METHOD OF TREATMENT AND PRONOSTIC OF ACUTE MYELOID LEUKEMIA

Abstract

The present invention relates to the treatment of AML. The inventors previously discovered a new epigenetic biomarker in a cohort of CN-AML patients; this consists in a strong enrichment in the H3K27me3 histone mark located on a 70 Kb part of the major histone cluster 1 (HIST1) that separates patients into two distinguishable groups defined as H3K27me3HIST1.sup.low and H3K27me3HIST1.sup.high. Patients harboring the H3K27me3 HIST1 epigenetic mark had a better event free survival. This first observation suggests that H3K27me3HIST1.sup.high patients may develop a less aggressive disease. Molecular characterisation of H3K27me3HIST1.sup.high patients showed that the linker histone H1d, but not the other histone H1 subtypes, was down-regulated in the H3K27me3 HIST1high group of patients. H1d knockdown primed ATRA differentiation, as assessed on CD11b/CD11c markers, morphological and gene expression analyses. These results suggested that targeting H1d could help to reverse the adverse immature phenotype of the H3K27me3 HIST1low group into the more favourable one of the H3K27me3 HIST1.sup.high group of patients and thus could be a good target in AML. Thus the invention relates to an H1d inhibitor for use in the treatment of acute myeloid leukemia (AML) in a patient in need thereof.

Claims

1. (canceled)

2. A method of sensitizing cancerous cells in a patient to therapeutic compounds used to treat AML, comprising administering to the patient an amount of an H1d inhibitor sufficient to sensitize the cancerous cells to the therapeutic compounds.

3-4. (canceled)

5. A method for treating AML in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an inhibitor of H1d.

6-8. (canceled)

9. A method of treatment of an AML in a patient in need thereof comprising the step of: a. determining if the patient as has a good or a bad prognosis by i) determining, in a sample obtained from the patient, the expression level of at least one gene selected in the group consisting in CYBB, FCN1, CLEC4 and ITGAM; and ii) comparing the expression level of the at least one gene determined at step i) with a predetermined reference value; and when the expression level determined at step i) is higher than the predetermined reference value, b. administering to said patient a therapeutically effective amount of a compound useful for the treatment of AML.

10-12. (canceled)

13. A method of treating an AML in a patient in need thereof comprising: a. determining if the patient is a good responder to a chemotherapeutic agent by i) determining in a sample obtained from the patient the histone tri-methylation profile level of H3K27, ii) comparing the histone tri-methylation profile level of H3K27 determined at step i) with a predetermined reference value and, when the histone tri-methylation profile level determined at step i) is higher than the predetermined reference value, b. administrating to said patient a therapeutically effective amount of the chemotherapeutic agent.

14. The method of claim 5, wherein the AML is a Cytogenetically normal AML (CT-AML), an acute promyelocytic leukemia (APL), an acute myeloid leukemia with trisomy 8 or an acute leukemia with MLL translocations.

15. The method of claim 5, wherein the H1d inhibitor is all-trans retinoic acid (ATRA), gemtuzumab or ozogamicin; or a combination of methotrexate, mercaptopurine and ATRA; or a demethylating agent.

16. The method of claim 5, further comprising administering to the patient a therapeutic compound used to treat AML or allograft.

17. The method of claim 16 wherein the H1d inhibitor and the compound used to treat AML or allograft are administered simultaneously, separately or sequentially.

18. The method claim 13, wherein the patient is suffering from CN-AML and has a NPM1 mutation.

19. The method claim 13, wherein the chemotherapeutic agent is selected from the group consisting of cytarabine (araC), volasertib, tozasertib (VX-680), nutlin 3 and olaparib.

Description

FIGURES

[0189] FIG. 1: H3K27me3 HIST1.sup.high is associated with a myelomonocytic GEP

[0190] (A) Expression of three genes associated with granulocytic functions according to H3K27me3 HIST1 status. CYBB (Cytochrome B-245 Beta Chain); FCN1 (Ficolin 1); CLEC4A (C-Type Lectin Domain Family 4 Member A). Data are represented in relative expression to HPRT (B) Expression analysis of patients from the TGCA and GSE 61804 cohorts separated according to the 3-HIST1 mRNA signature as described in the text (B) myelomonocytic genes TNFSF10 (TNF Superfamily Member 10), FCN1 (Ficolin 1), CLEC4A (C-Type Lectin Domain Family 4 Member A), ITGAM (Integrin Subunit Alpha M) were analyzed in 3-HIST1 mRNA.sup.low (N=114) and in 3-HIST1 mRNA.sup.high (N=79) patient samples.

