Abstract: Many cancer therapies induce high response rates, with some resulting in undetectable disease as assessed using standard clinical assays. This is particularly true in leukemia and lymphoma, in which patients often achieve deep remission yet ultimately experience relapse. These outcomes highlight a critical need to better understand ultrarare persistent cells that survive therapy but remain inaccessible to current techniques. Here, we developed Live-cell Pick-Seq (LiP-Seq), an advanced platform leveraging multiplexed live-cell imaging to identify and retrieve individual target cells for downstream analysis. LiP-Seq enables high-resolution transcriptomic profiling of single, viable lymphoma cells present at frequencies as low as 10−6, providing a technological window into the biology of these elusive reservoirs. Applying this method to patients with mantle cell lymphoma after treatment with CD19 chimeric antigen receptor T-cell (CAR-T) therapy, we identified recurrent upregulation of the immune modulator IFITM2 in the persistent cell fraction. Functional validation demonstrated that IFITM2 overexpression conferred protection against CAR-T cytotoxicity in vitro, implicating it as a potential survival mechanism under therapeutic pressure. Our results provide, to our knowledge, the first transcriptomic characterization of viable, ultrarare persistent cells using LiP-Seq, establishing a new paradigm for identifying and targeting features that may enable treatment evasion.

Chemotherapy resistance in acute myeloid leukemia (AML) remains a major clinical challenge. Integration of multiomic profiling and in vivo functional genomics revealed splicing dysregulation as a determinant of chemoresistance in AML. We uncovered a network involving the splicing regulator SRRM1 and the CLK1/4 and PAK1 kinase families as vulnerabilities in chemoresistant AML cells. Both kinase families are hyperactivated in chemoresistant cells, promoting SRRM1 phosphorylation and altering its scaffolding function. We also identified a relapse-associated PAK1 variant, c.1429G>T p.(Ala477→Ser), that confers chemotherapy resistance. Combined PAK1 and CLK1/4 inhibition recapitulated the splicing changes induced by SRRM1 loss, preferentially targeting chemoresistant AML and enhancing chemotherapy efficacy in cell lines, primary cells, and mouse models. Last, we pinpointed MAP2K5 as a critical downstream effector because missplicing of exons 17 and 18 of MAP2K5 upon SRRM1 depletion sensitized cells to chemotherapy. Our findings highlight a therapeutic strategy to overcome AML relapse by targeting splicing dysregulation.

Proteins that drive or support human disease phenotypes are attractive molecular targets for precision therapy, yet most are nominated by knockout studies and then targeted with drugs that inhibit core catalytic pockets. These strategies cannot resolve which residues are essential, whether non-catalytic sites offer better selectivity or potency, or identify on-target resistance mechanisms. We introduce a framework that integrates precision genome editing, mechanistically diverse therapeutics, and computational sequence-structure-function analysis to map protein essentiality and potential druggability at single amino acid resolution. Applying this framework across 9 cyclin-dependent kinases (CDKs) and 15 cancer therapeutics-including ATP-competitive inhibitors, PROTACs, and molecular glue degraders-we identify shared and CDK-specific residues critical for cell fitness and drug response, including known resistance mutations and dozens of new variants. The resulting functional maps resolve residue- and mechanism-specific differences in the resistance spectra among agents targeting the same protein. We show that this iterative strategy can also uncover higher order interactions by performing intra- and extragenic epistasis screens to identify residues that mediate on-target and within-family cell fitness and drug resistance. Finally, we find evidence of novel CDK6 mutations in breast cancer patients and concordance between experimental and clinical correlates of response to CDK4/6 inhibitors. By mapping residue-level essentiality and forecasting therapy resistance mutations, target-drug interaction maps could inform clinical treatment and guide design of more selective therapeutic molecules.

