Outer kinetochore assembly enables chromosome attachment to microtubules and spindle assembly checkpoint (SAC) signaling in mitosis. Aurora B kinase controls kinetochore assembly by phosphorylating the Mis12 complex (Mis12C) subunit Dsn1. Current models propose Dsn1 phosphorylation relieves autoinhibition, allowing Mis12C binding to inner kinetochore component CENP-C. Using Xenopus laevis egg extracts and biochemical reconstitution, we found that autoinhibition of the Mis12C by Dsn1 impedes its phosphorylation by Aurora B. Our data indicate that the INCENP central region increases Dsn1 phosphorylation by enriching Aurora B at inner kinetochores, close to CENP-C. Furthermore, centromere-bound CENP-C does not exchange in mitosis, and CENP-C binding to the Mis12C dramatically increases Dsn1 phosphorylation by Aurora B. We propose that the coincidence of Aurora B and CENP-C at inner kinetochores ensures the fidelity of kinetochore assembly. We also found that the central region is required for the SAC beyond its role in kinetochore assembly, suggesting that kinetochore enrichment of Aurora B promotes the phosphorylation of other kinetochore substrates.

Genomic sequencing of thousands of tumors has revealed many genes associated with specific types of cancer. Similarly, large scale CRISPR functional genomics efforts have mapped genes required for cancer cell proliferation or survival in hundreds of cell lines. Despite this, for specific disease subtypes, such as metastatic prostate cancer, there are likely a number of undiscovered tumor specific driver genes that may represent potential drug targets. To identify such genetic dependencies, we performed genome-scale CRISPRi screens in metastatic prostate cancer models. We then created a pipeline in which we integrated pan-cancer functional genomics data with our metastatic prostate cancer functional and clinical genomics data to identify genes that can drive aggressive prostate cancer phenotypes. Our integrative analysis of these data reveals known prostate cancer specific driver genes, such as AR and HOXB13, as well as a number of top hits that are poorly characterized. In this study we highlight the strength of an integrated clinical and functional genomics pipeline and focus on two top hit genes, KIF4A and WDR62. We demonstrate that both KIF4A and WDR62 drive aggressive prostate cancer phenotypes in vitro and in vivo in multiple models, irrespective of AR-status, and are also associated with poor patient outcome.

Cell state evolution underlies tumor development and response to therapy, but mechanisms specifying cancer cell states and intratumor heterogeneity are incompletely understood. Schwannomas are the most common tumors of the peripheral nervous system and are treated with surgery and ionizing radiation. Schwannomas can oscillate in size for many years after radiotherapy, suggesting treatment may reprogram schwannoma cells or the tumor microenvironment. Here we show epigenetic reprogramming shapes the cellular landscape of schwannomas. We find schwannomas are comprised of 2 molecular groups distinguished by reactivation of neural crest development pathways or misactivation of nerve injury mechanisms that specify cancer cell states and the architecture of the tumor immune microenvironment. Schwannoma molecular groups can arise independently, but ionizing radiation is sufficient for epigenetic reprogramming of neural crest to immune-enriched schwannoma by remodeling chromatin accessibility, gene expression, and metabolism to drive schwannoma cell state evolution and immune cell infiltration. To define functional genomic mechanisms underlying epigenetic reprograming of schwannomas, we develop a technique for simultaneous interrogation of chromatin accessibility and gene expression coupled with genetic and therapeutic perturbations in single-nuclei. Our results elucidate a framework for understanding epigenetic drivers of cancer evolution and establish a paradigm of epigenetic reprograming of cancer in response to radiotherapy.

Despite the abundance of somatic structural variations (SVs) in cancer, the underlying molecular mechanisms of their formation remain unclear. Here, we use 6,193 whole-genome sequenced tumors to study the contributions of transcription and DNA replication collisions to genome instability. After deconvoluting robust SV signatures in three independent pan-cancer cohorts, we detect transcription-dependent replicated-strand bias, the expected footprint of transcription-replication collision (TRC), in large tandem duplications (TDs). Large TDs are abundant in female-enriched, upper gastrointestinal tract and prostate cancers. They are associated with poor patient survival and mutations in TP53, CDK12, and SPOP. Upon inactivating or suppressing CDK12, cells display significantly more TRCs and R-loops. Inhibition of WEE1, a cell cycle regulator that promotes DNA repair, selectively inhibits the growth of cells with loss of CDK12. Our data suggest that large TDs in cancer form due to TRC, and their presence can be used as a biomarker for prognosis and treatment.