[0191] FIG. 2: H1d KD promotes granulocytic differentiation in ATRA-treated OCI-AML3 cell line

[0192] (A) OCI-AML cells were stably infected by a doxycycline inducible shCtrl or shH1d. Expression of the main histone H1 genes (HIST1H1B, HIST1H1C, HIST1H1D, HIST1H1E, HIST1H1F0 and HIST1HIFx) were analyzed by qPCR in shCtrl and sh #1 without (Dox−) or with 6-days-induction of doxycycline (Dox+). Data represent three independent Dox inductions. Results are normalized on HPRT and expressed in fold change (FC) between Doxycyline treated (Dox+) and non-treated conditions (Dox−). (B) Percentage of CD11b positive cells in shCtrl and sh #1 upon or not (Dox−) 6-days-induction with 2 μg/mL doxycycline (Dox+), without ATRA (wo ATRA) or with 72 hours of ATRA treatment (0.5 μM or 1 μM). (C) Percentage of CD11b-CD11c cells upon 72 h of ATRA-treatment (0.5 μM) in shCtrl and sh #1 conditions. Data represent three independent experiments. Statistical significance was estimated using Mann Whitney test * p<0.05; **p<0.005. (D) Cytological analysis of shCtrl and sh #1 clones upon or not (Dox−) 6-days-induction with doxycycline (Dox+) and after 96 h of ATRA treatment (0.5 μM). (D) May-Grunwald Giemsa coloration of shCtrl and sh #1 clones after 6-days-induction with doxycycline and 96 h hours of ATRA treatment. Black arrows are pointing cytoplasmic azurophilic granules. (E) Expression analysis of two genes associated with ATRA-induced differentiation: CYBB (Cytochrome B-245 Beta Chain) and ITGAM (Integrin Subunit Alpha M) in untreated OCI-AML3 and in ATRA-treated (1 μM) sh #1 without or with doxycycline induction. Data represent three independent experiments. Gene expression was normalized to two housekeeping genes (PGK1 and PPIA). Statistical significance was estimated using T-test (* one-tailed p value <0.05).

[0193] FIG. 3: Chemogram on H3K27me3HIST1.sup.high and H3K27me3HIST1.sup.low AML cells. (A) H3K27me3HIST1.sup.high and H3K27me3HIST1.sup.low AML cells were seeded in 96 well plates to test cell viability after drug treatment (B) 78 drugs have been tested, among witch 14 presented a fold-change >5 between H3K27me3HIST1.sup.high and H3K27me3HIST1.sup.low patients. 4 drugs showed a significant difference (p value>0,05; unpaired Two-tailed t test). (C) IC50 of Chemo drugs and (D) the 4 drugs with significant difference between H3K27me3HIST1.sup.high and H3K27me3HIST1.sup.low patients. Unpaired Two-tailed t test was performed. NS non-significant; *p<0,05; ** p<0,01; *** p<0,001.

[0194] FIG. 4: Detection of leukemic initiating cells after ARAC treatment. (A) H3K27me3HIST1.sup.high and H3K27me3HIST1.sup.low AML secondary transplants were injected in immunocompromised mice (NSG). At disease detection, mice were treated with a first round of ARAC (T1). Blasts in the peripheral blood were followed and a second ARAC treatment (T2) was administrated. (B) Frequency of leukemic initiating cells were analyzed in the BM of the transplanted mice. Percentages are calculated in relation to CD45 and CD33 positive cells detected.

EXAMPLE

[0195] Material & Methods

[0196] Patient Samples.

[0197] This study was performed after approval by our institutional review board. Patient's samples were obtained after informed consent in accordance with the Declaration of Helsinki and stored at Institut Paoli-Calmettes/Centre de Recherche en Cancérologie de Marseille (IPC/CRCM) Tumor Bank and in the Groupe Ouest Est d'Etude des Leucémies Aiguës et autres Maladies du Sang (GOELAMS) repository.

[0198] Histone Gene Nomenclature.

