Oncogenic mutations of SF3B1 are common in myeloid cancers, chronic lymphocytic leukemia (CLL) and select solid tumors. Their mechanistic basis for promoting oncogenesis has been investigated in detail, with the stereotyped missplicing of mRNA protein coding sequences most intensively studied. These changes, in genes such as MAP3K7, BRD9, and ABCB7, typically lead to loss of function, thus contributing to cancer pathogenesis. Here, we systematically analyzed the impact of mutant SF3B1 on non-coding regions of mRNA transcripts across disease types, in both cell lines and primary patient specimens. This identified numerous novel and highly reproducible splicing alterations in such regions. Studies of one target gene, DCAF16, revealed multiple complex mutation-induced alterations in its 5’ and 3’ untranslated regions (5’, 3’ UTRs). Remarkably, these were mechanistically associated with increased DCAF16 protein levels in SF3B1 mutant cells, representing the first time that oncogenic SF3B1 has been shown to increase levels of a target protein in a gain-of-function manner. DCAF16 is a substrate recognition adapter for the DDB1/CUL4 E3 ubiquitin ligase complex. Novel protein degrader small molecules which co-opt DCAF16 to degrade BRD4 as a neosubstrate demonstrated preferential selectivity for SF3B1 mutant cancers and CLL primary patient specimens due to increased DCAF16 protein levels. In turn, this reveals the therapeutic relevance of mutant SF3B1 dysregulation of transcript untranslated regions and uncovers a novel strategy for the treatment of these important neoplasms.

Oncogenic mutations of the core RNA splicing factor SF3B1 are common in myeloid cancers, chronic lymphocytic leukemia (CLL) and select solid tumors, and act as clear disease drivers. Despite multiple efforts, therapeutic modulation of RNA splicing has not yet demonstrated benefit for SF3B1 mutant cancers in the clinic, and there are no FDA approved disease-modifying therapies for cancers with this mutation. This highlights an urgent unmet medical need for targeted therapies. Here we identified the first known gain-of-function target of oncogenic SF3B1, i.e. DCAF16, and exploit it to selectively target SF3B1 mutant cancers – thus demonstrating a novel therapeutic approach to this oncogene. Mutant SF3B1 drives oncogenesis through a variety of complex mechanisms. Among the best-studied are its impact on the stereotyped missplicing of mRNA coding regions (CDS) in target genes like MAP3K7 and BRD9. Ordinarily, these misspliced gene targets yield loss-of-function, contributing to disease pathogenesis. Here we systematically analyzed the impact of mutant SF3B1 on mRNA untranslated regions (UTRs) o in wildtype (WT) vs. SF3B1 mutant cancers across primary myeloid, lymphoid and solid tumors, as well as isogenic cell lines. This identified numerous novel and highly reproducible missplicing events. Among the most prominent targets with UTR missplicing is DCAF16, a substrate recognition adapter for the DDB1/CUL4 E3 ubiquitin ligase complex and emerging pharmacologic target. RNA sequencing (RNA-seq), 3' end sequencing (3'-seq), and Nanopore long read sequencing (LRS) experiments revealed complex mutation-induced alterations in DCAF16 5' and 3' UTRs. Specifically, mutant SF3B1 mediates exclusion of exons 2 and 3 in the DCAF16 5' UTR to generate CDS isoforms with an alternative 5' UTR. Further, in other isoforms mutant SF3B1 promotes use of an alternative 5' splice site in exon 4, which ordinarily contains the CDS and 3' UTR. As a result, one novel DCAF16 transcript skips the CDS to encode a long noncoding RNA (lncRNA) by inclusion of exon 5 to generate an alternative mRNA 3' end. Remarkably, mutant SF3B1 is reproducibly associated with elevated DCAF16 protein levels in multiple isogenic hematologic and solid tumor SF3B1 mutant cell lines over WT, as well as primary myeloid cancers and CLL specimens vs. WT controls. Further, DCAF16 isoforms skipping exon 3 in the 5‘ UTR, or the DCAF16 associated lncRNA, both of which are promoted by mutant SF3B1, directly cause increased DCAF16 protein levels. These transcript species and the presence of mutant SF3B1 are not associated with changes in DCAF16 mRNA half-life, subcellular localization, expression levels of its CDS, or DCAF16 protein half-life. Conversely, the presence or absence of exon 3 in dual luciferase reporter systems, as well as polysome profiling of WT vs. SF3B1 mutant cells indicate that elements within exon 3 of the DCAF16 5‘ UTR, which are misspliced by mutant SF3B1, may regulate DCAF16 translation efficiency. Together, these studies represent the first time that oncogenic SF3B1 has been shown to increase levels of a target protein in a gain-of-function manner, by altering the composition of a target gene's untranslated regions. Finally, DCAF16 is a novel target for protein degrader therapeutics such as proteolysis targeting chimeras (PROTACs) and molecular glues which co-opt it to mediate the proteosomal degradation of neosubstrates of therapeutic importance. Small molecules that utilize DCAF16 to degrade key cancer protein targets including BRD4, DYRK1A and RPS6KA4 among others demonstrated preferential selectivity for SF3B1 mutant cancer cell lines and primary CLL patient specimens due to increased DCAF16 protein. The preferential sensitivity of SF3B1 mutant cells to DCAF16-based degraders was associated with accentuated loss of protein degradation targets such as BRD4, as well as consistent alterations in downstream targets such as MYC and anti-apoptotic BH3 family members in the case of DCAF16-BRD4 degraders. Comprehensive gene expression profiling confirmed reproducible effects of DCAF16-based degrader compounds. Further, solid tumors bearing SF3B1 mutations also exhibit enhanced sensitivity to DCAF16-based protein degraders over WT. This reveals the therapeutic relevance of mutant SF3B1 dysregulation of mRNA UTRs and uncovers targeting DCAF16 as a novel strategy for their selective treatment across a range of solid and hematologic tumors.

