Exquisite Sensitivity to Dual BRG1/BRM ATPase Inhibitors Reveals Broad SWI/SNF Dependencies in Acute Myeloid Leukemia.

Various subunits of mammalian SWI/SNF chromatin remodeling complexes display loss-of-function mutations characteristic of tumor suppressors in different cancers, but an additional role for SWI/SNF supporting cell survival in distinct cancer contexts is emerging. In particular, genetic dependence on the catalytic subunit BRG1/SMARCA4 has been observed in acute myelogenous leukemia (AML), yet the feasibility of direct therapeutic targeting of SWI/SNF catalytic activity in leukemia remains unknown. Here, we evaluated the activity of dual BRG1/BRM ATPase inhibitors across a genetically diverse panel of cancer cell lines and observed that hematopoietic cancer cell lines were among the most sensitive compared with other lineages. This result was striking in comparison with data from pooled short hairpin RNA screens, which showed that only a subset of leukemia cell lines display sensitivity to BRG1 knockdown. We demonstrate that combined genetic knockdown of BRG1 and BRM is required to recapitulate the effects of dual inhibitors, suggesting that SWI/SNF dependency in human leukemia extends beyond a predominantly BRG1-driven mechanism. Through gene expression and chromatin accessibility studies, we show that the dual inhibitors act at genomic loci associated with oncogenic transcription factors, and observe a downregulation of leukemic pathway genes, including MYC, a well-established target of BRG1 activity in AML. Overall, small-molecule inhibition of BRG1/BRM induced common transcriptional responses across leukemia models resulting in a spectrum of cellular phenotypes. IMPLICATIONS: Our studies reveal the breadth of SWI/SNF dependency in leukemia and support targeting SWI/SNF catalytic function as a potential therapeutic strategy in AML.

The Discovery of SWI/SNF Chromatin Remodeling Activity as a Novel and Targetable Dependency in Uveal Melanoma.

Uveal melanoma is a rare and aggressive cancer that originates in the eye. Currently, there are no approved targeted therapies and very few effective treatments for this cancer. Although activating mutations in the G protein alpha subunits, GNAQ and GNA11, are key genetic drivers of the disease, few additional drug targets have been identified. Recently, studies have identified context-specific roles for the mammalian SWI/SNF chromatin remodeling complexes (also known as BAF/PBAF) in various cancer lineages. Here, we find evidence that the SWI/SNF complex is essential through analysis of functional genomics screens and further validation in a panel of uveal melanoma cell lines using both genetic tools and small-molecule inhibitors of SWI/SNF. In addition, we describe a functional relationship between the SWI/SNF complex and the melanocyte lineage-specific transcription factor Microphthalmia-associated Transcription Factor, suggesting that these two factors cooperate to drive a transcriptional program essential for uveal melanoma cell survival. These studies highlight a critical role for SWI/SNF in uveal melanoma, and demonstrate a novel path toward the treatment of this cancer.

Degron mediated BRM/SMARCA2 depletion uncovers novel combination partners for treatment of BRG1/SMARCA4-mutant cancers.

Recent studies have highlighted that cancer cells with a loss of the SWI/SNF complex catalytic subunit BRG1 are dependent on the remaining ATPase, BRM, making it an attractive target for cancer therapy. However, an understanding of the extent of target inhibition required to arrest cell growth, necessary to develop an appropriate therapeutic strategy, remains unknown. Here, we utilize tunable depletion of endogenous BRM using the SMASh degron, and interestingly observe that BRG1-mutant lung cancer cells require near complete depletion of BRM to robustly inhibit growth both in vitro and in vivo. Therefore, to identify pathways that synergize with partial BRM depletion and afford a deeper response, we performed a genome-wide CRISPR screen and discovered a combinatorial effect between BRM depletion and the knockout of various genes of the oxidative phosphorylation pathway and the anti-apoptotic gene MCL1. Together these studies provide an important framework to elucidate the requirements of BRM inhibition in the BRG1-mutant state with implications on the feasibility of targeting BRM alone, as well as reveal novel insights into pathways that can be exploited in combination toward deeper anti-tumor responses.

Enzyme Substrate Specificity Conferred by Distinct Conformational Pathways.

Substrate recognition is one of the hallmarks of enzyme catalysis. Enzyme conformational changes have been linked to selectivity between substrates with little direct evidence. Aldolase, a glycolytic enzyme, must distinguish between two physiologically important substrates, fructose 1-phosphate and fructose 1,6-bisphosphate, and provides an excellent model system for the study of this question. Previous work has shown that isozyme specific residues (ISRs) distant from the active site are responsible for kinetic distinction between these substrates. Notably, most of the ISRs reside in a cluster of five surface α-helices, and the carboxyl-terminal region (CTR), and cooperative interactions among these helices have been demonstrated. To test the hypothesis that conformational changes are at the root of these changes, single surface-cysteine variants were created with the cysteine located on helices of the cluster and CTR. This allowed for site-specific labeling with an environmentally sensitive fluorophore, and subsequent monitoring of conformational changes by fluorescence emission spectrophotometry. These labeled variants revealed different spectra in the presence of saturating amounts of each substrate, which suggested the occurrence of different conformations. Emission spectra collected at various substrate concentrations showed a concentration dependence of the fluorescence spectra, consistent with binding events. Lastly, stopped-flow fluorescence spectrophotometry showed that the rate of these fluorescence changes was on the same time-scale as catalysis, thus suggesting a link between the different fluorescence changes and events during catalysis. On the basis of these results, we propose that different conformational changes may be a common mechanism for dictating substrate specificity in other enzymes with multiple substrates.

