Prostate cancer (PCa) is the second leading cause of cancer death among men in the United States. Current available treatments include chemical castration and therapies targeting androgen receptor (AR) signaling pathways. Although initial responses are observed, 30% of patients have primary resistance to both forms of treatment and the majority of patients progress from androgen-dependent PCa (ADPC) to castration-resistant PCa (CRPC). Studies on mechanisms of resistance suggest that AR signaling remains a key driver in PCa even after treatment with AR-targeted therapies. Co-treatment with therapies that modulate alternative signaling pathways involved in oncogenesis in PCa including epigenetic modifiers, have been proposed as a modality to overcome resistance. Enhancer of Zeste Homolog 2 (EZH2) is the enzymatic subunit of the polycomb repressive complex 2 and is one of the most highly upregulated genes in CRPC compared to localized PCa. Several reports suggest that EZH2 plays an oncogenic role both in ADPC and CRPC.Tazemetostat is a potent, selective, orally bioavailable, investigative first-in-class small molecule inhibitor of EZH2. Phase 2 clinical trials have demonstrated objective clinical responses in patients with B-cell lymphomas and molecularly defined solid tumors, including epithelioid sarcoma and other INI1-negative tumors. Given the implication of EZH2 in PCa, we explored the combination potential of tazemetostat with current second-line therapies, the androgen signaling inhibitors (ASIs) abiraterone and enzalutamide. Tazemetostat displayed both time- and dose-dependent antiproliferative activity in PCa cell lines dependent on AR signaling or those of the neuroendocrine subtype with IC50 values at or below 1 μM in 14-day assays. Synergistic activity was observed in vitro in a subset of cell lines when tazemetostast was combined with ASIs in a 7-day co-treatment model and this activity was further enhanced when cells were pretreated with tazemetostat prior to the cotreatment. To further validate these findings, we conducted in vivo studies in PCa cell line-derived xenograft models. Tazemetostat and enzalutamide showed single agent antitumor activity in a subcutaneous LNCaP xenograft mouse model. Co-administration of enzalutamide and tazemetostat markedly reduced tumor growth rate compared to either of the single agent(s). Similar results were observed with the combination of tazemetostat and abiraterone. Interestingly in the 22Rv1-derived in vivo xenograft model, a cell line that expresses AR-V7, a splice variant implicated in resistance to ASIs in CRPC, modest tumor growth inhibition was observed with tazemetostat and no significant antitumor activity was observed when given in combination with abiraterone or enzalutamide. A significant decrease in intratumoral H3K27me3 levels were observed in all treatment groups containing tazemetostat in both in vivo models, indicating an on-target activity of EZH2 inhibition. Current efforts are underway to investigate the mechanism of action and cellular inhibitory pathways for these combinations. In summary, our preclinical data demonstrates the importance of EZH2 inhibition in CRPC and provides a rationale for combination of tazemetostat with abiraterone and enzalutamide in the clinic. Citation Format: Vinny Motwani, Dorothy Brach, Chloe Pantano, Vania Estanek, Jeffrey A. Keats, Daniel T. Dransfield, Alejandra Raimondi. Synergistic activity of tazemetostat in combination with androgen signaling inhibitors in preclinical models of prostate cancer demonstrates potential for clinical expansion [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B011. doi:10.1158/1535-7163.TARG-19-B011
SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin subfamily A member 2 (SMARCA2) is an ATP-dependent DNA helicase and a catalytic component of the SWI/SNF complex. SWI/SNF-mediated nucleosome remodeling is a critical regulator of chromatin accessibility resulting in transcriptional regulation of gene sets that determine and maintain cell state. The role of SMARCA2 as the catalytic driver of SWI/SNF activity is mutually exclusive with that of its close paralog, SMARCA4. Loss of functional SMARCA4, as is reported in a subset of NSCLC and other cancer types generally confers a dependency on SMARCA2. Cancers lacking functional SMARCA4 have been shown to be sensitive to loss of SMARCA2 function either through genetic ablation or the use of a small molecule SMARCA2/4 inhibitor. We describe the identification and characterization of a small molecule SMARCA2/4 inhibitor Compound 1 and its selective activity in the SMARCA4-mutant setting both in in vitro and in vivo model systems. The compound demonstrates inhibition of SMARCA2 and SMARCA4 enzymatic activity in biochemical assays and broad selectivity against other helicases. Selective sensitivity in anti-proliferative assays of 15-fold was observed in NSCLC cell lines with SMARCA4 protein loss compared to those harboring WT SMARCA4 protein. Oral dosing demonstrates dose-dependent in vivo SMARCA2 inhibition and anti-tumor activity in SMARCA4-mutant NSCLC xenograft models. This compound is suitable for further exploration of the role of SMARCA2/4 and SWI/SNF in vitro and in vivo in cancer and other indications. Citation Format: Allison E. Drew, Suzanne L. Jacques, Lindsey W. Eichinger, Chloe Pantano, Vinny Motwani, Cuyue Tang, Cuyue Tang, Neil Farrow, Dorothy Brach, Selene Howe, Alejandra Raimondi, Daniel T. Dransfield, Kenneth W. Duncan, Kim Stickland, Liyue Huang, John Lampe. Identification of a potent, orally-available SMARCA2/4 inhibitor with in vitro and in vivo activity in preclinical models of SMARCA4-mutant NSCLC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1768.
SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin subfamily A member 2 (SMARCA2) is an ATP-dependent DNA helicase and a catalytic component of the SWI/SNF complex. SWI/SNF-mediated nucleosome remodeling is a critical regulator of chromatin accessibility resulting in transcriptional regulation of gene sets that determine and maintain cell state. SMARCA2's role as the catalytic driver of SWI/SNF activity is mutually exclusive with that of its close paralog, SMARCA4. Here, we show that the synthetic lethal relationship between SMARCA2 and SMARCA4 can be genetically engineered bi-directionally in SMARCA2/SMARCA4 wild type (WT) cell lines. While knockout (KO) of either protein individually minimally affects cell growth, a genetically engineered cell line with loss of either SMARCA2 or SMARCA4 becomes sensitive to loss of its paralog. While SMARCA2 is infrequently lost through genetic mutation, SMARCA4 mutations occur in a wide variety of cancer types. We present CRISPR-Cas9 pooled screening data showing that cell lines with SMARCA4 mutations have a selective sensitivity to loss of SMARCA2 across multiple cancer indications, including NSCLC and ovarian carcinoma. SMARCA2 and SMARCA4, though partially redundant, also play unique roles within the cell as is evidenced by the unique set of transcriptional changes that occur in the SMARCA2/4 isogeneic cell lines, including the differential modulation of genes involved in TGFB signaling, regulation of angiogenesis, and in determinants of epithelial or mesenchymal cell state. ChIP-seq studies show changes in SWI/SNF occupancy as well as in the chromatin landscape that are specific to the loss of either SMARCA2 or SMARCA4. These isogenic cell lines also respond differently to treatment with a novel SMARCA2/4 inhibitor, Compound 1, in transcriptional as well as functional effects, providing evidence that dual inhibition with a small molecule may not be equivalent to the complete genetic ablation of these two proteins The NCI-H358 NCSLC cell line expresses WT SMARCA2 and SMARCA4 and is weakly sensitive to treatment with Compound 1. CRISPR-Cas9 mediated KO of SMARCA4 confers several-fold greater sensitivity to treatment with the novel inhibitor compared to the parental cell line. Surprisingly, KO of SMARCA2 does not confer sensitivity to inhibitor treatment. This selectivity sensitivity to treatment with Compound 1 that we observe in the SMARCA4-KO cell line is also observed across a panel of SMARCA4-mutant NSCLC. The relationship between the transcriptional and phenotypic changes that occur in these isogenic knockout lines as well as in SMARCA4-mutated NSCLC cell lines in response to SMARCA2/4 inhibitor treatment have revealed novel mechanisms driven by specific SWI/SNF complexes. These data have allowed us to better understand the potential therapeutic utility of a SMARCA2/4 inhibitor in both a SMARCA4-mut and SMARCA4-wt NSCLC patient population. Citation Format: Lindsey W. Eichinger, Chloe Pantano, Vinny Motwani, Dorothy Brach, Selene Howe, Alejandra Raimondi, Daniel T. Dransfield, Kenneth W. Duncan, Kim Stickland, Cuyue Tang, Neil A. Farrow, John Lampe, Suzanne L. Jacques, Allison E. Drew. Aberrant SWI/SNF complexes lacking SMARCA2 or SMARCA4 differentially affect cell state and response to a novel SMARCA2/4 inhibitor [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2924.
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