Enhancing production of the anti-inflammatory cytokine interleukin-10 (IL-10) is a promising strategy to suppress pathogenic inflammation. To identify new mechanisms regulating IL-10 production, we conducted a phenotypic screen for small molecules that enhance IL-10 secretion from activated dendritic cells. Mechanism-of-action studies using a prioritized hit from the screen, BRD6989, identified the Mediator-associated kinase CDK8, and its paralog CDK19, as negative regulators of IL-10 production during innate immune activation. The ability of BRD6989 to upregulate IL-10 is recapitulated by multiple, structurally differentiated CDK8 and CDK19 inhibitors and requires an intact cyclin C–CDK8 complex. Using a highly parallel pathway reporter assay, we identified a role for enhanced AP-1 activity in IL-10 potentiation following CDK8 and CDK19 inhibition, an effect associated with reduced phosphorylation of a negative regulatory site on c-Jun. These findings identify a function for CDK8 and CDK19 in regulating innate immune activation and suggest that these kinases may warrant consideration as therapeutic targets for inflammatory disorders.

The RNF43_p.G659fs mutation occurs frequently in colorectal cancer, but its function remains poorly understood and there are no specific therapies directed against this alteration. In this study, we find that RNF43_p.G659fs promotes cell growth independent of Wnt signaling. We perform a drug repurposing library screen and discover that cells with RNF43_p.G659 mutations are selectively killed by inhibition of PI3K signaling. PI3K/mTOR inhibitors yield promising antitumor activity in RNF43659mut isogenic cell lines and xenograft models, as well as in patient-derived organoids harboring RNF43_p.G659fs mutations. We find that RNF43659mut binds p85 leading to increased PI3K signaling through p85 ubiquitination and degradation. Additionally, RNA-sequencing of RNF43659mut isogenic cells reveals decreased interferon response gene expression, that is reversed by PI3K/mTOR inhibition, suggesting that RNF43659mut may alter tumor immunity. Our findings suggest a therapeutic application for PI3K/mTOR inhibitors in treating RNF43_p.G659fs mutant cancers.

No abstract

Aneuploidy is a hallmark of human cancer, yet the cellular mechanisms that allow cells to cope with aneuploidy-induced cellular stresses remain largely unknown. Such coping mechanisms may present cellular vulnerabilities that can be harnessed for targeting cancer cells. Here, we induced aneuploidy in non-transformed RPE1-hTERT cells and derived multiple stable clones with various degrees of chromosome imbalances. We performed an unbiased genomic profiling of 6 isogenic clones, using whole-exome and RNA sequencing. We then functionally interrogated their cellular dependency landscapes, using genome-wide CRISPR/Cas9 screens and large-scale drug screens. We found that aneuploid clones activated the DNA damage response (DDR), and were consequently more resistant to further DNA damage induction. Interestingly, aneuploid cells also exhibited elevated RAF/MEK/ERK pathway activity, and were more sensitive to several clinically-relevant drugs targeting this pathway, and in particular to genetic and chemical CRAF inhibition. CRAF activity was functionally linked to the resistance to DNA damage induction, as CRAF inhibition sensitized aneuploid cells to DNA damage-inducing chemotherapies. The association between aneuploidy, RAF/MEK/ERK signaling, and DDR was independent of p53. The increased activity and dependency of aneuploid cells on the RAF/MEK/ERK pathway was validated in another isogenic aneuploid system, and across hundreds of human cancer cell lines, confirming their relevance to human cancer. Overall, our study provides a comprehensive resource for genetically-matched karyotypically-stable cells of various aneuploidy states, and reveals a novel therapeutically-relevant cellular dependency of aneuploid cells.

