BM transplantation studies were performed as previously described ((Yvan-Charvet et al., 2010)). After 5 weeks of reconstitution, mice were i.p injected with poly:IC (250 μg/injection with a cumulative dose of 750 μg/mice, Invivogen) to induce gene deletion/recombination. Mice were used between 3 and 5 months after the injections of poly:IC depending of the experiment.

[0118] Animal procedures were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committees at Mediterranean Center of Molecular Medicine (C3M). Mice were maintained on a 12 h light/12 h darkness lighting schedule. Animals had ad libitum access to both food and water.

[0119] Plasmids.

[0120] Mouse ABCA1 cDNA, with a homology of 97% to human ABCA1 cDNA, was used to generate P711L, A1291T, G1421R, P1423S and A2011T mutant cDNAs and cloned into pLKO lentiviral vectors to genetically perturb cells by lentiviral infection and avoid cross reactivity.

[0121] Lentiviral BM Transplantation.

[0122] The lentiviral BM transplant assay was performed as previously described (Gautier et al., 2013). In brief, Mx1-Cre.sup.+ and Mx1-Cre.sup.+ Tet2.sup.fl/fl mice were injected with 5-fluorouracile (3 mg/mice of 5-FU, F6627, Sigma) 3 days before the experiment to enrich HSPCs within the BM. Control, ABCA1-WT and ABCA1-mutant lentiviral particles (pLKO lentiviral vector containing a MSCV-IRES-EGFP sequence, Genecust) were tittered and used to transduce Mx1-Cre.sup.+ or Mx1-Cre.sup.+ Tet2.sup.fl/fl cells. BM cells were cultured for 24 h in transplantation media (RPMI+10% FBS+6 ng/ml IL-3 (Corning), 10 ng/ml IL-6, and 10 ng/ml stem cell factor (Milteny Biotech)) and treated with lentiviral particles (MOI of 5 in the presence of polybrene (Sigma)). After washing, the cells were used for BM transplantation into lethally irradiated WT or human apoA-1 transgenic recipient mice as indicated in the figure legends. The transduction efficiency ranged from 70-90% in LSK cells before implantation as previously described (Gough et al., 2003) (Pikman et al., 2006) (Westerterp et al., 2012). After 5 weeks of reconstitution, mice were i.p injected with poly:IC (250 μg/injection with a cumulative dose of 750 μg/mice, Invivogen) to induce gene deletion/recombination. Mice were used between 3 and 5 months after the injections of poly:IC depending of the experiment.

[0123] White Blood Cell Counts

[0124] Leukocytes, differential blood counts, platelets and erythrocytes were quantified from whole blood using a hematology cell counter (HEMAVET® 950).

[0125] Histopathology

[0126] Mice were euthanized and tissues were harvested and fixed in 4% paraformaldehyde. Spleen was serially paraffin sectioned using a Microm HM340E microtome (Microm Microtech, Francheville France) and stained with H&E for morphological analysis as previously described (Yvan-Charvet et al., 2010).

[0127] HEK293 cell transfection and culture. HEK293 cells (human embryonic kidney, CRL-1573, ATCC) at a density of 10.sup.6 cells/well were transiently transfected with similar amounts of control empty vector (pcDNA 3.1.sup.+), ABCA1-WT or mutant cDNA using LipofectAMINE 2000 according to the manufacturer's instructions (Invitrogen). Then, cells were incubated for different times in DMEM containing 10% FBS before treatments as indicated in the figure legends.

[0128] Human THP-1 monocytic leukemia cells and treatments. THP-1 monocytes (human acute monocytic leukemia cell line, TIB-202, ATCC) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) at 37° C. in 5% CO2. Non-adherent monocytes were transduced at MOI of 5 with control, ABCA1-WT and ABCA1-mutant lentiviral particles (pLKO lentiviral vector containing a CMV promoter, Genecust) in the presence of polybrene (Sigma) and used 3 days later for experiments as described in the figure legends. THP1 cells were treated for 16 hours with puromycin 24 hours after transfection to improve the transient transduction/transfection efficiency up to 60% to 80% (data not shown), which slowed down the proliferation rate of these cells. In some experiments, stable overexpressing ABCA1-WT and ABCA/-mutants THP-1 macrophages were generated after lentiviral transduction and GFP selection of a puromycin resistant pLKO vector containing ABCA/gene.

