Genome-wide CRISPR Screen Research Articles

Abstract 4266: Evaluation of integrin alpha v beta 5 as a novel target for non-small cell lung cancer via tumor intrinsic and host immune regulation

Abstract Lung cancer is the leading cause of cancer related deaths worldwide, with the most prevalent subtype being non-small cell lung cancer (NSCLC). As only a subset of patients exhibits durable clinical responses to current treatments, including targeted therapy and immunotherapy, new treatment options are needed. Integrins (ITGs) are a diverse class of heterodimeric, transmembrane receptors that bind extracellularly to the extracellular matrix (ECM) and intracellularly to the cytoskeleton to integrate signals bi-directionally between the extracellular environment and cell interior. One of 18 α subunits is paired with one of 6 β subunits to create the 24 known αβ ITGs in mammals. ITGs are expressed at low levels in normal tissue and can be upregulated in cancer cells in response to stress signals in the tumor microenvironment (TME). Altered expression of ITGs is associated with enhanced cell proliferation, metastasis, and immune evasion. Targeting of the diverse ITG family members by inhibitors not specific to individual isoforms has obscured understanding of potentially distinct functions of each heterodimer. Through data mining of a genome wide CRISPR screening publicly available at the DepMap portal, we have identified a novel cancer dependency to ITG αvβ5 in approximately 40% of NSCLC cell lines. Using small molecule ITG αv inhibitors, we have mirrored sensitivity of NSCLC cell lines to their ITG αvβ5 CRISPR scores, suggesting that these inhibitors function specifically through ITG αvβ5 in cell lines. RNA sequencing analysis following αvβ5 inhibition in human NSCLC cell lines revealed downregulation of YAP and E2F1 target genes with alterations in gene ontologies associated with cell cycling and cell death in sensitive, but not resistant, lines. This is consistent with the known function of YAP driving gene transcription promoting cell proliferation and survival, and of E2F1 being implicated in regulating the proliferation and self-renewal of cancer stem cell (CSC). Additional changes to immune processes, specifically perturbations to interferon (IFN) pathways, were identified in response to ITG αvβ5 inhibition, suggesting that ITG αvβ5 may play a role in host anti-tumor immune responses. Pharmacological inhibition of ITG αvβ5 also led to a reduction of tumor growth in NSCLC murine models accompanied with increased CD8 T cell activation. These findings support ITG αvβ5 as a unique target of cancer dependency in NSCLC whose inhibition promotes anti-tumor responses through both tumor intrinsic and host immune regulatory mechanisms. Citation Format: Bitta P. Kahangi, William P. Crosson, Ji-Ann Lee, Camelia Dumitras, Tianhao Zhang, Ramin Salehi-Rad, Linh M. Tran, Michael Palazzolo, Dylan Conklin, Steven M. Dubinett, Bin Liu. Evaluation of integrin alpha v beta 5 as a novel target for non-small cell lung cancer via tumor intrinsic and host immune regulation [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 4266.

Abstract 1413: BRCA1 loss is synthetic lethal with double stranded RNA: A genome wide CRISPR screen to identify mechanisms of RNASEL independent death

Abstract Double-stranded RNAs (dsRNAs) are potent immunostimulatory nucleic acids of viral origin but also physiologically produced by mammalian cells. Recent studies demonstrated that elevating dsRNA levels, either exogenously through dsRNA mimetics like Polyinosinic-polycytidylic acid (pI:C) or endogenously through epigenetic modulation of retroviral elements, can elicit anticancer effects. In response to dsRNA, cells elicit a dual response consisting in Interferon (IFN) production and translational shutdown associated, in some cells, with cell death. These two responses are linked in a feed-forward loop in which induced cytokines themselves enhance dsRNA sensing by the death-inducing pathway, complicating our understanding of the cell-intrinsic mechanisms at play. The 2,5-oligoadenylate synthetase (OAS)-RNASEL system is widely thought to be the main responsible for dsRNA-induced cell death, but it remains unclear how its activity leads to cell death.To elucidate these mechanisms, we decided to minimize the impact of interferon modulation by studying dsRNA-induced death in interferon-saturated conditions. RNASEL-KO A549 cells were, as expected, refractory to IFN+pI:C induced cell death; however, inhibiting protein synthesis or RNA transcription with Cycloheximide (CHX) or Actinomycin D (ActD) abolished the need for RNASEL causing RNASEL-KO cells to undergo apoptosis, although CHX/ActD alone had no effect. In addition, HT-29 cells revealed no clear dependence on RNASEL for dsRNA-induced cell death. Transient ablation through siRNA showed that this novel death-inducing pathway is independent of other canonical dsRNA sensors such as RIG-I, MDA5 and PKR. We then performed a genome-wide CRISPR screen for factors involved in dsRNA-induced death in our interferon-saturated conditions in HT29 cells. Pathway enrichment analysis of screen hits revealed that RNA surveillance, RNA Polymerase II Transcription Initiation, Mitochondrial translation and respiratory chain and DNA Repair genes have a protective role against dsRNA increase. In particular, we validated the synthetic lethality of IFN+pI:C with BRCA1 by CRISPR. Ongoing research is aimed at validating this interaction in vivo models.Our work shed light on potential new mechanisms involved in dsRNA dependent cell death, revealing a connection with DNA damage and BRCA1. If confirmed, these findings may pave the way for the use of dsRNA mimetics, possibly in conjunction with immune checkpoint inhibitors, in the treatment of BRCA1-mutated tumors. Citation Format: Deborah Trastulli, Gianluca Vozza, Veronica Pinamonti, Luca Mazzarella. BRCA1 loss is synthetic lethal with double stranded RNA: A genome wide CRISPR screen to identify mechanisms of RNASEL independent death [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 1413.