A common mechanism by which cancer cells acquire resistance to chemotherapeutics is through the overexpression of efflux pumps, enabling the removal of cytotoxic agents, such as anthracycline drugs. However, platinum anticancer agents that crosslink DNA and interact with proteins are poor efflux pump substrates. Here, we design dual warhead drug conjugates by tethering a platinum pharmacophore to the doxorubicin backbone. These drug conjugates retain the anticancer activity of anthracyclines and exhibit the ability to both circumvent drug efflux and delay the acquisition of drug resistance. In vivo experiments demonstrate that such drug conjugates extend survival in a preclinical organoid-based model of metastatic colon cancer in mice. Mechanistic studies indicate that these drug conjugates overcome resistance through covalent platinum-protein interactions, leading to significantly improved drug retention and alteration of subcellular drug distribution. This application of platinum offers many opportunities to confront issues related to chemoresistance and alternative pathways for augmenting conventional chemotherapeutics.

Chimeric antigen receptor (CAR) T-cell therapy has transformed the treatment of hematologic cancers. However, its efficacy in solid tumors, including pancreatic ductal adenocarcinoma (PDAC), has been limited. By integrating modular CRISPR screening with immunocompetent orthotopic models of PDAC, we identified unknown tumor-intrinsic modulators of CAR T-cell therapy response. Disruption of genes involved in oxidative and proteotoxic stress, particularly the Nrf2 target Slc33a1 , sensitizes PDAC tumors to CAR T-cell killing. Single cell gene expression analyses revealed that CAR-T resistant tumors exhibit reduced Nrf2 pathway activity. Mechanistically, we show that Nrf2 pathway hyperactivation by genetic ablation of Keap1 or expression of a tumor-derived Keap1 allele sensitized PDAC tumors to CAR T-cell therapy. Thus, cell-intrinsic molecular states accompanying malignant progression can sensitize tumor cells to cell-based immunotherapies. These molecular mechanisms could be exploited to augment both the efficacy of CAR-T cell therapy in solid malignancies, and may allow patient stratification by tumor genotype. Statement of significance:CAR T-cell therapy remains an unsolved challenge for pancreatic cancer. The discovery of tumor-intrinsic mechanisms of resistance has been largely limited by current experimental models. Using large-scale genomic screening in an orthotopic, immunocompetent model of pancreatic cancer, we uncover a role for cell-intrinsic metabolic states in regulating CAR T-cell response.

The mutagenic translesion synthesis (TLS) pathway, which is critically dependent on REV1’s ability to recruit inserter TLS polymerases and the POLζ extender polymerase, enables cancer cells to bypass DNA lesions while introducing mutations that likely contribute to the development of chemotherapy resistance and secondary malignancies. Targeting this pathway represents a promising therapeutic strategy. Here, we demonstrate that the expression of the C-terminal domain (CTD) of human REV1, a ca. 100 amino acid scaffold essential for TLS polymerase interactions, disrupts REV1/POLζ-dependent TLS in mammalian cells. Inducible expression of REV1-CTD in multiple human and murine cancer cell lines sensitizes cells to DNA-damaging agents such as cisplatin, benzo[a]pyrene diol epoxide, and methyl methanesulfonate, without intrinsic cytotoxicity. REV1-CTD expression increases genomic instability, decreases mutagenesis, and enhances G2 arrest following genotoxic stress. Mutational disruption of the CTD’s interaction interfaces abrogates these effects, confirming a dominant-negative mechanism via sequestration of TLS components. In a xenograft mouse model, REV1-CTD expression markedly enhances cisplatin efficacy, significantly reducing tumor burden. These findings establish the REV1-CTD as an effective dominant-negative inhibitor of TLS and support its development as a therapeutic agent delivered to cancer cells to enhance the efficacy of genotoxic chemotherapy.