[0199] The large cluster of histone gene HIST1 on human chromosome 6p22 is 2.1 Mb large and contains 55 histone genes. Five genes encode the canonical somatic histone linkers H1 (HIST1H1.A-E) while sets of 10-20 genes encode each of the core histone proteins (H2A, H2B, H3 and H4). Two non-canonical somatic histone linker genes, H1.0 and H1X are encoded outside the HIST1 cluster on 22q13 and 3q21, respectively. Each of these genes is translated into a unique mRNA with a distinct 5′ and 3′ and promoter, as well as slight nucleotide changes in the coding region. These genes are named according to their location in the cluster.14

[0200] ChIP-qPCR.

[0201] ChIP was performed as previously described.15 Quantification of ChIPed DNA was performed by real-time PCR using the SsoADV Univer SYBR Green Supermix (Biorad) and detected with a CFX96 Real-Time PCR Detection System (Biorad). IgG control “cycle over the threshold” Ct values were subtracted to Input or IP Ct values and converted into bound value by 2(−(IP Ct or input Ct-IgG IP Ct)). Data are expressed as % of bound/input and double normalization was done as previously described.15

[0202] Gene Expression Profiling.

[0203] RNA expression profiling of NPM1mut CN-AML was done with Affymetrix Human gene ST 2.0 DNA microarrays (see Supplemental data).

[0204] Protein Analysis.

[0205] Cellular fractionation was carried out using the subcellular protein fractionation kit for cultured cells (Thermofisher). Mass spectrometry procedures are explained in supplemental data. Immunoblot were performed as previously described.16 Antibodies used were anti-Histone H1.3 (H1D) antibody (Abcam, ab24174, 1/750), anti-H1Antiboy (pAb) (Active Motif, 39707, 1/2000) and anti-H3 (Active Motif, cat 39163, 1/10000).

[0206] Cell Culture, shRNA Lentiviral Infection, Stable H1d Knockdown and Treatments.

[0207] The OCI-AML3 cells were grown in MEMα medium supplemented with 20% fetal bovine serum, 100 U/mL penicillin and 100 U/mL streptomycin at 37° C. in humidified atmosphere containing 5% CO2. H1d knockdown was achieved using doxycylin-induced Dharmacon™SMARTvector™ short hairpin RNA (n° V3SH7669-229784413). A non-silencing sh RNA (piSMART VSC10730) was used as a control (shCtrl). Cells containing the SMARTvector™ were selected on puromycin (2 μg/mL) during one day and sorted using ARIAIII cytometer before clonal selection. Three independent cellular clones (sh #1; sh #2; sh #3) were selected. KD of H1d protein was obtained by the addition on doxycycline (2 μg/mL) during 5-7 days. All-trans-retinoic acid (ATRA; Sigma) was resuspended at 10 mM in DMSO and stored at −20° C. Intermediate dilutions were made in culture medium before adding to the cells.

[0208] Flow Cytometry.

[0209] Flow cytometry analyses were performed using a BD-LSRII cytometer and analyzed using BD-DIVA Version 6.1.2 software (BD Biosciences). Antibodies used were CD11b-PE (Mac-1), 3:100, Beckman Coulter; CD11b-APC (M1/70), 1:500, eBioscience; CD11c-PeCy7 (BU-15), 3:100, Beckman Coulter; DRAQ7™, 1:400; Biostatus.

[0210] Morphological Analyses.

[0211] Cytospins were prepared by centrifugation in 200p1 PBS at a speed of 500 rpm for 5 min using Superfrost slides. Cytospin slides were stained at room temperature with May-Grunwald Giemsa (Sigma-Aldrich). 100 cells were counted in duplicate for each condition and examined for cellular morphology using an structured light ApoTome™ microscope (Zeiss, Munich, Germany) equipped with a 63× 1.4 plan ApoChromat objective and an Axiocam™ MRc5 camera.

[0212] Statistical Analyses.

[0213] Statistical analyses were carried out using R software (version 2.15.2) (The Comprehensive R Archive Network. http://www.cran.r-project.org/) and Graph Pad Prism (Graph Pad Software, San Diego, Calif., USA) and the significance of the differences between groups was determined via unpaired T-test, Mann-Whitney test or exact Fisher test. Data were presented as the median ±SEM. Overall survival (OS) and Leukemia-free survival (LFS) were calculated from the date of diagnosis to the date of death or to the date of relapse, death or last follows up, respectively. Follow-up was measured from the date of diagnosis to the date of last news for living patients. Survivals were calculated using the Kaplan-Meier method and were compared with the log-rank test. Uni- and multivariate survival analyses were done using Cox regression analysis (Wald test). Variables with a p-value <0.05 were tested in multivariate analysis. All statistical tests were two-sided at the 5% level of significance, unless where clearly indicated.

