Targeting SWI/SNF Complexes in Cancer: Pharmacological Approaches and Implications.

SWI/SNF enzymes are heterogeneous multi-subunit complexes that utilize the energy from ATP hydrolysis to remodel chromatin structure, facilitating transcription, DNA replication, and repair. In mammalian cells, distinct sub-complexes, including cBAF, ncBAF, and PBAF exhibit varying subunit compositions and have different genomic functions. Alterations in the SWI/SNF complex and sub-complex functions are a prominent feature in cancer, making them attractive targets for therapeutic intervention. Current strategies in cancer therapeutics involve the use of pharmacological agents designed to bind and disrupt the activity of SWI/SNF complexes or specific sub-complexes. Inhibitors targeting the catalytic subunits, SMARCA4/2, and small molecules binding SWI/SNF bromodomains are the primary approaches for suppressing SWI/SNF function. Proteolysis-targeting chimeras (PROTACs) were generated by the covalent linkage of the bromodomain or ATPase-binding ligand to an E3 ligase-binding moiety. This engineered connection promotes the degradation of specific SWI/SNF subunits, enhancing and extending the impact of this pharmacological intervention in some cases. Extensive preclinical studies have underscored the therapeutic potential of these drugs across diverse cancer types. Encouragingly, some of these agents have progressed from preclinical research to clinical trials, indicating a promising stride toward the development of effective cancer therapeutics targeting SWI/SNF complex and sub-complex functions.

Epigenetic and pharmacological control of pigmentation via Bromodomain Protein 9 (BRD9).

Lineage-specific differentiation programs are activated by epigenetic changes in chromatin structure. Melanin-producing melanocytes maintain a gene expression program ensuring appropriate enzymatic conversion of metabolites into the pigment, melanin, and transfer to surrounding cells. During neuroectodermal development, SMARCA4 (BRG1), the catalytic subunit of SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complexes, is essential for lineage specification. SMARCA4 is also required for development of multipotent neural crest precursors into melanoblasts, which differentiate into pigment-producing melanocytes. In addition to the catalytic domain, SMARCA4 and several SWI/SNF subunits contain bromodomains which are amenable to pharmacological inhibition. We investigated the effects of pharmacological inhibitors of SWI/SNF bromodomains on melanocyte differentiation. Strikingly, treatment of murine melanoblasts and human neonatal epidermal melanocytes with selected bromodomain inhibitors abrogated melanin synthesis and visible pigmentation. Using functional genomics, iBRD9, a small molecule selective for the bromodomain of BRD9 was found to repress pigmentation-specific gene expression. Depletion of BRD9 confirmed a requirement for expression of pigmentation genes in the differentiation program from melanoblasts into pigmented melanocytes and in melanoma cells. Chromatin immunoprecipitation assays showed that iBRD9 disrupts the occupancy of BRD9 and the catalytic subunit SMARCA4 at melanocyte-specific loci. These data indicate that BRD9 promotes melanocyte pigmentation whereas pharmacological inhibition of BRD9 is repressive.

SWI/SNF Chromatin Remodeling Enzymes in Melanoma.

Melanoma is an aggressive malignancy that arises from the transformation of melanocytes on the skin, mucosal membranes, and uvea of the eye. SWI/SNF chromatin remodeling enzymes are multi-subunit complexes that play important roles in the development of the melanocyte lineage and in the response to ultraviolet radiation, a key environmental risk factor for developing cutaneous melanoma. Exome sequencing has revealed frequent loss of function mutations in genes encoding SWI/SNF subunits in melanoma. However, some SWI/SNF subunits have also been demonstrated to have pro-tumorigenic roles in melanoma and to affect sensitivity to therapeutics. This review summarizes studies that have implicated SWI/SNF components in melanomagenesis and have evaluated how SWI/SNF subunits modulate the response to current therapeutics.

Androgen up-regulation of Twist1 gene expression is mediated by ETV1.

Twist1, a basic helix-loop-helix transcription factor that regulates a number of genes involved in epithelial-to-mesenchymal transition (EMT), is upregulated in prostate cancer. Androgen regulation of Twist1 has been reported in a previous study. However, the mechanism of androgen regulation of the Twist1 gene is not understood because the Twist1 promoter lacks androgen receptor (AR)-responsive elements. Previous studies have shown that the Twist1 promoter has putative binding sites for PEA3 subfamily of ETS transcription factors. Our lab has previously identified Ets Variant 1 (ETV1), a member of the PEA3 subfamily, as a novel androgen-regulated gene that is involved in prostate cancer cell invasion through unknown mechanism. In view of these data, we hypothesized that androgen-activated AR upregulates Twist1 gene expression via ETV1. Our data confirmed the published work that androgen positively regulates Twist1 gene expression and further showed that this positive effect was directed at the Twist1 promoter. The positive effect of androgen on Twist1 gene expression was abrogated upon disruption of AR expression by siRNA or of AR activity by Casodex. More importantly, our data show that disruption of ETV1 leads to significant decrease in both androgen-mediated upregulation as well as basal level of Twist1, which we are able to rescue upon re-expression of ETV1. Indeed, we are able to show that ETV1 mediates the androgen upregulation of Twist1 by acting on the proximal region of Twist1 promoter. Additionally, our data show that Twist1 regulates prostate cancer cell invasion and EMT, providing a possible mechanism by which ETV1 mediates prostate cancer cell invasion. In conclusion, in this study we report Twist1 as an indirect target of AR and androgen regulation through ETV1.

