BRD9 at the crossroads of splicing, chromatin remodeling, and hematopoiesis.

The Baf Subunit Dpf2 Regulates Resolution of Inflammation By Controlling Macrophage Differentiation Transcription Factor Networks

BackgroundEpigenetic regulation is important in hematopoiesis, but the involvement of histone variants is poorly understood. Myelodysplastic syndromes (MDS) are heterogeneous clonal hematopoietic stem cell (HSC) disorders characterized by ineffective hematopoiesis. MacroH2A1.1 is a histone H2A variant that negatively correlates with the self-renewal capacity of embryonic, adult, and cancer stem cells. MacroH2A1.1 is a target of the frequent U2AF1 S34F mutation in MDS. The role of macroH2A1.1 in hematopoiesis is unclear.ResultsMacroH2A1.1 mRNA levels are significantly decreased in patients with low-risk MDS presenting with chromosomal 5q deletion and myeloid cytopenias and tend to be decreased in MDS patients carrying the U2AF1 S34F mutation. Using an innovative mouse allele lacking the macroH2A1.1 alternatively spliced exon, we investigated whether macroH2A1.1 regulates HSC homeostasis and differentiation. The lack of macroH2A1.1 decreased while macroH2A1.1 haploinsufficiency increased HSC frequency upon irradiation. Moreover, bone marrow transplantation experiments showed that both deficiency and haploinsufficiency of macroH2A1.1 resulted in enhanced HSC differentiation along the myeloid lineage. Finally, RNA-sequencing analysis implicated macroH2A1.1-mediated regulation of ribosomal gene expression in HSC homeostasis.ConclusionsTogether, our findings suggest a new epigenetic process contributing to hematopoiesis regulation. By combining clinical data with a discrete mutant mouse model and in vitro studies of human and mouse cells, we identify macroH2A1.1 as a key player in the cellular and molecular features of MDS. These data justify the exploration of macroH2A1.1 and associated proteins as therapeutic targets in hematological malignancies.