Abstract 5887: Identifying new immunotherapy targets using machine learning and ex vivo validation

Abstract Overview: Immunotherapy has revolutionized cancer treatment. However, existing immune checkpoint inhibitors (CPI) yield low response rates in most cancers, highlighting the need for new therapeutic options. Traditional immune-oncology (IO) target discovery relies on preclinical models, which struggle to recapitulate human tumor complexity, limiting translation potential. Attention is thus directed at harnessing multimodal patient molecular data with modern machine learning (ML) techniques to identify new IO targets which may have higher clinical viability. Approach: We posed IO target discovery as a binary classification task, training ML systems on known candidate drug targets that have progressed to stage I or higher clinical trials. We constructed a rich knowledge graph database to support model development. Graph nodes (n=11,919) comprised genes with edges linking genes involved in n=99,275 protein-protein interactions (PPIs; PMID: 28936969). Genes were labelled with n=6,387 gene-disease associations alongside bulk exome and transcriptome (RNAseq) features from n=1,317 CPI-treated patients (PMID: 33508232). We considered the role of different immune subsets by incorporating cell type-specific PPIs from single cell RNAseq atlases of n=350 samples (PMID: 31786210). To capture how antigen processing affects immunotherapy response, we also examined the immunopeptidome of n=60 patients (PMID: 30556813). Causal data on immune responses to genetic perturbation stemmed from n=7 publicly available CRISPR tumor-T cell co-cultures and n=15,442 SNP-phenotype links. Lastly, we used interpretability analysis to understand drivers of model predictions and elucidate critical genes that influence multiple IO target pathways. Results: Firstly, we developed an ensemble ML approach which achieved test ROC-AUC>0.75. Next, we used a graph-based ML framework which yielded superior performance (test ROC-AUC>0.90). Orthogonal validation confirmed these models can discriminate known targets by trial phase (p<0.001), predict patient response in new CPI trials (p<0.05), and identify genes that rank highly in unseen genome-wide CRISPR screens. Targets from both methods have entered experimental validation in patient-derived explants and organoid-immune co-cultures. Already, we have identified 2 novel targets predicted to relate to macrophage activity. Early data suggest that perturbing them leads to macrophage repolarization. Further validation experiments are ongoing. Conclusions: We will reveal candidate targets, showing that our method is effective at uncovering new IO targets and can identify critical nodes in biological networks that might be attractive hits. In addition, deciphering data types that drive model predictions provides a broader immunobiological understanding of anti-tumor immune responses. Thus, our results endorse using ML and multimodal data for novel IO target discovery. Citation Format: Marcellus Augustine, Nuno Rocha Nene, Krupa Thakkar, Danwen Qian, Evelyn Fitzsimons, Benjamin S. Simpson, Chris Watkins, Charles Swanton, Kevin Litchfield. Identifying new immunotherapy targets using machine learning and ex vivo validation [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 5887.

Abstract 2944: A high-throughput screening system for identifying therapeutic targets in immuno-oncology

