No abstract
SWI/SNF complexes play an important role in controlling gene expression by remodeling chromatin. SMARCA2 (BRM) and SMARCA4 (BRG1) are the core catalytic subunits of the SWI/SNF complexes, containing an ATPase domain and a DNA binding bromodomain. SMARCA4 protein expression is lost in some cancers due to nonsense mutations, and SMARCA4-deleted cancer cells are highly dependent on its paralog gene SMARCA2 for their survival. Therefore, targeting SMARCA2 in SMARCA4-deleted cancers using selective SMARCA2 degraders induces synthetic lethality while sparing SMARCA4 wild type (WT) normal cells. We have identified PRT3789, a potent and selective SMARCA2 targeted degrader, that selectively inhibits proliferation of SMARCA4-deleted cancer cells. Here, we describe the potential mechanism of action for PRT3789 at the molecular level and the in vitro and in vivo anti-tumor activity in SMARCA4-deleted cancer cells. To further elucidate the SMARCA2 degradation selectivity of PRT3789, we performed mass spectrometry to identify the selective SMARCA2 lysine residues ubiquitinated following treatment with PRT3789. This data, in combination with site-directed mutagenesis against these SMARCA2-specific ubiquitinated residues, has revealed important insights into the mechanism of action of PRT3789. In addition, to further understand the specific vulnerability of SMARCA2 in SMARCA4-deleted cells, we investigated whether PRT3789 affected the integrity of the residual SWI/SNF complex. Coimmunoprecipitation of SMARCC1 revealed that PRT3789 disrupts specific SWI/SNF complex subunits, including ACTL6A (BAF53). Functional genome-wide experiments are ongoing to evaluate the impact of this finding and the residual activity of the SWI/SNF complex. Furthermore, treatment with PRT3789 demonstrated robust inhibition of cell proliferation of SMARCA4-deleted non-small cell lung cancer (NSCLC) cells in vitro and NSCLC PDX tumors ex vivo, but not SMARCA4 WT cancer cells, in a concentration-dependent manner. Lastly, PRT3789 shows favorable pharmacokinetic properties in vivo, which correlate to its pharmacodynamics effects as evidenced by reduced SMARCA2 protein and KRT80 mRNA levels in tumor tissues. In subcutaneous cell-line derived xenograft (CDX) models of NSCLC, administration of PRT3789 demonstrated significant dose-related inhibition of SMARCA4-deleted NSCLC growth at tolerated doses, but no effect on the growth of SMARCA4 WT cancers. In summary, consistent with our previous validation studies and genomic perturbation analyses, our potent and selective SMARCA2 targeted degrader PRT3789 induces strong synthetic lethality in SMARCA4-deleted cancers in vitro and in vivo. Citation Format: Michael Hulse, Anjana Agarwal, Min Wang, Jack Carter, Monisha Sivakumar, Brian Vidal, Justin Brown, Andrew Moore, Alexander Grego, Neha Bhagwat, Joseph Rager, Liang Lu, Corey Basch, Klare Bersch, Chaofeng Dai, Philip Pitis, Andrew Combs, Bruce Ruggeri, Kris Vaddi, Peggy Scherle, Koichi Ito. Preclinical characterization of PRT3789, a potent and selective SMARCA2 targeted degrader [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3263.