The mammalian SWI/SNF (mSWI/SNF or BAF) complex is an ATP-dependent chromatin remodeler long thought to be a tumor suppressor because it is frequently inactivated in cell lines. It was definitively identified as a tumor suppressor in malignant rhabdoid tumors (MRTs), an aggressive pediatric cancer. In most of these tumors, the dedicated BAF subunit SMARCB1 (BAF47) undergoes loss-of-heterozygosity, with no other large-scale chromosomal instability, leading to the conclusion that the tumor-suppression mechanism in MRTs is “epigenetic” in nature, meaning that it leads to changes in gene expression without necessarily altering other sequences of DNA. However, these tumors account for a small fraction of all cancers containing mutations in BAF subunits, and other evidence implicates the BAF complex in DNA repair processes, making its tumor-suppression mechanism uncertain. We sought to determine the applicability of this epigenetic model of tumor suppression in the large number of other cancers bearing BAF subunit mutations. To answer this question, we analyzed exome sequencing and genomic copy-number data derived from 5,659 patient samples, together representing 22 cancer types and over 4,500,000 genetic alterations, to compare the genomic states of tumors with BAF subunit defects against similar tumors that lacked such defects. We find that BAF subunits collectively are mutated significantly more frequently than expected in 19 of the 22 cancer types examined, far more than expected. The increase in BAF subunit mutations was statistically robust, both in samples with low mutation rates (e.g., lower-grade glioma), as well as those with high rates (e.g., melanoma, tumors of the lung), indicating that mutation of the complex is under positive selection in the vast majority of cancers. We also performed an analysis of copy-number variation of BAF subunits in each cancer, and find recurrent patterns of biallelic deletions and amplifications. In many malignancies, mutation and biallelic deletion of BAF subunits occurs at rates comparable to or higher than well-known tumor suppressors, such as TP53, PTEN, and RB1. In the majority of cancer types, tumors with BAF subunit defects have higher mutational rates than cancers of the same tissue lacking such defects. By reconstructing the mutational processes active in each patient sample, we find that excess mutations in BAF-deficient cancers represent the signatures of defective mismatch repair, cytidine deamination, UV-damage response, DNA end joining, Pol epsilon function, and other enzymatic processes acting on DNA. In four cancer types where ChIP-seq tracks were available for the healthy cell type of origin, we tracked the locations of over 280,000 mutations, and find that excess mutations in BAF-deficient tumors are biased towards regions of elevated H3K36me3 and low H3K27me3 in their corresponding non-cancerous tissues. We hypothesized that these mutations resulted from inaccessibility for repair and maintenance factors when a BAF subunit is defective. We tested this hypothesis using topoisomerase II alpha binding as representative of enzymes that require access to DNA, and find that rapid conditional deletion of the BAF ATPase Brg led to an increase in genome-wide deposition of H3K27me3 at Brg-dependent topoisomerase II alpha sites from an initial low state, contributing to loss of enzyme accessibility at its target sites. Our data indicate that the tumor suppressive functions of BAF complexes arise from their contribution to maintaining genomic stability by providing access to DNA for repair and maintenance mechanisms. Consistent with this conclusion, we examined levels of P53 and phospho-P53 in mouse embryonic fibroblasts expressing a tamoxifen-inducible deletion of Brg, the ATPase of the BAF complex. Deletion of Brg causes a significant increase in the levels of P53 and phospho-P53 following DNA damage by a panel of conditions, confirming that genomic stability and DNA repair is compromised upon loss of BAF activity. Our data are consistent with a general model, wherein DNA accessibility is maintained at a steady state arising from a balance between silencing factors and factors (including the BAF complex) that maintain accessibility. Many repair and maintenance factors operate on DNA that require at least transient accessibility, making the BAF complex an upstream contributor to several activities. When BAF activity is compromised, the equilibrium is shifted towards a state that is less accessible to repair enzymes. This disruption of normal repair and maintenance function leads to accumulation of mutations. Our analysis of 5,659 patient genomes indicates that this is the mechanism of tumor suppression under positive selection in the majority of human malignancies. Citation Format: Courtney Hodges, Diana Hargreaves, Erik Miller, Gerald R. Crabtree. Chromatin accessibility underlies the tumor-suppression role of BAF (mSWI/SNF) complexes in many malignancies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr NG05. doi:10.1158/1538-7445.AM2015-NG05

Nucleosome sliding is a frequent result of energy-dependent nucleosome remodelling in vitro. This review discusses the possible roles for nucleosome sliding in the assembly and maintenance of dynamic chromatin and for the regulation of diverse functions in eukaryotic nuclei.

Deubiquitinases (DUBs) are enzymes that control the stability, interactions or localization of most cellular proteins by removing their ubiquitin modification. In recent years, some DUBs, such as USP7, USP9X and USP10, have been identified as promising therapeutic targets in hematological malignancies. Importantly, some potent inhibitors targeting the oncogenic DUBs have been developed, showing promising inhibitory efficacy in preclinical models, and some have even undergone clinical trials. Different DUBs perform distinct function in diverse hematological malignancies, such as oncogenic, tumor suppressor or context-dependent effects. Therefore, exploring the biological roles of DUBs and their downstream effectors will provide new insights and therapeutic targets for the occurrence and development of hematological malignancies. We summarize the DUBs involved in different categories of hematological malignancies including leukemia, multiple myeloma and lymphoma. We also present the recent development of DUB inhibitors and their applications in hematological malignancies. Together, we demonstrate DUBs as potential therapeutic drug targets in hematological malignancies.

