Mammalian SWI/SNF Chromatin Remodeling Complexes: Emerging Mechanisms and Therapeutic Strategies.

Small molecule-based targeting of chromatin regulatory factors has emerged as a promising therapeutic strategy in recent years. The development and ongoing clinical evaluation of novel agents targeting a range of chromatin regulatory processes, including DNA or histone modifiers, histone readers, and chromatin regulatory protein complexes, has inspired the field to identify and act upon the full compendium of therapeutic opportunities. Emerging studies highlight the frequent involvement of altered mammalian Switch/Sucrose-Nonfermentable (mSWI/SNF) chromatin-remodeling complexes (also called BAF complexes) in both human cancer and neurological disorders, suggesting new mechanisms and accompanying routes toward therapeutic intervention. Here, we review current approaches for direct targeting of mSWI/SNF complex structure and function and discuss settings in which aberrant mSWI/SNF biology is implicated in oncology and other diseases.

CREB5 Promotes Resistance to Androgen-Receptor Antagonists and Androgen Deprivation in Prostate Cancer.

Androgen-receptor (AR) inhibitors, including enzalutamide, are used for treatment of all metastatic castration-resistant prostate cancers (mCRPCs). However, some patients develop resistance or never respond. We find that the transcription factor CREB5 confers enzalutamide resistance in an open reading frame (ORF) expression screen and in tumor xenografts. CREB5 overexpression is essential for an enzalutamide-resistant patient-derived organoid. In AR-expressing prostate cancer cells, CREB5 interactions enhance AR activity at a subset of promoters and enhancers upon enzalutamide treatment, including MYC and genes involved in the cell cycle. In mCRPC, we found recurrent amplification and overexpression of CREB5. Our observations identify CREB5 as one mechanism that drives resistance to AR antagonists in prostate cancers.

Renal medullary carcinomas depend upon SMARCB1 loss and are sensitive to proteasome inhibition.

Renal medullary carcinoma (RMC) is a rare and deadly kidney cancer in patients of African descent with sickle cell trait. We have developed faithful patient-derived RMC models and using whole-genome sequencing, we identified loss-of-function intronic fusion events in one SMARCB1 allele with concurrent loss of the other allele. Biochemical and functional characterization of these models revealed that RMC requires the loss of SMARCB1 for survival. Through integration of RNAi and CRISPR-Cas9 loss-of-function genetic screens and a small-molecule screen, we found that the ubiquitin-proteasome system (UPS) was essential in RMC. Inhibition of the UPS caused a G2/M arrest due to constitutive accumulation of cyclin B1. These observations extend across cancers that harbor SMARCB1 loss, which also require expression of the E2 ubiquitin-conjugating enzyme, UBE2C. Our studies identify a synthetic lethal relationship between SMARCB1-deficient cancers and reliance on the UPS which provides the foundation for a mechanism-informed clinical trial with proteasome inhibitors.

Binding of TMPRSS2-ERG to BAF Chromatin Remodeling Complexes Mediates Prostate Oncogenesis.

Chromosomal rearrangements resulting in the fusion of TMPRSS2, an androgen-regulated gene, and the ETS family transcription factor ERG occur in over half of prostate cancers. However, the mechanism by which ERG promotes oncogenic gene expression and proliferation remains incompletely understood. Here, we identify a binding interaction between ERG and the mammalian SWI/SNF (BAF) ATP-dependent chromatin remodeling complex, which is conserved among other oncogenic ETS factors, including ETV1, ETV4, and ETV5. We find that ERG drives genome-wide retargeting of BAF complexes in a manner dependent on binding of ERG to the ETS DNA motif. Moreover, ERG requires intact BAF complexes for chromatin occupancy and BAF complex ATPase activity for target gene regulation. In a prostate organoid model, BAF complexes are required for ERG-mediated basal-to-luminal transition, a hallmark of ERG activity in prostate cancer. These observations suggest a fundamental interdependence between ETS transcription factors and BAF chromatin remodeling complexes in cancer.

