Genome-wide CRISPR-Cas9 Screen Research Articles

CHMP2A regulates broad immune cell-mediated antitumor activity in an immunocompetent in vivo head and neck squamous cell carcinoma model

BackgroundNatural killer (NK) cells are key effector cells of antitumor immunity. However, tumors can acquire resistance programs to escape NK cell-mediated immunosurveillance. Identifying mechanisms that mediate this resistance enables us...

Integrated drug profiling and CRISPR screening identify BCR::ABL1-independent vulnerabilities in chronic myeloid leukemia

BCR::ABL1-independent pathways contribute to primary resistance to tyrosine kinase inhibitor (TKI) treatment in chronic myeloid leukemia (CML) and play a role in leukemic stem cell persistence. Here, we perform exvivodrug screening of CML CD34+ leukemic stem/progenitor cells using 100 single drugs and TKI-drugcombinations and identify sensitivities to Wee1, MDM2, and BCL2 inhibitors. These agents effectively inhibit primitive CD34+CD38- CML cells and demonstrate potent synergies when combined with TKIs. Flow-cytometry-baseddrug screening identifies mepacrine to induce differentiation of CD34+CD38- cells. We employ genome-wide CRISPR-Cas9 screening for six drugs, and mediator complex, apoptosis, and erythroid-lineage-related genes are identified as key resistance hits for TKIs, whereas the Wee1 inhibitor AZD1775 and mepacrine exhibit distinct resistance profiles. KCTD5, a consistent TKI-resistance-conferring gene, is found to mediate TKI-induced BCR::ABL1 ubiquitination. In summary, we delineate potential mechanismsfor primary TKI resistance and non-BCR::ABL1-targeting drugs, offering insights for optimizing CML treatment.

Abstract 6000: A novel pathway controlling anticancer drug induced immunogenic cell death

Abstract Acting through estrogen receptor alpha (ERα), in orthotopic mouse xenograft models and a PDX, our non-competitive anticancer drug ErSO induces complete or near complete regression of primary and metastatic therapy-resistant ERα-positive breast, ovarian and endometrial tumors. ErSO is also highly effective in ovarian cancer PDOs from patient ascites. Unlike most anticancer agents which inhibit cancer cell proliferation or induce apoptosis, ErSO induces immune-cell-activating necrosis through sustained hyperactivation of the anticipatory unfolded protein response (a-UPR) pathway resulting in ATP depletion and cell swelling followed by membrane rupture. The medium from cancer cells killed by ErSO not only robustly activates mouse and human macrophages, but also dramatically increases monocyte migration. ErSO therefore has the potential to help extend the reach of immunotherapy to many solid tumors that do not express neoantigens. However, unlike apoptosis which has an established signaling pathway, how ErSO induces necrosis was unknown. From a genome-wide CRISPR-Cas9 screen in MCF-7 cells with negative selection against ErSO, we identified FGD3, a guanine exchange factor (GEF) of Rho GTPase Cdc42 as the top target. Consistent with the screen, through subsequent knockout and overexpression of FGD3 in human breast cancer cells, we found that the knockout cells have significant resistance to ErSO while the overexpressing cells exhibit increased sensitivity to killing by ErSO in both 2D cell culture and a 3D organoid model. Our data indicates that this protein is not an upstream regulator of the ErSO-induced necrosis pathway. Instead, it is a regulator of the last stage of necrosis - cell membrane rupture. Thus, we identified an actin reorganization signaling pathway with multiple components that plays a pivotal role in whether the breast cancer cell responds to a-UPR activation and swelling by membrane rupture and necrotic cell death or reorganizes its actin enabling the cancer cell to survive. RNA-seq and other studies indicate that by regulating actin reorganization, this axis is important in ErSO-persister breast cancer cells exhibiting long-term survival in ErSO. This work enables identification of breast cancer patients whose elevated levels of components of this signaling pathway make them most likely to benefit from this novel therapy. Notably, FGD3 knockout cells also show considerable resistance to necrosis or necroptosis induced by other anticancer drugs. Here, we describe a therapy that induces immunogenic cell death in ERα-positive breast, ovarian and endometrial cancer cells. We also show that, as cancer cells respond to agents that induce immunogenic cell death through membrane rupture, an actin regulating pathway plays a critical role in life-death decisions. Citation Format: Junyao Zhu, Santanu Ghosh, Darjan Duraki, Matthew Boudreau, Musarrat Jabeen, Chengjian Mao, Ben H. Park, Georgina Cheng, Erik R. Nelson, Paul J. Hergenrother, David J. Shapiro. A novel pathway controlling anticancer drug induced immunogenic cell 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 6000.

Abstract 5836: CRISPR/Cas9 screening identifies genes affecting the sensitivity to the copanlisib/venetoclax combination in lymphoma cells