Abstract INTRODUCTION: Over the last decade, cancer immunotherapy has revolutionized cancer treatment. However, more than half of patients don’t respond, and responders often lack lasting effects. Understanding how genetic variants in cancer cells influence their interaction with immune cells, especially T cells, is crucial to uncover underlying mechanisms. Genetic screens are powerful for uncovering responsible genes for various phenotypes, but challenges in obtaining ample samples to set up a proper immune and cancer co-culture system have limited their use for identifying immune modulators in cancer: primary mouse T cells expressing CD3 against a specific antigen (typically albumin) need to be extracted from transgenic mice and only syngeneic mouse cancer cell lines that match these T cells can be paired, restricting the utilization of human T cells and human cancer cells for this co-culture screening setup. METHODS: Here, we present a highly scalable co-culture system that enables genome-wide CRISPR screens on a pair of human primary T cells and any allogeneic human cancer cell lines. This system utilizes the expression of a membrane-targeted protein in mammalian cells, named as ImBridge, which directly engages and activates CD3+ immune cells. This mechanism bypasses the need for matched MHC on target cancer cells. RESULTS: We validated activation of CD3+ immune cells and selective cancer cell cytotoxicity by ImBridge. A genome-scale CRISPR screen utilizing the co-culture system unveiled known immune-oncology targets including PD-L1 and PTPN2 and revealed MEDiC_01 as a novel therapeutic target. Genetic loss of MEDiC_01 in cancer cells significantly sensitized them to cytotoxic cells, effectively eliminating cancers in the presence of human PBMCs. CONCLUSION: We demonstrated a scalable and efficient approach for conducting high-throughput functional screens to uncover genes that modulate interactions between effector T cells and target cancer cells. This can facilitate the identification of new immuno-oncology therapeutic targets, insights into underlying mechanisms, and genetic factors influencing immune susceptibility and evasion. Citation Format: Dippal Parikh, Soon Youn Choi, Ki Eun Pyo, Hong-Pyo Lee, Kyuho Han. A high-throughput screening system for identifying therapeutic targets in immuno-oncology [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 2944.

Abstract 5996: Genome-wide CRISPR screening reveals a central role for ferroptotic cell death in the anti-tumor response to mTOR inhibitors in HNSCC

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer globally, resulting in more than 300,000 deaths each year worldwide. Treatment options for HNSCC patients include surgery, radiation, chemotherapy and molecularly targeted therapies, and although immunotherapies have recently revolutionized the treatment landscape, <20% of HNSCC patients respond to immune check point blockade (ICB) therapies. The survival rate of HNSCC patients has changed only modestly over the past decades, and therefore HNSCC is a significant global health problem with high mortality and morbidity. Genomic alterations converging in the persistent activation of the PI3K/mTOR pathway (>80% cases) represent one of the most frequently altered signaling circuitries in HNSCC. This overreliance on PI3K/mTOR signaling for tumor growth may expose a cancer vulnerability that can be exploited therapeutically, as revealed in recent clinical trials using mTOR inhibitors (mTORi) in HNSCC in the adjuvant and neoadjuvant setting. Here, we took advantage of a whole-genome CRISPR screening approach to identify mechanisms of sensitivity and resistance to mTORi, aimed at increasing their clinical activity. We found that the activation of autophagy, a biological process that plays a paradoxical pro-survival or antitumoral role in a cancer specific fashion, is strictly required for mTORi cell growth inhibition in HNSCC. In depth analysis of our CRISPR screening also revealed multiple hits suggestive of a novel role for iron metabolism and ferroptosis downstream from mTORi. Ferroptosis is iron-dependent regulated cell death process caused by the peroxidation of polyunsaturated fatty acids. Indeed, we found that mTORi induces ferroptosis in HNSCC cells, and that inhibition of ferroptosis reduces the effect of mTORi. Evidence will be presented supporting a novel mechanism linking mTORi-triggered autophagy and the activation of ferroptotic death programs, as well as new synergistic combinations with mTORi by repurposing approved drugs that disable cellular ferroptotic defense mechanisms. Our studies uncovered how mTORi act in HNSCC, thereby revealing new multimodal precision therapies for HNSCC and many human malignancies displaying overactive PI3K/mTOR signaling. Citation Format: Keiichi Koshizuka, Xingyu Wu, Kuniaki Sato, Pham Thuy Vo, Gosia M. Murawska, Tomohiko Ishikawa, Zhiyong Wang, Alfredo A. Molinolo, Edward A. Dennis, Prashant Mali, J. Silvio Gutkind. Genome-wide CRISPR screening reveals a central role for ferroptotic cell death in the anti-tumor response to mTOR inhibitors in HNSCC [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 5996.