While targeted therapies have revolutionized cancer treatment, drug resistance remains a major barrier to their curative potential. We recently demonstrated biological proof-of-concept for selection gene drive circuits, a technology that overwrites disease evolution to proactively eliminate resistance in vivo, but translation requires a delivery method compatible with disseminated metastatic disease. Now, we demonstrate a clinically feasible delivery solution with novel tumor-targeting lentiviral vectors that selectively install these therapeutic circuits in tumor cells in situ. Systemic administration of these vectors demonstrated durable elimination of visible tumor burden and minimal body weight loss, validating the translational potential of a new class of genetic medicines for long-term control of resistance in cancer. ### Competing Interest Statement SML and JRP are co-founders of Red Ace Bio. National Cancer Institute, https://ror.org/040gcmg81, U01CA265709

Acquiring nucleotides is essential for all dividing cells, and failure to maintain sufficient levels and balance of nucleotides impairs DNA replication. Eukaryotic cells meet nucleotide demands through either de novo synthesis or salvage of nucleotide precursors from the extracellular environment. Although it is known that levels of salvageable nucleotide precursors vary across tissues, the contribution of nucleotide salvage to total nucleotide acquisition in malignant cells remains underexplored. Using a mouse model of B-cell acute lymphoblastic leukemia (B-ALL), we formulated a mouse-plasma-like medium (MPM) to investigate how cells balance nucleotide acquisition strategies in nutrient environments that mimic mouse plasma during leukemogenesis. We used stable-isotope tracing and LC-MS to measure the contribution of de novo synthesis and salvage pathways for nucleotide acquisition in B-ALL cells. We found that cells cultured in MPM preferentially acquire some nucleotide species through salvage pathways, and genetic perturbation of nucleotide salvage pathways leads to deoxynucleotide triphosphate (dNTP) depletion, induction of replication stress, and reduced proliferation. We found that the dependence on nucleotide salvage arises because physiological levels of the vitamin folate, a molecule used in de novo nucleotide synthesis, are low enough to constrain de novo nucleotide biosynthesis in physiological conditions, driving increased reliance on salvage pathways. Accordingly, culturing cancer cells in conventional cell culture media with folate levels found in physiological conditions attenuates cell proliferation due to decreased de novo nucleotide synthesis. In vivo, genetically perturbing pyrimidine nucleotide salvage slows B-ALL proliferation, and this is further exacerbated when mice were fed folate-depleted diets. On the other hand, dietary folate supplementation increased the proliferation of salvage deficient cells in vivo. Together, these findings demonstrate that physiological folate availability limits de novo nucleotide synthesis in B-ALL, creating dependence on nucleotide salvage pathways. This work informs potential combination therapy strategies targeting folate metabolism and nucleotide salvage. Ryan Elbashir, Keene L. Abbott, Ahmed Ali, Diya L. Ramesh, Michelle Wu, Brian T. Do, Anya Shevzov-Zebrun, Tenzin Kunchok, Millenia Waite, Wontaek Chung, Chelsea Zhang, Sharanya Sivanand, Azucena Ramos, Jacob A. Hansen, Raphael Ferreira, Alexander Muir, Michael H. Hemann, Matthew G. Vander Heiden. Physiological nutrient levels reveal nucleotide salvage as a dependency in B-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3800.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second leading cause of cancer death by 2030. PDAC lacks effective treatment options and innovative strategies for therapeutic intervention are urgently needed. Chimeric Antigen Receptor (CAR)-T cell therapy has shown remarkable success in treating hematologic malignancies, yet achieving efficacy in solid tumors remains a significant challenge. Early results from clinical trials show limited efficacy of CAR T-cell therapy in PDAC. To uncover tumor-intrinsic mechanisms of resistance to CAR T-cell therapy in vivo, we performed iterative CRISPR/Cas9-based pooled screens in a fully immunocompetent, orthotopic model of PDAC. We identified a variety of genes influencing CAR T-cell treatment efficacy. In particular, loss of genes involved in redox biology and oxidative stress sensitized PDAC tumors to CAR T-cell therapy in vivo. Additionally, single-cell RNA sequencing of PDAC tumors subjected to CAR T-cell therapy unveiled a distinct subset of tumor cells resilient to the treatment, characterized by changes in oxidative stress. Our findings indicate a potential avenue for therapeutic synergy by targeting pathways involved in oxidative stress to increase the efficacy of CAR T cell therapy in PDAC. Citation Format: Julia Froese, Charlie Whittaker, Paul Leclerc, Adam Langenbucher, Riley Hellinger, Daniel Goulet, Michael T. Hemann. Elucidating the tumor-driven mechanisms of resistance to immunotherapies in pancreatic cancer through in vivo CRISPR/Cas9 knockout screening [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr B039.