[0129] BM Harvest and Treatment

[0130] Briefly, femurs were flushed with ice-cold PBS and centrifugated for 5 min at 1,000 rpm to extract BM cells. After red blood cell lysis, over 90% of BM cells were CD45-positive cells of hematopoietic origin (Westerterp et al., 2012). Primary BM cells were resuspended in IMDM (Gibco) containing 10% FCS (STEMCELL Technologies) and cultured for 1 h in tissue culture flasks to remove adherent cells, including macrophages. The transduction rate of control, ABCA1-WT and ABCA 1-mutant lentiviral particles was determined after BM transplantation as described above. Suspended cells were then normalized to the same concentration and cultured for 72 h in the presence of 6 ng/mL IL-3 and 2 ng/mL GM-CSF (R&D Systems). In some experiments, the cyclodextrin (Sigma) was used at the final concentration of 5 mM, tempol (EMD Millipore) at 4 mM and anti-IL3Rbeta AF549 antibody (R&D Systems) at 50 μg/mL.

[0131] [.sup.3H]-Thymidine Proliferation Assay

[0132] For proliferation assays, cells were pulsed for 2 h with 2 μCi/ml [.sup.3H]-thymidine, and the radioactivity incorporated into the cells was determined by standard procedures using a liquid scintillation counter.

[0133] Isotopic Cholesterol Efflux Assay

[0134] THP-1 monocytes were treated with 100 nmol/L PMA (Phorbol myristate acetate) for 24 hour to facilitate differentiation into macrophages and cultured for 24 h in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) containing 2 μCi/ml of [3H]-cholesterol. Cholesterol efflux was performed for 6 h in 0.2% BSA DMEM containing 15 μg/mL apoA-I. The cholesterol efflux was expressed as the percentage of the radioactivity released from the cells in the medium relative to the total radioactivity in cells plus medium (Yvan-Charvet et al., 2010).

[0135] Cellular and Tissue Cholesterol Content

[0136] Total lipids were extracted with chloroform/methanol from total cell lysates. Cholesterol mass in cells was determined using colorimetric kits (Wako Chemicals).

[0137] Flow Cytometry Analysis.

[0138] BM cells, peripheral blood and splenocytes were collected from leg bones, blood and spleen cells after manual flushing or grinding, lysis to remove red blood cells and filtering through a 40-μm cell strainer as previously described (Yvan-Charvet et al., 2010). For peripheral blood leukocytes analysis, 100 μL of blood were collected into EDTA tubes before red blood cell lysis and filtration. Freshly isolated BM, spleen and blood cells were stained with the appropriate antibodies for 30 min on ice. Cellular cholesterol content was quantified using the Bodipy-cholesterol probe (Life Technologies). Phosphoflow staining was performed according to the manufacturer's instruction (BD Biosciences). HSPC and hematopoietic progenitor subsets and myeloid cell populations were analyzed by flow cytometry using an LSR Fortessa (Becton Dickinson) or sorted with a FACSAria II instrument (Becton Dickinson). All gating strategies are depicted in the Figures. Data were analyzed with FlowJo software (Tree Star).

[0139] Cholera Toxin staining

[0140] After a wash in complete growth medium, THP-1 transduced monocytes were stained 15 min at 37° C., in 1 μg/ml working solution of Cholera Toxin Subunit B, Alexa Fluor 594 conjugate (Invitrogen, C34777). Cells were then stained with 1 ng/ml working solution of DAPI (4′,6-diamidino-2-phenylindole) and washed 3 times in PBS1X. Immunostaining of cells was read on a Nikon Confocal MR microscope.