Abstract 1878: CTPS1: An unexplored vulnerability in breast and ovarian cancer

Abstract Breast and ovarian cancer represent the most common cancer types in women worldwide and are among the top fatal cancers. Despite the use of cytotoxic chemotherapy and targeted agents, recurrence is frequent and survival rates for patients with advanced metastatic disease are dismal. These realities highlight the need to identify novel biomarkers and therapeutic vulnerabilities in such tumors and to develop alternative treatment strategies. We took an unbiased approach to tackle this problem in which we leveraged publically available (DepMap) and in-house generated genome-wide CRISPR screens conducted in over 1000 cancer cell lines. We initially focused on the identification of essential genes that were unique to triple-negative breast cancer (TNBC) cells and whose expression levels were associated with worse patient outcomes. This interrogation revealed six genes (CTPS1, RHOA, PRKRA, RAD9A, HUS1, and RAD1) meeting these initial criteria. The essentiality of these genes was validated using gene-specific siRNAs in a panel of TNBC cells and knockdown of CTPS1 was shown to elicit the most inhibitory effect. CTPS1 depletion resulted in a rapid S-phase cell cycle arrest followed by apoptosis, not only in TNBC cells but also in highly aggressive ovarian cancer cells which share many molecular features with TNBC. CTPS1 converts uridine triphosphate to cytidine triphosphate, forming an essential nucleic acid required for multiple cellular processes. CTPS1 mRNA expression was substantially elevated in tumor vs. normal tissue, for both breast and ovarian cancer. Using over 40 breast and ovarian cancer cell lines, RT-PCR analyses confirmed increased expression of CTPS1 in TNBC and most ovarian cancer cell lines compared to other disease sub-types and “normal” cell line/tissue controls. Interestingly, chemoresistant and PARP inhibitor-resistant models exhibited the highest levels of CTPS1 among all cells analyzed. We subsequently identified and acquired STP938, a first-in-class highly selective CTPS1 inhibitor being developed by Step-Pharma. STP938 was found to elicit potent anti-cancer effects at nM concentrations across many cell line and patient-derived models, in both 2D and 3D culture systems. RNAseq analysis in aggressive cell line models following STP938 treatment or CTPS1 knockdown revealed significant regulation of genes related to DNA replication and the cell cycle pathways suggesting that combinatorial/sequential treatments of STP938 with replication stress/DNA damage response related drugs may be synergistic. STP938 is currently being developed for the treatment of lymphoma, and our findings support the repurposing of STP938 for breast and ovarian cancer, an avenue that we are currently developing. Citation Format: Xiyin Wang, Michael Emch, Lauren Voll, Rebecca Russell, Esther Rodman, Nicole Pearson, Xiaonan Hou, Scott Kaufmann, Saravut Weroha, Philip Beer, John Hawse. CTPS1: An unexplored vulnerability in breast and ovarian cancer [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 1878.

A genome-wide CRISPR screening uncovers that TOB1 acts as a key host factor for FMDV infection via both IFN and EGFR mediated pathways.

The interaction between foot-and-mouth disease virus (FMDV) and the host is extremely important for virus infection, but there are few researches on it, which is not conducive to vaccine development and FMD control. In this study, we designed a porcine genome-scale CRISPR/Cas9 knockout library containing 93,859 single guide RNAs targeting 16,886 protein-coding genes, 25 long ncRNAs, and 463 microRNAs. Using this library, several previously unreported genes required for FMDV infection are highly enriched post-FMDV selection in IBRS-2 cells. Follow-up studies confirmed the dependency of FMDV on these genes, and we identified a functional role for one of the FMDV-related host genes: TOB1 (Transducer of ERBB2.1). TOB1-knockout significantly inhibits FMDV infection by positively regulating the expression of RIG-I and MDA5. We further found that TOB1-knockout led to more accumulation of mRNA transcripts of transcription factor CEBPA, and thus its protein, which further enhanced transcription of RIG-I and MDA5 genes. In addition, TOB1-knockout was shown to inhibit FMDV adsorption and internalization mediated by EGFR/ERBB2 pathway. Finally, the FMDV lethal challenge on TOB1-knockout mice confirmed that the deletion of TOB1 inhibited FMDV infection in vivo. These results identify TOB1 as a key host factor involved in FMDV infection in pigs.

PRMT5 is an actionable therapeutic target in CDK4/6 inhibitor-resistant ER+/RB-deficient breast cancer

CDK4/6 inhibitors (CDK4/6i) have improved survival of patients with estrogen receptor-positive (ER+) breast cancer. However, patients treated with CDK4/6i eventually develop drug resistance and progress. RB1 loss-of-function alterations confer resistance to CDK4/6i, but the optimal therapy for these patients is unclear. Through a genome-wide CRISPR screen, we identify protein arginine methyltransferase 5 (PRMT5) as a molecular vulnerability in ER+/RB1-knockout breast cancer cells. Inhibition of PRMT5 blocks the G1-to-S transition in the cell cycle independent of RB, leading to growth arrest in RB1-knockout cells. Proteomics analysis uncovers fused in sarcoma (FUS) as a downstream effector of PRMT5. Inhibition of PRMT5 results in dissociation of FUS from RNA polymerase II, leading to hyperphosphorylation of serine 2 in RNA polymerase II, intron retention, and subsequent downregulation of proteins involved in DNA synthesis. Furthermore, treatment with the PRMT5 inhibitor pemrametostat and a selective ER degrader fulvestrant synergistically inhibits growth of ER+/RB-deficient cell-derived and patient-derived xenografts. These findings highlight dual ER and PRMT5 blockade as a potential therapeutic strategy to overcome resistance to CDK4/6i in ER+/RB-deficient breast cancer.