The multi-subunit BAF (SWI/SNF) complex is capable of using energy generated from ATP hydrolysis to reorder chromatin structure. Such chromatin changes are known to affect cell biological processes through gene expression regulation. Thus, BAF complex plays pivotal roles in many developmental event. As a focus of the studies present, we identified the BAF complex as a powerful regulator of coticogenesis and embryogenesis. In this study, we employed a novel mouse model system in which deletion of the BAF complex subunits BAF155 and BAF170 results in proteomic degradations of the entire BAF complex and resultant loss of its epigenetic function. The first part of the thesis, highlights how the BAF complex stability is dependent on the dual presence of the scaffolding subunits BAF155 and BAF170, and the disturbance in the H3K27me2/3 epigenetic landscape when they are ablated specifically in the forebrain under the control Foxg1-Cre activity or in the entire embryo using the ubiquitous-inducible CAG-Cre. Preliminary evidence of the involvement of BAF complex in cortical development was thus obtained in the first part of the studies. In the second part, we gathered conclusive evidence on how the BAF complex regulates cortical and hippocampal development. Our investigation in the dcKO_hGFAP_Cre developing cortex revealed the BAF complexes induces heterochromatin state at gene loci involved in neural progenitor proliferation and Wnt signaling; leading to their suppression. On the other hand, the BAF complex enhances the transcription of neuronal differentiation-related genes by promoting euchromatin formation at associated genomic regions. Together, we reported that the activity of the BAF complex ensures the appropriate proliferative capacity of neural progenitors and their neuronal output in late cortical development.

Genetic Validation of the Palmitoylation/Depalmitoylation Cycle As a Drug Target in NRAS Mutant Hematologic Malignancies In Vivo

The deregulation of epigenetic modulation has been well established as a common occurrence in cancer. However, the extent of its involvement in the development and progression of cancer was underscored by genome- and exome-wide sequencing studies over the last several years, which revealed a close association between the epigenome and the pathogenesis of cancer. A class of chromatin regulators, the ATP-dependent chromatin remodellers (particularly subunits of the mammalian BAF complex) are among the most frequently mutated genes across cancer types. These factors showed an alteration frequency in over 20% of all cancers. Among the BAF complex subunits, defects in ARID1A (AT-rich interactive domain-containing protein 1A) are the most frequently found and widespread across many cancer types. Mutations in ARID1A are usually frameshift or nonsense mutations which lead to a loss of its expression in these tumors. In fact, ARID1A is also one of the most frequently mutated chromatin regulators in human colorectal cancer (CRC). Several studies in cell culture and mouse models have shown that loss of ARID1A leads to increased proliferation and tumorigenesis in several cancer types, indicating a tumor suppressive function. Very interestingly, a study described the pivotal role of ARID1A in driving CRC in which its inactivation alone led to the formation of invasive adenocarcinomas in mice. Surprisingly, in contrast to the expected tumor suppressive role of ARID1A in CRC, we observed that the knockout (KO) of ARID1A in CRC cell lines leads to impaired proliferation. Moreover, subcutaneous xenografts in SCID mice using human ARID1A KO CRC cells did not form more aggressive tumors than their wildtype counterparts. Also, the generation of several mouse models in the literature of Arid1a deletion revealed that it can have oncogenic functions. These results indicate a context-dependent role of ARID1A in cancer. We observed an impairment in proliferation in two of the four cell lines in which we performed ARID1A KO. Strikingly, these cell lines harbor the KRASG13D mutation. Therefore, we sought to explore the transcriptional role that ARID1A plays downstream of this pathway. To uncover this, we utilized several publicly available ChIP-seq, mRNA-seq and ATAC-seq datasets and generated our own ChIP-seq dataset for ARID1A in the CRC cell line HCT116. We observed a substantial co-localization of the BAF complex with AP1 transcription factors, such as JunD, that act downstream of the MEK/ERK signaling pathway, suggesting cooperation between these factors. Analysis of the sites at which ARID1A binds showed an enrichment of AP1 transcription factor binding sequences. Most sites co-occupied by ARID1A/AP1 are distal to transcription start sites. Therefore, it is likely that these transcriptional regulators functionally interact at enhancers to elicit gene expression changes in CRC. To examine this, we next explored the MEK/ERK pathway. We identified some targets that are co-localized at distal regulatory sites for genes and are downregulated by ARID1A KO, Trametinib (a MEK/ERK pathway inhibitor) treatment and depletion of JunD. Strikingly, the occupancy of JunD and the acetylation of H3K27 (often an active enhancer mark) was also reduced at these distal regulatory sites upon Trametinib treatment and ARID1A KO. Conversely, the occupancy of ARID1A was reduced upon Trametinib treatment or JunD depletion. Thus, these regulatory regions are targets of the MEK/ERK pathway (through AP1) and are dependent on ARID1A as a co-factor. This effect does not seem to be mediated by the known chromatin remodeller functions of ARID1A since the accessibility of chromatin is not affected upon its loss. Thus, we were able to show that ARID1A is required for regulation of KRAS mutation-driven CRC by acting as a co-factor with AP1 transcription factors (TFs) which are downstream of the MEK/ERK pathway at distal regulatory elements. Importantly, this enables the identification of a strategy to stratify CRC by KRAS mutation status and to target the BAF complex in CRCs that are particularly dependent on this pathway.

