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Targeted protein degradation (TPD) is an emerging therapeutic modality with the potential to target previously undruggable targets. However, it has been more challenging to identify orally bioavailable TPD molecules due to the physicochemical properties of the large molecules and narrow structure-activity relationship (SAR) compared to conventional small molecule inhibitors. In the present study, we identified orally active TPD molecules that selectively and potently degrade SMARCA2 protein and induce synthetic lethality in SMARCA4-deficient cancer cells. SMARCA2 (BRM) and SMARCA4 (BRG1) are the two mutually exclusive catalytic core subunits of SWI/SNF complexes that play an important role in controlling gene expression by remodeling chromatin. The complexes are mutated in more than 20% of human cancers and subsets of solid tumors lose expression of SMARCA4 protein due to damaging mutations or gene deletion. The SMARCA4-deficient cancer cells are highly dependent on the paralog gene SMARCA2 for their survival and thus SMARCA2 has been suggested as an attractive therapeutic target for patients with SMARCA4-deficient cancers. We have recently identified SMARCA2 selective degraders that demonstrate oral bioavailability in mice with favorable pharmacokinetic properties, and acceptable DMPK and safety profiles in rodent studies. These SMARCA2 degraders show 50 to 300-fold DC50 selectivity for SMARCA2 over SMARCA4 in our cellular assays. When pre-treated with a proteasome inhibitor or neddylation inhibitor, the degradation of SMARCA2 was rescued, confirming that the degradation is mediated by the ubiquitin-proteasome-dependent pathway. These TPD molecules inhibit only SMARCA4-deficient cancer cell proliferation (NCI-H838, NCI-H1693, HT1080 SMARCA4 KO) with IC50 values ranging from 3-10 nM, but not SMARCA4 WT cells (Calu-6, NCI-H520, HT1080 WT). Oral administration of our SMARCA2 degraders resulted in significant tumor growth inhibition of SMARCA4-deficient lung cancer xenografts at well tolerated doses. The treated tumor tissues show robust SMARCA2 protein reduction for more than 72h post dosing, consistent with the SMARCA2 degradation kinetics-based pharmacodynamic prediction model. In summary, our orally bioavailable SMARCA2 degraders induce synthetic lethality in SMARCA4-deficient cancers in vitro and in vivo. Efforts to further evaluate these compounds in additional models and in combination with other agents are ongoing. Citation Format: Koichi Ito, Artem Shvartsbart, Joseph Rager, Anjana Agarwal, Michael Hulse, Komali Vykuntam, Min Wang, Justin Kurian, Miles Cowart, Joy Cote, Monisha Sivakumar, Jack Carter, Jessica Burtell, Alex Grego, Andrew Moore, Neha Bhagwat, Stefan Ruepp, Tom Emme, Liang Lu, Philip Pitis, Corey Basch, Klare Bersch, Song Mei, Raul Leal, John Rose, Danielle Roth, Ganfeng Cao, Kris Vaddi, Sandy Geeganage, Bruce Ruggeri, Andrew Combs, Peggy Scherle. Selective and orally bioavailable SMARCA2 targeted degraders induce synthetic lethality in SMARCA4- deficient solid tumor. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6277.

Richter’s Transformation (RT) is a poorly understood and fatal progression of chronic lymphocytic leukemia (CLL) manifesting histologically as diffuse large B-cell lymphoma. Protein arginine methyltransferase 5 (PRMT5) is implicated in lymphomagenesis, but its role in CLL or RT progression is unknown. We demonstrate herein that tumors uniformly overexpress PRMT5 in patients with progression to RT. Furthermore, mice with B-specific overexpression of hPRMT5 develop a B-lymphoid expansion with increased risk of death, and Eµ-PRMT5/TCL1 double transgenic mice develop a highly aggressive disease with transformation that histologically resembles RT; where large-scale transcriptional profiling identifies oncogenic pathways mediating PRMT5-driven disease progression. Lastly, we report the development of a SAM-competitive PRMT5 inhibitor, PRT382, with exclusive selectivity and optimal in vitro and in vivo activity compared to available PRMT5 inhibitors. Taken together, the discovery that PRMT5 drives oncogenic pathways promoting RT provides a compelling rationale for clinical investigation of PRMT5 inhibitors such as PRT382 in aggressive CLL/RT cases.

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.