[0141] Antibodies. TCR-β (H57-597), F4/80 (BM8), CD2 (RM2-5), CD3e (145-2C11), CD4 (GK1.5), CD8b (53-6.7), CD19 (eBio1D3), CD45R (B220, RA3-6B2), Gr-1 (Ly6G, RB6-8C5), Cd11b (Mac1, M1/70), Ter119 (Ly76) and NK1.1 (Ly53, PK136)-FITC were all from eBioscience and used for lineage determination. c-Kit (CD117, ACK2)-APCeFluor780 from eBioscience, Sca-1-Pacific blue from Biolegend, FcgRII/III-PE (CD16/32, 2.4G2), CD34 (RAM34)-AlexaFluor 647, CD135 (Flt3, A2F10)-PE, CD150 (Slamf1, TC15-12F 12.2)-PECy7 were from Biolegend and used to quantify HSPCs and progenitor subsets. Peripheral leukocytes were stained with CD115 (AFS98)-APC, CD45 (30-F11)-APCCy7 and Ly6C/G or Gr-1 (RB6-8C5)-PercPCy5.5 from eBioscience and BD Biosciences, respectively.

[0142] RNA analysis

[0143] Total RNA extraction, cDNA synthesis and real-time PCR were performed as described previously (16). m36B4 RNA expression was used to account for variability in the initial quantities of mRNA.

[0144] Western Blotting

[0145] The expression of ABCA1, TET2, phospho JAK2 and ERK were measured in BM cell by Western blot analysis. Briefly, cell extracts were electrophoresed on 4-20% gradient SDS-PAGE gels and transferred to 0.22-μm nitrocellulose membranes. The membrane was blocked in Tris-buffered saline, 0.1% Tween20 containing 5%(w/v) nonfat milk (TBST-nfm) at room temperature (RT) for 1 h and then incubated with the primary antibody (all from Cell Signaling) in TBST-nfm at RT for 4 h, followed by incubation with the appropriate secondary antibody coupled to horseradish peroxidase. Proteins were detected by ECL chemiluminescence (Pierce). Intensity of each protein strips was quantified using Image J software.

[0146] Statistical Analysis

[0147] Data are shown as mean±SEM. Statistical significance was performed using two-tailed parametric student's t test or by one-way analysis of variance (ANOVA, 4-group comparisons) with a Bonferroni multiple comparison post-test according to the dataset (GraphPad software, San Diego, Calif.). Results were considered as statistically significant when P<0.05.

[0148] Results

[0149] Identification of ABCA1 Somatic Mutations in CMML

[0150] Sequencing of full-length ABCA1, ABCG1 and NR1H2/3 (LXRs) in 26 CMML samples revealed a somatic mutational frequency of 19% of samples for ABCA1 (n=5) and 0% for ABCG1 and NR1H2/3. All mutations were somatic missense mutations with only one mutation observed in each patient sample (data not shown). The identity of the paired samples was verified by Sequenom SNP genotyping demonstrating that the likelihood of a match occurring by chance was <1×10-13 (data not shown). These ABCA1 mutations occur in evolutionarily conserved regions (data not shown). The ABCA1 mutations have not been previously described even though different ABCA1 mutations have been identified in Tangier Disease (Brunham et al., 2006; Sjöblom et al., 2006). Sequencing of other genes implicated in the pathogenesis of CMML in these same samples revealed that ABCA1 mutations co-existed with known oncogenic mutations in JAK2, Flt3, and N-Ras (Emanuel, 2008). We noted that (1) of the 4 genes sequenced, somatic non-synonymous mutations were found in only 2 of the 4 genes and (2) the somatic nonsynonymous mutation rate for ABCA1 was higher than the expected background silent mutation rate and higher than expected by chance alone by binomial tests (p-value of 3.6×10-10 for ABCA1), suggesting that mutations in ABCA1 do not represent passenger gene effects.