Abstract Background: Copanlisib, a PI3K inhibitor with a higher selectivity on PI3Kα and PI3Kδ, is currently being evaluated as a single agent and in combination for lymphoma and solid tumor patients. We and others reported the synergism achieved by adding the BCL2 inhibitor venetoclax, and the combination is in its early clinical assessment (NCT04939272, NCT04572763, NCT03886649). To explore genes affecting the response to the combination, we performed a genome-wide CRISPR Cas9 screen in a marginal zone B cell lymphoma (MZL) cell line under drug exposure. Methods: We generated, via lentiviral infection, a stable Cas9-expressing VL51 line, which was then infected (MOI 0.3) with the genome-wide Brunello guide RNA library, interrogating 19114 genes. After puromycin selection, 40M cells underwent DNA extraction (T0), 40M were exposed to DMSO ( control arm), 40M to copanlisib (0.5nM) + venetoclax (25nM) (experimental arm) for 2 weeks. DNA at T0 and extracted at 2 weeks underwent Illumina sequencing according to the Broad Institute Genetic Perturbation Platform protocol. MAGeCK-MLE was used to model sgRNA read counts and assess the sgRNAs statistical enrichment (P<0.05 FDR< 0.15), GSEA for genesets enrichments. Results: In the DMSO-treated cells, 160 genes had a positive β value (i.e., the sgRNAs knocking down the genes were positively enriched after 2 weeks), while 1152 had a negative β value (i.e., the sgRNAs knocking down the genes were negatively enriched after two weeks), for a total of 1152 differential enriched targets. In the cells exposed to the drug combination, 207 genes had positive and 1324 negative β, for a total of 1531 differentially enriched targets. Genes defined as essential, according to DepMap, represented 768/1152 (67%) and 843/1531 (55%) in control and experimental arms, respectively. The experimental arm did not massively differ from the control arm as shown by 815 genes (615, 74%, defined as essential by DepMap) overlapping between the 2 conditions and by highly significant enrichments at GSEA. A few genes were relevant only in VL51 exposed to copanlisib/venetoclax and not to DMSO. Selected hits were individually validated. Knocking down VKORC1L1, XCL1, RFK, MAP2K6, POC1A, and WTN5A improved the activity of the combination. WTN5A was also validated using recombinant protein (10 ng/mL), which reduced the effect of copanlisib/venetoclax. SPARCL1, MAPK4, ID2, and MSTN silencing reduced the activity of the combination. MSTN, coding for myostatin, was also validated by adding the recombinant protein (10 ng/mL), which increased the drugs anti-tumor activity. Conclusions: A genome-wide genetic screening identified genes modulating the lymphoma cells response to the copanlisib/venetoclax combination, providing novel therapeutic targets or biomarkers. Citation Format: Sara Napoli, Alberto J. Arribas, Eleonora Cannas, Federica Fuzio, Luciano Cascione, Andrea Rinaldi, Emanuele Zucca, Andrea Alimonti, Anastasios Stathis, Francesco Bertoni. CRISPR/Cas9 screening identifies genes affecting the sensitivity to the copanlisib/venetoclax combination in lymphoma cells [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 5836.

Abstract 1447: Development of advanced preclinical modeling for a comprehensive characterization of cardiac angiosarcoma biology

Abstract Cardiac angiosarcoma (CAS) is an extremely rare malignant cardiac tumor known for its propensity for early metastasis and poor prognosis. CAS frequency is around 1-2% of all angiosarcoma cases and mostly arise in the right atrium. At present, there are no effective treatments and the survival rate is less than 10% at 5 years. Furthermore, the dearth of preclinical models has limited the advancement of biological insights into CAS. To address this challenge, we developed a somatic mosaic genetically engineered mouse model (smGEMM) with the aim of accurately mirroring the biological features of CAS. This model will be a crucial instrument for acquiring in-depth insights into the disease and steering the development of impactful therapeutic strategies. To develop a CAS smGEMM, we leveraged an endothelial-specific CRE recombinase to activate a fluorescent reporter in a tissue-dependent manner and CRISPR/Cas9 technology to engineer somatic drivers of CAS tumorigenesis. Using this strategy, all endothelial cells express tdTomato fluorescent reporter and spCas9. We then administered adeno-associated viral (AAV) particles carrying selected tandem sgRNAs with two different approaches: a direct approach where AAV particles were administered by ultrasound-guided injection in the cardiac wall of the smGEMM; and an ex-vivo approach where primary endotheliocytes derived from cardiac tissue of our smGEMM were first transduced with AAV particles in vitro, and then transplanted orthotopically by ultrasound-guided injection. With the latter strategy, we successfully generated the first cardiac angiosarcoma model that faithfully recapitulates angiosarcoma features, including lung dissemination. Cell lines from primary and metastatic tumors were also established, characterized and utilized for genome-wide CRISPR Cas9 screening. Current efforts are focused on prioritizing hits for validation, aiming to glean insights that can inform potential therapeutic strategies. Citation Format: Rosalba Minelli, Luigi Perelli, Benjamin J. Bivona, Sanjana Srinivasan, Desiree Catania, Bianca E. Amador, Frederick S. Robinson, Johnathon L. Rose, Christopher T. Terranova, Alejandro Hernandez Martinez, Charles E. Deckard, Courtney N. Le, Kuo-Shun Hsu, Michael Peoples, Aparna Subramaniam, Hannah C. Beird, Ningping Feng, Joseph R. Daniele, Christopher Vellano, Joseph Marszalek, Vinod Ravi, Alexander J. Lazar, Andrew Futreal, Timothy P. Heffernan, Giannicola Genovese, Virginia Giuliani. Development of advanced preclinical modeling for a comprehensive characterization of cardiac angiosarcoma biology [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 1447.

Abstract 3042: Unraveling the mechanism of YAP toxicity in neuroendocrine solid tumors: Insights from Merkel cell carcinoma

Abstract The Hippo pathway, a pivotal regulator orchestrating organ size and tissue growth, has emerged as a central player in cancer biology. The downstream transcription factor YAP within this pathway has been implicated in diverse oncogenic processes. YAP activation contributes to drug resistance, metastasis, and poor clinical outcomes in many solid tumors. Conversely, YAP exhibits growth-suppressive effects in neuroendocrine (NE) solid cancers, a paradox that remains poorly understood. Analysis of RNAseq data from the Cancer Cell Line Encyclopedia (CCLE) revealed a distinct classification of tumors into YAP1-expressing (YAPon) and non-expressing (YAPoff) subgroups, with hematopoietic malignancies and NE solid tumors featured prominently in the latter. Merkel cell carcinoma (MCC) is a rare, high-grade, NE carcinoma of the skin with two distinct etiologies. Virus-positive MCC contains integrated Merkel cell polyomavirus that expresses the viral T antigens and virus-negative MCC caused by sunlight exposure and extensive UV damage. Bulk RNAseq performed with more than 60 MCC tumors revealed that YAP expression displayed an inverse correlation with an NE gene expression signature. To gain insight into why YAP expression is so low in MCC, we generated MCC cell lines with inducible expression of YAP. Expression of YAP led to growth arrest and cell death. We performed a genome-wide CRISPR-Cas9 screen to identify genes that when lost enable MCC cells to tolerate YAP expression. Functional enrichment analysis of the positively selected genes uncovered a variety of signaling pathways. Intriguingly, the CRISPR screen revealed a positive enrichment of non-canonical BAF complex members, including BRD9 and SMARCD1. To validate this finding, we treated MKL-1 cells with a BRD9 inhibitor, observing a substantial reduction in YAP toxicity. These findings open avenues for the development of targeted therapeutic strategies for high grade NE cancers, addressing a critical gap in our understanding of YAP biology in the context of MCC and beyond. Citation Format: Furkan Bahar, Thomas C. Frost, Varsha Ananthapadmanabhan, Julia L. Schnabel, Manisha Thakuria, James A. DeCaprio. Unraveling the mechanism of YAP toxicity in neuroendocrine solid tumors: Insights from Merkel cell carcinoma [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 3042.