CAR T cell therapy has revolutionized the treatment of a spectrum of blood-related malignancies. However, treatment responses vary among cancer types and patients. Accurate monitoring of CAR T cell dynamics is crucial for understanding and evaluating treatment efficacy. Positron emission tomography (PET) offers a comprehensive view of CAR T cell homing, especially in critical organs such as lymphoid structures and bone marrow. This information will help assess treatment response and predict relapse risk. Current PET imaging methods for CAR T require genetic modifications, limiting clinical use. To overcome this, we developed an antigen-based imaging approach enabling whole-body CAR T cell imaging. The probe detects CAR T cells in vivo without affecting their function. In an immunocompetent B cell leukemia model, CAR-PET signal in the spleen predicted early mortality risk. The antigen-based CAR-PET approach allows assessment of CAR T therapy responses without altering established clinical protocols. It seamlessly integrates with FDA-approved and future CAR T cell generations, facilitating broader clinical application.

In immunocompetent hosts, the ablation of FAS and CD3 in allogeneic chimeric antigen receptor T cells confers them with partial protection from rejection by T cells and natural killer cells.

BackgroundIntegrating molecular-targeted agents into combination chemotherapy is transformative for enhancing treatment outcomes in cancer. However, realizing the full potential of this approach requires a clear comprehension of the genetic dependencies underlying drug synergy. While the interactions between conventional chemotherapeutics are well-explored, the interplay of molecular-targeted agents with conventional chemotherapeutics remains a frontier in cancer treatment. Hence, we leveraged a powerful functional genomics approach to decode genomic dependencies that drive synergy in molecular-targeted agent/chemotherapeutic combinations in gastric adenocarcinoma, addressing a critical need in gastric cancer therapy.MethodsWe screened pharmacological interactions between fifteen molecular-targeted agent/conventional chemotherapeutic pairs in gastric adenocarcinoma cells, and examined the genome-scale genetic dependencies of synergy integrating genome-wide CRISPR screening with the shRNA-based signature assay. We validated the synergy in cell death using fluorescence-based and lysis-dependent inference of cell death kinetics assay, and validated the genetic dependencies by single-gene knockout experiments.ResultsOur combination screen identified SN-38/erlotinib as the drug pair with the strongest synergism. Functional genomics assays unveiled a genetic dependency signature of SN-38/erlotinib identical to SN-38. Remarkably, the enhanced cell death with improved kinetics induced by SN-38/erlotinib was attributed to erlotinib's off-target effect, inhibiting ABCG2, rather than its on-target effect on EGFR.ConclusionIn the era of precision medicine, where emphasis on primary drug targets prevails, our research challenges this paradigm by showcasing a robust synergy underpinned by an off-target dependency. Further dissection of the intricate genetic dependencies that underlie synergy can pave the way to developing more effective combination strategies in gastric cancer therapy.