[0151] Functional Analysis of ABCA1 Mutations In Vitro

[0152] Given the key role of ABCA1-dependent cholesterol efflux pathway in controlling myeloid expansion (Tall and Yvan-Charvet, 2015), we sought to test whether ABCA/CMML mutations affect cellular proliferation. We used site-directed mutagenesis to introduce each of these five somatic mutations individually into the ABCA1 cDNA. To compare the ability of ABCA1 mutants to control proliferation, we transiently transfected HEK293 cells with the ABCA1 cDNAs. Overexpression of WT-ABCA1 resulted in an approximately 1.7-fold decrease in cell proliferation compared with empty vector-transfected cells (data not shown). All mutants located in either the N- and C-terminal regions (P711L and A2011T), the PEST sequence (A1291T) or the apoA-I binding region (G1421R and P1423S) exhibited a significant reduction in anti-proliferative activity (FIG. 1A and data not shown). We also tested the relevance of these ABCA1 mutations in human THP-1 monocytic leukemia cell lines which express endogenous ABCA1 (FIG. 1B). ABCA1-P711L, ABCA1-A129 IT, ABCA1-G1421R, ABCA1-P1423S and ABCA1-A201IT displayed reductions in anti-proliferative activity, compared to ABCA1-WT in lentivirus transduced cells (FIG. 1C), consistent with observations in HEK293 cells. Although the proliferation rate of THP1 cells was slowed down by transient transfection, we showed a growth advantage of all mutations over a culture period of a week compared to WT-ABCA1 expression (FIG. 1D). Stable cell lines expressing ABCA1-G1421R and ABCA1-A201IT (data not shown) with normal proliferation rate showed growth and proliferative advantages compared to ABCA1-WT (data not shown). We also observed an activation of JAK-STAT signaling pathway in ABCA1 mutant-transduced cells compared to WT-ABCA1 as illustrated by higher levels of pSTAT5 quantified by flow cytometry (FIG. 1E). Because assessment of cholesterol efflux capacity is not practical in suspension cells, we next tested the dependence of ABCA1 mutations on their efflux capacity in differentiated THP-1 macrophages. Under this setting, three out of the five ABCA1 mutations (ABCA1-A129 IT, ABCA1-G1421R, ABCA1-P1423S) showed a decrease in the ability of apoA-1 to promote cholesterol efflux compared with WT-ABCA1 (FIG. 1F). Nevertheless, quantification of BODIPY (bore-dipyrromethene)-neutral lipid staining revealed higher neutral lipid accumulation in ABCA1 mutant-transduced cells compared to WT-ABCA1 (FIG. 1G). Quantification of cellular cholesterol content confirmed these findings (data not shown). The increased proliferation of these cells was also associated with increased cholera toxin subunit B (CTx-B) staining of ABCA1 mutant-transduced cells compared to WT-ABCA1 at the cell surface (ABCA1-A129 IT, ABCA1-G1421R, ABCA1-P1423S) or in intracellular endosomal-like structure (ABCA1-P711L, ABCA1-A20117) (data not shown)suggesting increased formation of cholesterol-rich lipid raft or perturbed intracellular cholesterol trafficking (Dietrich et al., 2001).