MCL1 is a member of the anti-apoptotic BCL2 family of proteins and plays a critical role in maintaining cellular homeostasis and promoting cell survival. MCL1 amplifications occur frequently in multiple tumor types. It has also been implicated in mediating resistance to chemotherapeutic agents and targeted therapies. We have previously described a novel, potent and orally bioavailable MCL1 inhibitor, PRT1419, that demonstrates anti-tumor efficacy in various preclinical models of cancer and is currently under evaluation in a Phase I clinical trial in patients with relapsed/refractory hematologic malignancies and advanced solid tumors. In an effort to identify novel biomarkers that might predict sensitivity to MCL1 inhibition, we conducted a gene dependency analysis using publicly available human cancer cell line data generated from genome-wide CRISPR/Cas9-mediated cell viability screens. We observed that mutations in the SWI/SNF complex, particularly in lung and ovarian cancer cell lines, conferred a strong functional dependency on MCL1. The mammalian SWI/SNF complex functions as a tumor suppressor in a number of cancers and regulates gene expression via chromatin-remodeling. It is comprised of multiple subunits, including one of two catalytic ATPases (SMARCA2 or SMARAC4), DNA-binding proteins ARID1A, ARID1B and ARID2, and other chromatin-binding subunits. Gene mutations in members of this complex occur in >20% of human cancers, and therapeutic agents targeting its function are under active clinical investigation. We and others have shown potent synthetic lethality with the use of SMARCA2 targeted protein degraders in SMARCA4 deleted lung cancer models. A previously published genome-wide CRISPR screen in SMARCA4-mut lung cancer cell lines demonstrated that loss of MCL1 could sensitize these cells to SMARCA2 degradation. Therefore, we evaluated PRT1419 in combination with a novel and selective SMARCA2 degrader, PRT3789, in SMARCA4 deleted lung cancer models. We observed a potent synergistic interaction in SMARCA4 deleted cell lines in vitro, whereas no additive benefit was seen in SMARCA4 WT lines. Further, combining PRT1419 and PRT3789 in vivo in cell line-derived xenograft models resulted in significant tumor growth inhibition, including tumor regressions. Additionally, we profiled PRT1419 ex vivo in a panel of lung cancer PDX models and observed significant, dose-dependent effects on cell viability in SMARCA4 deleted models with low SMARCA2 expression. In a broader lung cancer cell line viability screen conducted with PRT1419, we observed that the presence of multiple, co-occurring alterations in SWI/SNF family members such as SMARCA4, ARID1A/B mutations and loss of SMARCA2 protein were associated with sensitivity to PRT1419. Based on these findings, preclinical evaluation of PRT1419 in other tumor types with recurrent SWI/SNF mutations is ongoing. Citation Format: Norman Fultang, Neha Bhagwat, Diane Heiser, Alexander Grego, Michael Hulse, Venkat Thodima, Koichi Ito, Kris Vaddi, Bruce Ruggeri, Peggy Scherle. Combination of the MCL1 inhibitor PRT1419 and SMARCA2 degrader PRT3789 shows combinatorial benefit in SMARCA4 deleted lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 420.
<div>Abstract<p>Fulvestrant is an estrogen receptor (ER) antagonist administered to breast cancer patients by monthly intramuscular injection. Given its present limitations of dosing and route of administration, a more flexible orally available compound has been sought to pursue the potential benefits of this drug in patients with advanced metastatic disease. Here we report the identification and characterization of AZD9496, a nonsteroidal small-molecule inhibitor of ERα, which is a potent and selective antagonist and downregulator of ERα <i>in vitro</i> and <i>in vivo</i> in ER-positive models of breast cancer. Significant tumor growth inhibition was observed as low as 0.5 mg/kg dose in the estrogen-dependent MCF-7 xenograft model, where this effect was accompanied by a dose-dependent decrease in PR protein levels, demonstrating potent antagonist activity. Combining AZD9496 with PI3K pathway and CDK4/6 inhibitors led to further growth-inhibitory effects compared with monotherapy alone. Tumor regressions were also seen in a long-term estrogen-deprived breast model, where significant downregulation of ERα protein was observed. AZD9496 bound and downregulated clinically relevant ESR1 mutants <i>in vitro</i> and inhibited tumor growth in an ESR1-mutant patient-derived xenograft model that included a D538G mutation. Collectively, the pharmacologic evidence showed that AZD9496 is an oral, nonsteroidal, selective estrogen receptor antagonist and downregulator in ER<sup>+</sup> breast cells that could provide meaningful benefit to ER<sup>+</sup> breast cancer patients. AZD9496 is currently being evaluated in a phase I clinical trial. <i>Cancer Res; 76(11); 3307–18. ©2016 AACR</i>.</p></div>
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