Abstract 3357: Identifying mechanisms of acquired resistance to lorlatinib utilizing a genome-wide CRISPR-Cas9 screen

Abstract Survival rates for neuroblastoma (NB), a predominantly pediatric solid tumor arising from the sympathoadrenal neural crest, have improved incrementally from 25% to 50% over three decades with dramatic escalation in therapy intensity. Gain-of-function mutations in anaplastic lymphoma kinase (ALK), of which 85% occur at residues F1174, F1245, and R1275, confer inferior survival compared to patients with wild-type ALK. Lorlatinib, a third generation ALK inhibitor, has completed Phase 1 testing in the NANT Consortium and has moved to frontline therapy in the Children’s Oncology Group. The robust and sustained activity observed with minimal toxicity is transient in relapse patients harboring MYCN amplification and ALK activation. We aim to elucidate and target mechanisms of acquired resistance that emerge from lorlatinib treatment. We utilized the Brunello library, a genome-wide CRISPR-CAS9 loss of function screen targeting 19,114 genes (4 sgRNA per gene), which allows for a high confidence of detection of hits. We screened four clinically representative cell lines: Kelly (ALKF1174L, MYCN-amplified, p53P117T), SMS-SAN (ALKF1174L, MYCN-amplified), LAN5 (ALKR1275Q, MYCN-amplified), and SY5Y (ALKF1174L, MYCN-nonamplified). sgRNAs were ranked according to the degree of depletion or enrichment. We validated top depleted targets through cell-based assays and immunoblotting. We identified several actionable targets across multiple NB cell lines that, when depleted, increase the efficacy of lorlatinib, including FGFR2, BCL2L1, SOX11, SMARCA2, and SMARCD2. Genetic knockout of FGFR2 led to a 40% reduction (p<0.05) in KO cells upon lorlatinib treatment compared to control. PROTAC degradation of the anti-apoptotic BCL-xL (BCL2L1) revealed the combination of lorlatinib and the PROTAC led to a decrease in IC50 in vitro. Additionally, we identified conserved core components of the SWI/SNF complex, a subfamily of ATP-dependent chromatin remodelers, as well as SOX11, a regulator of the SWI/SNF complex and transcription factor of the core regulatory circuitry in adrenergic high-risk NB. In conclusion, we identified actionable targets across four NB cell lines that, when depleted, increase the efficacy of lorlatinib. Depletion of FGFR2 led to increased sensitivity to lorlatinib, however enzymatic inhibition showed minimal effect. Future investigation into dual targeting of ALK and FGFR2 through novel alternative methods is warranted. Furthermore, we observed a synergistic relationship between lorlatinib and a BCL-xL PROTAC in vitro. Ongoing in vivo studies will test the combinatorial efficacy in lorlatinib-naïve and resistant models. Finally, we are investigating epigenetic remodeling by the SWI/SNF complex and its regulators in NB cells during lorlatinib treatment through targeted genetic manipulation of SOX11 to understand its role as a master transcriptional regulator of MYCN and FGFR2. Citation Format: Joshua Renn Kalna, Smita Matkar, Emily O'Drisoll, Skye Balyasny, Matteo Calafatti, Mark Gerelus, Dave Groff, Tina Acholla, Colleen E. Casey, Paul Kamitsuka, Grant Li, Steven J. Pastor, Gabriela Witek, Kateryna Krytska, Jarrett Lindsay, Ophir Shalem, Yael P. Mossé. Identifying mechanisms of acquired resistance to lorlatinib utilizing a genome-wide CRISPR-Cas9 screen [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 3357.

Abstract 3913: Molecular mechanism of action and targets of glucocorticoids in lymphoma therapy

Abstract Prednisone is an anti-inflammatory glucocorticoid (GC) that is cytotoxic for normal and malignant B cells and, on this basis, has long been included in combination chemotherapies to treat aggressive B-cell lymphomas. However, the mechanisms by which GCs kill malignant lymphoma cells still largely remains elusive due to the pleiotropic consequences of GC binding to the glucocorticoid receptor (GR; NR3C1), a ligand-induced transcription factor. This prompted us to search for critical direct targets of GC action with the hypothesis that GCs may inhibit key survival pathways in lymphomas. To determine effector genes that increase glucocorticoid toxicity, we performed genome-wide CRISPR-Cas9 screens in the presence and absence of prednisolone. Screens in cell line models of Burkitt lymphoma (BL) and diffuse large B cell lymphoma (DLBCL) revealed strong synergy between GC treatment and inactivation of genes encoding components of the B cell receptor (BCR) signaling pathway (≥2 SD). To identify direct transcriptional GR targets and binding sites genome-wide, we performed the cleavage under targets and release using nuclease (CUT&RUN) assay and integrated this dataset with RNA-seq upon GC treatment. The pairwise comparison analysis (p<0.05) for GR-bound genes across BL and DLBCL cell lines revealed a significant enrichment of negative regulators of BCR/PI3K signaling and BCR-related tumor suppressors (LAPTM5, KLHL14, ARID5B, DDIT4, DAPP1, INPP5D, and CSK). Interestingly, expression of CSK, an inhibitor of all Src-family kinases (SFKs), was repressed while that of other BCR negative regulators were directly activated by binding of GR. Combinatorial deletion of activated genes counteracted the toxicity of GC up to 83.9%. In contrast, depletion of CSK paradoxically increased the cytotoxic effects of GC in BCR-dependent aggressive lymphoma with decreased proximal BCR signaling. To gain further insights on CSK function in aggressive lymphomas, we performed phospho-proteome and ubiquitinome profiling by treating lymphoma models with a small molecule inhibitor of CSK kinase activity (CSKi). The CSKi treatment initially increased constitutive BCR signaling, as expected, but then triggered exuberant ubiquitination of the LYN, HCK and BLK, leading to their proteasomal degradation. Consequently, the CSKi blocked BCR-dependent NF-κB activation in ABC DLBCL models and BCR-dependent PI3 kinase activation in models of GCB DLBCL and BL. In summary, inhibition of oncogenic BCR signaling is a major mode of action for GCs. Furthermore, small molecule inhibition of CSK kinase activity potentiated the effect of GCs on oncogenic BCR signaling and strongly synergized with GCs in killing ABC and GCB DLBCL models in vitro and preventing the growth of ABC and GCB DLBCL and patient-derived xenografts, warranting the development of clinical-grade CSK inhibitors for the treatment of these aggressive cancers. Citation Format: Jaewoo Choi, Michele Ceribelli, James D. Phelan, Björn Häupl, Da Wei Huang, George W. Wright, Tony Hsiao, Vivian Morris, Francesco Ciccarese, Boya Wang, Sean Corcoran, Sebastian Scheich, Xin Yu, Weihong Xu, Yandan Yang, Hong Zhao, Joyce Zhou, Grace Zhang, Jagan Muppidi, Giorgio Ga Inghirami, Thomas Oellerich, Craig J. Thomas, Wyndham H. Wilson, Louis M. Staudt. Molecular mechanism of action and targets of glucocorticoids in lymphoma therapy [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 3913.