[0214] Results

[0215] H3K27Me3 Level on HIST1 Locus Association with Clinical and Molecular Features in CN-AML.

[0216] To further characterize the H3K27me3 HIST1 mark, we performed H3K27me3 ChIP-qPCR on samples obtained from 44 de novo CN-AML patients included in GOELAMS multicenter clinical trials LAM2006IR (NCT00860639) or LAM2007SA (NCT00590837). All patients received conventional induction chemotherapy and their characteristics are depicted in Table S 1. H3K27me3 level was determined at five HIST1 genomic locations as described previously..sup.15 Heatmap showing H3K27me3 HIST1 gene enrichment of the GOELAMS patients confirmed the variation of H3K27me3 HIST1 level among CN-AML patients (data not shown). The mean of the 5 normalized H3K27me3 HIST1 values was calculated and this index showed a clear segregation of the H3K27me3 HIST1.sup.low and H3K27me3 HIST1.sup.high patients. With a cut off value at 15, approximately half of CN-AML patients displayed an H3K27me3 HIST1 enrichment mark (data not shown).

[0217] Concerning clinical and molecular features of patients, there was no association of H3K27me3 HIST1 status with age, gender, FLT3ITD, DNMT3A, IDH1, or CEBPA mutations. Nevertheless, we noted a strong association between NPM1 mutational status and H3K27me3 HIST1.sup.high (data not shown) confirming our first observation..sup.15 In addition we observed a significant association between H3K27me3 HIST1.sup.high and the presence of IDH2 R140 mutation (64.3% versus 4.7%, P=0.01).

[0218] H3K27me3 HIST1.sup.high patients had a significantly better OS and LFS as compared to H3K27me3 HIST1.sup.low patients with a median OS of 42 months versus 14.6 months (HR, 2.5 [1.5-5.5]; P=0.04) and a median LFS of 21 months versus 9 months (HR, 2.5 [1.5-4.9]; P=0.02) (FIG. 1C). The survival gain was independent in multivariate analyses taking age and FLT3-ITD status into account (data not shown). Again, this observation confirms in an independent cohort the association of H3K27me3 HIST1.sup.high with better prognosis that we previously reported.

[0219] Influence of H3K27Me3 HIST1 on Clinical Outcome of NPM1mut AML.

[0220] Given that NPM1 mutated AMLs represent a distinct entity in the World Health Organization (WHO) classification, commonly associated with a better risk prognosis.sup.5, we next analyzed the effect of the H3K27me3 HIST1 mark in the NPM1mut subgroup. We used the biological material obtained from NPM1mut patients, provided by the GOELAMS cell repository (n=33), and by the IPC tumor bank patients (these include 46 previously analyzed samples and 24 additional samples) (data not shown). Of these 103 NPM1mut CN-AML patients presented in figure S1, 75 (73%) were H3K27me3 HIST1.sup.high (data not shown). In terms of molecular abnormalities, NPM1mut H3K27me3 HIST1.sup.high group of patients was not enriched with DNMT3A or FLT3ITD mutations, two of the most frequently NPM1mut co-occurring alterations.sup.17. By contrast, IDH2 R140 was significantly overrepresented in the NPM1mut H3K27me3 HIST1.sup.high subgroup in comparison with NPM1mut H3K27me3 HIST1.sup.low (27.6% versus 7.6%, P=0.05). H3K27me3 HIST1.sup.high leukemic cells had a significantly lower CD34 expression in comparison to their H3K27me3 HIST1.sup.low counterparts (24.1% versus 71.4% CD34 positivity ≥2%, P=0.01) (Table 1).

[0221] In this group of NPM1mut-AMLs, OS and LFS were significantly better in H3K27me3 HIST1.sup.high patients as compared to H3K27me3 HIST1.sup.low patients, (median OS, 38.3 versus 15.7 months; HR, 2 [range, 1.0-3.0]; P=0.03; median LFS, 20.9 versus 10.6 months; HR, 2.7 [range, 1.3-5.7]; P=0.01) (FIG. 1D).

[0222] Influence of H3K27Me3 HIST1 on Clinical Outcome According to Age and Treatment Intensity.