A SWI/SNF-dependent transcriptional regulation mediated by POU2AF2/C11orf53 at enhancer

Recent studies have identified a previously uncharacterized protein C11orf53 (now named POU2AF2/OCA-T1), which functions as a robust co-activator of POU2F3, the master transcription factor which is critical for both normal and neoplastic tuft cell identity and viability. Here, we demonstrate that POU2AF2 dictates opposing transcriptional regulation at distal enhance elements. Loss of POU2AF2 leads to an inhibition of active enhancer nearby genes, such as tuft cell identity genes, and a derepression of Polycomb-dependent poised enhancer nearby genes, which are critical for cell viability and differentiation. Mechanistically, depletion of POU2AF2 results in a global redistribution of the chromatin occupancy of the SWI/SNF complex, leading to a significant 3D genome structure change and a subsequent transcriptional reprogramming. Our genome-wide CRISPR screen further demonstrates that POU2AF2 depletion or SWI/SNF inhibition leads to a PTEN-dependent cell growth defect, highlighting a potential role of POU2AF2-SWI/SNF axis in small cell lung cancer (SCLC) pathogenesis. Additionally, pharmacological inhibition of SWI/SNF phenocopies POU2AF2 depletion in terms of gene expression alteration and cell viability decrease in SCLC-P subtype cells. Therefore, impeding POU2AF2-mediated transcriptional regulation represents a potential therapeutic approach for human SCLC therapy.

In vivo genome-wide CRISPR screening identifies CITED2 as a driver of prostate cancer bone metastasis.

Most cancer deaths are due to metastatic dissemination to distant organs. Bone is the most frequently affected organ in metastatic prostate cancer and a major cause of prostate cancer deaths. Yet, our partial understanding of the molecular factors that drive bone metastasis has been a limiting factor for developing preventative and therapeutic strategies to improve patient survival and well-being. Although recent studies have uncovered molecular alterations that occur in prostate cancer metastasis, their functional relevance for bone metastasis is not well understood. Using genome-wide CRISPR activation and inhibition screens we have identified multiple drivers and suppressors of prostate cancer metastasis. Through functional validation, including an innovative organ-on-a-chip invasion platform for studying bone tropism, our study identifies the transcriptional modulator CITED2 as a novel driver of prostate cancer bone metastasis and uncovers multiple new potential molecular targets for bone metastatic disease.

Genome-wide CRISPR screen identifies ESPL1 limits the response of gastric cancer cells to apatinib

Apatinib was the first anti-angiogenic agent approved for treatment of metastatic gastric cancer (GC). However, the emergence of resistance was inevitable. Thus investigating new and valuable off-target effect of apatinib directly against cancer cells is of great significance. Here, we identified extra spindle pole bodies-like 1 (ESPL1) was responsible for apatinib resistance in GC cells through CRISPR genome-wide gain-of-function screening. Loss of function studies further showed that ESPL1 inhibition suppressed cell proliferation, migration and promoted apoptosis in vitro, and accordingly ESPL1 knockdown sensitized GC cells to apatinib. In addition, we found ESPL1 interacted with mouse double minute 2 (MDM2), a E3 ubiquitin protein ligase, and the combination of MDM2 siRNA with apatinib synergistically ameliorated the resistance induced by ESPL1 overexpression. In summary, our study indicated that ESPL1 played a critical role in apatinib resistance in GC cells. Inhibition of MDM2 could rescue the sensitivity of GC cells to apatinib and reverse ESPL1-mediated resistance.

A genome-wide CRISPR screen identifies BRD4 as a regulator of cardiomyocyte differentiation.

Human induced pluripotent stem cell (hiPSC) to cardiomyocyte (CM) differentiation has reshaped approaches to studying cardiac development and disease. In this study, we employed a genome-wide CRISPR screen in a hiPSC to CM differentiation system and reveal here that BRD4, a member of the bromodomain and extraterminal (BET) family, regulates CM differentiation. Chemical inhibition of BET proteins in mouse embryonic stem cell (mESC)-derived or hiPSC-derived cardiac progenitor cells (CPCs) results in decreased CM differentiation and persistence of cells expressing progenitor markers. In vivo, BRD4 deletion in second heart field (SHF) CPCs results in embryonic or early postnatal lethality, with mutants demonstrating myocardial hypoplasia and an increase in CPCs. Single-cell transcriptomics identified a subpopulation of SHF CPCs that is sensitive to BRD4 loss and associated with attenuated CM lineage-specific gene programs. These results highlight a previously unrecognized role for BRD4 in CM fate determination during development and a heterogenous requirement for BRD4 among SHF CPCs.

Host E3 ubiquitin ligase ITCH mediates Toxoplasma gondii effector GRA35-triggered NLRP1 inflammasome activation and cell-autonomous immunity.