Abstract 5578: Multi-omics approach reveals a SMAD4-deficiency signature, translational reprogramming and synthetic lethality in esophageal adenocarcinom

Abstract Esophageal adenocarcinoma (EAC) is a cancer of high mutation burden with negligible recurrent and druggable driver genes and, therefore, a paucity of options for targeted therapy. Mutation or loss of the tumor suppressor gene SMAD4 occurs in up to 20% of EAC cases, and we have previously shown that loss of SMAD4 is sufficient to promote EAC tumorigenesis in an in vivo xenograft model. The present study aimed to delineate the role of SMAD4 in EAC tumorigenesis and identify synthetic lethal interactions in SMAD4-deficient cancers as a novel approach for treating EAC. Our multi-omics methodology integrated RNA-sequencing, proteomics, polysome-sequencing, reverse-phase protein arrays, and genome-wide CRISPR-Cas9 screening to unveil a SMAD4-deficiency signature and new therapeutic avenues for EAC. These data reveal a complex interplay between SMAD4 loss and regulation of mTOR signaling, with specific downstream effects to reprogram translation. SMAD4-deficient cells express decreased levels of 4E-BP1, the inhibitory binding partner of EIF4E, which is the rate-limiting component of the cap-dependent translation initiation complex. This was accompanied by increased mTOR activity, including phosphorylation and inactivation of 4E-BP1. We found that SMAD4-deficient cells exhibit vastly different polysome traces compared to their wildtype counterparts and preferentially upregulate cap-dependent translation, acquiring an addiction to translation of oncogenic mRNAs. Furthermore, perturbation of additional negative regulators of mTOR signaling in combination with SMAD4 knockout exacerbated these effects and accelerated tumorigenesis in vivo and growth of patient-derived organoids in vitro. This work advances our knowledge of EAC tumorigenesis, provides new mechanistic insights into SMAD4-driven transformation as well as novel potential therapeutic avenues for SMAD4-deficient EAC. Citation Format: Julia V. Milne, Kenji Fujihara, Eric Kusnadi, Maree Pechlivanis, Niko Thio, Carlos Cabalag, Twishi Gulati, Jovana Gotovac, Kaylene Simpson, Cuong Duong, Luc Furic, Wayne Phillips, Nicholas Clemons. Multi-omics approach reveals a SMAD4-deficiency signature, translational reprogramming and synthetic lethality in esophageal adenocarcinom [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 5578.

Abstract 60: Functional mapping of glioblastoma recurrence reveals targetable dependencies in an axonal guidance pathway in highly invasive brain cancers

Abstract Resistance to genotoxic therapies and tumor recurrence are hallmarks of glioblastoma (GBM), an aggressive brain tumor. Here, we explore the functional drivers of post-treatment recurrent GBM. By conducting genome-wide CRISPR-Cas9 screens in patient-derived GBM models, we uncover distinct genetic dependencies in recurrent tumor cells that were absent in their patient-matched primary predecessors, accompanied by increased mutational burden and differential transcript and protein expression. These analyses map a multilayered genetic response to drive tumor recurrence, identifying protein tyrosine phosphatase 4A2 (PTP4A2) as a novel modulator of self-renewal, proliferation and tumorigenicity at GBM recurrence. Genetic perturbation or small molecule inhibition of PTP4A2 represses axon guidance activity through a dephosphorylation axis with roundabout guidance receptor 1 (ROBO1), exploiting a functional dependency on ROBO signaling. Importantly, engineered anti-ROBO1 single-domain antibodies also mimic effects of PTP4A2 inhibition. Since a pan-PTP4A inhibitor was limited by poor penetrance across the blood brain barrier (BBB) in vivo, a second-generation chimeric antigen receptor (CAR)-T cell therapy was engineered against ROBO1 that elicits specific and potent anti-tumor responses in vivo. A single dose of anti-ROBO1 CAR-T cells doubles median survival in patient-derived xenograft (PDX) models of recurrent glioblastoma, and also eradicates tumors in around 50% of mice engrafted with PDX models of lung-to-brain metastases and pediatric relapsed medulloblastoma. We conclude that functional reprogramming drives tumorigenicity and dependence on a multi-targetable PTP4A-ROBO1 signaling axis at GBM recurrence, with potential in other malignant brain tumors. Citation Format: Sheila Kumari Singh, Chirayu Chokshi, Vaseem Shaikh, Benjamin Brakel, Alisha Anand, Chitra Venugopal, Thomas Kislinger, Kevin Henry, Martin Rossotti, Kevin Brown, John Lazo, Jason Moffat. Functional mapping of glioblastoma recurrence reveals targetable dependencies in an axonal guidance pathway in highly invasive brain cancers [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 60.