Distinct organization and regulation of the outer kinetochore KMN network downstream of CENP-C and CENP-T.

The kinetochore provides a vital connection between chromosomes and spindle microtubules [1, 2]. Defining the molecular architecture of the core kinetochore components is critical for understanding the mechanisms by which the kinetochore directs chromosome segregation. The KNL1/Mis12 complex/Ndc80 complex (KMN) network acts as the primary microtubule-binding interface at kinetochores [3] and provides a platform to recruit regulatory proteins [4]. Recent work found that the inner kinetochore components CENP-C and CENP-T act in parallel to recruit the KMN network to kinetochores [5-8]. However, due to the presence of these dual pathways, it has not been possible to distinguish differences in the nature of kinetochore assembly downstream of CENP-C or CENP-T. Here, we separated these pathways by targeting CENP-C and CENP-T independently to an ectopic chromosomal locus in human cells. Our work reveals that the organization of the KMN network components downstream of CENP-C and CENP-T is distinct. CENP-C recruits the Ndc80 complex through its interactions with KNL1 and the Mis12 complex. In contrast, CENP-T directly interacts with Ndc80, which in turn promotes KNL1/Mis12 complex recruitment through a separate region on CENP-T, resulting in functional relationships for KMN network localization that are inverted relative to the CENP-C pathway. We also find that distinct regulatory paradigms control the assembly of these pathways, with Aurora B kinase promoting KMN network recruitment to CENP-C and cyclin-dependent kinase (CDK) regulating KMN network recruitment to CENP-T. This work reveals unexpected complexity for the architecture and regulation of the core components of the kinetochore-microtubule interface.

Kinetochore genes are coordinately up-regulated in human tumors as part of a FoxM1-related cell division program.

The key player in directing proper chromosome segregation is the macromolecular kinetochore complex, which mediates DNA-microtubule interactions. Previous studies testing individual kinetochore genes documented examples of their overexpression in tumors relative to normal tissue, leading to proposals that up-regulation of specific kinetochore genes may promote tumor progression. However, kinetochore components do not function in isolation, and previous studies did not comprehensively compare the expression behavior of kinetochore components. Here we analyze the expression behavior of the full range of human kinetochore components in diverse published expression compendia, including normal tissues and tumor samples. Our results demonstrate that kinetochore genes are rarely overexpressed individually. Instead, we find that core kinetochore genes are coordinately regulated with other cell division genes under virtually all conditions. This expression pattern is strongly correlated with the expression of the forkhead transcription factor FoxM1, which binds to the majority of cell division promoters. These observations suggest that kinetochore gene up-regulation in cancer reflects a general activation of the cell division program and that altered expression of individual kinetochore genes is unlikely to play a causal role in tumorigenesis.

Review series: The functions and consequences of force at kinetochores.

Chromosome segregation requires the generation of force at the kinetochore-the multiprotein structure that facilitates attachment of chromosomes to spindle microtubules. This force is required both to move chromosomes and to signal the formation of proper bioriented attachments. To understand the role of force in these processes, it is critical to define how force is generated at kinetochores, the contributions of this force to chromosome movement, and how the kinetochore is structured and organized to withstand and respond to force. Classical studies and recent work provide a framework to dissect the mechanisms, functions, and consequences of force at kinetochores.

CENP-T provides a structural platform for outer kinetochore assembly.

The kinetochore forms a dynamic interface with microtubules from the mitotic spindle during mitosis. The Ndc80 complex acts as the key microtubule-binding complex at kinetochores. However, it is unclear how the Ndc80 complex associates with the inner kinetochore proteins that assemble upon centromeric chromatin. Here, based on a high-resolution structural analysis, we demonstrate that the N-terminal region of vertebrate CENP-T interacts with the 'RWD' domain in the Spc24/25 portion of the Ndc80 complex. Phosphorylation of CENP-T strengthens a cryptic hydrophobic interaction between CENP-T and Spc25 resulting in a phospho-regulated interaction that occurs without direct recognition of the phosphorylated residue. The Ndc80 complex interacts with both CENP-T and the Mis12 complex, but we find that these interactions are mutually exclusive, supporting a model in which two distinct pathways target the Ndc80 complex to kinetochores. Our results provide a model for how the multiple protein complexes at kinetochores associate in a phospho-regulated manner.