Background: Alternative polyadenylation (APA) affects most human genes and is recurrently dysregulated in all studied cancers. However, the mechanistic origins of this dysregulation are incompletely understood. Results: We describe an unbiased analysis of molecular regulators of poly(A) site selection across The Cancer Genome Atlas and identify that colorectal adenocarcinoma is an outlier relative to all other cancer subtypes. This distinction arises from the frequent presence of loss-of-function APC mutations in colorectal adenocarcinoma, which are strongly associated with long 3 ' UTR expression relative to tumors lacking APC mutations. APC knockout similarly dysregulates APA in human colon organoids. By mining previously published APC eCLIP data, we show that APC preferentially binds G- and C-rich motifs just upstream of proximal poly(A) sites. Lastly, we find that reduced APC expression is associated with APA dysregulation in tumor types lacking recurrent APC mutations. Conclusions: As APC has been previously identified as an RNA-binding protein that preferentially binds 3 ' UTRs during mouse neurogenesis, our results suggest that APC promotes proximal poly(A) site use and that APC loss and altered expression contribute to pervasive APA dysregulation in cancers.

Alternative polyadenylation (APA) is strikingly dysregulated in many cancers. Although global APA dysregulation is frequently associated with poor prognosis, the importance of most individual APA events is controversial simply because few have been functionally studied. Here, we address this gap by developing a CRISPR-Cas9-based screen to manipulate endogenous polyadenylation and systematically quantify how APA events contribute to tumor growth in vivo. Our screen reveals individual APA events that control mouse melanoma growth in an immunocompetent host, with concordant associations in clinical human cancer. For example, forced Atg7 3′ UTR lengthening in mouse melanoma suppresses ATG7 protein levels, slows tumor growth, and improves host survival; similarly, in clinical human melanoma, a long ATG7 3′ UTR is associated with significantly prolonged patient survival. Overall, our study provides an easily adaptable means to functionally dissect APA in physiological systems and directly quantifies the contributions of recurrent APA events to tumorigenic phenotypes.