[0153] ABCA1 mutants associated with CMML fail to suppress myelopoiesis in vivo. Previous studies have suggested that loss of ABCA1 function alone is insufficient to promote prominent myelopoiesis in hypercholesterolemic mice (Yvan-Charvet et al., 2010). We hypothesized that proliferative effects of ABCA1 mutants observed in CMML might become more evident when combined with other CMML mutant alleles. Tet2 inactivation through loss-of-function mutation is commonly found in CMML (Bowman and Levine, 2017; Solary et al., 2014). Therefore, to assess the in vivo effects of ABCA1 mutants, bone marrow (BM) cells from WT or Mx1-Cre.sup.+Tet2.sup.fl/fl mice (i.e, mice bearing the conditional Tet2 allele and the interferon inducible Cre transgene) were transduced with pLKO-Puro-GFP lentiviral vectors containing WT-ABCA1 or ABCA1 mutants and transplanted into lethally irradiated C57BL/6J mice (data not shown). Animals were analyzed 5 weeks after BM reconstitution (T0) and at the indicated time point following polyinosinic:polycytidylic acid (PIPC) injection (data not shown). Consistent with earlier works (Moran-Crusio et al., 2011; Quivoron et al., 2011), we observed loss of Tet2 expression in the BM of WT recipient mice transplanted with Mx1-Cre.sup.+Tet2.sup.fl/fl BM compared to Mx1-Cre.sup.+ BM (data not shown) and ablation of the gene was paralleled by a significant reduction of 5-hydroxylation of methycytosine (5hmC) in a pool of peripheral blood cells, which reflect the enzymatic activity of TET2 (data not shown). Quantification of Abca1 mRNA expression confirmed similar levels of overexpression of ABCA1-WT and mutants in the BM of Mx1-Cre.sup.+Tet2.sup.fl/fl recipients (FIG. 2A) and controls (data not shown). Despite similar leukocyte counts (data not shown), overexpression of WT-ABCA1 on a Tet2 deficient background caused a marked reduction in myeloid cells (Gr-1.sup.highCD11b.sup.high) in blood over time (FIG. 2B) reflecting mainly lower inflammatory Ly6C.sup.hi monocyte and neutrophil counts (FIG. 2C and data not shown). In contrast, ABCA1 mutants-expressing animals on a Tet2 deficient background exhibited higher peripheral myeloid cells (both monocytes and neutrophils) compared to ABCA1-WT-transduced animals (FIG. 2B, 2C and data not shown). These effects were not observed when ABCA1-WT or mutants were transduced on a WT background (data not shown). T- and B-cell numbers and hematocrit and platelet counts were normal on both backgrounds (data not shown). These data indicate that ABCA1 mutants impede the protective effect of ABCA1-WT in preventing myeloid expansion on a Tet2 deficient background. Importantly, the five ABCA1 mutations identified in CMML patients were found to be loss-of-function mutations as demonstrated by their failure to suppress blood leukocyte counts in the setting of Tet2 deficiency.

[0154] ABCA1 Mutants Fail to Prevent CMML-Associated Extramedullary Hematopoiesis and Splenomegaly.

[0155] Overexpression of ABCA1-WT suppressed the splenomegaly of animals transplanted with Tet2 deficient BM (FIG. 2D data not shown). Despite a variability within the groups, this was statistically not observed in ABCA1 mutant-transduced animals on a Tet2 deficient background (FIG. 2D). Pathological examination revealed robust infiltration of myeloid cells, including significant destruction of normal spleen architecture in ABCA1 mutant-transduced animals on a Tet2 deficient background compared to ABCA1-WT transduced animals, similar to what was observed in empty vector-transduced animals (data not shown). Flow analysis of the spleen confirmed an increased proportion and number of CD11b.sup.+GrI.sup.+ myeloid cells in ABCA1 mutant-transduced animals on a Tet2 deficient background and to some extent on a WT background compared to ABCA1-WT-transduced animals (FIG. 2E and data not shown). An increased percentage of hematopoietic stem/progenitor cells (LSK cells, Lineage.sup.−Sca1.sup.+ c-Kit.sup.+) was also observed in the spleen of ABCA1 mutant-transduced animals compared to ABCA1-WT-transduced animals (data not shown). Thus, unlike WT ABCA1, specific ABCA1 mutants associated with human CMML were unable to limit the increased extramedullary hematopoiesis and splenomegaly that are classical features of CMML.