Sickle cell disease (SCD) is an inherited disorder of hemoglobin (Hb); approximately 300,000 babies are born worldwide with SCD each year. In SCD, fibers of polymerized sickle Hb (HbS) form in red blood cells (RBCs), which cause RBCs to develop their characteristic “sickled” shape, resulting in hemolytic anemia and numerous vascular complications including vaso-occlusive crises. The development of novel antisickling compounds will provide new therapeutic options for patients with SCD. We developed a high-throughput “sickling assay” that is based on an automated high-content imaging system to quantify the effects of hypoxia on the shape and size of RBCs from HbSS SCD patients (SS RBCs). We used this assay to screen thousands of compounds for their ability to inhibit sickling. In the assay, voxelotor (an FDA-approved medication used to treat SCD) prevented sickling with a z ′-factor > 0.4, suggesting that the assay is capable of identifying compounds that inhibit sickling. We screened the Broad Repurposing Library of 5393 compounds for their ability to prevent sickling in 4% oxygen/96% nitrogen. We identified two compounds, SNS-314 mesylate and voxelotor itself, that successfully prevented sickling. SNS-314 mesylate prevented sickling in the absence of oxygen, while voxelotor did not, suggesting that SNS-314 mesylate acts by a mechanism that is different from that of voxelotor. The sickling assay described in this study will permit the identification of additional, novel antisickling compounds, which will potentially expand the therapeutic options for SCD.

Introduction: RNF43 is a transmembrane E3 ubiquitin ligase and WNT signaling suppressor that is commonly mutated in colorectal cancer (CRC). A C-terminal RNF43 hotspot mutation, RNF43_G659fs, occurs in approximately 36% (55/151) of microsatellite-instability (MSI)-high CRCs, but its underlying mechanism and function remain poorly understood. This study investigated the functional role of RNF43_G659fs in order to evaluate potential novel therapeutic approaches for tumors harboring this mutation. Methods: Isogenic RNF43117mut and RNF43659mut cell line models were generated using the CRISPR/Cas9 system to evaluate CRC tumorigenesis and WNT dependency. RNF43659mut was screened with a novel high-throughput drug repurposing library that employed a set of 5363 small molecules to identify compounds capable of selectively inhibiting RNF43659mut cell growth. Small molecules that selectively killed the RNF43659mut cells were validated in organoid models. Proteomic analysis, RNA-Seq and gene set enrichment analysis (GSEA) were performed to characterize mechanistic interactions and related signaling pathways of RNF43659mut in CRC. Results: Unlike N-terminal RNF43 frameshift mutations, we observed that RNF43659mut conferred a growth advantage over RNF43WT cells independent of WNT signaling. Furthermore, RNF43659mut and RNF43WT exhibited differential drug responses in the high-throughput drug repurposing screen which revealed that RNF43659mut cells were vulnerable to PI3K/AKT/mTOR inhibitors, including BYL-719 (Alpelisib). Enhanced AKT and mTOR activation was observed in RNF43659mut cell and attenuated by BYL-719 treatment in a dose-dependent manner. These results were subsequently validated in patient-derived organoid models. Furthermore, immunoprecipitation and proteomic analysis revealed interactions between RNF43_G659fs and p85, a negative regulator of PI3K. We also demonstrated that the RNF43_G659fs mutant activated PI3K/AKT/mTOR signaling through binding and degradation of p85. Consistent with the role of PI3K in immunomodulation, our RNA-Seq results showed that the RNF43_G659fs mutation was positively related to NF-kB activation (Normalized Enrichment Score=1.842, p<0.01) and inversely related to Interferon-alpha/Interferon-gamma response pathways (Normalized Enrichment Score= -1.992, p<0.01; Normalized Enrichment Score= -1.577, p<0.01, respectively), indicating its role in tumor microenvironment remodeling. Conclusion: This study confirms that RNF43659mut is an essential driver mutation in CRC and provides evidence that patients harboring RNF43_G659fs-mutant tumors may respond favorably to PI3K inhibition. Citation Format: Lishan Fang, Dane Ford-Roshon, Max Russo, Casey O'Brien, Carino Gurjao, Maximilien Grandclaudon, Steven M. Corsello, Srivatsan Raghavan, Namrata Udeshi, James Berstler, Ewa Sicinska, Kimmie Ng, Marios Giannakis. RNF43 G659fs is an oncogenic mutation in colorectal cancer and sensitizes tumor cells to PI3K/mTOR inhibition [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 960.