Abstract Alternative polyadenylation (APA) is markedly dysregulated across cancer types. Although this phenomenon is well established and associated with altered patient prognosis, the functional importance of most dysregulated polyadenylation to cancer phenotypes remains unknown, as does the potential therapeutic impact of dysregulated APA. Recent work by our lab and others to address this gap has systematically identified specific APA events that alter key tumor phenotypes, including malignant cell growth and tumor recognition by cytotoxic T cells. Motivated by these studies, we sought to identify small molecules that modulate APA for therapeutic intent. We created a sensitive reporter of APA and used this reporter to conduct a molecular phenotypic reporter screen for APA-altering compounds. This screen revealed a diversity of compounds that altered APA transcriptome-wide. Phenotypic characterization of the strongest hit emerging from this screen revealed that it strongly induced cell differentiation and death of myeloid leukemia cells, with negligible toxicity for normal hematopoietic stem cells. Overall, these results highlight the untapped but promising therapeutic potential of targeting APA in cancer. Citation Format: Toshihiro Banjo, Satoshi Kaito, Austin M Gabel, Omar Abdel-Wahab, Robert K Bradley. Alternative polyadenylation as a therapeutic target in cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr I002.

Hypertensive disorder of pregnancy (HDP) is associated with an increased risk for later-life cardiovascular disease (CVD). Whether the HDP pregnancy itself confers risk towards CVD later in life is suggested in several epidemiologic studies. Given this connection and that the HDP exposure itself may play a role, understanding whether markers associated with cardiovascular risk vary based on HDP history in the years following pregnancy may assist with risk stratification and development of targeted interventions. We measured 77 proteins (CVD-associated and inflammatory markers) in n=22 individuals with a history of HDP and n=43 matched controls with no HDP history at a median of 4 years after pregnancy. Several CVD-associated proteins (fibrinogen, fetuin-A, L-selectin, and alpha-1-acid glycoprotein) were significantly elevated, by orders of magnitude, in individuals with a history of HDP compared to normotensive pregnancies (all p<0.0001). In multivariable linear regression models controlling for age, body mass index, chronic hypertension, and diabetes, a history of HDP remained associated with higher levels of CVD-associated proteins (all p<0.0001). We clustered samples based on global patterns of CVD protein expression and found a significant difference in CVD protein expression patterns between post-Normal and post-HDP samples. Conversely, differences in circulating inflammatory markers were largely insignificant or more subtle than that observed with the CVD-associated proteins. Identification of biomarkers associated with CVD in the intervening years after HDP but before evident CVD is critical to understanding post-HDP cardiovascular risk to provide insight for the development of therapeutic interventions that mitigate CVD event risk in this high-risk population.

Abstract Alternative polyadenylation (APA) affects most human genes and is recurrently dysregulated in cancers. However, the mechanistic origins of this dysregulation are incompletely understood. We completed an unbiased computational analysis of molecular regulators of poly(A) site selection across The Cancer Genome Atlas and identified that colorectal adenocarcinoma is distinct from all other cancer subtypes. We linked this distinction to the frequent presence of loss-of-function APC mutations in colorectal adenocarcinoma, which were strongly associated with expression of long 3′ UTRs relative to tumors lacking APC mutations. APC knockout similarly dysregulated APA in human colon organoids, and reduced APC expression was associated with APA dysregulation in tumor types lacking APC mutations. Building on previous work that identified APC as an RNA-binding protein that preferentially binds 3′ UTRs during mouse neurogenesis, we found that APC binding is most significantly enriched just upstream of proximal poly(A) sites within 3′ UTRs. Our results suggest that APC promotes proximal poly(A) site use and that APC loss contributes to pervasive APA dysregulation in human cancers. Citation Format: Austin M Gabel, Andrea E Belliville, James D Thomas, Jose Mario B Pineda, Robert K Bradley. APC mutations dysregulate alternative polyadenylation in cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A005.

Many of the most highly conserved elements in the human genome are "poison exons," alternatively spliced exons that contain premature termination codons and permit post-transcriptional regulation of mRNA abundance through induction of nonsense-mediated mRNA decay (NMD). Poison exons are widely assumed to be highly conserved due to their presumed importance for organismal fitness, but this functional importance has never been tested in the context of a whole organism. Here, we report that a poison exon in Smndc1 is conserved across mammals and plants and plays a molecular autoregulatory function in both kingdoms. We generated mouse and A. thaliana models lacking this poison exon to find its loss leads to deregulation of SMNDC1 protein levels, pervasive alterations in mRNA processing, and organismal size restriction. Together, these models demonstrate the importance of poison exons for both molecular and organismal phenotypes that likely explain their extraordinary conservation.