[0156] ABCA1 Mutants Fail to Suppress Expansion and Myeloid Bias of Tet2 Deficient HSPCs

[0157] Tet2 loss or defective cholesterol efflux pathways leads to BM hematopoietic stem/progenitor cell expansion (HSPCs) and differentiation toward a myeloid lineage fate in vivo (Moran-Crusio et al., 2011; Quivoron et al., 2011; Yvan-Charvet et al., 2010). Analysis of the BM HSPCs showed a reduction of the LSK cells in ABCA1-WT-transduced animals on a Tet2 deficient background compared to empty control-transduced animals. This effect was lost in ABCA1 mutant-transduced animals (FIG. 3A). Although ABCA1 mutants barely altered the percentage of BM megakaryocyte-erythrocyte progenitors (MEPs, Lineage.sup.−Sca1.sup.− c-Kit.sup.+CD34.sup.lowFcγR.sup.low) (data not shown), the granulocyte-monocyte progenitors (GMPs, Lineage.sup.− Sca1.sup.− c-Kit.sup.+CD34.sup.hiFcγR.sup.hi) and the common myeloid progenitors (CMPs, Lineage.sup.− Sca1.sup.− c-Kit.sup.+CD34.sup.hiFcγR.sup.hi) were significantly increased in ABCA1 mutant-transduced BM compared to ABCA1-WT-transduced BM both on WT (data not shown) and Tet2 deficient background (FIG. 3B). To determine whether ABCA1 mutants engage HSPCs to differentiate into the myelomonocytic lineages, BM cells from ABCA1 mutant-transduced animals were cultured ex vivo in the presence of IL-3 and GM-CSF. BM cultures showed that ABCA1 mutants disengaged the suppressive effects of ABCA1 on HSPC expansion (FIG. 3C) and myeloid lineage expansion (FIG. 3D) in the Tet2 deficient background. These findings suggest that in contrast to ABCA1-WT, ABCA1 mutants allow Tet2 deficient HSPCs to proliferate and to favor myelopoiesis as a result of increased IL-3/GM-CSF signaling. Of note, in secondary transplants (data not shown), ABCA1 mutant-transduced animals had a significantly higher percentage of peripheral Gr-1.sup.highCD11b.sup.high myeloid cells compared to ABCA1-WT-transduced animals (data not shown) and failed to suppress splenomegaly (FIG. 3E) or splenic cholesterol accumulation (FIG. 3F). These findings confirm that ABCA1 inactivation not only confers a significant competitive advantage to Tet2 deficient HSPCs in vivo but also a transplantable myeloproliferative disorder.

[0158] ABCA1 deficiency cooperate with Tet2 loss to propagate myeloid transformation In parallel, we crossed Mx1-Cre.sup.+Tet2.sup.fl/fl mice (Moran-Crusio et al., 2011; Quivoron et al., 2011) to Abca1.sup.fl/fl mice (Yvan-Charvet et al., 2010) to generate Mx1-Cre.sup.+Tet2.sup.fl/flAbca1.sup.fl/fl (referred to subsequently as DKO.sup.ΔHSC) mice. BM cells from these mice were subsequently transplanted into lethally irradiated C57BL/6J mice (data not shown). Animals were analyzed 5 weeks after BM reconstitution (T0) and at the indicated time point following polyinosinic:polycytidylic acid (PIPC) injection (data not shown). We confirmed the excision of both Tet2 and Abca1 mRNA expression in the BM of Mx1-Cre.sup.+Tet2.sup.fl/fl mice and Mx1-Cre.sup.+Abca1.sup.fl/fl mice, respectively (FIG. 4A). DKO.sup.ΔHSC mice also had a marked increase in peripheral myeloid cells compared to single Abca1 or Tet2 deficient mice (FIG. 4B, 4C and data not shown). These findings show a cooperative effect between ABCA1 and Tet2 mutations on myeloid expansion. Additionally, Abca1 deficiency exacerbated the splenomegaly of Tet2-deficient animals while having no effect on its own. (FIG. 4D and data not shown). DKO.sup.ΔHSC mice also exhibited higher myeloid cell infiltration compared to single knockout mice after pathological examination (data not shown) with an increased percentage and number of eosinophil, neutrophil, monocyte and red pulp macrophages (RPMs) determined by flow cytometry (FIG. 4E and data not shown). Thus complete deletion of ABCA1 in Tet2-deficient mice increased spleen size and enhanced extramedullary myelopoiesis. Consistently, DKO.sup.ΔHSC mice showed an increased frequency of LSK cells compared with WT or Tet2 deficient mice (FIG. 4F). There was also an additive effect of ABCA1 and TET2 deficiency in promoting BM myeloid progenitor expansion (FIG. 4G). Altogether, these data indicate that concurrent ABCA1 deficiency and Tet2 loss in hematopoietic cells exacerbated myeloid transformation supporting the finding that ABCA1 mutations identified in CMML patients were loss-of-function mutations.