There are hundreds of genes typically overexpressed in breast cancer cells and it's often assumed that their overexpression contributes to cancer progression. However, the precise proportion of these overexpressed genes contributing to tumorigenicity remains unclear. To address this gap, we undertook a comprehensive screening of a diverse set of seventy-two genes overexpressed in breast cancer. This systematic screening evaluated their potential for inducing malignant transformation and, concurrently, assessed their impact on breast cancer cell proliferation and viability. Select genes including ALDH3B1, CEACAM5, IL8, PYGO2, and WWTR1, exhibited pronounced activity in promoting tumor formation and establishing gene dependencies critical for tumorigenicity. Subsequent investigations revealed that CEACAM5 overexpression triggered the activation of signaling pathways involving β-catenin, Cdk4, and mTOR. Additionally, it conferred a growth advantage independent of exogenous insulin in defined medium and facilitated spheroid expansion by inducing multiple layers of epithelial cells while preserving a hollow lumen. Furthermore, the silencing of CEACAM5 expression synergized with tamoxifen-induced growth inhibition in breast cancer cells. These findings underscore the potential of screening overexpressed genes for both oncogenic drivers and tumor dependencies to expand the repertoire of therapeutic targets for breast cancer treatment.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second leading cause of cancer death by 2030. PDAC lacks effective treatment options and innovative strategies for therapeutic intervention are urgently needed. Chimeric Antigen Receptor (CAR)-T cell therapy has shown remarkable success in treating hematologic malignancies, yet achieving efficacy in solid tumors remains a significant challenge. Early results from clinical trials show limited efficacy of CAR T-cell therapy in PDAC. To uncover tumor-intrinsic mechanisms of resistance to CAR T-cell therapy in vivo, we performed iterative CRISPR/Cas9-based pooled screens in a fully immunocompetent, orthotopic model of PDAC. We identified a variety of genes influencing CAR T-cell treatment efficacy. In particular, loss of genes involved in redox biology and oxidative stress sensitized PDAC tumors to CAR T-cell therapy in vivo. Additionally, single-cell RNA sequencing of PDAC tumors subjected to CAR T-cell therapy unveiled a distinct subset of tumor cells resilient to the treatment, characterized by changes in oxidative stress. Our findings indicate a potential avenue for therapeutic synergy by targeting pathways involved in oxidative stress to increase the efficacy of CAR T cell therapy in PDAC. Citation Format: Julia Fröse, Charlie Whittaker, Paul Leclerc, Adam Langenbucher, Riley Hellinger, Daniel R. Goulet, Michael T. Hemann. Unveiling resistance mechanisms to CAR T-cell therapy in pancreatic cancer through in vivo CRISPR/Cas9 knockout screening [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B060.

Abstract Glioblastoma (GBM) is the most common and deadly adult brain cancer with a post-diagnosis survival of 8-15 months even with treatment, highlighting a desperate need for new therapeutic strategies. One promising strategy involves the use of chimeric antigen receptor T-cell (CAR-T) therapy, created by engineering patient T-cells to express a CAR that recognizes a tumor-specific surface antigen thus allowing for directed tumor cell killing. While CAR-T therapy has experienced unprecedented success in treating hematologic malignancies, its application in solid tumors has proven challenging. However, early clinical studies in GBM have shown promise including a case in which a patient with disseminated GBM experienced a complete response. Unfortunately, the patient relapsed highlighting both potential therapeutic efficacy and our limited knowledge of resistance and relapse mechanisms. Established resistance mechanisms include tumor target antigen loss or intrinsic CAR-T failure, but there are certainly others that have yet to be identified. Furthermore, solid tumors present unique challenges and may possess distinctive resistance mechanisms related to hostile, immunosuppressive tumor microenvironments and vast inter-tumoral heterogeneity. Ultimately, improved understanding could lead to increased therapeutic efficacy through CAR-T combination therapies or direct CAR-T cell modifications. Beyond the promise of CAR-T however, there still remains a scarcity of effective treatment options for GBM, underscoring a critical need to gain a better fundamental understanding in order to create additional novel therapeutic strategies. In order to both understand mechanisms of resistance to CAR-T therapy in GBM and uncover novel tumor dependencies for therapeutic exploitation, I took an unbiased, functional genomics approach. I conducted a series of in vivo genome-wide screens in an orthotopic, immunocompetent mouse model of GBM using a novel, modular, small-pooled CRISPR/Cas9 single guide (sgRNA) library. Cas9-expressing tumor cells with library sgRNAs were intracranially injected into mice that subsequently received no treatment or direct intertumoral injection of non-targeting control or targeting CAR-T cells. Top depletion and enrichment hits were used to generate 2 separate validation libraries one containing CAR-T sensitization or resistance hits and the other containing hits essential for in vivo tumor growth and therefore potentially exploitable as novel therapeutic targets. Validation libraries were screened in the same manner as the original genome-wide screen and top hits are actively being validated with planned in-depth mechanistic follow up. Importantly, to date, these are the first in vivo genome-wide CRISPR/Cas9 screens conducted in an orthotopic, immunocompetent mouse model of GBM. Ultimately, these results could aid not only in the identification of novel therapeutic strategies for GBM and potential improvements in CAR-T clinical efficacy, but also in a broader understanding of the fundamental biology that underlies treatment resistance in GBM. Citation Format: Catherine E. Koch, Charles A. Whittaker, Michael T. Hemann. In vivo genome-wide CRISPR/Cas9 screens conducted in an immunocompetent mouse model of glioblastoma identify novel in vivo tumor liabilities and potential mechanisms of resistance to chimeric antigen receptor T-cell (CAR-T) therapy [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr PR-003.

