HNRNPM controls circRNA biogenesis and splicing fidelity to sustain cancer cell fitness

High spliceosome activity is a dependency for cancer cells, making them more vulnerable to perturbation of the splicing machinery compared to normal cells. To identify splicing factors important for prostate cancer (PCa) fitness, we performed pooled shRNA screens in vitro and in vivo. Our screens identified HNRNPM as a regulator of PCa cell growth. RNA- and eCLIP-sequencing identified HNRNPM binding to transcripts of key homeostatic genes. HNRNPM binding to its targets prevents aberrant exon inclusion and back-splicing events. In both linear and circular mis-spliced transcripts, HNRNPM preferentially binds to GU-rich elements in long flanking proximal introns. Mimicry of HNRNPM dependent linear splicing events using splice-switching-antisense-oligonucleotides (SSOs) was sufficient to inhibit PCa cell growth. This suggests that PCa dependence on HNRNPM is likely a result of mis-splicing of key homeostatic coding and non-coding genes. Our results have further been confirmed in other solid tumors. Taken together, our data reveal a role for HNRNPM in supporting cancer cell fitness. Inhibition of HNRNPM activity is therefore a potential therapeutic strategy in suppressing growth of PCa and other solid tumors

Splice‐switch oligonucleotide‐based combinatorial platform prioritizes synthetic lethal targets CHK1 and BRD4 against MYC‐driven hepatocellular carcinoma

Abstract Deregulation of MYC is among the most frequent oncogenic drivers in hepatocellular carcinoma (HCC). Unfortunately, the clinical success of MYC‐targeted therapies is limited. Synthetic lethality offers an alternative therapeutic strategy by leveraging on vulnerabilities in tumors with MYC deregulation. While several synthetic lethal targets of MYC have been identified in HCC, the need to prioritize targets with the greatest therapeutic potential has been unmet. Here, we demonstrate that by pairing splice‐switch oligonucleotide (SSO) technologies with our phenotypic‐analytical hybrid multidrug interrogation platform, quadratic phenotypic optimization platform (QPOP), we can disrupt the functional expression of these targets in specific combinatorial tests to rapidly determine target–target interactions and rank synthetic lethality targets. Our SSO‐QPOP analyses revealed that simultaneous attenuation of CHK1 and BRD4 function is an effective combination specific in MYC‐deregulated HCC, successfully suppressing HCC progression in vitro. Pharmacological inhibitors of CHK1 and BRD4 further demonstrated its translational value by exhibiting synergistic interactions in patient‐derived xenograft organoid models of HCC harboring high levels of MYC deregulation. Collectively, our work demonstrates the capacity of SSO‐QPOP as a target prioritization tool in the drug development pipeline, as well as the therapeutic potential of CHK1 and BRD4 in MYC‐driven HCC

RBFOX2 deregulation promotes pancreatic cancer progression and metastasis through alternative splicing

Abstract RNA splicing is an important biological process associated with cancer initiation and progression. However, the contribution of alternative splicing to pancreatic cancer (PDAC) development is not well understood. Here, we identify an enrichment of RNA binding proteins (RBPs) involved in splicing regulation linked to PDAC progression from a forward genetic screen using Sleeping Beauty insertional mutagenesis in a mouse model of pancreatic cancer. We demonstrate downregulation of RBFOX2, an RBP of the FOX family, promotes pancreatic cancer progression and liver metastasis. Specifically, we show RBFOX2 regulates exon splicing events in transcripts encoding proteins involved in cytoskeletal remodeling programs. These exons are differentially spliced in PDAC patients, with enhanced exon skipping in the classical subtype for several RBFOX2 targets. RBFOX2 mediated splicing of ABI1, encoding the Abelson-interactor 1 adapter protein, controls the abundance and localization of ABI1 protein isoforms in pancreatic cancer cells and promotes the relocalization of ABI1 from the cytoplasm to the periphery of migrating cells. Using splice-switching antisense oligonucleotides (AONs) we demonstrate the ABI1 ∆Ex9 isoform enhances cell migration. Together, our data identify a role for RBFOX2 in promoting PDAC progression through alternative splicing regulation