[0159] Cholesterol Accumulation Links ABCA1 Mutants and Tet2 Loss to IL3-Receptor Signaling Hypersensitivity

[0160] We next sought to identify mechanisms responsible for the lack of tumor suppressor function of ABCA1 mutants in Tet2 deficient BM cells. Increased cholesterol accumulation and reduced expression of ABCA1 have been repeatedly observed in cancer cells (Bovenga et al., 2015; Lin and Gustafsson, 2015). Thus, taking advantage of publicly available gene expression datasets (Kunimoto et al., 2018), we first interrogated whether Tet2 deficient LSK, CMP and GMPs cells could transcriptionally regulate cholesterol metabolic pathways. We didn't observe major transcriptional regulation of genes involved in cholesterol metabolism (<10% overall changes) including liver X receptor (LXR) target genes and ABCA1 in Tet2 deficient hematopoietic progenitors (data not shown). The functional behavior of these cells was next assessed by quantifying neutral lipid accumulation in single knockout and DKO.sup.ΔHSC HSPCs by flow cytometry using BODIPY staining. An increase in cellular neutral lipid content in single knockout and DKO.sup.ΔHSC HSPCs and committed myeloid progenitors (i.e, GMP and CMP) was observed compared to controls (FIGS. 5A and 5B). Combined deficiency of Tet2 and Abca1 also led to a cumulative BODIPY-neutral lipid staining per cell (expressed as mean fluorescence intensity) in myeloid progenitors compared to single knockout cells (FIG. 5B) but not in HSPCs (FIG. 5A). Consistently, overexpression of WT-ABCA1 reduced BODIPY staining in Tet2 deficient CMP progenitors but not in Tet2 deficient HSPCs (FIGS. 5C and 5D). However, ABCA1 mutants failed to reduce BODIPY staining on a Tet2 deficient background (FIGS. 5C and 5D) and to some extent on a control background (i.e, ABCA1-A1291T, G1421R and P1423S) (data not shown). The anti-tumor activity of ABCA1 could be linked to inhibition of SREBP-2 and cholesterol biosynthesis target genes that has recently been linked to reduced expression of cell growth facilitating mevalonate products in a solid tumor (Moon et al., 2018). However, the accumulation of cholesterol in ABCA1 mutants-transduced Tet2 deficient BM cells was rather associated with reduced expression of genes in the mevalonate pathway (data not shown), which is a well-known feed-back regulatory mechanism by which sterols accumulation in the ER limit SREBP-2 processing and expression of its target genes (Brown and Goldstein, 2009; Spann and Glass, 2013).