Background and Significance: Measurable residual disease (MRD) represents the fundamental driver of relapse and mortality in acute myeloid leukemia (AML). However, there are currently no established approaches to address this unmet need. Our central hypothesis is that targeting vulnerabilities associated with specific leukemia genotypes will eradicate MRD and prevent disease relapse. Break Through Cancer is a collaboration between our centers aimed at making progress in the deadliest cancers by stimulating radical clinical and laboratory research collaboration. The menin-KMT2A interaction is a critical dependency in acute leukemias caused by rearrangement of the Lysine methyl transferase (KMT2Ar) or Nucleoporin 98 (NUP98r) genes, or mutation of the Nucleophosmin 1 gene (NPM1mt). Revumenib (previously SNDX-5613), is a potent, oral, selective inhibitor of this interaction with an established safety and efficacy in refractory leukemias with these genotypes (Issa GC, Nature 2023). Additionally, KMT2Ar or NPM1mt leukemias can undergo BCL2-dependent apoptosis, and dual Bcl-2 and menin inhibition led to synergistic activity in KMT2Ar or NPM1mt leukemia models (Carter BZ, Blood 2021; Fiskus W, BCJ 2022). Therefore, we designed this study investigating the combination of revumenib and venetoclax to eradicate MRD in these AML subtypes (NCT06284486). Study Design and Methods: This is a single arm, open label, multicenter, phase I/II investigator-initiated study. Patients (pts) age ≥ 12 years with weight ≥ 45Kg, and known history of NPM1mt, or KMT2Ar, or NUP98r AML with MRD ≥ 0.1% identified by multiparameter flow cytometry (MFC) using central testing would be eligible; no morphologic evidence of AML (blasts <5%) in first remission following high intensity therapy or at least 2 cycles of low intensity therapy, or in second remission following any therapy. Up to 12 pts will be enrolled on the phase I, using 3+3, with escalating doses of revumenib and a target dose of 163 mg PO Q12h (with strong CYP3A inhibitor) or 276 mg PO Q12h (without strong CYP3A inhibitor) days 1-28, with venetoclax 400 mg (target dose) PO daily, days 1-14. The primary objective of the phase I is to determine safety, and the recommended phase II dose. The primary objective of the phase II is to assess the efficacy of venetoclax and revumenib in clearance of MRD (conversion to undetectable by central MFC) within 8 cycles. Up to 14 pts will be included on the phase II portion of the study. With a sample size of 20 pts (Phase II + RP2D in Phase I), the power is 76% assuming an MRD clearance of 30% (based on QUAZAR trial, Roboz et al. Blood 2022) against a null rate of 8%, by a two-sided Fisher's exact test at a significance level of 0.05. We plan to monitor futility and toxicity where enrollment will be stopped early if >95% probability that the MRD conversion rate is < 30% or there is >90% probability that the unacceptable toxicity rate >20%. Secondary objectives include assessment of duration of response, event-free and overall survival and concordance of genetic and flow MRD. This trial includes longitudinal collection of samples, with exploratory objectives focused on improving MRD detection using cell-free DNA, single-cell mutation analysis and cytometry by time of flight. In addition, we aim to use various models in addition to patient samples to improve understanding of MRD biology and identify novel susceptibilities. Accrual is planned at MD Anderson, Dana Farber, Memorial Sloan Kettering, and Johns Hopkins. This study may identify a novel strategy to eradicate MRD using combination targeted therapies which may decrease recurrence and improve remission duration. In addition, this study could improve detection of MRD, and our understanding of MRD biology.

Supplementary Figures 1-6 and Supplementary Methods