Background: The success of chimeric antigen receptor (CAR) T-cells against B-cell neoplasms has spurred investigation of such therapies in acute myeloid leukemia (AML). However, their clinical efficacy has been limited. These treatment failures are often attributed to the lack of an ideal target antigen that is universally expressed. Indeed, our preclinical models show that despite improved survival after CAR T-cell treatment, heterogeneous antigen expression predisposes to relapse with antigen-negative disease. Notably, the presence of a subset of tumor cells that lack the target antigen does not preclude complete response to CAR T-cells in other diseases such as B-cell acute lymphoblastic leukemia. These observations prompted our investigation of factors that allow CAR T-cells to clear disease when target expression is not universal. In some tumors, malignant cells with insufficient target antigen can be killed by T-cells primed by antigen-positive cells. It is unknown if this occurs in AML, and we aimed to develop models to study these functions and potential resistance mechanisms. Methods: CD33 and CLL-1 CAR T-cells were generated from donor T-cells by CD3/CD28 bead activation, lentiviral delivery of CAR transgenes, fluorescence-activated cell sorting (FACS) and IL-7/15 expansion. Where applicable, T-cell receptor (TCR) knockout (KO) was performed with ribonucleoprotein (RNP) CRISPR/Cas9 editing. CD33- and/or CLL-1-expressing human AML cell lines were selected for use, and target KO variants were created using RNP. Clonal lines were established by FACS, and we verified that target KO cells did not independently induce CAR T-cell activation. Each cell line was transduced to express a unique fluorescent protein and luciferase to allow differential quantitation of tumor burden using flow cytometry (FC) and bioluminescence imaging. For instance, in vitro killing assays were performed by coculture of labeled CD33KO AML blasts with unlabeled CD33 wild type (WT) AML, and CD33 CAR T-cells or media control. Cytotoxicity was calculated by FC enumeration of viable fluorescent protein-expressing events in CAR T-cell conditions relative to controls. We performed CRISPR KO screening using lentiviral delivery of individually cloned sgRNAs to Cas9-expressing CD33KO AML cells. Transduced cells were FACS enriched, pooled and cultured in quadruplicate with unmodified CD33WT AML cells, and CD33 CAR T-cells or media control. Integrated sgRNAs were PCR amplified and sequenced, and fold change was computed by count comparison with control conditions. Enrichment was determined relative to non-targeting sgRNAs using two-sided Student's t test. Results: We discovered that CD33KO AML blasts are lysed in vitro by CD33 CAR T-cells only after activation by engagement with CD33WT AML (PRIMECAR killing). This mechanism is independent of the TCR and is not unique to CD33 CARs- we observed similar findings using a CLL-1 CAR T-cell system. Importantly, hematopoietic stem cells appear resistant to these effector mechanisms. To determine if PRIMECAR killing contributes to control of AML burden in vivo, we developed a murine model in which CD33KO and CD33WT AML xenografts could be individually monitored over time. Here, NOD.SCID.IL2rg-/- (NSG) mice were injected with individually labeled CD33WTand CD33KO AML cell lines and then treated with CD33 or negative control CAR T-cells. CD33 CAR T-cells not only improved control of CD33WT AML compared to control groups, but also significantly reduced CD33KO burden. Using transwell assays, we showed that although PRIMECAR killing of AML occurs via soluble factors, the killing is greatly enhanced by proximity. As death receptor (DR) apoptotic signaling is a T-cell mechanism enhanced by cell-cell contact, we performed pooled CRISPR KO screening to study DR pathways essential for PRIMECAR killing in AML. The TNFRSF1a KO phenotype was resistant to PRIMECAR killing (p=8.1e-05) indicating that TNFα apoptotic signaling is critical. As validation, anti-TNFα monoclonal antibodies significantly reduced PRIMECAR killing in vitro and in vivo. Conclusion: Our findings indicate that cognate antigen-engaged CAR T-cells use DRs to mediate the clearance of proximal antigen-negative AML. Impaired apoptotic TNFα signaling is one resistance mechanism in AML, and further study of this pathway can inform strategies to improve the effectiveness of CAR T-cells in this disease.