[0223] H3K27me3 HIST1.sup.high positive impact on survival was found both in young and older patients (>60 years) although more pronounced in older patients, probably due to the lesser proportion of allogeneic hematopoietic stem-cell transplantation HSCT in the older group of patients (11% vs 37,5%; P=0.002) (data not shown). HSCT represents a highly effective consolidation treatment proposed to patients with low comorbidities, according to their disease characteristics. In our series, 24 patients underwent HSCT (data not shown). As expected, the LFS difference was more apparent when censoring patients at HSCT (FIG. 1E). Moreover, H3K27me3 HIST1high favorable prognosis was predominant in the non-HSCT group of patients, contrary to the HSCT group of patients, suggesting that HSCT could salvage the H3K27me3 HIST1.sup.low patient pejorative prognosis (data not shown).

[0224] H3K27Me3 HIST1 Impacts the NPM1mut/FLT3wt Patient Outcome.

[0225] Given that the outcome of NPM1mut disease treatment is influenced by co-occurring mutations (FLT3-ITD, DNMT3A).sup.4,18, we tested the influence of the H3K27me3 HIST1.sup.high on survival depending on these mutations. As shown in FIG. 1E, H3K27me3 HIST1 status had a significant impact on survival in the FLT3wt/NPM1mut subgroup (n=53, median OS, 23.2 months versus 111.6 months; P=0.03; median LFS, 13.9 months versus 44.1 months; P=0.01, for H3K27me3 HIST1.sup.low and H3K27me3 HIST1.sup.high, respectively) but not in the FLT3ITD/NPM1mut subgroup (data not shown). No significant impact was found in the DNMT3wt/NPM1mut nor in the DNMT3Amut/NPM1mut subgroups in univariate analyses (data not shown). In multivariate analyses, the prognostic significance of H3K27me3 HIST1.sup.high was independent of known clinical and molecular risk factors (data not shown).

[0226] All together our results suggest that H3K27me3 HIST1 status is an independent marker that could help to refine prognostic classification of NPM1mut CN-AML. This is particularly relevant in NPM1mut/FLT3wt patients.

[0227] Histone mRNA Gene Expression is Anti-Correlated to H3K27Me3 HIST1 Level and Predicts Patients Outcome of NPM1mut CN-AML Patients

[0228] To analyze the anti-correlation of histone mRNA level and the presence of the H3K27me3 mark, we selected three histone genes (HIST1H1D, HIST1H2BG and HIST1H1BH) spread over the H3K27me3 HIST1 islet and associated with clinical outcome in public data (see below) and measured their mRNA levels. Expression of these 3 genes was lower in H3K27me3 HIST1high patients (n=34) than in H3K27me3 HIST1low patients (n13) (data not shown).

[0229] We next asked whether expression of these genes, as a consequence of H3K27me3 repressive mark, was associated with patient survival. Given the small size of our cohort, we analyzed HIST1 gene expression in two published cohorts with publicly accessible clinical and mRNA expression data: TCGA3 and Metzeler17. NPM1mut CN-AML patients were identified by using a published gene expression signature that predicts the NPM1 mutational status 18 (see supplemental methods). Association of histone expression with survival was first tested for each of the 11 histone genes covered by the H3K27me3 HIST1 mark. This highlighted three histone genes, HIST1H1D HIST1H2BG and HIST1H2BH, for which high expression was associated with poor outcome (P=0.004, 0.015 and 0.044 respectively, data not shown). Then, we tested this 3-HIST1 mRNA signature in univariate analysis; 3-HIST1 mRNAlow patients had a favorable OS with a median OS of 17.7 months versus 9.6 months HR=1.66, Range, 1.13-2.42, P=0.009 (data not shown). Multivariate analyses showed that the 3-HIST1 mRNAlow status was associated with a better prognosis (HR=1.60, Range 1.60-2.31, P=0.01), independently of other markers including age, FAB classification and FLT3 status (Data not shown).

[0230] These results show that H3K27me3 HIST1.sup.high is associated with a lower expression of histone genes, and that 3-HIST1 mRNAlow signature defines NPM1mut AML patients with better outcome.

[0231] GEP Associated with H3K27Me3 HIST1.sup.high Identifies a “Mature Like” Phenotype.

