KDM6 demethylases integrate DNA repair gene regulation and loss of KDM6A sensitizes human acute myeloid leukemia to PARP and BCL2 inhibition.

Acute myeloid leukemia (AML) is a heterogeneous, aggressive malignancy with dismal prognosis and with limited availability of targeted therapies. Epigenetic deregulation contributes to AML pathogenesis. KDM6 proteins are histone-3-lysine-27-demethylases that play context-dependent roles in AML. We inform that KDM6-demethylase function critically regulates DNA-damage-repair-(DDR) gene expression in AML. Mechanistically, KDM6 expression is regulated by genotoxic stress, with deficiency of KDM6A-(UTX) and KDM6B-(JMJD3) impairing DDR transcriptional activation and compromising repair potential. Acquired KDM6A loss-of-function mutations are implicated in chemoresistance, although a significant percentage of relapsed-AML has upregulated KDM6A. Olaparib treatment reduced engraftment of KDM6A-mutant-AML-patient-derived xenografts, highlighting synthetic lethality using Poly-(ADP-ribose)-polymerase-(PARP)-inhibition. Crucially, a higher KDM6A expression is correlated with venetoclax tolerance. Loss of KDM6A increased mitochondrial activity, BCL2 expression, and sensitized AML cells to venetoclax. Additionally, BCL2A1 associates with venetoclax resistance, and KDM6A loss was accompanied with a downregulated BCL2A1. Corroborating these results, dual targeting of PARP and BCL2 was superior to PARP or BCL2 inhibitor monotherapy in inducing AML apoptosis, and primary AML cells carrying KDM6A-domain mutations were even more sensitive to the combination. Together, our study illustrates a mechanistic rationale in support of a novel combination therapy for AML based on subtype-heterogeneity, and establishes KDM6A as a molecular regulator for determining therapeutic efficacy.

Pbrm1 Steers Mesenchymal Stromal Cell Osteolineage Differentiation by Integrating PBAF-Dependent Chromatin Remodeling and BMP/TGF-ß Signaling.

Bone morphogenic protein (BMP)/transforming growth factor ß (TGF-ß) signaling determines mesenchymal-stromal-cell (MSC) osteolineage commitment and tissue identity. However, molecular integration of developmental signaling with MSC-intrinsic chromatin regulation remains incompletely understood. SWI/SNF-(BAF) is an ATP-dependent chromatin remodeler implicated in multi-cellular development. We show that BMPs and long-term osteogenic signals in MSCs selectively induce expression of polybromo BAF (PBAF) components Pbrm1, Arid2, and Brd7. Loss of Pbrm1/Arid2/Brd7 profoundly impairs osteolineage gene expression and osteogenesis without compromising adipogenesis. Pbrm1 loss attenuates MSC in vivo ossification. Mechanistically, Pbrm1/PBAF deficiency impairs Smad1/5/8 activation through locus-specific epi-genomic remodeling, involving Pbrm1 bromodomains, along with transcriptional downregulation of Bmpr/TgfßrII affecting BMP-early-responsive gene expression. Gain of function of BmprIß, TgfßrII in PBAF-deficient MSCs partly restores Smad1/5/8 activation and osteogenesis. Pbrm1 loss further affects hematopoietic stem and progenitor activity through non-cell-autonomous regulation of microenvironment and niche-factor expression. Together, these findings reveal a link illustrating epi-genomic feedforward control of BMP/TGF-ß signaling to transcriptional and cellular plasticity in the mesenchymal microenvironment and account for stromal-SWI/SNF in hematopoiesis.

MBD3/NuRD loss participates with KDM6A program to promote DOCK5/8 expression and Rac GTPase activation in human acute myeloid leukemia.

