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Epigenetic regulation is essential for the maintenance of the hematopoietic system, and its deregulation is implicated in hematopoietic disorders. In this study, UTX, a demethylase for lysine 27 on histone H3 (H3K27) and a component of COMPASS-like and SWI/SNF complexes, played an essential role in the hematopoietic system by globally regulating aging-associated genes. Utx-deficient (UtxΔ/Δ) mice exhibited myeloid skewing with dysplasia, extramedullary hematopoiesis, impaired hematopoietic reconstituting ability, and increased susceptibility to leukemia, which are the hallmarks of hematopoietic aging. RNA-sequencing (RNA-seq) analysis revealed that Utx deficiency converted the gene expression profiles of young hematopoietic stem-progenitor cells (HSPCs) to those of aged HSPCs. Utx expression in hematopoietic stem cells declined with age, and UtxΔ/Δ HSPCs exhibited increased expression of an aging-associated marker, accumulation of reactive oxygen species, and impaired repair of DNA double-strand breaks. Pathway and chromatin immunoprecipitation analyses coupled with RNA-seq data indicated that UTX contributed to hematopoietic homeostasis mainly by maintaining the expression of genes downregulated with aging via demethylase-dependent and -independent epigenetic programming. Of note, comparison of pathway changes in UtxΔ/Δ HSPCs, aged muscle stem cells, aged fibroblasts, and aged induced neurons showed substantial overlap, strongly suggesting common aging mechanisms among different tissue stem cells.

The chromosomal translocation t(7; 11)(p15;p15), observed in human myeloid leukemia, results in a NUP98 and HOXA9 gene fusion. We generated a transgenic mouse line that specifically expressed the chimeric NUP98-HOXA9 gene in the myeloid lineage. While only 20% of the transgenic mice progressed to leukemia after a latency period, myeloid progenitor cells from nonleukemic transgenic mice still exhibited increased proliferative potential. This suggested that the NUP98-HOXA9 fusion induced a preleukemic phase, and other factors were required for complete leukemogenesis. NUP98-HOXA9 expression promoted the onset of retrovirus-induced BXH2 myeloid leukemia. This phenomenon was used to identify cooperative disease genes as common integration sites (CISs). Meis1, a known HOX cofactor, was identified as a CIS with a higher integration frequency in transgenic than in wild-type BXH2 mice. By the same means we identified further 4 candidate cooperative genes, Dnalc4, Fcgr2b, Fcrl, and Con1. These genes cooperated with NUP98-HOXA9 in transforming NIH 3T3 cells. The system described here is a powerful tool to identify cooperative oncogenes and will assist in the clarification of the multistep process of carcinogenesis.

There is a critical need for new agents with novel therapeutic targets and improved safety profiles in high-risk acute lymphoblastic leukemia (ALL), which is a significant cause of morbidity and mortality in pediatric and adult populations. Phenotypic high-throughput chemical screens allow for discovery of small molecules that modulate complex phenotypes and provide lead compounds for novel therapies; however, identification of their mechanistically relevant targets remains a major experimental challenge. We applied a chemical genetics approach involving sequential unbiased high-throughput chemical and ultra-complex, genome-scale shRNA screens to address this challenge and identify novel agents in ALL. A cell-based phenotypic high-throughput chemical screen of 115,000 compounds identified 640 compounds that inhibited growth of one or both ALL cell lines with high-risk Mixed Lineage Leukemia (MLL) genetic abnormalities, but did not inhibit the growth of a cell line lacking MLL rearrangement. The most potent and selective 64 were tested on an expanded panel of eight human B-ALL cell lines to identify lead compound STF-118804. STF-118804 inhibited the growth of most B-ALL cell lines with high potency demonstrating IC50 values in the low nanomolar range. Leukemic samples from five pediatric ALL patients were also sensitive to STF-118804 in the low nanomolar range. STF-118804 displayed 5–10 fold more potency against most leukemias in comparison to cycling human (lineage-negative cord blood) and murine (c-kit+ bone marrow) progenitor cells, demonstrating a therapeutic index. STF-118804 displays distinctive cytotoxicity by inducing apoptosis without causing a phase-specific cell cycle arrest. To discover the molecular target of STF-118804, a functional genomic screen was performed to identify shRNAs that conferred sensitivity or resistance to STF-118804, utilizing an ultra-complex (∼25 shRNAs per gene) library targeting in total ∼9300 human genes and 1000s of negative control shRNAs. NAMPT was the most statistically significant gene to confer sensitivity to STF-118804, suggesting that STF-118804 functioned as a NAMPT inhibitor. NAMPT encodes nicotinamide phosphoribosyl transferase, a rate-limiting enzyme in the biosynthesis of nicotinamide adenine dinucleotide (NAD+), a crucial cofactor in many biochemical processes. STF-118804 was confirmed as a novel class of NAMPT inhibitor through metabolic rescue, enzymatic, and genetic studies. STF-118804 displayed strong inhibitory activity in in vitro NAMPT enzymatic assays. Over-expression of wild-type or mutant NAMPT in cells indicated that STF-118804 cytotoxicity is a result of its ability to inhibit NAMPT, and that STF-118804 does not have significant off-target effects on cell viability. The potential efficacy of STF-118804 in vivo was assessed in an orthotopic xenograft model of ALL. Sublethally irradiated immunodeficient mice were transplanted with human ALL cells engineered to constitutively express firefly luciferase. Dosing of STF-118804 was initiated two weeks post-transplant when ALL cells had engrafted and bioluminescent signal was detectable. Mice treated with STF-118804 showed regression of leukemia by bioimaging and significantly extended survival. The leukemia initiating cell (LIC) frequency in STF-118804 treated mice was significantly lower (∼8 fold) than vehicle treated mice, showing that STF-118804 was effective in reducing LICs. In summary, tandem high-throughput screening identified a highly-specific, potent, and structurally novel small molecule inhibitor of NAMPT that is active in ALL. Tandem high throughput screening using chemical and ultra-complex shRNA libraries provides a rapid chemical genetics approach for seamless progression from small molecule lead identification to target discovery and validation. Disclosures: No relevant conflicts of interest to declare.