Although mutations in DNA are the best-studied source of neoantigens that determine response to immune checkpoint blockade, alterations in RNA splicing within cancer cells could similarly result in neoepitope production. However, the endogenous antigenicity and clinical potential of such splicing-derived epitopes have not been tested. Here, we demonstrate that pharmacologic modulation of splicing via specific drug classes generates bona fide neoantigens and elicits anti-tumor immunity, augmenting checkpoint immunotherapy. Splicing modulation inhibited tumor growth and enhanced checkpoint blockade in a manner dependent on host T cells and peptides presented on tumor MHC class I. Splicing modulation induced stereotyped splicing changes across tumor types, altering the MHC I-bound immunopeptidome to yield splicing-derived neoepitopes that trigger an anti-tumor T cell response in vivo. These data definitively identify splicing modulation as an untapped source of immunogenic peptides and provide ameans to enhance response to checkpoint blockade that is readily translatable to the clinic.

Immune checkpoint blockade therapy has revolutionized cancer care, including the treatment of advanced metastatic disease. However, most patients derive little or no clinical benefit from these therapies and many cancer types are notoriously non-responsive. Motivated by (1) the correlation between tumor neoantigen abundance and anti-tumor immunity and (2) that most cancers are characterized by widespread dysregulation of RNA processing, we reasoned that pharmacologic modulation of RNA splicing might increase cancer cell immunogenicity via the generation of splicing-derived neoantigens. We demonstrated that two compounds which modulate RNA splicing via distinct mechanisms, inhibited tumor growth and enhanced response to immune checkpoint blockade in a manner dependent on host T cells and peptides presented on tumor MHC class I. Critical for their clinical translatability, therapeutic doses of splicing inhibitors were non-toxic, tolerated by the host immune system, and did not affect T cell activation, proliferation, and anti-cancer killing activities. Mechanistically, splicing modulation induced stereotyped, dose-dependent “splicing failure” — dramatic intron retention, alternative exon skipping, etc. — that was consistent across multiple mouse and human tumor types. By combining RNA-seq-based peptide predictions and mass spectrometry of the MHC I-bound immunopeptidome, we identified drug-induced, splicing-derived peptides that promote the expansion of antigen-specific CD8+ T cells and trigger anti-tumor T cell responses in vivo. These data definitively identify splicing modulation as an untapped source of immunogenic peptides and provide a means to enhance response to checkpoint blockade that is readily translatable to the clinic. Citation Format: James D. Thomas, Sydney X. Lu, Emma De Neef, Erich Sabio, Benoit Rousseau, Mathieu Gigoux, David A. Knorr, Benjamin Greenbaum, Yuval Elhanati, Simon J. Hogg, Andrew Chow, Arnab Ghosh, Abigail Xie, Dmitriy Zamarin, Daniel Cui, Caroline Erickson, Michael Singer, Hana Cho, Eric Wang, Bin Lu, Benjamin H. Durham, Harshal Shah, Diego Chowell, Austin M. Gabel, Yudao Shen, Jing Liu, Jian Jin, Matthew C. Rhodes, Richard E. Taylor, Henrik Molina, Jedd D. Wolchok, Taha Merghoub, Luis A. Diaz Jr, Omar Abdel-Wahab, Robert K. Bradley. Pharmacologic modulation of RNA splicing enhances anti-tumor immunity. [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 5742.

Many cancers carry recurrent, change-of-function mutations affecting RNA splicing factors. Here, we describe a method to harness this abnormal splicing activity to drive splicing factor mutation-dependent gene expression to selectively eliminate tumor cells. We engineered synthetic introns that were efficiently spliced in cancer cells bearing SF3B1 mutations, but unspliced in otherwise isogenic wild-type cells, to yield mutation-dependent protein production. A massively parallel screen of 8,878 introns delineated ideal intronic size and mapped elements underlying mutation-dependent splicing. Synthetic introns enabled mutation-dependent expression of herpes simplex virus–thymidine kinase (HSV–TK) and subsequent ganciclovir (GCV)-mediated killing of SF3B1-mutant leukemia, breast cancer, uveal melanoma and pancreatic cancer cells in vitro, while leaving wild-type cells unaffected. Delivery of synthetic intron-containing HSV–TK constructs to leukemia, breast cancer and uveal melanoma cells and GCV treatment in vivo significantly suppressed the growth of these otherwise lethal xenografts and improved mouse host survival. Synthetic introns provide a means to exploit tumor-specific changes in RNA splicing for cancer gene therapy. Synthetic introns tailored for specific splice-factor mutations enable targeted cancer gene therapy.