Cancer-Specific Retargeting of BAF Complexes by a Prion-like Domain.

Alterations in transcriptional regulators can orchestrate oncogenic gene expression programs in cancer. Here, we show that the BRG1/BRM-associated factor (BAF) chromatin remodeling complex, which is mutated in over 20% of human tumors, interacts with EWSR1, a member of a family of proteins with prion-like domains (PrLD) that are frequent partners in oncogenic fusions with transcription factors. In Ewing sarcoma, we find that the BAF complex is recruited by the EWS-FLI1 fusion protein to tumor-specific enhancers and contributes to target gene activation. This process is a neomorphic property of EWS-FLI1 compared to wild-type FLI1 and depends on tyrosine residues that are necessary for phase transitions of the EWSR1 prion-like domain. Furthermore, fusion of short fragments of EWSR1 to FLI1 is sufficient to recapitulate BAF complex retargeting and EWS-FLI1 activities. Our studies thus demonstrate that the physical properties of prion-like domains can retarget critical chromatin regulatory complexes to establish and maintain oncogenic gene expression programs.

Going beyond genetics to discover cancer targets.

Two recent studies demonstrate the power of integrating tumor genotype information with epigenetic and proteomic studies to discover potential therapeutic targets in breast cancer.

RAG-mediated DNA double-strand breaks activate a cell type-specific checkpoint to inhibit pre-B cell receptor signals.

DNA double-strand breaks (DSBs) activate a canonical DNA damage response, including highly conserved cell cycle checkpoint pathways that prevent cells with DSBs from progressing through the cell cycle. In developing B cells, pre-B cell receptor (pre-BCR) signals initiate immunoglobulin light (Igl) chain gene assembly, leading to RAG-mediated DNA DSBs. The pre-BCR also promotes cell cycle entry, which could cause aberrant DSB repair and genome instability in pre-B cells. Here, we show that RAG DSBs inhibit pre-BCR signals through the ATM- and NF-κB2-dependent induction of SPIC, a hematopoietic-specific transcriptional repressor. SPIC inhibits expression of the SYK tyrosine kinase and BLNK adaptor, resulting in suppression of pre-BCR signaling. This regulatory circuit prevents the pre-BCR from inducing additional Igl chain gene rearrangements and driving pre-B cells with RAG DSBs into cycle. We propose that pre-B cells toggle between pre-BCR signals and a RAG DSB-dependent checkpoint to maintain genome stability while iteratively assembling Igl chain genes.

Loss of DAP12 and FcRγ drives exaggerated IL-12 production and CD8(+) T cell response by CCR2(+) Mo-DCs.

Dap12 and FcRγ, the two transmembrane ITAM-containing signaling adaptors expressed in dendritic cells (DC), are implicated in the regulation of DC function. Several activating and adhesion receptors including integrins require these chains for their function in triggering downstream signaling and effector pathways, however the exact role(s) for Dap12 and FcRγ remains elusive as their loss can lead to both attenuating and enhancing effects. Here, we report that mice congenitally lacking both Dap12 and FcRγ chains (DF) show a massively enhanced effector CD8(+) T cell response to protein antigen immunization or West Nile Virus (WNV) infection. Thus, immunization of DF mice with MHCI-restricted OVA peptide leads to accumulation of IL-12-producing monocyte-derived dendritic cells (Mo-DC) in draining lymph nodes, followed by vastly enhanced generation of antigen-specific IFNγ-producing CD8(+) T cells. Moreover, DF mice show increased viral clearance in the WNV infection model. Depletion of CCR2+ monocytes/macrophages in vivo by administration anti-CCR2 antibodies or clodronate liposomes completely prevents the exaggerated CD8+ T cell response in DF mice. Mechanistically, we show that the loss of Dap12 and FcRγ-mediated signals in Mo-DC leads to a disruption of GM-CSF receptor-induced STAT5 activation resulting in upregulation of expression of IRF8, a transcription factor. Consequently, Dap12- and FcRγ-deficiency exacerbates GM-CSF-driven monocyte differentiation and production of inflammatory Mo-DC. Our data suggest a novel cross-talk between DC-ITAM and GM-CSF signaling pathways, which controls Mo-DC differentiation, IL-12 production, and CD8(+) T cell responses.

