Therapeutic modulation of ROCK overcomes metabolic adaptation of cancer cells to OXPHOS inhibition and drives synergistic anti-tumor activity.

Genomic studies have identified frequent mutations in subunits of the SWI/SNF chromatin remodeling complex including SMARCA4 and ARID1A in non-small cell lung cancer. Previously, we and others have identified that SMARCA4-mutant lung cancers are highly dependent on oxidative phosphorylation (OXPHOS). Despite initial excitements, therapeutics targeting metabolic pathways such as OXPHOS have largely been disappointing due to rapid adaptation of cancer cells to inhibition of single metabolic enzymes or pathways, suggesting novel combination strategies to overcome adaptive responses are urgently needed. Here, we performed a functional genomics screen using CRISPR-Cas9 library targeting genes with available FDA approved therapeutics and identified ROCK1/2 as a top hit that sensitizes cancer cells to OXPHOS inhibition. We validate these results by orthogonal genetic and pharmacologic approaches by demonstrating that KD025 (Belumosudil), an FDA approved ROCK inhibitor, has highly synergistic anti-cancer activity in vitro and in vivo in combination with OXPHOS inhibition. Mechanistically, we showed that this combination induced a rapid, profound energetic stress and cell cycle arrest that was in part due to ROCK inhibition-mediated suppression of the adaptive increase in glycolysis normally seen by OXPHOS inhibition. Furthermore, we applied global phosphoproteomics and kinase-motif enrichment analysis to uncover a dynamic regulatory kinome upon combination of OXPHOS and ROCK inhibition. Importantly, we found converging phosphorylation-dependent regulatory cross-talk by AMPK and ROCK kinases on key RHO GTPase signaling/ROCK-dependent substrates such as PPP1R12A, NUMA1 and PKMYT1 that are known regulators of cell cycle progression. Taken together, our study identified ROCK kinases as critical mediators of metabolic adaptation of cancer cells to OXPHOS inhibition and provides a strong rationale for pursuing ROCK inhibitors as novel combination partners to OXPHOS inhibitors in cancer treatment.

Discovery of Novel, Potent and Orally Bioavailable SMARCA2 PROTACs with Synergistic Anti-tumor Activity in Combination with KRAS G12C Inhibitors.

Cancer genomic studies have identified frequent mutations in subunits of the SWI/SNF chromatin remodeling complex including SMARCA4 in non-small cell lung cancer with a frequency of up to 33% in advanced stage disease, making it the most frequently mutated complex in lung cancer. We and others have identified SMARCA2 to be synthetic lethal to SMARCA4, indicating SMARCA2 is a high value therapeutic target. Here, we disclose the discovery and characterization of potent, selective and orally bioavailable Cereblon-based SMARCA2 PROTACs. Biochemically, YDR1 and YD54 are potent SMARCA2 degraders with an average DC 50 of 7.7nM and 3.5nM respectively in SMARCA4 mutant lung cancer cells. Phenotypically, both YDR1 and YD54 selectively inhibited growth of SMARCA4 mutant cancer cells. Further, we showed anti-tumor growth inhibitory activity of YDR1 and YD54 in SMARCA4 mutant xenograft models of lung cancer. Finally, we show that YDR1 and YD54 synergize with the KRAS G12C inhibitor sotorasib to inhibit growth of SMARCA4 and KRAS G12C co-mutant lung cancer cells. These findings provide additional evidence for the utility of single agent or combination regimens containing SMARCA2 PROTACs as synthetic lethal therapeutics against SMARCA4 mutant cancers.

SMARCA4 mutation induces tumor cell-intrinsic defects in enhancer landscape and resistance to immunotherapy.