The BAF (mammalian SWI/SNF) complexes are a family of multi-subunit ATP-dependent chromatin remodelers that use ATP hydrolysis to alter chromatin structure. Distinct BAF complex compositions are possible through combinatorial assembly of homologous subunit families and can serve non-redundant functions. In mammalian neural development, developmental stage-specific BAF assemblies are found in embryonic stem cells, neural progenitors and postmitotic neurons. In particular, the neural progenitor-specific BAF complexes are essential for controlling the kinetics and mode of neural progenitor cell division, while neuronal BAF function is necessary for the maturation of postmitotic neuronal phenotypes as well as long-term memory formation. The microRNA-mediated mechanism for transitioning from npBAF to nBAF complexes is instructive for the neuronal fate and can even convert fibroblasts into neurons. The high frequency of BAF subunit mutations in neurological disorders underscores the rate-determining role of BAF complexes in neural development, homeostasis, and plasticity.

The brain is one of the complex organs designed under the control of a plethora of molecular and cellular regulatory mechanisms, including transcriptional and epigenetic pathways. Brain organogenesis proceeds in a stepwise manner involving both embryonic and postnatal cell biological processes. Such crucial processes include patterning, neurogenesis, and neuronal migration. Patterning of the brain ensures early regional subdivision of the telencephalon and functional areal sculpting of the cortex, neurogenesis is the process of neuronal production, and newly generated neurons migrate to their structural and functional maturation site in the cortex via the process of migration. The ATP-dependent chromatin remodeling BAF complex is one of the powerful epigenetic regulators of several aspects of brain development, although some of its functions thereof are only implicated and not clearly defined. How the BAF complex regulates patterning of the dorsal aspect of the telencephalon and radial glia fiber-dependent neuron migration, are the main themes of this dissertation aimed at expanding the neurodevelopmental importance of the chromatin remodelers during cortical histogenesis. By adopting a conditional genetic manipulation scheme of deleting the two BAF complex scaffolding subunits BAF155 and BAF170, we were able to generate mouse mutants that lacked the expression of BAFs in the cortex. It was observed that early patterning of the dorsal telencephalon that leads to delineation of the midline telencephalic structures such as the hem and hippocampus from the dorsolateral telencephalon (neocortex) is perturbed in the absence of BAF complex. The BAF complex-deficient dorsal telencephalon displayed an expanded hem, no hippocampal primordium, and marked medialization of the ensuing cortex. The BAF complex was identified to cooperate with the transcription and patterning factor LHX2 to drive the regional patterning of the dorsal telencephalon. Strikingly, neurons are utterly misplaced in the mis-patterned BAF complex-ablated dorsal telencephalon, raising the possibility of cortical laminar mis-patterning at later stages of forebrain development. Indeed, the late embryonic and early postnatal developing BAF complex mutant cortices are improperly laminated. Abnormal radial neuronal migration was identified to underscore the disturbed layer formation which specifically stemmed from the loss of radial glia fibers/scaffolds, depletion of cell adhesion, and sub-optimal neuronal polarization following deletion of BAF complex in neural progenitors and/or postmitotic neurons. Interestingly, the WNT signaling was shown to be modulated by the BAF complex to afford normal radial migration of cortical neurons. Altogether, these current investigations have provided insights into how the chromatin remodeling BAF complex contributes to cortical morphogenesis through regulating the regional subdivision of the dorsal telencephalon and neuronal placement during the cytoarchitectural organization of the cortex. Our current findings further clarify the multifaceted means by which the BAF complex regulates brain development and offers additional potential targets for therapeutic consideration in neurodevelopmental disorders imputable to malfunction of the BAF complex in the mammalian brain.