SWI/SNF complexes play an important role in controlling gene expression by remodeling chromatin. SMARCA2 (BRM) and SMARCA4 (BRG1) are the core subunits of the SWI/SNF complexes which contain ATPase domain and DNA binding bromodomain. SMARCA4 protein expression is lost in some cancers due to damaging mutations (e.g. nonsense, frameshift deletion, splice site mutations) and SMARCA4-deleted cancer cells are highly dependent on its paralog gene SMARCA2 for their survival. Therefore, targeting SMARCA2 in SMARCA4-deleted cancers through the use of selective SMARCA2 degraders induces synthetic lethality. We designed and synthesized a variety of novel SMARCA2 degraders and tested them for degradation potency and selectivity by high-throughput in-cell western blot and probed for both SMARCA2 and SMARCA4 proteins in SMARCA4 WT expressing non-small cell lung cancer (NSCLC) NCI-H520 cells. We identified a unique series of potent SMARCA2 degraders with 20-40 fold greater selectivity for SMARCA2 vs SMARCA4. Global proteomics analysis confirmed that these degraders are highly selective for SMARCA2, and demonstrate minimal degradation of any other protein except PBRM1, another bromodomain VIII family protein. Our data suggest that the greater degradation selectivity observed is a result of preferential SMARCA2 ternary complex formation and/or poly-ubiquitination processing. Global RNA expression analysis shows that treatment with SMARCA2 degraders downregulates gene signatures associated with the cell cycle, cell proliferation, apoptosis and cell adhesion in SMARCA4-deleted NSCLC NCI-H1693 cells. Specifically, we found a significant reduction of gene expression of AXL, SMAD3, MMP2, and KRT80, all of which are important factors for tumor cell signaling and invasion. Additionally, protein set enrichment analysis (PSEA) shows that SMARCA2 degrader-treated NCI-H1693 cells downregulate expression of cell cycle and DNA replication related protein signatures as well as upregulated antigen processing and presentation pathway. A cell line panel analysis for cell viability and clonogenicity demonstrated that our selective SMARCA2 degraders effectively inhibit proliferation of SMARCA4-deleted cancer cells with nanomolar potencies, but not SMARCA4 WT or SMARCA2/4 null cancer cells. Our SMARCA2 degraders also significantly suppressed the growth of patient derived SMARCA4-deleted NSCLC tumor cells in primary 3D culture assay. In summary, consistent with previous studies and genomic perturbation analyses, our potent and selective SMARCA2 degraders induce synthetic lethality in SMARCA4-deleted cancers in vitro. Citation Format: Koichi Ito, Anjana Agarwal, Philip Pitis, Min Wang, Jack Carter, Miles Cowart, Chaofeng Dai, William Gowen-MacDonald, Eliza Elliot, Brian Vidal, Jacob Spruance, Hsin-Yao Tang, Bruce Ruggeri, Liang Lu, Andrew Combs, Hong Lin, Peggy Scherle, Kris Vaddi. Potent SMARCA2 targeted degraders induce genetic synthetic lethality in SMARCA4 deleted cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1139.

The concept of "ligand bias" at G protein coupled receptors has been introduced to describe ligands which preferentially stimulate one intracellular signaling pathway over another. There is growing interest in developing biased G protein coupled receptor ligands to yield safer, better tolerated, and more efficacious drugs. The classical μ opioid morphine elicited increased efficacy and duration of analgesic response with reduced side effects in β-arrestin-2 knockout mice compared to wild-type mice, suggesting that G protein biased μ opioid receptor agonists would be more efficacious with reduced adverse events. Here we describe our efforts to identify a potent, selective, and G protein biased μ opioid receptor agonist, TRV130 ((R)-30). This novel molecule demonstrated an improved therapeutic index (analgesia vs adverse effects) in rodent models and characteristics appropriate for clinical development. It is currently being evaluated in human clinical trials for the treatment of acute severe pain.

The concept of ligand bias at G protein-coupled receptors broadens the possibilities for agonist activities and provides the opportunity to develop safer, more selective therapeutics. Morphine pharmacology in β-arrestin-2 knockout mice suggested that a ligand that promotes coupling of the μ-opioid receptor (MOR) to G proteins, but not β-arrestins, would result in higher analgesic efficacy, less gastrointestinal dysfunction, and less respiratory suppression than morphine. Here we report the discovery of TRV130 ([(3-methoxythiophen-2-yl)methyl]({2-[(9R)-9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]ethyl})amine), a novel MOR G protein-biased ligand. In cell-based assays, TRV130 elicits robust G protein signaling, with potency and efficacy similar to morphine, but with far less β-arrestin recruitment and receptor internalization. In mice and rats, TRV130 is potently analgesic while causing less gastrointestinal dysfunction and respiratory suppression than morphine at equianalgesic doses. TRV130 successfully translates evidence that analgesic and adverse MOR signaling pathways are distinct into a biased ligand with differentiated pharmacology. These preclinical data suggest that TRV130 may be a safer and more tolerable therapeutic for treating severe pain.