Abstract 5960: Targeting CDK8: A translatable therapeutic approach for fusion-positive aRMS

Abstract Alveolar rhabdomyosarcoma (aRMS), characterized by poor overall survival and limited advancements in therapy over the past four decades, poses a great challenge in childhood cancer treatment. More than 60% of high-risk aRMS is driven by the oncofusion protein PAX3-FOXO1. The direct targeting of transcription factor fusions, however, is quite difficult. Given that PAX3-FOXO1 is reported to drive disease by controlling transcriptional programs to maintain an undifferentiated myoblastic state, an alternative approach would be to target the transcriptional machinery recruited by the fusion. To identify potential novel targets in aRMS, we utilized the Broad Institute’s Cancer Dependency Map dataset, a collection of genome-wide CRISPR-Cas9 screens performed in over 1000 cancer cell line models. We focused on druggable dependencies that are selective to fusion-positive aRMS and intersected this data with PAX3-FOXO1-associated transcriptional complexes. Among these candidates, we identified CDK8 as a top selective dependency. Importantly, other components of the CDK8 kinase module in the mediator complex, such as CCNC, also scored as a strong selective dependency in fusion-positive aRMS. CDK8 expression is notably elevated in aRMS tumors compared to other pediatric tumors, and aRMS cell lines exhibit significantly higher CDK8 protein levels than non-transformed primary skeletal muscle cells. We next validated these in silico results through the application of shRNA knockdown, CRISPR knockout, and the use of CDK8 kinase inhibitory small molecules, including BI-1347, SEL-120-34A, and JH-XII-178, which demonstrated on-target activity as assessed by STAT1Ser727 phosphorylation. Both genetic loss of CDK8 function and kinase inhibition impaired aRMS cell line growth in vitro. In vivo studies demonstrated a significant inhibition of xenograft tumor growth with both doxycycline inducible CDK8-directed shRNAs and with pharmacologic inhibition of CDK8 with SEL120-34A treatment. To understand the mechanisms by which CDK8 inhibition impairs aRMS growth, we evaluated the transcriptional consequences of CDK8 inhibition by RNA-sequencing in aRMS, as well as the chromatin accessibility by ATAC-sequencing. We discovered that loss of CDK8 led to gain of chromatin accessibility and the upregulation of hallmark genes involved in myogenesis, such as MYH3, MYOG, MEF2C, and MYBPH, as well as alteration of PAX3-FOXO1 gene expression programs. We next validated this differentiation phenotype both in vitro through detection of increased myogenin staining by immunofluorescence and in vivo by visualization of myofibrils in the xenograft tumors and increased myoglobin quantified by qRT-PCR after CDK8 inhibition. In conclusion, this work identifies CDK8 as a novel and readily translatable therapeutic target in fusion-positive aRMS laying a robust foundation for further mechanistic and preclinical studies. Citation Format: Susu Zhang, Clare Malone, Brian Guedes, Kathleen Engel, Marissa Just, Kenneth Ross, Assil Fahs, Giulia Digiovanni, Diyana Granum, Rex Bentley, Leen Barbar, Christian Cerda-Smith, Elizabeth Mendes, Ozgun Erdogan, Alexander Kovach, Kristianne Oristian, Seth Zimmerman, Jack F. Shern, Kris Wood, Chris Counter, Corinne M. Linardic, Kimberly Stegmaier. Targeting CDK8: A translatable therapeutic approach for fusion-positive aRMS [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 5960.

The herpesvirus UL49.5 protein hijacks a cellular C-degron pathway to drive TAP transporter degradation.

The transporter associated with antigen processing (TAP) is a key player in the major histocompatibility class I-restricted antigen presentation and an attractive target for immune evasion by viruses. Bovine herpesvirus 1 impairs TAP-dependent antigenic peptide transport through a two-pronged mechanism in which binding of the UL49.5 gene product to TAP both inhibits peptide transport and triggers its proteasomal degradation. How UL49.5 promotes TAP degradation has, so far, remained unknown. Here, we use high-content siRNA and genome-wide CRISPR-Cas9 screening to identify CLR2KLHDC3 as the E3 ligase responsible for UL49.5-triggered TAP disposal. We propose that the C terminus of UL49.5 mimics a C-end rule degron that recruits the E3 to TAP and engages the cullin-RING E3 ligase in endoplasmic reticulum-associated degradation.

Genetic determinants of micronucleus formation in vivo

Genomic instability arising from defective responses to DNA damage1 or mitotic chromosomal imbalances2 can lead to the sequestration of DNA in aberrant extranuclear structures called micronuclei (MN). Although MN are a hallmark of ageing and diseases associated with genomic instability, the catalogue of genetic players that regulate the generation of MN remains to be determined. Here we analyse 997 mouse mutant lines, revealing 145 genes whose loss significantly increases (n = 71) or decreases (n = 74) MN formation, including many genes whose orthologues are linked to human disease. We found that mice null for Dscc1, which showed the most significant increase in MN, also displayed a range of phenotypes characteristic of patients with cohesinopathy disorders. After validating the DSCC1-associated MN instability phenotype in human cells, we used genome-wide CRISPR–Cas9 screening to define synthetic lethal and synthetic rescue interactors. We found that the loss of SIRT1 can rescue phenotypes associated with DSCC1 loss in a manner paralleling restoration of protein acetylation of SMC3. Our study reveals factors involved in maintaining genomic stability and shows how this information can be used to identify mechanisms that are relevant to human disease biology1.

7-Dehydrocholesterol dictates ferroptosis sensitivity.