Treatment advances for soft-tissue sarcomas have been slow. This is, in part, due to their rarity (accounting for 0.7% of all cancers) and heterogeneity (over 50 different diseases fall under this category). Moreover, preclinical models are scarce, often exhibiting slow growth kinetics, which limits their study by large genetic and pharmacological libraries. Here, we present an update on our efforts to harness the power of patient-partnered research to create a platform for rare cancer drug target discovery as a broadly available community resource. We developed a patient-partnered tissue donation pipeline to enable patients anywhere in the United States to participate and piloted our approach for CTNNB1-driven desmoid tumors. To overcome challenges in tissue heterogeneity during ex vivo culture, we optimized a multiplexed sequencing protocol to quantitatively track changes in tumor cell fraction across hundreds of media formulations. Following this strategy, we were able to verify and expand three cell lines that preserve the CTNNB1 mutations at high purity. To identify potential therapeutics, we completed a 6,750-drug repurposing screen, at 2.5uM in duplicate, in two verified cell line models. After extensive quality control assessments and data integration steps to leverage the power of other large scale drug screens, we selected 263 compounds for follow-up based on potency, selectivity, and association with molecular features associated with desmoid tumors. Approximately 70% of selected compounds were validated by an 8-point, 2-fold dilution, dose-response format with a top concentration of 10uM. Of the confirmed active compounds, 80 showed a strong pattern of selectivity, 20 are FDA approved drugs and 13 investigational compounds show a statistical association with CTNNB1 hotspot mutation status or transcriptomic features associated with desmoid tumors. To prioritize potential therapeutic targets, we tested an efficient CRISPR/Cas9 all-in-one library design. The reduction of the CRISPR/Cas9 library size was achieved via multiple gene- and guide-level strategies, which enables statistically powered gene essentiality interrogation in slow-growing patient-derived models. We tested several plating and infection parameters and developed an optimized pipeline for the rapid introduction of this library into early patient-derived samples. Established cell lines of mesenchymal and non-mesenchymal origin, which have previously been tested by genome-wide libraries, were used to control for library and lineage effects. We are developing a biologist-friendly web portal that will enable the research community to easily interact with models and data produced by this effort. Our study provides evidence that a systematic patient-powered approach can facilitate discovery of therapeutic hypotheses for these understudied diseases. Citation Format: Mushriq Al-Jazrawe, Kathryn Cebula, Elisabeth A. Abeyta, Haley S. Curtis, Jane K. McIninch, Jaime H. Cheah, James Berstler, Lisa Miller, James Neiswender, Lisa Brenan, Mike Burger, Francisca Vazquez, Jesse S. Boehm. Drug repurposing and genetic screening strategies for effective treatment discovery in soft-tissue sarcomas. [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 5324.

INTRODUCTION: We have observed that approximately 26% of recurrent gliomas acquire hypermutation following treatment with temozolomide (TMZ). Intriguingly, 91% of these tumors harbor mutations in mismatch repair (MMR) genes. Since MMR deficiency confers resistance to TMZ, strategies to target MMR-deficient gliomas stand to impact many patients. METHODS: We ablated the MMR genes MSH2, MSH6, MLH1, and PMS2 using an all-in-one sgRNA-CRISPR/Cas9 expression vector to generate isogenic MMR knockouts in patient-derived glioma cell lines. We characterized the gene expression profiles of these MMR-deficient glioma models and leveraged high-throughput drug screens and genome-scale CRISPR/Cas9 dropout screens to identify therapeutic vulnerabilities induced by loss of MMR. RESULTS: We show that loss of each major MMR gene confers resistance to TMZ. Gene set enrichment analysis of our MMR-deficient knockouts shows enrichment of several hallmark gene sets including DNA repair and G2M checkpoint signatures, and our genome-wide CRISPR dropout screen reveals that MMR-deficient cells are preferentially dependent on a number of genes involved in DNA repair and cell cycle, along with several other pathways. Lastly, the high-throughput drug repurposing (REPO) screen shows that loss of MMR confers differential dependencies to small molecule inhibitors. CONCLUSIONS: Using CRISPR/Cas9 to knock out individual MMR pathway members allows us to systematically study the response of MMR-deficient cells to alkylating agents in an isogenic context. Importantly, these isogenic models reveal that MMR-deficient glioma cells possess novel genetic dependencies and sensitivities to small molecules, which may inform future therapies for MMR-deficient tumors.