ENPP1 expression correlates with poor prognosis in many cancers, and we previously discovered that ENPP1 is the dominant hydrolase of extracellular cGAMP: a cancer-cell-produced immunotransmitter that activates the anticancer STING pathway. However, ENPP1 has other catalytic activities and the molecular and cellular mechanisms contributing to its tumorigenic effects remain unclear. Here, using single cell RNA-seq (scRNA-seq), we show that ENPP1 overexpression drives primary breast tumor growth and metastasis by synergistically dampening extracellular cGAMP-STING mediated antitumoral immunity and activating immunosuppressive extracellular adenosine (eADO) signaling. In addition to cancer cells, stromal and immune cells in the tumor microenvironment (TME) also express ENPP1 that restrains their response to tumor-derived cGAMP. Enpp1 loss-of-function in both cancer cells and normal tissues slowed primary tumor initiation and growth and prevented metastasis in an extracellular cGAMP- and STING-dependent manner. Selectively abolishing the cGAMP hydrolysis activity of ENPP1 phenocopied total ENPP1 knockout, demonstrating that restoration of paracrine cGAMP-STING signaling is the dominant anti-cancer mechanism of ENPP1 inhibition. Strikingly, we find that breast cancer patients with low ENPP1 expression have significantly higher immune infiltration and improved response to therapeutics impacting cancer immunity upstream or downstream of the cGAMP-STING pathway, like PARP inhibitors and anti-PD1. Altogether, selective inhibition of ENPP1's cGAMP hydrolase activity alleviates an innate immune checkpoint to boost cancer immunity and is therefore a promising therapeutic approach against breast cancer that may synergize with other cancer immunotherapies.

Somatic structural variations (SVs) are common in cancer. Although a small fraction of SVs in breast and ovarian cancers can be attributed to homologous recombination deficiency, the underlying molecular mechanisms for the vast majority of somatic SVs remain unclear. Here, we focus on the roles of transcription and DNA replication collisions in genomic instability in cancer. Such collisions are unavoidable in cells since both transcription and replication use the same DNA as template. We hypothesized that transcription replication collisions (TRCs), if not properly repaired, would lead to collapsed replication forks and result in SVs. To this end, we studied somatic SVs in 5994 high-coverage whole-genome sequenced primary and metastatic tumors from three independent pan-cancer cohorts. A total of 12 conserved SV signatures, representing independent molecular mechanisms, were deconvoluted from these cohorts using non-negative matrix factorization approach. We detected replicated-strand bias, the expected footprint of transcription-replication collision, in large tandem duplications (TDs) across multiple cohorts. This bias was only observed in expressed genes, consistent with TRCs depending on transcription activity. Large TDs were abundant in female-specific (breast, ovarian and uterus), upper gastric-intestinal tract and prostate cancers. They were associated with worse patient survival and TP53 and CDK12 mutations. CDK12 is a cyclin-dependent kinase (CDK) and a key regulator of transcription elongation. Deleting or suppressing CDK12 using CRISPR-Cas9 in prostate cancer cell lines not only increased RNA:DNA hybrids (R-loops), but also promoted TRCs, suggesting a mechanism by which dysregulation of a transcriptional CDK may lead to genomic instability. Finally, using existing large-scale drug screening data, we found that cancer cell lines with abundant large TDs were significantly more sensitive to the WEE1 inhibitor, MK-1775, which we experimentally validated in prostate cancer cells lacking CDK12. In summary, our data suggest that large TDs in cancer form due to impaired TRC repair and can be used as a biomarker for prognosis and treatment. Citation Format: Yang Yang, Michelle Badura, Patrick O’Leary, Emily Egusa, Troy Robinson, Xiaoming Zhong, Jason Swinderman, Minkyu Kim, Haolong Li, Alan Ashworth, Felix Feng, Jonathan Chou, Lixing Yang. Large tandem duplications in cancer resulting from transcription and DNA replication collisions [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 299.

DNA methylation profiling provides robust classification of nervous system tumors, but mechanisms driving epigenetic identity of individual tumor types are incompletely understood. Integrating DNA methylation profiling (n=76), RNA sequencing (n=24), single-cell RNA-sequencing (n=9), and mass cytometry (n=9), we discovered vestibular schwannomas are comprised of two epigenetic groups distinguished by neural crest development pathways or repair and regeneration pathways driving immune infiltration. Analyses of preoperative magnetic resonance imaging studies (n=66) or paired primary and recurrent schwannomas (n=13) suggested radiotherapy was sufficient but not necessary for epigenetic reprogramming of neural crest enriched schwannomas into immune enriched schwannomas. In support of this hypothesis, DNA methylation profiling, RNA sequencing, single-cell RNA sequencing, proteomic mass spectrometry, and lymphocyte migration assays demonstrated radiotherapy epigenetically reprogramed viable schwannoma cells to secrete immunomodulatory signals and recruit lymphocytes in vitro. Genome-wide CRISPRi screens identified histone acetyltransferases or DNA methyltransferases driving schwannoma radiotherapy responses, including the epigenetic regulators KDM5C or KDM1A. CRISPRi and lymphocyte migration assays ± radiotherapy confirmed KDM5C drives schwannoma immune infiltration whereas KDM1A inhibits schwannoma immune infiltration. To define genomic mechanisms underlying epigenetic group identity, we performed pooled CRISPRi screening coupled with single-cell RNA sequencing (Perturb-seq) of 44 schwannoma markers. In parallel, we developed single nuclei profiling of chromatin accessibility through paired ATAC sequencing and RNA sequencing coupled with pooled CRISPRi screening (snARC-seq) of 54 epigenetic regulators identified by our genome-wide CRISPRi screen. Functional genomic approaches revealed the tyrosine phosphatase PTPRG as a regulator of survival, and KDM5C and KDM1A as regulators of inflammation. In summary, we report two epigenetic groups of schwannomas and mechanisms underlying epigenetic group identity using a new functional genomic technique allowing for simultaneous interrogation of single-cell epigenetic and gene expression changes in the context of genetic and therapeutic perturbations. These data elucidate a novel epigenetic mechanism of action of radiotherapy.