Ferroptosis, a form of regulated cell death that is driven by iron-dependent phospholipid peroxidation, has been implicated in multiple diseases, including cancer1-3, degenerative disorders4 and organ ischaemia-reperfusion injury (IRI)5,6. Here, using genome-wide CRISPR-Cas9 screening, we identified that the enzymes involved in distal cholesterol biosynthesis have pivotal yet opposing roles in regulating ferroptosis through dictating the level of 7-dehydrocholesterol (7-DHC)-an intermediate metabolite of distal cholesterol biosynthesis that is synthesized by sterol C5-desaturase (SC5D) and metabolized by 7-DHC reductase (DHCR7) for cholesterol synthesis. We found that the pathway components, including MSMO1, CYP51A1, EBP and SC5D, function as potential suppressors of ferroptosis, whereas DHCR7 functions as a pro-ferroptotic gene. Mechanistically, 7-DHC dictates ferroptosis surveillance by using the conjugated diene to exert its anti-phospholipid autoxidation function and shields plasma and mitochondria membranes from phospholipid autoxidation. Importantly, blocking the biosynthesis of endogenous 7-DHC by pharmacological targeting of EBP induces ferroptosis and inhibits tumour growth, whereas increasing the 7-DHC level by inhibiting DHCR7 effectively promotes cancer metastasis and attenuates the progression of kidney IRI, supporting a critical function of this axis in vivo. In conclusion, our data reveal a role of 7-DHC as a natural anti-ferroptotic metabolite and suggest that pharmacological manipulation of 7-DHC levels is a promising therapeutic strategy for cancer and IRI.

CRISPR-Cas9 library screening combined with an exosome-targeted delivery system addresses tumorigenesis/TMZ resistance in the mesenchymal subtype of glioblastoma.

Rationale: The large-scale genomic analysis classifies glioblastoma (GBM) into three major subtypes, including classical (CL), proneural (PN), and mesenchymal (MES) subtypes. Each of these subtypes exhibits a varying degree of sensitivity to the temozolomide (TMZ) treatment, while the prognosis corresponds to the molecular and genetic characteristics of the tumor cell type. Tumors with MES features are predominantly characterized by the NF1 deletion/alteration, leading to sustained activation of the RAS and PI3K-AKT signaling pathways in GBM and tend to acquire drug resistance, resulting in the worst prognosis compared to other subtypes (PN and CL). Here, we used the CRISPR/Cas9 library screening technique to detect TMZ-related gene targets that might play roles in acquiring drug resistance, using overexpressed KRAS-G12C mutant GBM cell lines. The study identified a key therapeutic strategy to address the chemoresistance against the MES subtype of GBM. Methods: The CRISPR-Cas9 library screening was used to discover genes associated with TMZ resistance in the U87-KRAS (U87-MG which is overexpressed KRAS-G12C mutant) cells. The patient-derived GBM primary cell line TBD0220 was used for experimental validations in vivo and in vitro. Chromatin isolation by RNA purification (ChIRP) and chromatin immunoprecipitation (ChIP) assays were used to elucidate the silencing mechanism of tumor suppressor genes in the MES-GBM subtype. The small-molecule inhibitor EPIC-0412 was obtained through high-throughput screening. Transmission electron microscopy (TEM) was used to characterize the exosomes (Exos) secreted by GBM cells after TMZ treatment. Blood-derived Exos-based targeted delivery of siRNA, TMZ, and EPIC-0412 was optimized to tailor personalized therapy in vivo. Results: Using the genome-wide CRISPR-Cas9 library screening, we found that the ERBIN gene could be epigenetically regulated in the U87-KRAS cells. ERBIN overexpression inhibited the RAS signaling and downstream proliferation and invasion effects of GBM tumor cells. EPIC-0412 treatment inhibited tumor proliferation and EMT progression by upregulating the ERBIN expression both in vitro and in vivo. Genome-wide CRISPR-Cas9 screening also identified RASGRP1(Ras guanine nucleotide-releasing protein 1) and VPS28(Vacuolar protein sorting-associated protein 28) genes as synthetically lethal in response to TMZ treatment in the U87-KRAS cells. We found that RASGRP1 activated the RAS-mediated DDR pathway by promoting the RAS-GTP transformation. VPS28 promoted the Exos secretion and decreased intracellular TMZ concentration in GBM cells. The targeted Exos delivery system encapsulating drugs and siRNAs together showed a powerful therapeutic effect against GBM in vivo. Conclusions: We demonstrate a new mechanism by which ERBIN is epigenetically silenced by the RAS signaling in the MES subtype of GBM. Restoration of the ERBIN expression with EPIC-0412 significantly inhibits the RAS signaling downstream. RASGRP1 and VPS28 genes are associated with the promotion of TMZ resistance through RAS-GDP to RAS-GTP transformation and TMZ efflux, as well. A quadruple combination therapy based on a targeted Exos delivery system demonstrated significantly reduced tumor burden in vivo. Therefore, our study provides new insights and therapeutic approaches for regulating tumor progression and TMZ resistance in the MES-GBM subtype.

Cancer Stem Cells: Current Challenges and Future Perspectives.

Despite major advances in health care including improved diagnostic tools, robust chemotherapeutic regimens, advent of precision, adjuvant and multimodal therapies, there is a major proportion of patients that still go on to experience tumor progression and recurrence. Cancer stem cells (CSCs) are shown to be responsible for tumor persistence and relapse. This subpopulation of cancer cells possessnormal stem celllike traitsof self-renewal, proliferation, and multilineage differentiation. Currently, they are isolated and enriched based on the cell surface markers that can be detected and sorted through fluorescence and magnetic-based cell sorting. In this chapter, we review the current challenges and limitations often encountered in CSC research, including the identification of universal markers, therapy resistance, and new drug development. Current and future perspectives are discussed to address these challenges including utilization of cutting-edge technologies such as next-generation sequencing to elucidate the genome, epigenome, and transcriptome on a single-cell level and genome-wide CRISPR-Cas9 screens to identify novel pathway-based targeted therapies. Further, we discuss the future of precision medicine and the need for the improvement of clinical trial designs.