[0161] Given the activation of the IL3-receptor β canonical pathway in myeloid cells with defective cholesterol efflux pathway (Yvan-Charvet et al., 2010) and the hypersensitivity of Tet2 deficient myeloid cells to GM-CSF (Kunimoto et al., 2018), we next assessed whether the myelo-suppressive function of ABCA1 on a Tet2 deficient background could be attributed to its role in removing excess cellular cholesterol in committed myeloid progenitors. Excess cellular cholesterol was removed by treatment with cyclodextrin in ABCA1 mutant-transduced Tet2 deficient BM cells cultured in presence or absence of IL-3 and GM-CSF. We first validated our ex vivo BM culture proliferation assay by showing that inhibition of the IL-3Rβ signaling pathway using IL-3Rβ blocking antibody prevented BM proliferation on IL-3 and GM-CSF treatment (FIG. 5E). We next showed that both IL-3Rβ blocking antibody and cyclodextrin prevented the proliferation of ABCA1 mutant-transduced Tet2 deficient BM cells similar to the effect of ABCA1-WT overexpression (FIG. 5E), without affecting cell viability (data not shown). Consistently, Western blot analysis showed higher levels of pErk1/2 or pJak2 in ABCA1 mutant-transduced Tet2 deficient BM cells compared to ABCA1-WT-transduced cells (data not shown). mRNA expression analysis also revealed higher cyclin D1 and PU.1 gene expression in ABCA1 mutant-transduced Tet2 deficient BM cells (data not shown). These genes are IL-3Rβ signaling targets involved in proliferation and myeloid lineage commitment. In contrast, we did not observe changes in the expression of the key transcription factor C/EBPα or in negative regulators of RAS signaling including dual specificity phosphatase 1 (DUSP1) or sprouty-related, EEVH1 domain-containing protein 1 (Spred1) (Kunimoto et al., 2018). These findings suggest that the tumor suppressor function of ABCA1 relies on its capacity to modulate cholesterol-dependent IL-3Rβ signaling and downstream MAPK and JAK2 signaling. We and others recently showed that reduced autophagy and enhanced mitochondrial reactive oxygen species (ROS) production were two major metabolic events promoting HSPC expansion and myeloid commitment downstream of the IL-3Rβ signaling pathway (Lum et al., 2005; Sarrazy et al., 2016). Thus, we estimated autophagic activity using the Cyto-ID probe by flow cytometry. Reduced autophagic activity was observed in Tet2 and Abca1 deficient myeloid progenitors (data not shown) and also in ABCA1 mutant-transduced CMP and GMP cells compared to ABCA1-WT-transduced BM both on WT and Tet2 deficient (data not shown). Finally, suppression of mitochondrial ROS with tempol prevented the basal proliferation and IL-3/GM-CSF hypersensitivity of ABCA1 mutants-transduced Tet2 deficient BM cells (FIG. 5E). These data confirm that ABCA1 mutations impair the myelo-suppressive function of ABCA1 by increasing the IL-3/GM-CSF hypersensitivity and downstream metabolic sequelae in Tet2 deficient myeloid progenitors.

[0162] Increased HDL Reverses Increased Myelopoiesis and Splenomegaly Caused by ABCA1 Mutants

[0163] Given the ability of increased HDL to suppress HSPC myeloid lineage commitment and rescue the myeloproliferative disorder of mice with defective cholesterol efflux (Yvan-Charvet et al., 2010), we hypothesized that raising HDL would alleviate some of the myeloproliferative phenotypes of ABCA1 mutant-transduced Tet2 deficient animals. First, HDL treatment ex vivo reduced the proliferation rates (FIG. 6A) and BODIPY cholesterol staining (FIG. 6B) of ABCA1 mutant-transduced Tet2 deficient BM cells. We next explored the efficacy of the apoA-1 transgene to increase HDL levels in vivo (FIGS. 6C and 6D). Transduction of ABCA1-A1291T, G1421R and A2011T were selected as proof of concept and compared to BM transduced with empty or ABCA1-WT. Similar to ABCA1-WT-transduced BM cells, the human apoA-1 transgene prevented the peripheral myeloid expansion of mice transplanted with ABCA1 mutants-transduced Tet2 deficient BM (FIG. 6E) and also abolished their splenomegaly (FIG. 6F). These data demonstrate that increased apoA-1 production and increased HDL show robust preclinical efficacy in leukemia driven by ABCA1 and TET2 mutants.

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