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Proteasome inhibitors, such as bortezomib and carfilzomib, have shown efficacy in anti-cancer therapy in hematological diseases but not in solid cancers. Here, we found that liposarcomas (LPS) are susceptible to proteasome inhibition, and identified drugs that synergize with carfilzomib, such as selinexor, an inhibitor of XPO1-mediated nuclear export. Through quantitative nuclear protein profiling and phospho-kinase arrays, we identified potential mode of actions of this combination, including interference with ribosome biogenesis and inhibition of pro-survival kinase PRAS40. Furthermore, by assessing global protein levels changes, FADS2, a key enzyme regulating fatty acids synthesis, was found down-regulated after proteasome inhibition. Interestingly, SC26196, an inhibitor of FADS2, synergized with carfilzomib. Finally, to identify further combinational options, we performed high-throughput drug screening and uncovered novel drug interactions with carfilzomib. For instance, cyclosporin A, a known immunosuppressive agent, enhanced carfilzomib’s efficacy in vitro and in vivo. Altogether, these results demonstrate that carfilzomib and its combinations could be repurposed for LPS clinical management. Electronic supplementary material The online version of this article (10.1007/s00018-020-03620-w) contains supplementary material, which is available to authorized users.

Recurrent loss-of-function mutations of spliceosome gene, ZRSR2, occur in myelodysplastic syndromes (MDS). Mutation/loss of ZRSR2 in human myeloid cells primarily causes impaired splicing of the U12-type introns. In order to further investigate the role of this splice factor in RNA splicing and hematopoietic development, we generated mice lacking ZRSR2. Unexpectedly, Zrsr2-deficient mice developed normal hematopoiesis with no abnormalities in myeloid differentiation evident in either young or ≥1-year old knockout mice. Repopulation ability of Zrsr2-deficient hematopoietic stem cells was also unaffected in both competitive and non-competitive reconstitution assays. Myeloid progenitors lacking ZRSR2 exhibited mis-splicing of U12-type introns, however, this phenotype was moderate compared to the ZRSR2-deficient human cells. Our investigations revealed that a closely related homolog, Zrsr1, expressed in the murine hematopoietic cells, but not in human cells contributes to splicing of U12-type introns. Depletion of Zrsr1 in Zrsr2 KO myeloid cells exacerbated retention of the U12-type introns, thus highlighting a collective role of ZRSR1 and ZRSR2 in murine U12-spliceosome. We also demonstrate that aberrant retention of U12-type introns of MAPK9 and MAPK14 leads to their reduced protein expression. Overall, our findings highlight that both ZRSR1 and ZRSR2 are functional components of the murine U12-spliceosome, and depletion of both proteins is required to accurately model ZRSR2-mutant MDS in mice. Ministry of Education (MOE) Ministry of Health (MOH) National Medical Research Council (NMRC) National Research Foundation (NRF) Published version This work was funded by the Leukemia and Lymphoma Society, the Singapore Ministry of Health’s National Medical Research Council (NMRC) under its Singapore Translational Research (STaR) Investigator Award to HPK (NMRC/STaR/0021/2014), the NMRC Center Grant awarded to the National University Cancer Institute of Singapore (NMRC/CG/012/2013) and the National Research Foundation Singapore and the Singapore Ministry of Education under its Research Centers of Excellence initiatives. This research is also supported by the RNA Biology Center at the Cancer Science Institute of Singapore, NUS, as part of funding under the Singapore Ministry of Education’s Tier 3 grants, grant number MOE2014-T3-1-006. We thank the Melamed Family for their generous support.

Precise regulation of chromatin architecture is vital to physiological processes including hematopoiesis. ARID1A is a core component of the mammalian SWI/SNF complex, which is one of the ATP-dependent chromatin remodeling complexes. To uncover the role of ARID1A in hematopoietic development, we utilized hematopoietic cell-specific deletion of Arid1a in mice. We demonstrate that ARID1A is essential for maintaining the frequency and function of hematopoietic stem cells and its loss impairs the differentiation of both myeloid and lymphoid lineages. ARID1A deficiency led to a global reduction in open chromatin and ensuing transcriptional changes affected key genes involved in hematopoietic development. We also observed that silencing of ARID1A affected ATRA-induced differentiation of NB4 cells, suggesting its role in granulocytic differentiation of human leukemic cells. Overall, our study provides a comprehensive elucidation of the function of ARID1A in hematopoiesis and highlights the central role of ARID1A-containing SWI/SNF complex in maintaining chromatin dynamics in hematopoietic cells.