The critical molecular determinants for leukemia stem cell (LSC) generation and maintenance are largely unknown. While the majority of studies focus on investigating key transcription factors and cytokines involved in hematological malignancies, very little is known about the roles of recently discovered microRNAs in leukemogenesis. Bioinformatic analysis of the Mouse Retroviral Tagged Cancer Gene Database (Neal Copeland et al.) revealed the 5′ regulatory region of the miR-142 gene as a recurrent target for retroviral integrations in a variety of mouse leukemia and lymphoma cancer models, including MLL-induced leukemia, thereby implicating miR-142 as a potential collaborating oncomir in hematologic malignancies. Subsequent expression analyses of differentially expressed microRNAs in LSC-enriched cell fractions (c-kit+) from MLL leukemias in comparison with various stages of normal hematopoietic progenitors indicated that the miR-17-5p family of microRNAs is highly expressed in LSC-enriched fractions and their possible normal counterparts, granulocyte-macrophage progenitors. Moreover, the expression of miR-142 and miR-17-5p family microRNAs was substantially reduced upon differentiation and loss of self-renewal in cells transformed by the conditional MLL-ENL-ER oncogene following withdrawal of tamoxifen. Forced expression of the miR-17-19b polycistron or miR-142 substantially shortened the latency for MLL leukemia development. In particular, leukemias expressing high levels of the miR-17-19b polycistron displayed a higher frequency of LSCs due to an efficient block of differentiation and enhanced proliferation associated with reduced expression of p21, a cyclin dependent kinase inhibitor previously implicated as a direct target of miR-17-19b microRNAs. Knock down of p21 in MLL oncogene transduced cells recapitulates the MLL-leukemic phenotype induced by over-expression of the miR-17-19b cluster including a significant decrease in MLL leukemia latency. This observation validates p21 as a biologically relevant in vivo target of the miR-17-19b polycistron in MLL leukemia. Thus, microRNAs are pathogenically important for quantitatively modulating the self-renewing LSC compartment in a murine model of MLL leukemia. Potential requirements of the miR-17-19b microRNA cluster for induction and/or maintenance of MLL leukemia are currently under study.

The RUNX1/AML1 gene is essential for hematopoiesis and is most frequently involved in human leukemias. Loss-of-function of RUNX1 is the common underlying mechanism of RUNX leukemias: those carrying t(8;21), inv(16) and RUNX1 point mutations. As the weakest mode, haploinsufficiency of RUNX1/AML1 causes familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML). Mice heterozygous for Runx1 null mutation, Runx1+/−, appear genetically equivalent to human FPD/AML patients, but showed no apparent defects and did not develop spontaneous leukemia, implying that additional genetic alterations are required for leukemia. To identify such abnormalities, retroviral insertional mutagenesis was employed by using BXH2 mice that have spontaneously transmittable ecotropic retrovirus. BXH2-Runx1+/− mice showed a shorter disease latency and more myeloid-specific phenotype than wild-type littermates. Therefore, BXH2-Runx1+/− mice appear to serve as the mouse model for FPD/AML to investigate myeloid leukemia. The genes selectively affected in BXH2-Runx1+/− mice by retroviral integration are likely to cooperate with Runx1+/−. c-Kit locus was involved twice in 24 BXH2-Runx1+/− but neither in 17 wild-type littermates nor in 135 inbred BXH2 mice reported in the RTCGD database, suggesting that c-KIT mutation cooperates with RUNX alteration in human leukemia. In fact, constitutively active mutants of c-KIT or functionally equivalent FLT3, were found in 10 out of 25 (40%) of human RUNX leukemias. Together, BXH2-Runx1+/− system appears useful to identify the genes cooperating with RUNX1 abnormalities.