Multiple Levels of Regulation of Sororin by Cdk1 and Aurora B.

The cohesin complex holds sister chromatids together until all chromosomes are properly attached to the mitotic spindle. Cleavage of the cohesin subunit Rad21 at the metaphase to anaphase transition allows separation of sister chromatids and is fundamental for the creation of identical daughter cells. Sororin blocks removal of cohesin from chromosomes from S phase until mitosis. In mitosis, Sororin is phosphorylated by Cdk1 releasing it from the cohesin complex. Aurora B phosphorylation of Sororin may play a similar role as Cdk1. Using PhosTag electrophoresis, we detect multiple Sororin species suggesting that phosphorylation of Sororin in mitosis is heterogeneous. Mutating the Cdk1 consensus site S21 to alanine eliminates many of the phosphorylated species suggesting that S21 is a key site of phosphorylation in vivo. Inhibiting Aurora B reduces phosphorylation of Sororin in cells, but only if Cdk1 sites are intact suggesting that some phosphorylation events on Sororin may be sequential. Surprisingly, mutating Aurora B consensus sites in Sororin causes it to relocalize to the nucleolus during interphase and blocks its interaction with chromosomes in Aurora B-inhibited cells. These observations indicate that phosphorylation plays unexpected roles in regulating the subcellular localization of Sororin.

Effects of phosphatase and proteasome inhibitors on Borealin phosphorylation and degradation.

The chromosomal passenger complex (CPC) senses tension defects at the kinetochore to activate the spindle assembly checkpoint, and helps to position the cleavage furrow. The CPC, consisting of INCENP, Survivin, Borealin and Aurora B localizes to the inner centromere at metaphase and re-localizes to the spindle midzone at anaphase; several CPC functions are regulated by post-translational modification. Borealin is phosphorylated at multiple sites and phosphorylation at S219 causes Borealin to migrate more slowly upon electrophoresis. Here we find that Cdk1 can induce a mobility shift of Borealin, suggesting that S219 phosphorylation is under Cdk1 control. However, Cdk1 is inefficient at phosphorylating purified Borealin in vitro. A yeast orthologue of Borealin, Npl1, is dephosphorylated by the phosphatase Cdc14. We find no difference in the mobility shift of Borealin in human cells lacking either Cdc14A or Cdc14B. In contrast, the phosphatase inhibitor okadaic acid does delay the dephosphorylation of Borealin as cells exit mitosis. The proteasome inhibitor MG132 reduces Borealin phosphorylation in mitosis and increases the steady-state level of Borealin, especially in mutants lacking the C-terminus. However, a second, structurally unrelated proteasome inhibitor, lactacystin did not up-regulate Borealin. These results suggest that the effect of MG132 on Borealin is due to the inhibition of an intracellular protease other than the proteasome.

Regulation of sororin by Cdk1-mediated phosphorylation.

Tumor cells are commonly aneuploid, a condition contributing to cancer progression and drug resistance. Understanding how chromatids are linked and separated at the appropriate time will help uncover the basis of aneuploidy and will shed light on the behavior of tumor cells. Cohesion of sister chromatids is maintained by the multi-protein complex cohesin, consisting of Smc1, Smc3, Scc1 and Scc3. Sororin associates with the cohesin complex and regulates the segregation of sister chromatids. Sororin is phosphorylated in mitosis; however, the role of this modification is unclear. Here we show that mutation of potential cyclin-dependent kinase 1 (Cdk1) phosphorylation sites leaves sororin stranded on chromosomes and bound to cohesin throughout mitosis. Sororin can be precipitated from cell lysates with DNA-cellulose, and only the hypophosphorylated form of sororin shows this association. These results suggest that phosphorylation of sororin causes its release from chromatin in mitosis. Also, the hypophosphorylated form of sororin increases cohesion between sister chromatids, suggesting that phosphorylation of sororin by Cdk1 influences sister chromatid cohesion. Finally, phosphorylation-deficient sororin can alleviate the mitotic block that occurs upon knockdown of endogenous sororin. This mitotic block is abolished by ZM447439, an Aurora kinase inhibitor, suggesting that prematurely separated sister chromatids activate the spindle assembly checkpoint through an Aurora kinase-dependent pathway.

Short and long-term tumor cell responses to Aurora kinase inhibitors.

Aurora kinases are essential for mitosis and are candidate targets of novel chemotherapeutic agents. The inhibitors ZM447439, MK-0457 (VX-680) as well as Hesperadin have been used to dissect the roles of Aurora kinases in the cell cycle and have been tested clinically for the treatment of cancer. Here we have carried out a detailed kinetic analysis of two isogenic cell lines differing in p53 function and have compared the effects of ZM447439 and VE-465 (related to MK-0457). We find that p53 is needed for efficient cell cycle arrest when Aurora kinases are inhibited by either ZM447439 or VE-465. However, the p53-induced cell cycle block is neither immediate nor absolute. ZM447439 induced the localized accumulation of gammaH2A.X indicating that p53 induction by this drug occurs in response to DNA damage. Our analysis of the long-term effects of ZM447439 indicates that cells can evade killing by the drug, but not via a classical drug-resistance mechanism. Several mechanisms to explain how cells may evade killing by Aurora kinase inhibitors are described.