Drug-tolerant Persister Cells Research Articles

Focal adhesion kinase-YAP signaling axis drives drug-tolerant persister cells and residual disease in lung cancer

Targeted therapy is effective in many tumor types including lung cancer, the leading cause of cancer mortality. Paradigm defining examples are targeted therapies directed against non-small cell lung cancer (NSCLC) subtypes with oncogenic alterations in EGFR, ALK and KRAS. The success of targeted therapy is limited by drug-tolerant persister cells (DTPs) which withstand and adapt to treatment and comprise the residual disease state that is typical during treatment with clinical targeted therapies. Here, we integrate studies in patient-derived and immunocompetent lung cancer models and clinical specimens obtained from patients on targeted therapy to uncover a focal adhesion kinase (FAK)-YAP signaling axis that promotes residual disease during oncogenic EGFR-, ALK-, and KRAS-targeted therapies. FAK-YAP signaling inhibition combined with the primary targeted therapy suppressed residual drug-tolerant cells and enhanced tumor responses. This study unveils a FAK-YAP signaling module that promotes residual disease in lung cancer and mechanism-based therapeutic strategies to improve tumor response.

Intratumor transforming growth factor-β signaling with extracellular matrix-related gene regulation marks chemotherapy-resistant gastric cancer

Drug-tolerant persister (DTP) cells remain following chemotherapy and can cause cancer relapse. However, it is unclear when acquired resistance to chemotherapy emerges. Here, we compared the gene expression profiles of gastric cancer patient-derived cells (GC PDCs) and their respective xenograft tumors with different sensitivities to 5-fluorouracil (5-FU) by using immunodeficient female BALB/c-nu mice. RNA sequencing analysis of 5-FU-treated PDCs demonstrated that DNA replication/cell cycle-related genes were transiently induced in the earlier phase of DTP cell emergence, while extracellular matrix (ECM)-related genes were sustainably upregulated during long-term cell survival in 5-FU-resistant residual tumors. NicheNet analysis, which uncovers cell-cell signal interactions, indicated the transforming growth factor-β (TGF-β) pathway as the upstream regulator in response to 5-FU treatment. This induced ECM-related gene expression in the 5-FU-resistant tumor model. In the 5-FU-resistant residual tumors, there was a marked upregulation of cancer cell-derived TGF-β1 expression and increased phosphorylation of SMAD3, a downstream regulator of the TGF-β receptor. By contrast, these responses were not observed in a 5-FU-sensitive tumor model. We further found that TGF-β-related upregulation of ECM genes was preferentially observed in non-responders to chemotherapy with 5-FU and/or oxaliplatin among 22 patient-derived xenograft tumors. These observations suggest that chemotherapy-induced activation of the TGF-β1/SMAD3/ECM-related gene axis is a potential biomarker for the emergence of drug resistance in GCs.

ATF3 characterizes aggressive drug-tolerant persister cells in HGSOC

High-grade serous ovarian cancer (HGSOC) represents the most common and lethal subtype of ovarian cancer. Despite initial response to platinum-based standard therapy, patients commonly suffer from relapse that likely originates from drug-tolerant persister (DTP) cells. We generated isogenic clones of treatment-naïve and cisplatin-tolerant persister HGSOC cells. In addition, single-cell RNA sequencing of barcoded cells was performed in a xenograft model with HGSOC cell lines after platinum-based therapy. Published single-cell RNA-sequencing data from neo-adjuvant and non-treated HGSOC patients and patient data from TCGA were analyzed. DTP-derived cells exhibited morphological alterations and upregulation of epithelial-mesenchymal transition (EMT) markers. An aggressive subpopulation of DTP-derived cells showed high expression of the stress marker ATF3. Knockdown of ATF3 enhanced the sensitivity of aggressive DTP-derived cells to cisplatin-induced cell death, implying a role for ATF3 stress response in promoting a drug tolerant persister cell state. Furthermore, single cell lineage tracing to detect transcriptional changes in a HGSOC cell line-derived xenograft relapse model showed that cells derived from relapsed solid tumors express increased levels of EMT and multiple endoplasmic reticulum (ER) stress markers, including ATF3. Single cell RNA sequencing of epithelial cells from four HGSOC patients also identified a small cell population resembling DTP cells in all samples. Moreover, analysis of TCGA data from 259 HGSOC patients revealed a significant progression-free survival advantage for patients with low expression of the ATF3-associated partial EMT genes. These findings suggest that increased ATF3 expression together with partial EMT promote the development of aggressive DTP, and thereby relapse in HGSOC patients.