Lack of oxygen (hypoxia and anoxia) is detrimental to cell function and survival and underlies many disease conditions. Hence, metazoans have evolved mechanisms to adapt to low oxygen. One such mechanism, metabolic suppression, decreases the cellular demand for oxygen by downregulating ATP-demanding processes. However, the molecular mechanisms underlying this adaptation are poorly understood. Here, we report on the role of ndrg1a in hypoxia adaptation of the anoxia-tolerant zebrafish embryo. ndrg1a is expressed in the kidney and ionocytes, cell types that use large amounts of ATP to maintain ion homeostasis. ndrg1a mutants are viable and develop normally when raised under normal oxygen. However, their survival and kidney function is reduced relative to WT embryos following exposure to prolonged anoxia. We further demonstrate that Ndrg1a binds to the energy-demanding sodium-potassium ATPase (NKA) pump under anoxia and is required for its degradation, which may preserve ATP in the kidney and ionocytes and contribute to energy homeostasis. Lastly, we show that sodium azide treatment, which increases lactate levels under normoxia, is sufficient to trigger NKA degradation in an Ndrg1a-dependent manner. These findings support a model whereby Ndrg1a is essential for hypoxia adaptation and functions downstream of lactate signaling to induce NKA degradation, a process known to conserve cellular energy.

Synthetic lethal therapies are a promising approach to expand therapeutic options for cancer patients. Since synthetic lethal therapies target tumor cells specifically, they have fewer off-target effects than oncogene targeted approaches. To identify new cancer drug targets, we focused on paralogs, ancestrally-duplicated genes that frequently retain redundant or overlapping functions. To find synthetic lethal human paralogs, we developed paired guide RNAs for Paralog gENetic interaction mapping (pgPEN), a pooled CRISPR-Cas9 single and double knockout approach targeting over 2,000 paralogs. We applied pgPEN to two cancer cell lines and found that 12% of human paralogs exhibit synthetic lethality in at least one context. To our knowledge, pgPEN represents the largest experimental assessment of human paralog synthetic lethality to date. We next identified paralog pairs to prioritize for follow-up study. A key drawback to synthetic lethal therapies is that many genetic interactions are context-dependent. To identify likely penetrant interactions, we compared our data to other published paralog screens and computational predictions of paralog synthetic lethality. We found that over 75% (n=96) of pgPEN hits were predicted to be broadly synthetic lethal, and nearly 10% (n=10) of pairs were synthetic lethal in multiple paralog screens. Finally, we prioritized paralogs targeted by existing small molecule therapies. Of the 122 synthetic lethal pairs identified by pgPEN, 16% (n=20) are currently druggable. We mined drug repurposing data from DepMap to find pairs where a paralog-targeting drug showed a stronger effect in cell lines with low target gene copy number or expression relative to cell lines with normal target gene copy number or expression. These drugs could selectively target cancer cells in cases where one or both paralogs is lost in tumors but retained in normal tissue. We also leveraged The Cancer Genome Atlas tumor sequencing data to find paralog pairs where one member is recurrently lost in cancer. Taken together, these studies identify druggable, highly penetrant synthetic lethal paralog interactions. In combination with tumor sequencing data, we identify high-priority paralog drug targets that can be further tested and translated to the clinic. Paralog synthetic lethal therapies could provide a relatively low-toxicity therapeutic approach to improve the efficacy of cancer treatments and prevent the emergence of acquired resistance. Citation Format: Phoebe C. Parrish, James D. Thomas, Austin M. Gabel, Shriya Kamlapurkar, Robert K. Bradley, Alice H. Berger. Expanding cancer therapy options by leveraging synthetic lethal interactions between druggable paralogs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2278.

CRISPR screens have accelerated the discovery of important cancer vulnerabilities. However, single-gene knockout phenotypes can be masked by redundancy among related genes. Paralogs constitute two-thirds of the human protein-coding genome, so existing methods are likely inadequate for assaying a large portion of gene function. Here, we develop paired guide RNAs for paralog genetic interaction mapping (pgPEN), a pooled CRISPR-Cas9 single- and double-knockout approach targeting more than 2,000 human paralogs. We apply pgPEN to two cell types and discover that 12% of human paralogs exhibit synthetic lethality in at least one context. We recover known synthetic lethal paralogs MEK1/MEK2, important drug targets CDK4/CDK6, and other synthetic lethal pairs including CCNL1/CCNL2. Additionally, we identify ten tumor suppressor paralog pairs whose compound loss promotes cell proliferation. These findings nominate drug targets and suggest that paralog genetic interactions could shape the landscape of positive and negative selection in cancer.

