Abstract CRISPR-based functional genomics screens are a powerful tool for identifying synthetically lethal cancer drug targets. Current strategies for analyzing pooled CRISPR screens usually rely on signals from single guide RNAs (sgRNA) with differential relative abundance between two experimental conditions. However, conventional approaches are susceptible to false positives and false negatives driven by outlier cell clones, since the sgRNA abundance does not account for the heterogeneous phenotypes resulting from different editing outcomes of the same sgRNA. To overcome this, we added DNA barcodes to each sgRNA to create unique molecular identifiers (UMIs) for CRISPR libraries and developed a companion analytical platform that enables robust, industry-scale CRISPR screens. Here, we present UMIBB, a novel nonparametric Bayesian approach for analyzing UMI-CRISPR data. The number of UMIs with normalized count depletion or enrichment compared to the control experimental condition for each sgRNA is modeled by a beta-binomial distribution. The gene level statistics are derived by combining z-scores of the sgRNAs level posterior probabilities weighted by the number of UMIs in each sgRNA. This approach minimizes the impact of outlier cell clones on statistics and prioritizes genes with consistent count differentials across multiple UMIs in each gene. To assess the power of UMIBB, we benchmarked it on a low coverage (200X) genome-scale negative-selection screen, comparing with results from a high coverage (1000X) screen. These screens were conducted on KRAS mutant cancer celllines (A549) treated with trametinib or vehicle control. Despite the high noise level usually observed in lower coverage screens, our method was able to uncover most of the validated sensitizer genes for trametinib and achieved the highest sensitivity compared to conventional methods. Furthermore, we applied UMIBB on a genome-scale positive-selection screen and successfully identified novel genes (RAD18 and UBE2K) as key mediators of USP1 dependency in BRCA1 mutant cell lines. Our studies demonstrate that UMIBB is highly robust against false positives due to clonal heterogeneity and is more likely to identify true genetic interactions. Citation Format: Ashley Choi, Samuel Meier, Silvia Fenoglio, Tianshu Feng, Justin Engel, Binzhang Shen, Shangtao Liu, Teng Teng, Tenzing Khendu, Alan Huang, Jannik Andersen, Xuewen Pan, Yi Yu. UMIBB: A novel nonparametric Bayesian method improves robustness and sensitivity of analysis in pooled CRISPR-Cas9 screens leveraging unique molecular identifiers [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 1224.
Abstract MTA-cooperative PRMT5 inhibitors are an emerging treatment option for patients with one of the 10-15% of all human cancers harboring MTAP homozygous deletion. To identify potential regulators of sensitivity to PRMT5 inhibitors, we performed genome-wide CRISPR knockout screens in the presence and absence of an MTA-cooperative PRMT5 inhibitor. Knockout of CAAP1 and AKAP17A were among the strongest sensitizing hits across multiple MTAP-deleted cancer cell lines representing different histologies. Strikingly, the CAAP1 gene co-localizes with MTAP and CDKN2A on chromosome 9p21. Co-deletion of CAAP1 is reported in 20 percent of MTAP-deleted cancers in the TCGA PanCancer Atlas. CAAP1 or AKAP17A knockout in MTAP-deleted cancer cell lines sensitized the cells to PRMT5 inhibitors including the clinical stage MTA-cooperative inhibitors, TNG908 and TNG462, and the non-MTA-cooperative inhibitor, GSK3326595. Moreover, we discovered that CAAP1 and AKAP17A protein levels are interdependent, as knockout of either gene caused decreased protein levels for the other. Consistent with this finding, CAAP1 reconstitution in CAAP1-deleted cell lines led to increased AKAP17A levels. Endogenous CAAP1 and AKAP17A protein levels are positively correlated across a panel of cancer cell lines and MTAP-deleted patient-derived xenograft models. Consistent with a previous report (Ni et al., 2023), exogenous CAAP1 and AKAP17A co-immunoprecipitation studies suggest that the proteins form a protein complex. AKAP17A and CAAP1 are not well-characterized proteins, but PRMT5 inhibitors induce global alternative splicing events (ASEs) in cancer cells, and based on preliminary studies a possible function for the CAAP1/AKAP17A complex could be to mitigate ASEs induced by PRMT5 inhibition. Collectively, these data indicate that CAAP1 and AKAP17A exist interdependently and mediate sensitivity to PRMT5 inhibitors. The colocalization and 20 percent incidence of CAAP1 deletion in the setting of MTAP deletion may suggest that such patients will have improved responses to PRMT5-targeted therapy. S. Yoda and M. R. Tonini contributed equally. Citation Format: Satoshi Yoda, Matthew R. Tonini, Hilary E. Nicolson, Samuel Poleretzky, Silvia Fenoglio, Steven Lombardo, Lauren Grove, Samuel R. Meier, Ashley Choi, Yi Yu, Kevin M. Cottrell, John P. Maxwell, Teng, Jannik N. Andersen, Kimberly J. Briggs. Genome-wide drug anchor screens identify CAAP1 and AKAP17A as regulators of PRMT5 inhibitor sensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4636.