[0232] We next characterized the gene expression profile (GEP) of H3K27me3 HIST1.sup.high samples (n=16) in comparison to H3K27me3 HIST1.sup.low samples (n=11) from the IPC cohort (data not shown). Eighty-one genes were differentially expressed (p<0.05, fold-change>1.5) between the two groups, 58 being up- and 23 being down-regulated in the H3K27me HIST1 high group (data not shown). Gene Set Enrichment Analysis (GSEA) identified upregulated genes in pathways active in myelomonocytic differentiation such as immune or inflammatory responses in H3K27me3 HIST1.sup.high patients (data not shown). Down-regulated genes in these patients belong to cell cycle and chromatin regulation categories, including histone genes from the HIST1 cluster (data not shown). Using qPCR, we confirmed the higher expression of three genes involved in mature granulocyte functions, CYBB, FCN1 and CLEC4A.sup.21-23 in H3K27me3 HIST1.sup.high patients (FIG. 1A). These results are in line with the loss of CD34 observed in H3K27me3 HIST1.sup.high patients (data not shown).

[0233] To further validate the relation between low mRNA level of HIST1 genes and the expression of granulocytic markers, we tested mRNA expression of myelomonocytic maturation genes (CYBB, FCN1, CLEC4 and ITGAM) in the TAGC and Metzeler cohorts of patients stratified with the previously defined 3-HIST1 mRNA signature. Results showed that the 3-HIST1 mRNA.sup.low patient group over-expressed the differentiation genes in comparison to the 3-HIST1 mRNA.sup.high group (FIG. 1B) corroborating our previous observation (FIG. 1A). Equally, genes such as SOCS2, CDK6, LAPTM4B and NGFRAP1—that were recently described as associated with a leukemic stem cell signature-.sup.24 were less expressed in the 3-HIST1 mRNA.sup.low patient group (data not shown).

[0234] Taken together, these results suggest that HIST1 mRNA down regulation by the H3K27me3 mark is associated with a more differentiated phenotype related to a committed state of leukemic cells.

[0235] Histone Protein Expression in AML Patients

[0236] To study the role of histones on AML clinical and biological features, we studied the effect of H3K27me3 HIST1 epigenetic silencing on the level of histone linker H1d encoded by HIST1HID. We chose to analyze specifically H1d because its mRNA level is affected by H3K27me3 HIST1 status (data not shown) and it is the leading gene for the mRNA signature (data not shown). In addition, H1 histone subtypes are heterogeneous in amino acid composition.sup.23, which probably reflects a subtype-specific function. First we looked at proportions of total histones and of each histone subtype (data not shown) in chromatin-bound fractions extracted from a series of 12 patient samples (six in each group) using Intensity Based Absolute Quantification (iBAQ) approach. Normalized quantities of total linker histone H1 and core histones H2A, H2B, H3 and H4 were similar in both H3K27me3 HIST1.sup.high and H3K27me3 HIST1.sup.low patients (data not shown) suggesting that H3K27me3 HIST1.sup.high status did not globally modify histone protein abundance.

[0237] Yet, when looking at the H1 subtype abundance, we observed that the H1d subtype was decreased in the H3K27me3 HIST1.sup.high group (normalized iBAQ value (Log 2)=6.09 vs 4.74; P=0.04) whereas the other H1 subtypes, H1b, Hic H1F0 and H1FX were unaffected (data not shown). These results are consistent with the HIST1H1D mRNA expression decrease observed in AML samples harboring the H3K27me3 HIST1 mark. We confirmed the lower expression of H1d observed in H3K27me3 HIST1.sup.high group in comparison to H3K27me3 HIST1.sup.low group of patients by western blot using pan H1 and specific H1d antibodies (data not shown).

[0238] In conclusion, as a consequence of the presence of an H3K27me3 islet, NPM1mut CN-AML H3K27me3 HIST1.sup.high patients express low level of H1d.