Neoantigen-specific T cells are strongly implicated as being critical for effective immune checkpoint blockade treatment (ICB) (e.g., anti-PD-1 and anti-CTLA-4) and are being targeted for vaccination-based therapies. However, ICB treatments show uneven responses between patients, and neoantigen vaccination efficiency has yet to be established. Here, we characterize neoantigen-specific CD8+ T cells in a tumor that is resistant to ICB and neoantigen vaccination. Leveraging the use of mass cytometry combined with multiplex major histocompatibility complex (MHC) class I tetramer staining, we screened and identified tumor neoantigen-specific CD8+ T cells in the Lewis Lung carcinoma (LLC) tumor model (mRiok1). We observed an expansion of mRiok1-specific CD8+ tumor-infiltrating lymphocytes (TILs) after ICB targeting PD-1 or CTLA-4 with no sign of tumor regression. The expanded neoantigen-specific CD8+ TILs remained phenotypically and functionally exhausted but displayed cytotoxic characteristics. When combining both ICB treatments, mRiok1-specific CD8+ TILs showed a stem-like phenotype and a higher capacity to produce cytokines, but tumors did not show signs of regression. Furthermore, combining both ICB treatments with neoantigen vaccination did not induce tumor regression either despite neoantigen-specific CD8+ TIL expansion. Overall, this work provides a model for studying neoantigens in an immunotherapy nonresponder model. We showed that a robust neoantigen-specific T-cell response in the LLC tumor model could fail in tumor response to ICB, which will have important implications in designing future immunotherapeutic strategies.

CRISPR-based cancer dependency maps are accelerating advances in cancer precision medicine, but adequate functional maps are limited to the most common oncogenes. To identify opportunities for therapeutic intervention in other rarer subsets of cancer, we investigate the oncogene-specific dependencies conferred by the lung cancer oncogene, RIT1. Here, genome-wide CRISPR screening in KRAS, EGFR, and RIT1-mutant isogenic lung cancer cells identifies shared and unique vulnerabilities of each oncogene. Combining this genetic data with small-molecule sensitivity profiling, we identify a unique vulnerability of RIT1-mutant cells to loss of spindle assembly checkpoint regulators. Oncogenic RIT1M90I weakens the spindle assembly checkpoint and perturbs mitotic timing, resulting in sensitivity to Aurora A inhibition. In addition, we observe synergy between mutant RIT1 and activation of YAP1 in multiple models and frequent nuclear overexpression of YAP1 in human primary RIT1-mutant lung tumors. These results provide a genome-wide atlas of oncogenic RIT1 functional interactions and identify components of the RAS pathway, spindle assembly checkpoint, and Hippo/YAP1 network as candidate therapeutic targets in RIT1-mutant lung cancer.

While RNA-seq has enabled comprehensive quantification of alternative splicing, no correspondingly high-throughput assay exists for functionally interrogating individual isoforms. We describe pgFARM (paired guide RNAs for alternative exon removal), a CRISPR–Cas9-based method to manipulate isoforms independent of gene inactivation. This approach enabled rapid suppression of exon recognition in polyclonal settings to identify functional roles for individual exons, such as an SMNDC1 cassette exon that regulates pan-cancer intron retention. We generalized this method to a pooled screen to measure the functional relevance of ‘poison’ cassette exons, which disrupt their host genes’ reading frames yet are frequently ultraconserved. Many poison exons were essential for the growth of both cultured cells and lung adenocarcinoma xenografts, while a subset had clinically relevant tumor-suppressor activity. The essentiality and cancer relevance of poison exons are likely to contribute to their unusually high conservation and contrast with the dispensability of other ultraconserved elements for viability. pgFARM (paired guide RNAs for alternative exon removal) is a CRISPR–Cas9-based approach to manipulate alternative splicing and identify functional roles for individual exons, including poison exons with essential and tumor-suppressor roles.