CRISPR-based functional genomics screening can be designed to identify novel cancer cell intrinsic targets that increase tumor immunogenicity. Using a FACS-based CRISPR sorting screen for PD-L1 expression, we identified euchromatic histone-lysine-N-methyltransferase 1 and 2 (EHMT1/2) as negative modulators of the interferon signaling pathways. EHMT1 and EHMT2 are histone methyltransferases that mono- and di-methylate lysine 9 of histone H3 to repress gene transcription of defined target genes. A novel and selective small molecule inhibitor of EHMT1/2, TNG917 was therefore developed as a therapy to increase anti-tumor immunity.
Methods
The PK/PD relationship of TNG917 was evaluated in MC38 tumor-bearing female C57BL/6 mice. Tumor PD following a QDx5 PO dosing regimen of TNG917 at 10, 30, 100 mg/kg was assessed 2 and 8 hours post last dose. The anti-tumor activity of TNG917 was evaluated in both HT29 human colon carcinoma tumor-bearing immune deficient NOG mice and CT26 in tumor-bearing BALB/C female mice. The immunological MOA was further evaluated in an immune phenotyping study in the CT26 tumor model. Tumors were collected at Day 7 of the study and analyzed by flow cytometry and nanostring.
Results
Gene knockout or pharmacological inhibition of EHMT1/2 in cancer cells resulted in de-repression of gene promoters, upregulation of interferon-stimulated genes (ISGs), and secretion of pro-inflammatory cytokines. Here, we present the preclinical characterization of TNG917 - an oral and highly selective EHMT1/2 inhibitor with low nanomolar cellular potency and favorable pharmacodynamic and pharmacokinetic properties. In humanized and syngeneic mouse models, treatment with TNG917 in combination with anti-PD1 promoted a T-cell-infiltrated tumor microenvironment, led to significant anti-tumor activity and resulted in survival benefit. In addition, TNG917 induced CD45 infiltration and CXCL10 cytokine secretion in human patient-derived xenograft (PDX) organoids co-cultured with human dermal fibroblasts (HDF) and PBMCs.
Conclusions
In summary, our in vitro and in vivo studies provide a rationale for the clinical development path of TNG917, in combination with checkpoint inhibition, in patients with immune-cold tumors.
