Functional mapping of PHF6 complexes in chromatin remodeling, replication dynamics, and DNA repair.

The Plant Homeodomain 6 gene (PHF6) encodes a nucleolar and chromatin-associated leukemia tumor suppressor with proposed roles in transcription regulation. However, specific molecular mechanisms controlled by PHF6 remain rudimentarily understood. Here we show that PHF6 engages multiple nucleosome remodeling protein complexes, including nucleosome remodeling and deacetylase, SWI/SNF and ISWI factors, the replication machinery and DNA repair proteins. Moreover, after DNA damage, PHF6 localizes to sites of DNA injury, and its loss impairs the resolution of DNA breaks, with consequent accumulation of single- and double-strand DNA lesions. Native chromatin immunoprecipitation sequencing analyses show that PHF6 specifically associates with difficult-to-replicate heterochromatin at satellite DNA regions enriched in histone H3 lysine 9 trimethyl marks, and single-molecule locus-specific analyses identify PHF6 as an important regulator of genomic stability at fragile sites. These results extend our understanding of the molecular mechanisms controlling hematopoietic stem cell homeostasis and leukemia transformation by placing PHF6 at the crossroads of chromatin remodeling, replicative fork dynamics, and DNA repair.

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.

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.

Epigenetic reversal of hematopoietic stem cell aging in Phf6-knockout mice.

Aging is characterized by an accumulation of myeloid-biased hematopoietic stem cells (HSCs) with reduced developmental potential. Genotoxic stress and epigenetic alterations have been proposed to mediate age-related HSC loss of regenerative and self-renewal potential. However, the mechanisms underlying these changes remain largely unknown. Genetic inactivation of the plant homeodomain 6 (Phf6) gene, a nucleolar and chromatin-associated factor, antagonizes age-associated HSC decline. Immunophenotyping, single-cell transcriptomic analyses and transplantation assays demonstrated markedly decreased accumulation of immunophenotypically defined HSCs, reduced myeloid bias and increased hematopoietic reconstitution capacity with preservation of lymphoid differentiation potential in Phf6-knockout HSCs from old mice. Moreover, deletion of Phf6 in aged mice rejuvenated immunophenotypic, transcriptional and functional hallmarks of aged HSCs. Long-term HSCs from old Phf6-knockout mice showed epigenetic rewiring and transcriptional programs consistent with decreased genotoxic stress-induced HSC aging. These results identify Phf6 as an important epigenetic regulator of HSC aging.

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.

Transcriptomic Analysis Identifies RNA Binding Proteins as Putative Regulators of Myelopoiesis and Leukemia.

Acute myeloid leukemia (AML) is a common and aggressive hematological malignancy. Acquisition of heterogeneous genetic aberrations and epigenetic dysregulation lead to the transformation of hematopoietic stem cells (HSC) into leukemic stem cells (LSC), which subsequently gives rise to immature blast cells and a leukemic phenotype. LSCs are responsible for disease relapse as current chemotherapeutic regimens are not able to completely eradicate these cellular sub-populations. Therefore, it is critical to improve upon the existing knowledge of LSC specific markers, which would allow for specific targeting of these cells more effectively allowing for their sustained eradication from the cellular milieu. Although significant milestones in decoding the aberrant transcriptional network of various cancers, including leukemia, have been achieved, studies on the involvement of post-transcriptional gene regulation (PTGR) in disease progression are beginning to unfold. RNA binding proteins (RBPs) are key players in mediating PTGR and they regulate the intracellular fate of individual transcripts, from their biogenesis to RNA metabolism, via interactions with RNA binding domains (RBDs). In this study, we have used an integrative approach to systematically profile RBP expression and identify key regulatory RBPs involved in normal myeloid development and AML. We have analyzed RNA-seq datasets (GSE74246) of HSCs, common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), monocytes, LSCs, and blasts. We observed that normal and leukemic cells can be distinguished on the basis of RBP expression, which is indicative of their ability to define cellular identity, similar to transcription factors. We identified that distinctly co-expressing modules of RBPs and their subclasses were enriched in hematopoietic stem/progenitor (HSPCs) and differentiated monocytes. We detected expression of DZIP3, an E3 ubiquitin ligase, in HSPCs, knockdown of which promotes monocytic differentiation in cell line model. We identified co-expression modules of RBP genes in LSCs and among these, distinct modules of RBP genes with high and low expression. The expression of several AML-specific RBPs were also validated by quantitative polymerase chain reaction. Network analysis identified densely connected hubs of ribosomal RBP genes (rRBPs) with low expression in LSCs, suggesting the dependency of LSCs on altered ribosome dynamics. In conclusion, our systematic analysis elucidates the RBP transcriptomic landscape in normal and malignant myelopoiesis, and highlights the functional consequences that may result from perturbation of RBP gene expression in these cellular landscapes.

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.

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.