ALBERT

Description: Ten-eleven translocation 2 (TET2) dioxygenase mutations resulting in enzymatic deficiency are among the most frequently reported molecular aberrations in myeloid neoplasms. Accumulating evidence indicates that biological interactions between TET2 deficiency and other molecular lesions are important drivers of myeloid disorder. For example, microbial-mediated innate immune signaling was demonstrated to play a critical role in promoting myeloproliferation in mice with TET2 deficiency. Histone demethylase KDM6B is an innate immune signal activator downstream of Toll like receptors (TLR) and is overexpressed in bone marrow (BM) hematopoietic stem and progenitor cells (HSPC) from patients with myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML). We previously demonstrated that overexpression of KDM6B in the hematopoietic compartment of mice led to a MDS-like phenotype and hyperactivation of innate immune signals (Wei Blood Advances 2018). These observations suggest that KDM6B overexpression and consequent innate immune deregulation may cooperatively interact with TET2 deficiency to drive myeloid disorders. In support of this hypothesis, transcriptomic analysis in the BM HSPCs of patients with MDS and CMML revealed that patients with concurrent KDM6B overexpression and TET2 deficiency had significantly increased activation of innate immune genes and signals. To investigate the interaction between KDM6B overexpression and TET2 deficiency, we developed a "double lesion" mouse model with both KDM6B overexpression and TET2 deficiency by crossing Vav-KDM6B mice with TET2flox/flox/Vav-Cre mice. Double lesion mice exhibited significant monocytosis compared to Vav-Cre or TET2 deficient mice (p=0.02 and p=0.03). Double lesion mice also experience reduced hemoglobin (p=0.01 and p=0.01) and splenomegaly (p=0.004 and p=0.01). Myeloid skewing was observed in the BM of double lesion mice as large expansile aggregates of immature myelomonocytic precursors, increase of Gr-1+ myeloid cells (p=0.001 and p=0.01), and decrease of Ter119+ erythroid cells (p=0.01 and p=0.02). An increase in the BM Lin-/Sca1+/cKit+ (LSK) population was also observed in double lesion mice (p=0.004 and p=0.05) particularly in aged mice (50-60 week old). Moreover, the BM of double lesion mice exhibited increased repopulating function, illustrated by higher chimerism of CD45.2 donor cells in CD45.1 recipient mice 6 months following competitive transplantation (p=0.004 and p=0.04). Overall, the MDS/CMML-like hematopoietic phenotype observed in double lesion mice, which was more significant than that observed in TET2 deficient single lesion mice, indicated that overexpression of KDM6B and TET2 deficiency cooperatively altered hematopoiesis and promoted development of myeloid disorders. To identify the biological mechanisms underlying the cooperative impact of KDM6B overexpression and TET2 deficiency in BM HSPCs, we performed transcriptomic analysis on LSKs collected from double lesion, TET2 deficient, Vav-KDM6B, and Vav-Cre mice. RNA-Seq illustrated hyper-activation of multiple innate immune pathways in the LSK of double-lesion mice compared to the other groups. Consistently, peripheral blood cytokine levels of TNF, IL-1, and IL-6 were significantly elevated in double lesion mice. RNA-Seq also revealed that the LSKs of double-lesion mice displayed significant down-regulation of cell cycle checkpoint and DNA repair signals. This finding was confirmed by the observation of increased phosphorylated γH2AX and aneuploidy in hematopoietic cells from double lesion mice. Finally, we applied GSK-J4, an inhibitor KDM6 proteins, to the BM LSKs from each mouse cohort and performed methylcellulose medium supported colony formation assays. Significantly reduced colony formation was observed in both the double lesion and TET2 deficiency groups (p