Chromosomal translocation t(8;21)(q22;q22) which leads to the generation of oncogenic RUNX1-RUNX1T1 (AML1-ETO) fusion is observed in approximately 10% of acute myelogenous leukemia (AML). To identify somatic mutations that co-operate with t(8;21)-driven leukemia, we performed whole and targeted exome sequencing of an Asian cohort at diagnosis and relapse. We identified high frequency of truncating alterations in ASXL2 along with recurrent mutations of KIT, TET2, MGA, FLT3, and DHX15 in this subtype of AML. To investigate in depth the role of ASXL2 in normal hematopoiesis, we utilized a mouse model of ASXL2 deficiency. Loss of ASXL2 caused progressive hematopoietic defects characterized by myeloid hyperplasia, splenomegaly, extramedullary hematopoiesis, and poor reconstitution ability in transplantation models. Parallel analyses of young and >1-year old Asxl2-deficient mice revealed age-dependent perturbations affecting, not only myeloid and erythroid differentiation, but also maturation of lymphoid cells. Overall, these findings establish a critical role for ASXL2 in maintaining steady state hematopoiesis, and provide insights into how its loss primes the expansion of myeloid cells.

Maturation of granulocytes is dependent on controlled gene expression by myeloid lineage restricted transcription factors. CEBPE is one of the essential transcription factors required for granulocytic differentiation. Identification of downstream targets of CEBPE is vital to understand better its role in terminal granulopoiesis. In this study, we have identified Card10 as a novel target of CEBPE. We show that CEBPE binds to regulatory elements upstream of the murine Card10 locus, and expression of CARD10 is significantly reduced in Cebpe knock-out mice. Silencing Card10 in a human cell line and in murine primary cells impaired granulopoiesis, affecting expression of genes involved in myeloid cell development and function. Taken together, our data demonstrate for the first time that Card10 is expressed in granulocytes and is a direct target of CEBPE with functions extending to myeloid differentiation.

Acute promyelocytic leukemia (APL) is a unique subtype of acute myeloid leukemia (AML). The disease is identified by distinctive morphology and is distinguished by a balanced reciprocal translocation between chromosomes 15 and 17. This aberration leads to the fusion between promyelocytic leukemia (PML) gene located on chromosome 15q21, and retinoic acid receptor α (RARA) gene from chromosome 17q21, leading to the resultant chimeric onco-fusion protein PML-RARA, which is detectable in more than 95% patients and disturbs proper promyelocytic differentiation. All-trans retinoic acid (ATRA) can induce granulocytic differentiation in APL and is used to treat APL patients. Genes containing PML-RARA-targeted promoters are transcriptionally suppressed in APL and most likely constitute a major mechanism of transcriptional repression occurring in APL. A growing body of evidence points to the role of distal regulatory elements, including enhancers, in the control of gene expression. In order to understand the unique sets of enhancers that might be under the control of PML-RAR and crucial for granulocytic differentiation of NB4 cells, we analysed the enhancer landscape of control and ATRA treated NB4 cells. H3K9Ac mapping identified a repertoire of enhancers that were gained in NB4 cells treated with ATRA. Closer investigation of these enhancer elements revealed enrichment of H3K9Ac signals around major drivers of myeloid differentiation. Of note, we identified a gain in enhancer signature for a region about 7kb downstream of the CEBPE gene. Our previous studies identified a novel enhancer for CEBPE in murine hematopoietic cells, which was 6 downstream of CEBPE core promoter. It appears that the +7kb region we identified in human APL cells may be analogous to the murine enhancer. We also observed that PML-RAR binds this +7kb region and ATRA treatment of NB4 cells displaced binding of PML-RAR from the + 7kb region, suggestive of a transcriptional repressive effect of PML-RAR at such enhancer elements. To test the transcription regulating potential of this +7kb region, we used catalytically inactive Cas9 fused to Krüppel associated box (KRAB) domain (dCas9-KRAB). We designed three guide RNAs covering this regulatory region. The sgRNAs effectively repressed expression of CEBPE accompanied by lowered granulocytic differentiation of these guide RNA targeted NB4 cells after ATRA treatment. To explore transcription factor (TF) occupancy at this +7 kb region, we analysed public available ChIP-seq datasets for hematopoiesis-specific factors. Analysis revealed that the +7kb region was marked by an open chromatin signature, accompanied by binding of a majority of hematopoietic TFs around this putative regulatory element with concurrent binding of EP300. Strikingly we noticed binding of CEBPA, CEBPB and CEBPE at this regulatory element. To assess whether binding of these members of the CEBP family of TFs is functionally relevant, luciferase reporter and electrophoretic mobility shift assays (EMSA) were performed. Co expression of the CEBP TFs led to significant induction of luciferase expression, and this data was further confirmed using EMSA assays. Based on these observations, we propose that PML-RAR blocks granulocytic differentiation by occupying this +7kb enhancer of CEBPE, hinders binding of other cell type/lineage specific TFs, and blocks CEBPE expression. When cells are stimulated with ATRA, PML-RAR is displaced from the CEBPE enhancer, allowing for efficient binding of myeloid-specific TFs. This results in increased CEBPE expression, which in turn promotes efficient granulocytic differentiation. The findings from our study expands our current understanding of the mechanism of differentiation therapy, the role of onco-fusion proteins in inhibiting myeloid differentiation, and may provide new therapeutic approaches to many acute myeloid leukemias. Disclosures Ong: National University of Singapore: Other: Royalties.