Early forebrain patterning entails the correct regional designation of the neuroepithelium, and appropriate specification, generation, and distribution of neural cells during brain development. Specific signaling and transcription factors are known to tightly regulate patterning of the dorsal telencephalon to afford proper structural/functional cortical arealization and morphogenesis. Nevertheless, whether and how changes of the chromatin structure link to the transcriptional program(s) that control cortical patterning remains elusive. Here, we report that the BAF chromatin remodeling complex regulates the spatiotemporal patterning of the mouse dorsal telencephalon. To determine whether and how the BAF complex regulates cortical patterning, we conditionally deleted the BAF complex scaffolding subunits BAF155 and BAF170 in the mouse dorsal telencephalic neuroepithelium. Morphological and cellular changes in the BAF mutant forebrain were examined using immunohistochemistry and in situ hybridization. RNA sequencing, Co-immunoprecipitation, and mass spectrometry were used to investigate the molecular basis of BAF complex involvement in forebrain patterning. We found that conditional ablation of BAF complex in the dorsal telencephalon neuroepithelium caused expansion of the cortical hem and medial cortex beyond their developmental boundaries. Consequently, the hippocampal primordium is not specified, the mediolateral cortical patterning is compromised, and the cortical identity is disturbed in the absence of BAF complex. The BAF complex was found to interact with the cortical hem suppressor LHX2. The BAF complex suppresses cortical hem fate to permit proper forebrain patterning. We provide evidence that BAF complex modulates mediolateral cortical patterning possibly by interacting with the transcription factor LHX2 to drive the LHX2-dependent transcriptional program essential for dorsal telencephalon patterning. Our data suggest a putative mechanistic synergy between BAF chromatin remodeling complex and LHX2 in regulating forebrain patterning and ontogeny.