Description: Myelodysplastic syndromes (MDS) are a group of heterogeneous hematopoietic malignancies characterized by the defective production of mature blood cells and the risk of progression to myeloid leukemia. Hypomethylating agents (HMA) are currently the first-line therapy for high-risk MDS and induce hematological improvement in 50% of the patients. However, complete responses occur in less than 20% of these patients, and a majority of MDS patients treated with HMA will eventually relapse, with a subsequently fatal prognosis. Although the presence of mutations in certain genes, such as Tet2, has been reported to predict response to HMA, the causes of resistance and relapse have yet to be described. MDS research has been traditionally a slow-moving field owing to the lack of well-established MDS cell lines and animal models. This landscape recently changed with the development of transgenic mouse models that mirror the phenotype of high-risk MDS patients. In mice from the 4th/5th generations (G4/G5) of our group's telomere-dysfunctional TERTER/ER mouse model, the DNA-damage response triggered by telomere erosion causes profound cell-intrinsic abnormalities in hematopoietic stem and progenitor cells (HSPC) that lead to myeloid-skewed hematopoiesis. These mice exhibit peripheral blood (PB) cytopenias, age-related granulocytosis and myeloid infiltration of the BM and the spleen (Colla et al. Cancer Cell 2015). Similarly, the conditional deletion of Tet2 in mouse hematopoietic cells generates a phenotype of HSC expansion and myeloid skewing of hematopoiesis. These defects, which are also cell-intrinsic, induce progressive PB neutrophilia and granulocytosis, with myeloid infiltration of the bone marrow (BM) and spleen and severe splenomegaly (Moran-Crusio et al. Cancer Cell 2011). Using the TERTER/ER and Tet2-/- mouse models as a platform for cellular and molecular studies, we have investigated the mechanism of action of HMA in MDS, with a focus on the characterization of response by the different HSPC populations and the mechanisms involved in resistance and relapse. Our results show that the administration of the HMA azacytidine (AZA) for 7 days induces in both mouse models a 20-40% drop in white blood cell counts and a 17-24% decrease in hemoglobin levels that mimic patient responses to AZA. After the last day of treatment and during the 3 last weeks of the cycle, cell counts and hemoglobin levels returned to normal values. These changes in PB were explained by an 80-86% decrease in the number of BM common myeloid progenitors (CMP; n = 8, P = 0.0046) and granulo-monocytic progenitors (GMP; n = 8, P

Description: Myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML)aremyeloid neoplasms characterized by abnormal bone marrow hematopoiesis and increased risk of transformation to acute myelogenous leukemia (AML). Epigenetic dysregulation and inflammatory hyper-activation have been recognized as key molecular abnormalities in the bone marrow (BM) hematopoietic stem and progenitor cells (HSPC) of MDS and CMML, which implies that key modulators of epigenetic and inflammatory regulation play an important role in the pathophysiology of these diseases, which could also serve as effective therapeutic targets. We recently identified such a candidate molecule: the histone demethylase KDM6B (JMJD3). We demonstrated that KDM6B is significantly overexpressed in the BM HSPCs of patients with MDS and CMML, and the overexpression of KDM6B mediates aberrant epigenetic activation of innate immune/inflammatory signals and consequent differentiation skewing in BM HSPCs of MDS, which can be reversed by targeting KDM6B. Importantly, systematic analysis of the global transcriptomic and genomic data of patients indicates that, although KDM6B is overexpressed in MDS and CMML, genetic lesions in this gene are very rare, and higher KDM6B expression correlates with TET2 mutation. These results imply that constitutive expression of KDM6B potentially interacts with more common genetic lesions during the development of MDS and CMML. To further investigate the effects of KDM6B overexpression on hematopoiesis and its role in myeloid disorders, we developed a novel hematopoietic KDM6B transgenic (Tg) mouse model that overexpresses KDM6B under the control of the murine hematopoietic specific Vav promoter (Vav-KDM6B). Long-term monitoring of the peripheral blood counts of the mice indicates that, although younger Vav-KDM6B mice display only minor changes in whole white blood cells (WBC), monocytes, and platelets, aged KDM6B mice (〉1 year old) have significant increases of WBC (by 22%, p