Mutations in genes encoding components of the RNA splicing machinery constitute the leading class of genetic alterations in myelodysplastic syndromes (MDS). Somatic inactivating alterations of splicing factor, ZRSR2, are observed in ~10% of MDS patients. Mutations/loss of ZRSR2 in human myeloid cells causes aberrant splicing, primarily impairing splicing of the U12-type introns. However, the precise role of ZRSR2 in splicing and hematopoietic development needs further exploration. To understand the function of ZRSR2 in hematopoietic development and splicing, we generated Zrsr2 knockout mice. RNA-Seq of sorted myeloid precursor populations (common myeloid progenitors, granulocyte monocyte progenitors and megakaryocyte erythrocyte progenitors) revealed aberrant retention of U12-type introns in ZRSR2-deficient murine cells, similar to our previous observation in ZRSR2 mutant MDS bone marrow cells (Madan et al, Nat Commun 2015). However, despite complete loss of ZRSR2 in our mouse model, the effect on splicing of U12-type introns was modest compared to the ZRSR2 mutant MDS bone marrow and ZRSR2 knockdown human AML cells (K562 and TF1). Moreover, our comprehensive analyses of hematopoietic compartment in ZRSR2-deficient (Zrsr2Δ/Y) compared to WT (Zrsr2+/Y) male mice, indicated that ZRSR2 was dispensable for hematopoietic differentiation. We did not observe any significant difference in peripheral blood counts, bone marrow cellularity, proportion of hematopoietic stem cells (HSCs) and myeloid precursors (CMP, GMP, MEP) in the bone marrow of both young (7-10 weeks) and old (>1 year) mice of either genotypes. Competitive and non-competitive reconstitution assays also demonstrated that loss of ZRSR2 does not affect repopulation potential of HSCs. Collectively, our data demonstrate that ZRSR2 is not essential for hematopoietic development in mice. Further, to understand the underlying determinant for our unexpected observation that deletion of Zrsr2 in mice did not impact hematopoietic development and affected modestly splicing of U12-type introns, we investigated the role of a closely-related homolog, ZRSR1, in splicing of U12-type introns. We utilized shRNA-mediated silencing of ZRSR1 in murine myeloid precursors (Lin−Kit+ bone marrow cells), and assessed splicing using RNA-Sequencing. Wildtype myeloid cells expressing Zrsr1 shRNA did not display significant mis-splicing of the U12-type introns. However, knockdown of Zrsr1 in ZRSR2-deficient myeloid cells exacerbated mis-splicing of the U12-introns. This suggested that ZRSR1 contributes to regulation of U12-spliceosome in murine hematopoietic cells. Increased mis-splicing of U12-type introns in ZRSR1/ZRSR2-deficient cells was validated using quantitative RT-PCR. Further to verify the compensatory role of murine ZRSR1 in splicing of U12-type introns, we used CRISPR/Cas9 technology to generate 32D cells lacking either one or both ZRSR proteins. Quantitative RT-PCR analysis showed notable aberrant retention of U12-type introns in Zrsr1/Zrsr2 double-deficient 32D cells compared to either WT or single KO cells. We also demonstrated that aberrant retention of U12-type introns of MAPK9 and MAPK14 caused by deficiency of ZRSR proteins led to their reduced protein expression in both human and murine myeloid cells. Taken together, our study highlights functional role of murine ZRSR1 in splicing of U12-type introns in murine hematopoietic cells where it can partially compensate for the deficiency of ZRSR2. Therefore, deficiency of ZRSR2 alone is insufficient to impact extensively RNA splicing in mice, and further studies with concurrent deficiency of ZRSR1 and ZRSR2 are warranted to replicate complete loss of ZRSR activity. Disclosures Chng: Novartis: Honoraria; Abbvie: Honoraria; Amgen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.