[0239] H1d Knockdown Confers a More Mature Phenotype in OCI-AML3 Cell Line

[0240] To investigate the effect of low H1d expression on AML, we performed H1d knockdown (KD) in OC1-AML3 cells, an AML cell line expressing a NPM1 mutated allele..sup.26 Efficiency and specificity of our KD were assessed by testing the different mRNA H1 subtypes expression by q-PCR (FIG. 2A) and by measuring H1d protein level after doxycycline induction (data not shown). Consequences of H1d KD on differentiation were evaluated by measuring CD11b and CD11c expression in OCI-AML3 after doxycycline induction. H1d KD did not induce a significant increase in CD11b (FIG. 2B) nor CD11c levels (data not shown). As OCI-AML3 can differentiate in vitro in the presence of ATRA, albeit at low efficiency,.sup.27,28 we further analyzed the effect of H1d KD in combination with ATRA treatment. Two different doses of ATRA (0.5 μM and 1 μM) induced a significant increase in CD11b expression, with a marked increase at 0.5 μM (22.6%±2.5 vs 41%±4.3; P=0.008) (FIG. 2C). We also observed an increase in the proportion of the double positive CD11b/CD11c populations in ATRA-treated H1d KD cells (29.8%±1.3 vs 42.5%±2.1; P=0.003) (FIG. 2B). That result suggests that a lower expression of H1d sensitizes AML cells to ATRA treatment. Next, we assessed the cellular morphology by May-Grunwald Giemsa coloration; at 96 h of ATRA treatment (0.5 μM), cytoplasmic granules that reflects the beginning of a maturation process appeared upon H1d KD (data not shown). Quantification revealed a higher proportion of cells with more than two cytoplasmic granules in the H1d KD (9.7%±2.6 vs 31.5±3.7; P=0.009), (FIG. 2D). Finally, mRNA expression levels of two ATRA-induced genes, CYBB and ITGAM.sup.29 were tested under H1d KD condition with ATRA-treatment; H1d down-regulation increased the amplitude of ATRA-induced upregulation of these two genes (FIG. 2E). Altogether these results suggest that down regulation of histone H1D induces ATRA-sensitization, and provide hypotheses to explain the more mature phenotype found in H3K27me3 HIST1.sup.high leukemia and suggest that ATRA could be a more efficient differentiating agent in NPM1mut AML with low H1d expression.

[0241] Implication of the H3K27meHIST1 Signature in NMP1-Mut CN-AML Progression and Sensitivity to Treatment

[0242] We questioned the implication of the H3K27meHIST1 signature in NMP1-mut CN-AML progression and sensitivity to treatment by testing a panel of drugs (78 FDA-approved and/or investigational drug compounds including chemo and epidrugs) on H3K27me3HIST1.sup.high versus H3K27me3HIST1.sup.low patient cells (FIG. 3A). Our preliminary results obtained with 4 H3K27me3HIST1.sup.high and 3 H3K27me3HIST1.sup.low AML patient cells suggested that H3K27me3HIST1.sup.high condition was more sensitive to chemotherapy (FIG. 3B-D), which reflected the better survival observed in patients (Garciaz, Clin Epigenetics, 2019, 11:141). In addition, we identified 4 interesting drugs that were more effective in H3K27me3HIST1.sup.high than in H3K27me3HIST1.sup.low AMLs (FIG. 3B-D). Altogether, these results suggest that H3K27me3HIST1.sup.high confers vulnerability to AML cells.

[0243] We determined the response to chemotherapy according to H3K27me3 status. This was tested in vivo using patient-derived xenograft (PDX) treated with chemotherapeutic drugs (Ara-C). We used secondary transplantation from 1 H3K27me3HIST1low and 2 H3K27me3HIST1high NPM1mut AML patients. Experimental groups of 6 mice/groups of H3K27me3 HIST1high versus H3K27me3 HIST1low were studied. Mice were treated for one week and progression of leukemia was evaluated by counting hCD45+ cells in blood and after euthanasia in bone marrow. Mouse maintenance and experimental procedures were performed in accordance with protocols approved and compliance with policies approved by the local Committee for Animal Experimentation of Marseille (CAE of Provence number 14), France (2-091009).

[0244] Response to chemotherapy was obtained in all mice and recovery was in the same range of time for all the PDX tested, independently of H3K27me3HIST1 status. We performed a second round of ARAC treatment and analyzed the presence of leukemic cells 4-day post treatment. Numbers of blasts were variable depending on the patient with an accumulation in the BM of leukemic Blast in one of the PDX. The leukemic initiating cell (LIC) has been shown to be responsible of treatment resistance and relapse. Thus we analyzed the % of LIC in the 2-round ARAC treated mice in comparison to untreated ones (FIG. 4A). Percentage of LIC was identical before and after treatment in the H3K27me3HIST1high PDX while the percentage of LIC was increased in the H3K27me3HIST1low PDX (FIG. 4B). This suggests that ARAC treatment increased the LIC percentage in the leukemia bulk of H3K27me3HIST1low AML samples.

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