Cancer genomic studies have identified frequent alterations in components of the SWI/SNF (SWItch/Sucrose Non- Fermenting) chromatin remodeling complex including SMARCA4 and ARID1A . Importantly, clinical reports indicate that SMARCA4 -mutant lung cancers respond poorly to immunotherapy and have dismal prognosis. However, the mechanistic basis of immunotherapy resistance is unknown. Here, we corroborated the clinical findings by using immune-humanized, syngeneic, and genetically engineered mouse models of lung cancer harboring SMARCA4 deficiency. Specifically, we show that SMARCA4 loss caused decreased response to anti-PD1 immunotherapy associated with significantly reduced infiltration of dendritic cells (DCs) and CD4+ T cells into the tumor microenvironment (TME). Mechanistically, we show that SMARCA4 loss in tumor cells led to profound downregulation of STING, IL1ß and other components of the innate immune system as well as inflammatory cytokines that are required for efficient recruitment and activity of immune cells. We establish that this deregulation of gene expression is caused by cancer cell-intrinsic reprogramming of the enhancer landscape with marked loss of chromatin accessibility at enhancers of genes involved in innate immune response such as STING, IL1ß, type I IFN and inflammatory cytokines. Interestingly, we observed that transcription factor NF-κB binding motif was highly enriched in enhancers that lose accessibility upon SMARCA4 deficiency. Finally, we confirmed that SMARCA4 and NF-κB co-occupy the same genomic loci on enhancers associated with STING and IL1ß, indicating a functional interplay between SMARCA4 and NF-κB. Taken together, our findings provide the mechanistic basis for the poor response of SMARCA4 -mutant tumors to anti-PD1 immunotherapy and establish a functional link between SMARCA4 and NF-κB on innate immune and inflammatory gene expression regulation.

A Lung Cancer Mouse Model Database.

Lung cancer, the leading cause of cancer mortality, exhibits diverse histological subtypes and genetic complexities. Numerous preclinical mouse models have been developed to study lung cancer, but data from these models are disparate, siloed, and difficult to compare in a centralized fashion. Here we established the Lung Cancer Mouse Model Database (LCMMDB), an extensive repository of 1,354 samples from 77 transcriptomic datasets covering 974 samples from genetically engineered mouse models (GEMMs), 368 samples from carcinogen-induced models, and 12 samples from a spontaneous model. Meticulous curation and collaboration with data depositors have produced a robust and comprehensive database, enhancing the fidelity of the genetic landscape it depicts. The LCMMDB aligns 859 tumors from GEMMs with human lung cancer mutations, enabling comparative analysis and revealing a pressing need to broaden the diversity of genetic aberrations modeled in GEMMs. Accompanying this resource, we developed a web application that offers researchers intuitive tools for in-depth gene expression analysis. With standardized reprocessing of gene expression data, the LCMMDB serves as a powerful platform for cross-study comparison and lays the groundwork for future research, aiming to bridge the gap between mouse models and human lung cancer for improved translational relevance.

3-Phosphoinositide-dependent kinase 1 drives acquired resistance to osimertinib.

Osimertinib sensitive and resistant NSCLC NCI-H1975 clones are used to model osimertinib acquired resistance in humanized and non-humanized mice and delineate potential resistance mechanisms. No new EGFR mutations or loss of the EGFR T790M mutation are found in resistant clones. Resistant tumors grown under continuous osimertinib pressure both in humanized and non-humanized mice show aggressive tumor regrowth which is significantly less sensitive to osimertinib as compared with parental tumors. 3-phosphoinositide-dependent kinase 1 (PDK1) is identified as a potential driver of osimertinib acquired resistance, and its selective inhibition by BX795 and CRISPR gene knock out, sensitizes resistant clones. In-vivo inhibition of PDK1 enhances the osimertinib sensitivity against osimertinib resistant xenograft and a patient derived xenograft (PDX) tumors. PDK1 knock-out dysregulates PI3K/Akt/mTOR signaling, promotes cell cycle arrest at the G1 phase. Yes-associated protein (YAP) and active-YAP are upregulated in resistant tumors, and PDK1 knock-out inhibits nuclear translocation of YAP. Higher expression of PDK1 and an association between PDK1 and YAP are found in patients with progressive disease following osimertinib treatment. PDK1 is a central upstream regulator of two critical drug resistance pathways: PI3K/AKT/mTOR and YAP.