Abstract C097: Cancer-selective metabolic vulnerabilities in MYC-amplified medulloblastoma

Abstract Background: MYC-amplified group 3 (G3) medulloblastoma (MB) represents a clinically aggressive subgroup of MB that is defined by leptomeningeal metastases, disease recurrence and particularly poor survivorship in comparison to other MB subgroups. There is an urgent need for a more thorough molecular understanding of G3 MB to develop more effective therapeutic modalities that will improve the durability of remission. Methods: We mapped the functional G3 MB metabologenomic landscape via parallel genome-wide CRISPR-Cas9 screening and untargeted liquid chromatography-mass spectrometry (LC-MS) metabolomics. The tractability of potential therapeutic targets was assessed using patient-derived orthotopic preclinical models of G3 MB. Transcriptomics, functional genomics, and metabolomics were employed to discern a mechanism whereby inhibition of DHODH (dihydroorotate dehydrogenase), which is a mitochondrial-bound enzyme that facilitates de novo pyrimidine biosynthesis, selectively impairs G3 MB tumor cell fitness. Results: Untargeted metabolomic profiling shows enrichment of de novo pyrimidine biosynthesis and depletion of salvage pyrimidine intermediates in G3 MB tumor cells in comparison to neural stem cells (NSC), which are the prospective cell-of-origin of MB. In agreement with reprogrammed nucleotide metabolism, DHODH was identified as a corresponding ‘druggable hit’ in the genetic screen. We demonstrate that genetic or pharmacological inhibition of DHODH selectively targets G3 MB brain tumor initiating cells (BTIC) while sparing normal NSC. We further show that MYC-amplified G3 MB tumors harbor subgroup-specific transcriptomic signatures that delineate enrichment of de novo pyrimidine biosynthesis, which sustains c-Myc activity via a metabolite-protein interaction. Significance: Despite clear evidence favoring an altered metabolic landscape in MYC-driven cancers, there have been few reports into the role of metabolic reprogramming in MYC-amplified G3 MB. This metabolic vulnerability is hence not exclusive to MB and has recently been identified in other primary brain malignancies; current efforts are focused on establishing relevant biomarkers that will facilitate clinical translation. Citation Format: Sheila Singh, William Gwynne, Yujin Suk, Jeremy Chan, Andrew Qualie, Laura Escudero, Kevin Brown, Chitra Venugopal, Jason Moffat, Rafael Montenegro-Burke. Cancer-selective metabolic vulnerabilities in MYC-amplified medulloblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C097.

Abstract C042: Integrative analyses of CRISPR and drug screens identify a selective and potent compound for oral squamous cell carcinoma

Abstract Mechanism-guided drug repurposing can accelerate the development of therapeutic strategies for oral squamous cell carcinoma (OSCC), which has an unmet clinical need. We integrated cell line sensitivity data for 339 anti-cancer drugs with genome-wide CRISPR-Cas9 screen gene essentiality data for 21 unique OSCC cell lines, derived primarily from Asian patients. We identified a significant correlation between AZD5582 sensitivity (an antagonist of the inhibitor of apoptosis (IAP) family proteins), and dependency on several members of the NF kappa-B pathway (RNF31, MAP3K7 and IKBKG). Dependent OSCC lines are more sensitive to AZD5582, with nanomolar range half-maximal inhibitory drug concentrations (IC50). We confirmed the on-target specificity and potency of AZD5582 in vitro and in vivo. Furthermore, AZD5582 sensitivity was associated with high levels of tumor necrosis factor (TNF) and either functional apoptotic or necroptotic pathways. In summary, our approach of integrating pharmacological and CRISPR screen data in a unique cohort of OSCC cell lines data has enabled the identification of specific dependencies and drug repurposing candidates. Citation Format: Annie Wai Yeeng Chai, Yee Hua Tan, Shiyin Ooi, Pei San Yee, Shi Mun Yee, Howard Lightfoot, Syd Bathorpe, Mathew J Garnett, Sok Ching Cheong. Integrative analyses of CRISPR and drug screens identify a selective and potent compound for oral squamous cell carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C042.

Exceptional Response to BTK Inhibitors in Aggressive Lymphomas Linked to Chronic Selective Autophagy

Diffuse large B cell lymphoma (DLBCL) is an aggressive cancer that is profoundly heterogeneous, both molecularly and phenotypically, presenting a challenge for precision medicine. Inhibitors of Bruton tyrosine kinase (BTK) block B cell receptor (BCR)-dependent NF-κB signaling and are particularly effective in DLBCL with mutations in the BCR subunit CD79B and MYD88 (MCD DLBCL). MCD tumors are enriched for a multiprotein supercomplex, termed the My-T-BCR, that is nucleated by MYD88 L265P, TLR9 and the BCR, and serves as a central hub of NF-κB signaling. The integrity of the My-T-BCR complex is rapidly compromised following BTK inhibition, but the molecular mechanisms responsible for the dissolution of this molecular machine have not been elucidated. To investigate the underlying mechanisms regulating the My-T-BCR, we used genome wide CRISPR-Cas9 screens in MCD DLBCL cell line models treated with the BTK inhibitors (BTKi) and compared the results to screens with inhibitors of SYK, IKK, mTOR, and BRD4. We identified several drug resistance genes encoding known negative regulators of BCR, NF-κB, and PI3 kinase signaling that were recurrently inactivated by mutation and/or deletion in DLBCL biopsies. Unexpectedly, we identified multiple autophagy-related genes involved autophagosome formation ( ATG9A, ATG101, ATG13, RB1CC1 and ATG14) and autophagosome membrane expansion ( ATG2A, WIPI2, WDR45) that strongly counteracted the toxicity of BTKi (≥3 SD). Notably, these autophagy genes did not promote resistance to the mTOR inhibitors, which induces classical autophagy. To gain further insight, we generated BTKi-resistant cell lines deficient in ATG9A or ATG101 (ATG KO) and performed genome-wide CRISPR screens and RNA-seq with or without BTKi. In doing so, we observed 11 ATG genes that displayed epistatic interactions, no longer conferring BTKi resistance in ATG KO MCD cells (p≤0.05). We also observed the buffering of many NF-κB negative regulators (p≤0.001) and the increased sensitivity to loss of NF-κB positive regulators (p≤0.05). Furthermore, gene expression studies showed a marked decrease of BCR, MYD88 and NF-κB signatures in control BTKi-treated cells, whereas ATG KOs demonstrated a rescue of these same gene signatures and displayed higher levels of nuclear NF-κB localization upon BTKi treatment. MCD patients display the highest levels of NF-κB gene expression compared to other genetic subtypes. Interestingly, MCD patients also displayed the lowest gene expression profile for a subset of the ATG genes (p≤2.7x10 -6), suggesting that this pathway may be counter-selected during MCD pathogenesis. As autophagy promotes the lysosomal degradation of proteins, and disruption of autophagy promoted NF-κB signaling in MCD cell lines, we assessed global protein levels and localization of proteins to the My-T-BCR in ATG KO cells using mass spectrometry. Selective autophagy receptors TAX1BP1, NBR1 and p62 were significantly upregulated in ATG KOs and displayed significant enrichment of proximity to MYD88 L265Pin ATG KOs (p≤0.01), suggesting MYD88 L265P is targeted for degradation by selective autophagy. To test this, we engineered MCD cell lines with dual fluorescent autophagy reporters (GFP-RFP fusions) for TAX1BP1, NBR1, p62, or MYD88 L265P. Each reporter displayed an accumulation of GFP relative to RFP in ATG KOs or upon treatment with the autophagy blocking v-ATPase inhibitor bafilomycin. To identify genes that modulate selective autophagy of MYD88 L265P, we performed a CRISPR screen in MCD cells stably expressing a MYD88 L265P-RFP-GFP reporter. We observed deletion of the same epistatic interactors of ATG9A in BTKi survival screens also blocked MYD88 L265P autophagic degradation. Conversely, deletion of BTK, mTORC1-related genes and IRF4 were all among the top-ranked genes that promoted MYD88 L265P autophagic degradation. We validated these findings using chemical inhibitors of each gene alone or in combination and observed synergy for the promotion of MYD88 L265P autophagic degradation. Collectively, we identified a non-canonical form of selective autophagy that chronically degrades MYD88 L265P, is counter-selected in MCD tumors, and is promoted by BTK inhibitors. Our findings help to elucidate the exceptional benefit of BTK-targeted therapies in the MCD DLBCL subtype and offer a rationally designed combination therapy regimen to specifically degrade this mutant allele of MYD88.