Cancer genome sequencing studies have focused on identifying oncogenic mutations. However, mutational profiling alone may not always help dissect underlying epigenetic dependencies in tumorigenesis. Nucleosome remodeling and deacetylase (NuRD) is an ATP-dependent chromatin remodeling complex that regulates transcriptional architecture and is involved in cell fate commitment. We demonstrate that loss of MBD3, an important NuRD scaffold, in human primary acute myeloid leukemia (AML) cells associates with leukemic NuRD. Interestingly, CHD4, an intact ATPase subunit of leukemic NuRD, coimmunoprecipitates and participates with H3K27Me3/2-demethylase KDM6A to induce expression of atypical guanine nucleotide exchange factors, dedicator of cytokinesis (DOCK) 5 and 8 (DOCK5/8), promoting Rac GTPase signaling. Mechanistically, MBD3 deficiency caused loss of histone deacytelase 1 occupancy with a corresponding increase in KDM6A, CBP, and H3K27Ac on DOCK5/8 loci, leading to derepression of gene expression. Importantly, the Cancer Genome Atlas AML cohort reveals that DOCK5/ 8 levels are correlated with MBD3 and KDM6A, and DOCK5/ 8 expression is significantly increased in patients who are MBD3 low and KDM6A high with a poor survival. In addition, pharmacological inhibition of DOCK signaling selectively attenuates AML cell survival. Because MBD3 and KDM6A have been implicated in metastasis, our results may suggest a general phenomenon in tumorigenesis. Collectively, these findings provide evidence for MBD3-deficient NuRD in leukemia pathobiology and inform a novel epistasis between NuRD and KDM6A toward maintenance of oncogenic gene expression in AML.-Biswas, M., Chatterjee, S. S., Boila, L. D., Chakraborty, S., Banerjee, D., Sengupta, A. MBD3/NuRD loss participates with KDM6A program to promote DOCK5/8 expression and Rac GTPase activation in human acute myeloid leukemia.

SWI/SNF subunit expression heterogeneity in human aplastic anemia stem/progenitors.

Acquired aplastic anemia (AA) is a bone marrow (BM) failure associated with autoimmune destruction of hematopoietic stem cells (HSCs). Although somatic mutations have been identified in AA patients, mutations alone do not explain AA pathophysiology. SWI/SNF is an evolutionarily conserved, multi-subunit, ATP-dependent chromatin-remodeling protein complex that plays an important role in mammalian hematopoiesis. Herein, gene expression analysis identified a significant loss of the SWI/SNF core component SMARCC1, along with ARID1B, ACTL6A, and SMARCD1, in human AA BM CD34+ HSCs and hematopoietic stem and progenitor cells (HSPCs) compared with normal HSPCs. However, expression of SMARCA4, SMARCB1, SMARCD3, and DPF2 remained intact in our AA cohort. PBRM1, BRD7, and SMARCA2 expression were significantly upregulated in both untreated and follow-up AA patients. Clonal hematopoiesis in AA is associated with evolution to late clonal disorders, including myelodysplastic syndromes (MDS). Apart from SMARCD1 loss, we did not observe significant alteration of SWI/SNF expression in MDS HSPCs, indicating SWI/SNF differential expression in AA and MDS. In addition, except for ACTL6A, SWI/SNF expression was unaltered in aged HSPCs. Importantly, our results provide evidence for loss of SWI/SNF in AA, and may implicate AA HSPC-autonomous defective SWI/SNF regulation as an integral component of BM failure, in addition to autoimmune destruction of AA HSCs. These findings illustrate for the first time SWI/SNF subunit expression heterogeneity in human AA HSPCs and require prognostic validation in a larger cohort.

SMARCB1 Deficiency Integrates Epigenetic Signals to Oncogenic Gene Expression Program Maintenance in Human Acute Myeloid Leukemia.

SWI/SNF is an evolutionarily conserved multi-subunit chromatin remodeling complex that regulates epigenetic architecture and cellular identity. Although SWI/SNF genes are altered in approximately 25% of human malignancies, evidences showing their involvement in tumor cell-autonomous chromatin regulation and transcriptional plasticity are limiting. This study demonstrates that human primary acute myeloid leukemia (AML) cells exhibit near complete loss of SMARCB1 (BAF47 or SNF5/INI1) and SMARCD2 (BAF60B) associated with nucleation of SWI/SNFΔ SMARCC1 (BAF155), an intact core component of SWI/SNFΔ, colocalized with H3K27Ac to target oncogenic loci in primary AML cells. Interestingly, gene ontology (GO) term and pathway analysis suggested that SMARCC1 occupancy was enriched on genes regulating Rac GTPase activation, cell trafficking, and AML-associated transcriptional dysregulation. Transcriptome profiling revealed that expression of these genes is upregulated in primary AML blasts, and loss-of-function studies confirmed transcriptional regulation of Rac GTPase guanine nucleotide exchange factors (GEF) by SMARCB1. Mechanistically, loss of SMARCB1 increased recruitment of SWI/SNFΔ and associated histone acetyltransferases (HAT) to target loci, thereby promoting H3K27Ac and gene expression. Together, SMARCB1 deficiency induced GEFs for Rac GTPase activation and augmented AML cell migration and survival. Collectively, these findings highlight tumor suppressor role of SMARCB1 and illustrate SWI/SNFΔ function in maintaining an oncogenic gene expression program in AML.Implications: Loss of SMARCB1 in AML associates with SWI/SNFΔ nucleation, which in turn promotes Rac GTPase GEF expression, Rac activation, migration, and survival of AML cells, highlighting SWI/SNFΔ downstream signaling as important molecular regulator in AML. Mol Cancer Res; 16(5); 791-804. ©2018 AACR.