Toxoplasma gondii is an intracellular parasite that can activate the NLRP1 inflammasome leading to macrophage pyroptosis in Lewis rats, but the underlying mechanism is not well understood. In this study, we performed a genome-wide CRISPR screen and identified the dense granule proteins GRA35, GRA42, and GRA43 as the Toxoplasma effectors mediating cell death in Lewis rat macrophages. GRA35 localizes on the parasitophorous vacuole membrane, where it interacts with the host E3 ubiquitin ligase ITCH. Inhibition of proteasome activity or ITCH knockout prevented pyroptosis in Toxoplasma-infected Lewis rat macrophages, consistent with the "NLRP1 functional degradation model." However, there was no evidence that ITCH directly ubiquitinates or interacts with rat NLRP1. We also found that GRA35-ITCH interaction affected Toxoplasma fitness in IFNγ-activated human fibroblasts, likely due to ITCH's role in recruiting ubiquitin and the parasite-restriction factor RNF213 to the parasitophorous vacuole membrane. These findings identify a new role of host E3 ubiquitin ligase ITCH in mediating effector-triggered immunity, a critical concept that involves recognizing intracellular pathogens and initiating host innate immune responses.IMPORTANCEEffector-triggered immunity represents an innate immune defense mechanism that plays a crucial role in sensing and controlling intracellular pathogen infection. The NLRP1 inflammasome in the Lewis rats can detect Toxoplasma infection, which triggers proptosis in infected macrophages and eliminates the parasite's replication niche. The work reported here revealed that host E3 ubiquitin ligase ITCH is able to recognize and interact with Toxoplasma effector protein GRA35 localized on the parasite-host interface, leading to NLRP1 inflammasome activation in Lewis rat macrophages. Furthermore, ITCH-GRA35 interaction contributes to the restriction of Toxoplasma in human fibroblasts stimulated by IFNγ. Thus, this research provides valuable insights into understanding pathogen recognition and restriction mediated by host E3 ubiquitin ligase.

A genome-wide CRISPR screen identified host genes essential for intracellular Brucella survival.

Brucella is a zoonotic intracellular bacterium that poses threats to human health and economic security. Intracellular infection is a hallmark of the agent Brucella and a primary cause of distress, through which the bacterium regulates the host intracellular environment to promote its own colonization and replication, evading host immunity and pharmaceutical killing. Current studies of Brucella intracellular processes are typically premised on bacterial phenotype such as intracellular bacterial survival, followed by biochemical or molecular biological approaches to reveal detailed mechanisms. While such processes can deepen the understanding of Brucella-host interaction, the insights into host alterations in infection would be easily restricted to known pathways. In the current study, we applied CRISPR Cas9 screen to identify host genes that are most affected by Brucella infection on cell viability at the genomic level. As a result of CRISPR screening, we firstly identified that knockout of the negatively selected genes GOLGA6L6, DEFB103B, OR4F29, and ERCC6 attenuate the viability of both the host cells and intracellular Brucella, suggesting these genes to be potential therapeutic targets for Brucella control. In particular, knockout of DEFB103B diminished Brucella intracellular survival by altering host cell autophagy. Conversely, knockout of positive screening genes promoted intracellular proliferation of Brucella. In summary, we screened host genes at the genomic level throughout Brucella infection, identified host genes that are previously not recognized to be involved in Brucella infection, and provided targets for intracellular infection control.IMPORTANCEBrucella is a Gram-negative bacterium that infects common mammals causing arthritis, myalgia, neuritis, orchitis, or miscarriage and is difficult to cure with antibiotics due to its intracellular parasitism. Therefore, unraveling the mechanism of Brucella-host interactions will help controlling Brucella infections. CRISPR-Cas9 is a gene editing technology that directs knockout of individual target genes by guided RNA, from which genome-wide gene-knockout cell libraries can be constructed. Upon infection with Brucella, the cell library would show differences in viability as a result of the knockout and specific genes could be revealed by genomic DNA sequencing. As a result, genes affecting cell viability during Brucella infection were identified. Further testing of gene function may reveal the mechanisms of Brucella-host interactions, thereby contributing to clinical therapy.

Genome-wide ATAC-see screening identifies TFDP1 as a modulator of global chromatin accessibility.

Chromatin accessibility is a hallmark of active regulatory regions and is functionally linked to transcriptional networks and cell identity. However, the molecular mechanisms and networks that govern chromatin accessibility have not been thoroughly studied. Here we conducted a genome-wide CRISPR screening combined with an optimized ATAC-see protocol to identify genes that modulate global chromatin accessibility. In addition to known chromatin regulators like CREBBP and EP400, we discovered a number of previously unrecognized proteins that modulate chromatin accessibility, including TFDP1, HNRNPU, EIF3D and THAP11 belonging to diverse biological pathways. ATAC-seq analysis upon their knockouts revealed their distinct and specific effects on chromatin accessibility. Remarkably, we found that TFDP1, a transcription factor, modulates global chromatin accessibility through transcriptional regulation of canonical histones. In addition, our findings highlight the manipulation of chromatin accessibility as an approach to enhance various cell engineering applications, including genome editing and induced pluripotent stem cell reprogramming.

