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Identification of synthetic lethal genetic interactions of SWI/SNF complex subunits

Hypothesizing the existence of functional genomic interactions that affect cellular viability, we have performed a genome-wide CRISPR-Cas9 knockout screen on pairs of SMARCA4 mutant and WT isogenic lung cancer cell lines (Fig. 1). The hits, putative synthetic lethal partners of SMARCA4, will be validated by orthogonal approaches in vitro and in vivo. Prioritization will be given based on the therapeutic accessibility of targets to aid in faster translation of discoveries. One such hit is SMARCA2 which we intend to validate further.

Importantly, we have developed potent chemical probes to degrade SMARCA2, a synthetic lethal partner of SMARCA4 and top hit from our screens. If successful, these studies will provide novel lung cancer therapeutic targets.

Fig. 1 Genome-wide CRISPR knockout screen is a powerful approach to discover synthetic lethal partners in isogenic SMARCA4 mutant and WT cell lines.?

Discover and develop proteolysis targeting chimeras (PROTACs) as cancer therapeutics

An important aspect of our research interest is the development of chemical probes to supplement genetic tools to understand chromatin biology in cancer. Proteolysis targeting chimeras (PROTACs) are exciting new molecular entities that have unique and potent pharmacologic properties. We have developed several PROTACs that induce selective degradation of target oncoproteins of interest including SMARCA2 as a therapeutic for SMARCA4 mutant lung cancer and glucocorticoid receptor (GR) as a combination therapy to circumvent resistance to chemotherapy in lung cancer (Fig. 2). These studies will provide first-in-class lung cancer therapeutics for clinical development.

Fig. 2 Chemical structures of some GR and SMARCA2 PROTACs synthesized in collaboration with talented and dedicated medicinal chemists (e.g. J.T Link on GR PROTACs).

Discovery of therapeutic vulnerabilities in genetically?defined cancer subtypes

Previous?work in our lab identified a metabolic vulnerability in targeting oxidative?phosphorylation (OXPHOS) in SMARCA4-mutant lung cancer.? However, OXPHOS inhibitors are still not?approved to treat cancer due to modest efficacy and adverse effects. This?strongly suggests that novel, efficacious and tolerated agents that synergize?with OXPHOS inhibition are urgently needed. To this end, our lab employed?functional genomic screens by utilizing a CRISPR-Cas9 library targeting genes?with available FDA approved therapeutics in genetically defined lung cancer?cell lines. Importantly, we utilized low doses of the OXPHOS inhibitor?IACS-10759 that are known to be well tolerated in patients. Among our top?validated hits was ROCK1/2 kinases and we demonstrate that Belumasudil, a clinically approved ROCK inhibitor with a robust safety
and tolerability profile, displays synergistic anti-tumor activity with?IACS-10759 in vitro and in vivo using multiple mouse xenograft and human PDX?models.?

Mechanistically,?metabolic profiling indicates that the combination of Belumasudil and?IACS-10759 induced a profound energetic stress primarily due to ROCK inhibition-mediated?suppression of the normally observed adaptive increase in glycolysis upon?OXPHOS inhibition via impediment of glucose? uptake. To further deepen our understanding, we performed?quantitative proteomics and kinase activity analysis of the global?phosphoproteome by TMT mass spectrometry. Through this strategy, we identified?significant cross-talk between the energy sensor AMPK and several Rho/ROCK?GTPase signaling substrates involved in actin cytoskeleton dynamics and cell?cycle progression.? Future studies are?exploring these potential cross-talk mechanisms.

?In summary, our lab leveraged several?multi-omic approaches to identify a key role of ROCK kinases in metabolic?adaptation of cancer cells and provides a strong rationale for pursuing ROCK inhibitors?as novel combination agents with OXPHOS inhibition. We intend to further?optimize this dose combination regimen for clinical trial testing.?

Chromatin remodeling, innate immunity and cancer immunotherapy response

Cancer genomic studies have identified frequent alterations in genes encoding components of the SWI/SNF chromatin remodeling complex, including SMARCA4 and ARID1A. Importantly, clinical reports indicate that SMARCA4-mutant lung cancers respond poorly to immunotherapy and have dismal prognosis. Here, we corroborated the clinical findings by using immune-humanized, syngeneic, and genetically engineered mouse models of lung cancer harboring SMARCA4 deficiency. Specifically, models with SMARCA4 loss showed decreased response to anti-PD1 immunotherapy associated with significantly reduced infiltration of dendritic cells and CD4+T cells into the tumor microenvironment. SMARCA4 loss in tumor cells led to profound downregulation of STING1, IL1¦Â, and other components of the innate immune system, as well as inflammatory cytokines that are required for efficient recruitment and activity of immune cells. The deregulation of gene expression was 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 STING1, IL1¦Â, type I IFN and inflammatory cytokines. Interestingly, the transcription factor NF-¦ÊB binding motif was enriched in enhancers that lose accessibility upon SMARCA4 deficiency. Furthermore, SMARCA4 and NF-¦ÊB co-occupied the same genomic loci on enhancers associated with STING1 and IFN¦Â, indicating a functional interplay between SMARCA4 and NF-¦ÊB. Taken together, these findings provide the mechanistic basis for the poor response of SMARCA4-mutant tumors to immunotherapy and establish a functional link between SMARCA4 and NF-¦ÊB in innate immune and inflammatory gene expression regulation.

We intend to build on these results and pursue chromatin remodeling as a viable cancer therapeutic strategy.