Abstract CRISPR-based functional genomic screening is a powerful approach for identifying novel classes of synthetic lethal drug targets. Here, we define the deubiquitinase USP1 as a synthetic lethal target in cancers with underlying DNA repair vulnerabilities. A highly potent and selective small molecule USP1 inhibitor conferred a viability defect in BRCA1-mutant, but not WT cell lines by activating replication stress. Genome-wide CRISPR screening uncovered RAD18 and UBE2K, which promote PCNA mono- and poly-ubiquitination respectively, as key mediators of USP1-BRCA1 dependency. Increased cellular mono- and poly-ubiquitination reduced PCNA protein levels, and restoration of PCNA protein expression rescued USP1 inhibitor sensitivity. USP1 dependency is associated with upregulated RAD18 and UBE2K expression, suggesting that elevated PCNA ubiquitination in the context of BRCA1 deficiency mediates USP1 synthetic lethality. Interestingly, USP1, but not PARP1 inhibition, elicited a viability defect in a subset of BRCA1/2 WT lung cancer cell lines, indicative of novel synthetic lethal interactions unique to USP1. Moreover, dual inhibition of PARP1 and USP1 are strongly synergistic in PARP1 inhibitor-responsive cell line models. Strong in vivo anti-tumor activity across multiple tumor models was demonstrated with USP1 inhibition alone and in combination with the PARP1 inhibitor olaparib. Our studies suggest that USP1 and PARP1 inhibitors target BRCA1-mutant cancer though distinct yet synergistic mechanisms. As such, USP1 inhibitors may provide novel treatment strategies for PARP1 inhibitor-resistant and -naïve BRCA1-mutant cancer. Citation Format: Justin Engel, Madhavi Bandi, Antione Simoneau, Katherine Lazarides, Deepali Gotur, Truc Pham, Shangtao Liu, Samuel Meier, Ashley Choi, Hongxiang Zhang, Binzhang Shen, Fang Li, Douglas Whittington, Shanzhong Gong, Xuewen Pan, Yi Yu, Lina Gu, Scott Throner, John Maxwell, Yingnan Chen, Alan Huang, Jannik Andersen, Tianshu Feng. USP1 inhibitor synthetic lethality in BRCA1-mutant cancer is driven by PCNA ubiquitination [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 2603.
Abstract Defective maintenance of genomic integrity is a hallmark of cancer cells that can result from oncogene-induced replication stress and by loss of DNA repair mechanisms. DNA repair deficiencies and elevated replication stress present targetable vulnerabilities for cancer treatment. Notably, BRCA1/2 mutant and homologous recombination deficient (HRD) tumors cannot repair double-strand breaks by homologous recombination and rely on alternative pathways of DNA repair. PARP inhibitors (PARPi), which are a standard of care in many BRCA1/2 mutant tumors, cause synthetic lethality with BRCA1/2 mutation by inhibiting the DNA base excision repair pathway. Despite the clinical benefit of PARPi, they are not effective in every HRD tumor and the acquisition of PARPi resistance limits long-term response. TNG348, a selective allosteric inhibitor of the deubiquitinating enzyme USP1, was specifically designed to target HRD vulnerabilities through an alternative mechanism. We previously showed that the anti-tumor activity of USP1 inhibition results from disruption of the translesion synthesis DNA damage tolerance pathway, a mechanism of action that is functionally distinct from base excision repair targeted by PARPi. Our preclinical studies show that TNG348 is active in HRD models and strongly synergizes with PARP inhibitors to drive strong anti-tumor responses. We have identified replication stress as a predictive biomarker of TNG348 response using cell line profiling and genome-wide CRISPR screens. For example, overexpression of oncogenes known to induce replication stress sensitized to the TNG348 and PARPi combination both in vitro and in vivo. These data indicate that cancer-specific elevated DNA replication stress could contribute to tumor sensitivity to TNG348 and provide additional patient stratification strategies and opportunities for indication expansion. Citation Format: Antoine Simoneau, Hsin-Jung Wu, Charlotte Pratt, Grace Comer, Shangtao Liu, Samuel Meier, Tenzing Khendu, Ashley Choi, Hongxiang Zhang, Binzhang Shen, Douglas Whittington, Sirimas Sudsakorn, Wenhai Zhang, Yi Yu, Yingnan Chen, Brian Haines, Adam Crystal, Jannik N. Andersen, John Maxwell, Scott Throner. TNG348 is synergistic with PARP inhibitors in tumor models with elevated replication stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4527.
'/%3e%3cuse xlink:href='%23a' transform='rotate(23.036 .093 25.536)'/%3e%3cuse xlink:href='%23a' transform='rotate(45.87 1.273 16.18)'/%3e%3cuse xlink:href='%23a' transform='rotate(69.945 .996 12.078)'/%3e%3cuse xlink:href='%23a' transform='rotate(20.66 -19.689 31.932)'/%3e%3c/svg%3e)