Epigenetic regulation of CD38/CD48 by KDM6A mediates NK cell response in multiple myeloma

Anti-CD38 monoclonal antibodies like Daratumumab (Dara) are effective in multiple myeloma (MM); however, drug resistance ultimately occurs and the mechanisms behind this are poorly understood. Here, we identify, via two in vitro genome-wide CRISPR screens probing Daratumumab resistance, KDM6A as an important regulator of sensitivity to Daratumumab-mediated antibody-dependent cellular cytotoxicity (ADCC). Loss of KDM6A leads to increased levels of H3K27me3 on the promoter of CD38, resulting in a marked downregulation in CD38 expression, which may cause resistance to Daratumumab-mediated ADCC. Re-introducing CD38 does not reverse Daratumumab-mediated ADCC fully, which suggests that additional KDM6A targets, including CD48 which is also downregulated upon KDM6A loss, contribute to Daratumumab-mediated ADCC. Inhibition of H3K27me3 with an EZH2 inhibitor resulted in CD38 and CD48 upregulation and restored sensitivity to Daratumumab. These findings suggest KDM6A loss as a mechanism of Daratumumab resistance and lay down the proof of principle for the therapeutic application of EZH2 inhibitors, one of which is already FDA-approved, in improving MM responsiveness to Daratumumab.

Genome-wide CRISPR screen reveals the synthetic lethality between BCL2L1 inhibition and radiotherapy.

Radiation therapy (RT) is one of the most commonly used anticancer therapies. However, the landscape of cellular response to irradiation, especially to a single high-dose irradiation, remains largely unknown. In this study, we performed a whole-genome CRISPR loss-of-function screen and revealed temporal inherent and acquired responses to RT. Specifically, we found that loss of the IL1R1 pathway led to cellular resistance to RT. This is in part because of the involvement of radiation-induced IL1R1-dependent transcriptional regulation, which relies on the NF-κB pathway. Moreover, the mitochondrial anti-apoptotic pathway, particularly the BCL2L1 gene, is crucially important for cell survival after radiation. BCL2L1 inhibition combined with RT dramatically impeded tumor growth in several breast cancer cell lines and syngeneic models. Taken together, our results suggest that the combination of an apoptosis inhibitor such as a BCL2L1 inhibitor with RT may represent a promising anticancer strategy for solid cancers including breast cancer.

Abstract PR08: Intratumoral heterogeneity drives resistance to Antibody Drug Conjugate therapy: Analysis of the NeoSTAR trial of neoadjuvant Sacituzumab govitecan for localized TNBC

Abstract Antibody Drug Conjugates (ADCs) are proving to be groundbreaking therapeutics for all breast cancer subtypes. Sacituzumab govitecan (SG), an ADC targeting the TROP2 tumor antigen and harboring a topoisomerase 1 inhibitor payload, improves overall survival in advanced Hormone Receptor positive (HR+) and Triple Negative Breast Cancer (TNBC). However, SG has not been tested in early-stage breast cancer, and response predictors and mechanisms of resistance to SG in any setting remain to be defined. We thus designed the NeoSTAR trial, a Phase 2 study of response-guided neoadjuvant SG for localized TNBC, in which 50 patients received single-agent SG followed by imaging and tumor bed biopsy, with subsequent therapy determined by clinical and pathologic response. We collected fresh pre-treatment and post-SG biopsies and carried out analysis employing single-cell RNAseq, exome sequencing, and spatial analysis. As anticipated, cell composition of TNBCs between subjects was highly heterogenous but revealed non-tumor cell types predictive of pathologic complete response (pCR) to SG alone. As in the metastatic setting, tumor cell TROP2 levels were variable both within and between TNBCs, and were not predictive of response to SG. In contrast, we identified tumor cell-intrinsic pathways predictive of response that were the same as those identified in parallel genome-wide CRISPR screens for SG response pathways in TNBC. Analysis of matched pre/post treatment specimens demonstrated substantial clonal selection and clonal evolution post SG. Of particular interest were small subpopulations of tumor cells with shared properties that were present in multiple tumors in very small numbers pre-treatment but dramatically expanded post-treatment, suggesting the presence of a common resistance phenotype. Ongoing work to be presented focuses on defining the nature of the shared resistant subpopulations, and on comprehensively assessing the genomic correlates of response to SG versus standard chemotherapy. In summary, detailed single-cell and genomic analysis of single-agent ADC therapy in treatment-naïve primary TNBC reveals how intratumoral heterogeneity and subclonal resistance phenotypes shape the landscape of treatment response. These observations provide new insights relevant to the clinical application of this complex class of therapeutics. Citation Format: Laura M. Spring, Bogang Wu, Ting Liu, Jacob Geisberg, Simona Cristea, Veerle Bossuyt, Rachel Occhiogrosso Abelman, Nicole Peiris, James Coates, Siang-Boon Koh, Mengran Zhang, Lianne Ryan, Beverly Moy, Steven J. Isakoff, Sara M. Tolaney, Franziska Michor, Aditya Bardia, Leif W. Ellisen. Intratumoral heterogeneity drives resistance to Antibody Drug Conjugate therapy: Analysis of the NeoSTAR trial of neoadjuvant Sacituzumab govitecan for localized TNBC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr PR08.