Description: The mechanisms of HMA failure in MDS remain unclear, as recent advances in sequencing approaches did not enable the molecular characterization of the cells that survive therapy and drive resistance and disease progression. Here, through combined functional and transcriptomic analyses of highly-purified hematopoietic populations isolated from the BM of 132 MDS patients enrolled in clinical trials of single drug HMA therapy, we show that 2 immunophenotypically and molecularly distinct cell types maintain the disease and expand at progression, and we propose therapeutic approaches to overcome MDS evolution. Unsupervised hierarchical clustering followed by principal component analysis of 101 untreated MDS samples based on the frequency of immunophenotypically defined stem and myeloid progenitor cell populations identified the frequencies of the lymphoid-primed multipotent progenitors (LMPPs) and granulo-monocytic progenitors (GMPs) as the main sources of variation across the samples. Further logistic regression analysis enabled the systematic stratification of the samples in 2 main groups. "CMP pattern" MDS was characterized by the prevalence of common myeloid progenitors (CMPs) in the progenitor compartment while "GMP pattern" MDS was characterized by the increased frequency of GMPs in the progenitors and by increased LMPPs and decreased long-term (LT)-HSC frequencies in the BM (Fig A, B). Functional analysis of the CMPs by immunophenotypic characterization of lineage-primed fractions and colony assays revealed that this population was myeloid-biased only in "CMP pattern" MDS but not in "GMP pattern" MDS, suggesting that 2 distinct hierarchical differentiation routes underlie disease manifestation (Fig C). Further logistic regression analysis of 31 MDS samples from patients with progressive disease showed that whereas "CMP pattern" MDS patients with blast expansion were characterized by a significant increase in the BM frequency of LT-HSCs, "GMP pattern" MDS patients were characterized by the expansion of the LMPPs (Fig D). To investigate the molecular pathways underlying the 2 different hierarchical patterns of progression, we analyzed the transcriptional profiling of the HSCs that selectively expanded in the 2 groups of MDS patients. RNA-Seq analysis revealed that, compared with those isolated from patients at baseline, LT-HSCs isolated from "CMP pattern" MDS patients with blast expansion had significantly upregulated genes involved in promoting cell proliferation and survival, including the anti-apoptotic regulator BCL2. In contrast, genes involved in the response to TNF-a were significantly upregulated in the LMPPs from "GMP pattern" MDS patients with progressive disease as compared with the LMPPs isolated at baseline (Fig E). Then, we hypothesized that, despite genetic dissimilarities, the HSCs that expanded at progression were addicted to the molecular pathways that were upregulated and that targeting these pathways represented a potential therapeutic strategy to overcome HMA resistance. As proof-of-principle, we treated CD34+ cells from "CMP pattern" MDS isolated after HMA failure, co-cultured over a layer of stromal cells, with the BCL2 inhibitor ABT-199 for 72 hours. ABT-199, in combination with 5-azacytidine (AZA), significantly decreased the number of LT-HSCs from MDS patients with progressive disease (Fig F), but did not affect those from MDS patients in whom HMA therapy failed because of persistent MDS. These data suggest that ABT-199 selectively targets the LT-HSCs from "CMP pattern" MDS with progressive disease, which depend on increased levels of BCL2 to support their expansion. Treatment of WT/vav-cre-TET2L/L BM chimeras with ABT-199 therapy showed selective apoptosis and depletion of the MDS-like LT-HSCs and the concomitant proliferation of the WT LT-HSCs. This suggests that ABT-199 allows selective therapeutic targeting that eliminates abnormal HSCs while restoring normal hematopoiesis (Fig G). Furthermore, ABT-199 treatment of xenografts generated by transplanting the MDS-L cell line in NSGS mice reduced tumoral burden by depleting the human blast population (Fig H). Taken together, these findings demonstrate that targeting commonly deregulated pathways in MDS HSCs is a feasible approach to tackling disease progression and provide a rationale for the selective inhibition of BCL2 in "CMP pattern" MDS with progressive disease. Figure. Figure. Disclosures Giuliani: Celgene Italy: Other: Avisory Board, Research Funding; Takeda Pharmaceutical Co: Research Funding; Janssen Pharmaceutica: Other: Avisory Board, Research Funding. Konopleva:Stemline Therapeutics: Research Funding. Colla:Abbvie: Research Funding.