ISWI chromatin remodeling ATPase SMARCA5 (SNF2H) is a well-known factor for its role in regulation of DNA access via nucleosome sliding and assembly. SMARCA5 transcriptionally inhibits the myeloid master regulator PU.1. Upregulation of SMARCA5 was previously observed in CD34+ hematopoietic progenitors of acute myeloid leukemia (AML) patients. Since high levels of SMARCA5 are necessary for intensive cell proliferation and cell cycle progression of developing hematopoietic stem and progenitor cells in mice, we reasoned that removal of SMARCA5 enzymatic activity could affect the cycling or undifferentiated state of leukemic progenitor-like clones. Indeed, we observed that CRISPR/cas9-mediated SMARCA5 knockout in AML cell lines (S5KO) inhibited the cell cycle progression. We also observed that the SMARCA5 deletion induced karyorrhexis and nuclear budding as well as increased the ploidy, indicating its role in mitotic division of AML cells. The cytogenetic analysis of S5KO cells revealed the premature chromatid separation. We conclude that deleting SMARCA5 in AML blocks leukemic proliferation and chromatid cohesion.

Development of lymphoid progenitors requires a coordinated regulation of gene expression, DNA replication, and gene rearrangement. Chromatin-remodeling activities directed by SWI/SNF2 superfamily complexes play important roles in these processes. In this study, we used a conditional knockout mouse model to investigate the role of Smarca5, a member of the ISWI subfamily of such complexes, in early lymphocyte development. Smarca5 deficiency results in a developmental block at the DN3 stage of αβ thymocytes and pro-B stage of early B cells at which the rearrangement of Ag receptor loci occurs. It also disturbs the development of committed (CD73⁺) γδ thymocytes. The αβ thymocyte block is accompanied by massive apoptotic depletion of β-selected double-negative DN3 cells and premitotic arrest of CD4/CD8 double-positive cells. Although Smarca5-deficient αβ T cell precursors that survived apoptosis were able to undergo a successful TCRβ rearrangement, they exhibited a highly abnormal mRNA profile, including the persistent expression of CD44 and CD25 markers characteristic of immature cells. We also observed that the p53 pathway became activated in these cells and that a deficiency of p53 partially rescued the defect in thymus cellularity (in contrast to early B cells) of Smarca5-deficient mice. However, the activation of p53 was not primarily responsible for the thymocyte developmental defects observed in the Smarca5 mutants. Our results indicate that Smarca5 plays a key role in the development of thymocytes undergoing β-selection, γδ thymocytes, and also B cell progenitors by regulating the transcription of early differentiation programs. Key Points ISWI ATPase Smarca5 guides developmental mRNA program of β-selected thymocytes. Inactivation of Smarca5 gene in developing thymocytes activates p53 and apoptosis. Smarca5 regulates cell fate decisions of γδ thymocytes and survival of pro-B cells. Visual Abstract

Multiple myeloma (MM) is characterized by malignant proliferation of plasma cells (PC) which accumulate in the bone marrow (BM). The advent of new drugs has changed the course of the disease from incurable to treatable, but most patients eventually relapse. One group of MM patients (10-15%) is considered high-risk because they relapse within 24 months. Recently, extramedullary relapse of MM (EM) has been observed more frequently. Due to its aggressivity and shorter survival, EM is also considered high-risk. The goal of this study was to determine if the so-called high-risk genes published by the University of Arkansas group (UAMS) are even more deregulated in EM patients than in high-risk MM patients and if these patients may be considered high-risk. Nine samples of bone marrow plasma cells from MM patients as well as 9 tumors and 9 bone marrow plasma cells from EM patients were used. Quantitative real-time PCR was used for evaluation of expression of 15 genes connected to the high-risk signature of MM patients. Comparison of high-risk plasma cells vs extramedullary plasma cells revealed 4 significantly deregulated genes (CKS1B, CTBS, NADK, YWHAZ); moreover, comparison of extramedullary plasma cells vs extramedullary tumors revealed significant differences in 9 out of 15 genes. Of these, 6 showed significant changes as described by the UAMS group (ASPM, SLC19A1, NADK, TBRG4, TMPO and LARS2). Our data suggest that increasing genetic abnormalities as described by the gene expression data are associated with increased risk for EM relapse.

The transcription factor PU.1 (Purine-rich DNA binding, SPI1) is a key regulator of hematopoiesis, whose level is influenced by transcription through its enhancers and its post-transcriptional degradation via microRNA-155 (miR-155). The degree of transcriptional regulation of the PU.1 gene is influenced by repression via DNA methylation, as well as other epigenetic factors, such as those related to progenitor maturation status, which is modulated by the transcription factor Myeloblastosis oncogene (MYB). In this work, we show that combinatorial treatment of acute myeloid leukemia (AML) cells with DNA methylation inhibitors (5-Azacytidine), MYB inhibitors (Celastrol), and anti-miR-155 (AM155) ideally leads to overproduction of PU.1. We also show that PU.1 reactivation can be compensated by miR-155 and that only a combined approach leads to sustained PU.1 derepression, even at the protein level. The triple effect on increasing PU.1 levels in myeloblasts stimulates the myeloid transcriptional program while inhibiting cell survival and proliferation, leading to partial leukemic differentiation.