CRISPR screening identifies BET and mTOR inhibitor synergy in cholangiocarcinoma through serine glycine one carbon.

Patients with cholangiocarcinoma have poor clinical outcomes due to late diagnoses, poor prognoses, and limited treatment strategies. To identify drug combinations for this disease, we have conducted a genome-wide CRISPR screen anchored on the bromodomain and extraterminal domain (BET) PROTAC degrader ARV825, from which we identified anticancer synergy when combined with genetic ablation of members of the mTOR pathway. This combination effect was validated using multiple pharmacological BET and mTOR inhibitors, accompanied by increased levels of apoptosis and cell cycle arrest. In a xenograft model, combined BET degradation and mTOR inhibition induced tumor regression. Mechanistically, the 2 inhibitor classes converged on H3K27ac-marked epigenetic suppression of the serine glycine one carbon (SGOC) metabolism pathway, including the key enzymes PHGDH and PSAT1. Knockdown of PSAT1 was sufficient to replicate synergy with single-agent inhibition of either BET or mTOR. Our results tie together epigenetic regulation, metabolism, and apoptosis induction as key therapeutic targets for further exploration in this underserved disease.

Abstract C034: Genome-wide CRISPR screen identifies novel immunotherapeutic targets in pancreatic cancer

Abstract Background: Cancer-cell-intrinsic properties dictate the immune contexture of tumors, impacting the outcome of immunotherapy. Despite the increasing value of CRISPR/Cas9-based loss-of-function (LOF) genetic screens for systematically assessing gene knockout phenotype in tumor cells, it still present challenges, particularly when it comes to genomic editing in the context of large scale experiments and the identification of immunomodulatory genes in in vivo application. To address this, we have established an AsCpf1 (Cas12a)-based screen platform to enable comprehensive whole genome in vivo LOF screening and uncover novel immunotherapeutic targets for pancreatic cancer, which is notoriously resistant to current immunotherapies. Methods: We designed and constructed the optimized minimal CRISPR/AsCpf1 library that targets the entire mouse genome and is only one-fourth the size of the existing genome-wide Cas9-based library. To identify novel immune-related gene vulnerabilities in pancreatic cancer, we infected cell cultures derived from the classic p48-Cre KrasLSL-G12D/+ p53+/- genetically engineered pancreatic cancer mouse model with our AsCpf1 library for in vivo LOF screen in immune-competent and -incompetent mice. Next generation sequencing analysis was performed with the resulting tumors. The potential immunomodulatory hits were called out through BAGEL analysis, using a false discovery rate (FDR) < 0.05, and by comparing between the two mouse groups. Results: Our AsCpf1-based screen identified highly confident hits with potential immunomodulation properties. Notably, we observed depletions of various components of the MHC complex, including H13, specifically in the immune-competent background, implicating their requirement for the regulation of anti-tumor immunity. Depletion of H13 with CRISPR/Cas9 system significantly suppressed pancreatic tumor growth in T-cell dependent manner. Further immune profiling revealed that the loss of H13 triggered potent immune response which was featured by a substantial increase in effective CD8+ T cells and reduction in immunosuppressive macrophages in both subcutaneous and orthotopic models of pancreatic cancer. These findings are in accordance with our clinically relevant analysis showing that low expression of H13 is correlated with improved patient survival in pancreatic cancer and augmented responsiveness to immune checkpoint therapies in several cancer types. Conclusions: Our AsCpf1-based LOF screening platform enables unbiased exploration of in vivo functional players at whole-genome level, which can be applied to diverse disease context and models. In our lead screen, we have identified H13 as a promising immunomodulatory target for patients with pancreatic cancer. Citation Format: Xiaofei Wang, Jintan Liu, Giulio Draetta, Haoqiang Ying, Wantong Yao. Genome-wide CRISPR screen identifies novel immunotherapeutic targets in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C034.