The XPO7/Npat Axis Inactivation Is a Therapeutic Vulnerability for TP53-Mutated AML

In acute myeloid leukemia (AML), TP53 mutations are the most prognostically unfavorable genetic alterations, as they confer resistance to various therapeutic agents and form relapsing clones, underscoring the critical need to devise a novel therapeutic strategy for TP53-mutated AML. To identify genes or pathways whose loss is vulnerable to TP53 deficiency in AML cells, we performed genome-wide CRISPR-Cas9 screens using Trp53-knockout (KO) and wild-type (WT) mouse AML cells. We employed two distinct KO screen libraries, through which we identified Xpo7, a putative nuclear/cytoplasmic transporter, as a common factor necessary for Trp53-KO AML survival. Interestingly, Trp53-KO cells were vulnerable to Xpo7 depletion, while Xpo7 functioned as a Trp53-dependent tumor suppressor in Trp53-WT AML cells. Further probing this phenomenon, we performed a CRISPR-Cas9 saturation mutagenesis scan on all Xpo7 exons. Strikingly, sgRNAs targeting Xpo7 coding regions were significantly enriched only in the Trp53-WT AMLs. In stark contrast, these same sgRNAs were mostly depleted in Trp53-KO AMLs. To determine the role of Xpo7 depletion on the expansion of Trp53 deficient clones, we prepared AML cells containing a small fraction of Trp53-KO cells both in the Xpo7-WT and KO backgrounds and assessed the proportion over time in vitro or in vivo. Remarkably, the expansion of Trp53-KO clones, noticeable in the Xpo7-WT setting, was significantly reduced under the Xpo7-KO background, prolonging the mice's survival. These data suggest that Xpo7 depletion effectively impedes Trp53-KO clone expansion. Since Xpo7 is presumed to mediate the nuclear import and export of proteins, we hypothesized that Xpo7 contributes to retaining Trp53 protein within the nucleus in Trp53-WT AMLs. As expected, nuclear Trp53 protein and mRNA levels of Trp53-targeting genes decreased significantly upon Xpo7 depletion. These data suggest that Xpo7 retains WT-Trp53 in the nucleus and functions as a Trp53-dependent tumor suppressor in Trp53-WT AMLs. Next, to elucidate the underlying mechanisms causing the toxic effects in Trp53-KO AMLs upon Xpo7 depletion, we set out to identify proteins whose localization was potentially regulated by Xpo7 in the Trp53-KO state. Employing mass spectrometry on an immunoprecipitate using anti-Xpo7 and cytoplasmic/nuclear protein extracts from both Xpo7-WT and KO AML cells, we found that Npat, an activator of histone transcription in the G1/S transition, interacted with Xpo7 and showed a significant shift towards cytoplasmic localization upon Xpo7 depletion in Trp53-KO AMLs. Strikingly, Npat protein levels displayed a notable upregulation by Trp53 depletion in AML cells. To evaluate the relevance of the XPO7/NPAT axis in human TP53-mutated AML cells, we examined the correlation between XPO7 and NPAT expression levels and AML subtypes and genetic background using publicly available datasets. XPO7 and NPAT mRNA levels were significantly upregulated in TP53-mutated AMLs in the TCGA datasets. Moreover, XPO7 and NPAT mRNAs levels were remarkably high in acute erythroid leukemia (AEL) cases, where TP53 mutations are frequently observed (Tyner et al. Nature, 2018; Iacobucci et al. Nat Genet. 2019). The Xpo7 and Npat protein expression evaluation using primary AML samples corroborated these findings, revealing overexpression in TP53-mutated AML cases. Finally, we assessed the therapeutic effect of Npat depletion in Trp53-deficient AML cells, using the dTAG system to induce Npat protein degradation by knock-in FKBP12 F36V into Npat loci. dTAG v-1 treatment immediately degraded endogenous Npat protein and led to delayed G1/S transition progression. Moreover, Npat degradation induced DNA damage, particularly in the presence of a WEE1 inhibitor or etoposide, and subsequent apoptosis. Transplantation of these AML cells into immunocompromised mice followed by dTAGv1 treatment for one week demonstrated suppressing leukemia progression in vivo. Furthermore, shRNA-mediated XPO7 or NPAT knockdown significantly suppressed the proliferation of HEL, a human AEL cell line harboring TP53 mutation, in vitro and in vivo. In summary, we identified a synthetic lethal relationship between TP53 and XPO7. We unveiled the novel regulatory mechanism of protein localization by XPO7, depending on TP53 mutational state. Our data suggest that XPO7/NPAT inactivation is a therapeutic vulnerability for TP53-mutated AML.