KDM6 and KDM4 histone lysine demethylases emerge as molecular therapeutic targets in human acute myeloid leukemia.

Acute myeloid leukemia (AML) remains an aggressive hematopoietic malignancy that is caused by proliferation of immature myeloid cells and is frequently characterized by perturbations in chromatin-modifying enzymes. Emerging evidence indicates that histone demethylases play a role in tumorigenesis. However, due to the complexity of this enormous family of histone-modifying enzymes, substrate redundancy, and context-specific roles, the contribution of each member remains ambiguous and targeting them remains challenging. Here, we analyzed expression of histone-3-lysine (H3K) demethylases and their cognate substrates in a cohort of de novo AML patients, which demonstrated that the expression of H3K27Me3/2-demethylases and selected members of H3K9Me3/2/1-demethylases are significantly increased in AML. KDM6 upregulation is associated with a global decrease in H3K27Me3 level. Importantly, our data show that pharmacological inhibition of H3K27Me3/2-demethylases or H3K9Me3/2-demethylases, either alone or in combination, could be considered an interesting molecular therapeutic modality in human AML independent of its subtype.

Primary extramedullary plasmacytoma of ovary: Report of a rare neoplasm.

Primary extramedullary plasmacytoma is a rare plasma cell neoplasm. Extramedullary plasmacytomas are most commonly found in head and neck region, but it can occur at other sites occasionally. Involvement of ovary by this tumor is exceedingly rare. Here, we report a case of primary ovarian plasmacytoma in a 47-year-old woman. The patient presented with a lower abdominal pain and a left ovarian mass (12 cm × 10 cm) was detected during the ultrasonographic examination. The patient underwent hysterectomy with bilateral salpingo-oophorectomy. Subsequent histopathologic, immunohistochemistry, bone marrow examination, and other relevant examinations established the diagnosis of primary ovarian plasmacytoma. The patient did not receive the postoperative chemotherapy and 6 months follow-up was uneventful.

Is there any role of mast cell density and microvessel density in cervical squamous cell carcinoma? A histologic study with special reference to CD-34 immunomarker staining.

BACKGROUND: Mast cells are involved in induction of angiogenesis in the early-stages of tumor development and in modulating blood vessel growth in the later stages of tumor progression. AIMS AND OBJECTIVES: This study was carried out to evaluate the association between mast cell density (MCD) and microvessel density (MVD) in carcinoma in situ (CIS), microinvasive carcinoma (CA) and invasive squamous cell CA of cervix. MATERIALS AND METHODS: Six cases of CIS, four cases of microinvasive CA and 38 cases of invasive CA were studied over a period of 2 years from August, 2011 to June, 2013. Ten control samples were included in the study. Routine histologic examination was done. Toluidine blue stain was used for MCD determination. Immunohistochemical analysis with CD-34 was done for assessing MVD. Student's t-test was used to calculate the statistical significance of MCD and MVD. RESULTS: Both MCD and MVD increased from normal samples through CIS to invasive cervical CA. In the four cases of microinvasive CA, the MCD and MVD were more than that of the control samples, but less than that of the six cases of CIS. CONCLUSION: There is a correlation between mast cell accumulation and angiogenesis in CIS, microinvasive CA and invasive cervical squamous cell CA. MCD and MVD in invasive CA exceed those in CIS and microinvasive CA. It gives us an opportunity to postulate that therapeutic strategies against mast cell mediators and angiogenesis may be of benefit in patients of early-stage cervical CA.