Polarity gene discs large homolog 1 regulates the generation of memory T cells.

Mammalian ortholog of Drosophila cell polarity protein, Dlg1, plays a critical role in neural synapse formation, epithelial cell homeostasis, and urogenital development. More recently, it has been proposed that Dlg1 may also be involved in the regulation of T-cell proliferation, migration, and Ag-receptor signaling. However, a requirement for Dlg1 in development and function of T lineage cells remains to be established. In this study, we investigated a role for Dlg1 during T-cell development and function using a combination of conditional Dlg1 KO and two different Cre expression systems where Dlg1 deficiency is restricted to the T-cell lineage only, or all hematopoietic cells. Here, using three different TCR models, we show that Dlg1 is not required during development and selection of thymocytes bearing functionally rearranged TCR transgenes. Moreover, Dlg1 is dispensable in the activation and proliferative expansion of Ag-specific TCR-transgenic CD4(+) and CD8(+) T cells in vitro and in vivo. Surprisingly, however, we show that Dlg1 is required for normal generation of memory T cells during endogenous response to cognate Ag. Thus, Dlg1 is not required for the thymocyte selection or the activation of primary T cells, however it is involved in the generation of memory T cells.

Cutting edge: cell-autonomous control of IL-7 response revealed in a novel stage of precursor B cells.

During early stages of B-lineage differentiation in bone marrow, signals emanating from IL-7R and pre-BCR are thought to synergistically induce proliferative expansion of progenitor cells. Paradoxically, loss of pre-BCR-signaling components is associated with leukemia in both mice and humans. Exactly how progenitor B cells perform the task of balancing proliferative burst dependent on IL-7 with the termination of IL-7 signals and the initiation of L chain gene rearrangement remains to be elucidated. In this article, we provide genetic and functional evidence that the cessation of the IL-7 response of pre-B cells is controlled via a cell-autonomous mechanism that operates at a discrete developmental transition inside Fraction C' (large pre-BII) marked by transient expression of c-Myc. Our data indicate that pre-BCR cooperates with IL-7R in expanding the pre-B cell pool, but it is also critical to control the differentiation program shutting off the c-Myc gene in large pre-B cells.

Novel mechanism of tumor suppression by polarity gene discs large 1 (DLG1) revealed in a murine model of pediatric B-ALL.

Drosophila melanogaster discs large (dlg) is an essential tumor suppressor gene (TSG) controlling epithelial cell growth and polarity of the fly imaginal discs in pupal development. A mammalian ortholog, Dlg1, is involved in embryonic urogenital morphogenesis, postsynaptic densities in neurons, and immune synapses in lymphocytes. However, a potential role for Dlg1 as a mammalian TSG is unknown. Here, we present evidence that loss of Dlg1 confers strong predisposition to the development of malignancies in a murine model of pediatric B-cell acute lymphoblastic leukemia (B-ALL). Using mice with conditionally deleted Dlg1 alleles, we identify a novel "pre-leukemic" stage of developmentally arrested early B-lineage cells marked by preeminent c-Myc expression. Mechanistically, we show that in B-lineage progenitors Dlg1 interacts with and stabilizes the PTEN protein, regulating its half-life and steady-state abundance. The loss of Dlg1 does not affect the level of PTEN mRNAs but results in a dramatic decrease in PTEN protein, leading to excessive phosphoinositide 3-kinase signaling and proliferation. Our data suggest a novel model of tumor suppression by a PDZ domain-containing polarity gene in hematopoietic cancers.