CCAAT/enhancer binding protein ε (CEBPE) is an essential transcription factor for granulocytic differentiation. Mutations of CEBPE occur in individuals with neutrophil-specific granule deficiency (SGD), which is characterized by defects in neutrophil maturation. Cebpe-knockout mice also exhibit defects in terminal differentiation of granulocytes, a phenotype reminiscent of SGD. Analysis of DNase I hypersensitive sites sequencing data revealed an open chromatin region 6 kb downstream of the transcriptional start site of Cebpe in murine myeloid cells. We identified an interaction between this +6-kb region and the core promoter of Cebpe using circular chromosome conformation capture sequencing (4C-seq). To understand the role of this putative enhancer in transcriptional regulation of Cebpe, we targeted it using catalytically inactive Cas9 fused to Krüppel-associated box (KRAB) domain and observed a significant downregulation of transcript and protein levels of CEBPE in cells expressing guide RNA targeting the +6-kb region. To further investigate the role of this novel enhancer further in myelopoiesis, we generated mice with deletion of this region using CRISPR/Cas9 technology. Germline deletion of the +6-kb enhancer resulted in reduced levels of CEBPE and its target genes and caused a severe block in granulocytic differentiation. We also identified binding of CEBPA and CEBPE to the +6-kb enhancer, which suggests their role in regulating the expression of Cebpe. In summary, we have identified a novel enhancer crucial for regulating expression of Cebpe and required for normal granulocytic differentiation.

ARID1A is a key component of ATP-dependent SWI/SNF complex involved in chromatin remodeling. Chromatin remodeling mediated by SWI/SNF complex is crucial for gene expression and affects a broad range of biological processes including hematopoietic development. ARID1A is frequently mutated across several solid tumors as well as hematopoietic malignancies, including Burkitt's lymphoma, diffuse large B-cell lymphoma and acute promyelocytic leukemia. Nevertheless, function of ARID1A in adult hematopoiesis and implications of its deficiency in development and progression of hematopoietic diseases has not been explored. In this study, we used a murine model of ARID1A deficiency to establish its essential function in maintaining normal hematopoietic development. Germline loss of Arid1a is embryonic lethal; therefore, we generated mice with deletion of Arid1a specifically in the hematopoietic compartment using Vav-iCre and Mx1-Cre transgenic mice. Arid1afl/fl;Vav-iCre+ mice occurred at a lower than expected frequency, suggesting some perinatal mortality. For the Mx1-Cre model, Arid1a exon 9 was excised by administrating poly(I:C) to adult mice and hematopoiesis was evaluated using flow cytometry. An increase in both percentage and absolute number of long-term hematopoietic stem cells (LTHSCs) defined as Lin-Sca1+Kit+CD34-FLT3- or Lin-Sca1+Kit+CD48-CD150+ occurred in the bone marrow using both models of Arid1a deficiency. RNA-sequencing of sorted LTHSCs from Arid1a KO bone marrow revealed dysregulated expression of several genes involved in cell cycle, G2/M checkpoint and related pathways. In vivo BrdU incorporation assays showed a substantially lower proportion of quiescent hematopoietic stem cells in Arid1a deficient bone marrow. To assess the reconstitution ability of ARID1A deficient HSCs, sorted KO or WT LTHSCs were transplanted into irradiated congenic recipient mice in competitive repopulation assays. Proportion of donor-derived cells in recipients transplanted with KO cells was strikingly lower compared to wild-type cells, suggesting poor reconstitution ability of Arid1a KO LTHSCs. Also, differentiation of both myeloid and lymphoid lineages was impaired in Arid1a KO mice compared to WT controls. To investigate the mechanism of perturbed differentiation of the myeloid and erythroid lineages, RNA-Seq was performed on sorted CMPs, GMPs and MEPs from WT and Arid1a KO BM. Our analysis showed significant decrease in expression of several transcription factors (Runx1, Gata2, Cebpa), which play a crucial role in lineage differentiation. To determine how Arid1a deficiency alters chromatin accessibility in myeloid precursors, Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-Seq) was performed on sorted Lin-Kit+ BM cells from both Arid1a KO and WT mice. A global reduction in open chromatin in Arid1a KO cells was noted compared to WT cells. A substantial overlap occurred between down regulated genes (RNA-seq) and reduced chromatin accessibility in Arid1a KO myeloid progenitors. Motifs for PU.1, RUNX1, GATA and CEBPA were significantly enriched in loci with reduced ATAC-seq signals in Arid1a KO cells. Our findings demonstrate an indispensable function of Arid1a in hematopoietic development and underline the importance of precise chromatin dynamics maintained by ARID1A-containing SWI/SNF complex in hematopoiesis. Disclosures No relevant conflicts of interest to declare.