Background: Cancer metabolism represents an emerging field of novel cancer target discovery. Somatic point mutations in the metabolic enzymes isocitrate dehydrogenase 1/2 (IDH1/2) confer a novel gain-of-function in cancer cells, which results in the accumulation and secretion of the onco-metabolite, R-2-hydroxyglutarate (2-HG). High levels of 2-HG have been shown to inhibit α᠄KG dependent dioxygenases including histone and DNA demethylases, which regulate the epigenetic state of cells and result in altered cellular differentiation. IDH2 mutations have been identified in a spectrum of solid tumors and hematologic malignancies including chondrosarcoma, glioblastoma, acute myeloid leukemia (AML), and myelodyplastic syndromes (MDS). AG-221 is the first IDH mutant inhibitor in clinical trials; it is an oral, potent, reversible, and selective inhibitor of the mutated IDH2 protein. In a primary human AML xenograft model, AG-221 treatment reduced 2-HG levels and demonstrated a dose dependent survival benefit. Early results of the ongoing first in human phase I study of AG-221 in patients with advanced IDH2 mutant positive hematologic malignancies are reported here. Study Methods: This phase I study of oral AG-221 is designed to evaluate the safety, pharmacokinetics (PK), pharmacodynamics (PD) including assessment of 2-HG levels, and clinical activity in patients with advanced hematologic malignancies. AG-221 is administered orally 2 times per day (BID) in continuous 28-day cycles. Sequential cohorts of up to 5 PK-evaluable patients are enrolled at higher dose levels, followed by multiple planned expansion cohorts. The eligible patient population includes those with relapsed or refractory AML, myelodysplastic syndromes (MDS,) and elderly untreated AML that harbor an IDH2 mutation. Blood and bone marrow is collected at multiple time points for determination of the PK and PD effects of AG-221. Response assessments via bone marrow examination are performed on Days 15, 29, 57, and every 56 days (2 cycles) thereafter. Study Status and Results: The study was activated in September 2013. As of February 26th 2014, a total of 19 patients have been enrolled; 18 with AML and 1 with MDS. All patients were IDH2-mutant positive by local testing. AG-221 doses administered included 30 mg BID (n=7), 50 mg BID (n=5), 75 mg BID (n=5), and 100 mg QD (n=2). Two patients were added to the 30 mg BID cohort to replace PK-unevaluable patients. Fourteen of 19 patients remain on study drug treatment. Therapy has been well tolerated; with no dose-limiting toxicities reported. The maximum tolerated dose has not been reached. Possible drug-related severe adverse events were reported in two patients: grade 2 hyperleukocytosis and grade 3 confusion. In the first cohort there were three deaths due to sepsis within 30 days of study drug termination. One of these was attributed as possibly related to study drug treatment. Preliminary analysis of PK at 30 and 50 mg doses demonstrated excellent oral AG-221 exposure and a mean plasma half-life of greater than 40 hours. Evaluation of the PD response demonstrated sustained reduction in 2-HG plasma levels of up to 97% following AG-221 dosing in cohort 1 and 2. Ten AML patients are evaluable for efficacy at this time: (N=5 at 30 mg BID, N=5 at 50 mg BID), 5 men and 5 women, with a median age (range) of 62.5 years (53-74). Eight patients had an R140Q mutation and two had a R172K mutation. The median number of prior regimens was 2 (1-4) including one patient who had relapsed after an allogeneic bone marrow transplant. Currently, 6 of 10 patients have had objective responses, including 2 complete remissions defined by the International Working Group Criteria (1 at 30mg BID and 1 at 50mg BID). The four other responses are ongoing and will be updated. Marked differentiation of myeloblasts into mature forms, consistent with preclinical models, was associated with responses. Only one patient experienced disease progression. One patient with a CR was removed from study to undergo allogeneic BMT. Five of the 6 responding patients remain on AG-221. Dose escalation continues along with exploration of a once daily dosing regimen. Expansion cohorts are being planned. Conclusions: AG-221, a novel, oral, potent IDH2 mutant inhibitor is well tolerated and shows promising initial clinical and pharmacodynamic activity in patients with relapsed and refractory IDH2 mutant hematologic malignancies, even in the lowest dose cohort. These data provide early validation of mutant IDH2 as a therapeutic target in hematologic malignancies. Additional safety and efficacy data from the ongoing study will be presented. Clinical Trial Information: NCT01915498 Citation Format: Eytan Stein, Martin Tallman, Daniel A. Pollyea, Ian W. Flinn, Amir T. Fathi, Richard M. Stone, Ross L. Levine, Samuel Agresta, David Schenkein, Hua Yang, Bin Fan, Kate Yen, Stephane De Botton. Clinical safety and activity in a phase I trial of AG-221, a first in class, potent inhibitor of the IDH2-mutant protein, in patients with IDH2 mutant positive advanced hematologic malignancies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT103. doi:10.1158/1538-7445.AM2014-CT103

Increased ACTL6A occupancy within mSWI/SNF chromatin remodelers drives human squamous cell carcinoma

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells that accumulate during pathological conditions such as cancer and are associated with a poor clinical outcome. MDSC expansion hampers the host anti-tumor immune response by inhibition of T cell proliferation, cytokine secretion, and recruitment of regulatory T cells. In addition, MDSC exert non-immunological functions including the promotion of angiogenesis, tumor invasion, and metastasis. Recent years, MDSC are considered as a potential target in solid tumors and hematological malignancies to enhance the effects of currently used immune modulating agents. This review focuses on the characteristics, distribution, functions, cell–cell interactions, and targeting of MDSC in hematological malignancies including multiple myeloma, lymphoma, and leukemia.

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