Diverse functions of cytochrome c in cell death and disease.

The redox-active protein cytochrome c is a highly positively charged hemoglobin that regulates cell fate decisions of life and death. Under normal physiological conditions, cytochrome c is localized in the mitochondrial intermembrane space, and its distribution can extend to the cytosol, nucleus, and extracellular space under specific pathological or stress-induced conditions. In the mitochondria, cytochrome c acts as an electron carrier in the electron transport chain, facilitating adenosine triphosphate synthesis, regulating cardiolipin peroxidation, and influencing reactive oxygen species dynamics. Upon cellular stress, it can be released into the cytosol, where it interacts with apoptotic peptidase activator 1 (APAF1) to form the apoptosome, initiating caspase-dependent apoptotic cell death. Additionally, following exposure to pro-apoptotic compounds, cytochrome c contributes to the survival of drug-tolerant persister cells. When translocated to the nucleus, it can induce chromatin condensation and disrupt nucleosome assembly. Upon its release into the extracellular space, cytochrome c may act as an immune mediator during cell death processes, highlighting its multifaceted role in cellular biology. In this review, we explore the diverse structural and functional aspects of cytochrome c in physiological and pathological responses. We summarize how posttranslational modifications of cytochrome c (e.g., phosphorylation, acetylation, tyrosine nitration, and oxidation), binding proteins (e.g., HIGD1A, CHCHD2, ITPR1, and nucleophosmin), and mutations (e.g., G41S, Y48H, and A51V) affect its function. Furthermore, we provide an overview of the latest advanced technologies utilized for detecting cytochrome c, along with potential therapeutic approaches related to this protein. These strategies hold tremendous promise in personalized health care, presenting opportunities for targeted interventions in a wide range of conditions, including neurodegenerative disorders, cardiovascular diseases, and cancer.

Abstract 5839: Identification of low-dose ligand dependent drug-resistance in ALK/ROS1 positive NSCLC via CRISPR library screening

Abstract Tyrosine kinase inhibitor (TKI) therapy has displayed significant effectiveness in treating patients with anaplastic lymphoma kinase (ALK) and ROS proto-oncogene 1 (ROS1)-positive non-small cell lung cancer (NSCLC). Nonetheless, the emergence of acquired resistance in most patients presents a major challenge. Drug-tolerant persister (DTP) cells have recently gained attention as potential sources of resistance, highlighting the need for a deeper understanding of drug resistance mechanisms. To address this issue, a comprehensive genome-wide CRISPR-Cas9 knockout screening was conducted using an ALK-positive NSCLC cell line derived from pleural effusion in a patient who had not been exposed to ALK-TKI treatment. Following a 9-day ALK-TKI treatment, sequencing analysis was performed to identify sgRNAs in DTP cells. Notably, this analysis detected the involvement of tumor suppressor genes, including NF2, BAX, and ERRFI1.NF2 depletion was found to induce a substantial resistance to ALK-TKIs, and a combination therapy involving mTOR inhibitors showed promise in partially overcoming this resistance. Moreover, knockout of ERRFI1 or BAX led to an increase in DTP cells, drawing particular attention to ERRFI1, also known as MIG6, which is known for negatively regulating EGFR signaling. One intriguing discovery was that the loss of MIG6 induced resistance to ALK-TKIs when exposed to a relatively low dose of EGF, comparable to concentrations in plasma of healthy adults. This resistance was attributed to the upregulation of the MAPK and PI3K/Akt/mTOR pathways. Similar to ALK-rearranged NSCLC, ROS1 fusion-positive NSCLC cell lines also showed resistance by MIG6 knockout. Interestingly, some cell lines with reduced MIG6 expression tended to develop resistance with low-dose EGF, even without prior treatment, and overexpression of MIG6 restored sensitivity to TKIs. We further explored the potential of combination therapy, combining ALK-TKIs with anti-EGFR antibodies, and this approach effectively addressed acquired resistance in both in vivo and in vitro models. Furthermore, an analysis of clinical samples revealed that individuals resistant to ALK-TKIs displayed decreased mRNA MIG6 levels in comparison to their sensitive counterparts. Investigating the mechanisms behind MIG6 expression, we confirmed that ALK-TKI therapy suppressed MIG6 expression levels, while factors such as corticosteroid usage or exposure to hypoxia induced the upregulation of MIG6, potentially influencing the formation of DTP cells. In conclusion, this research has unveiled a novel factor contributing to resistance in ALK and ROS1-TKI therapies by activating the EGFR pathway through exposure to low-dose ligands, shedding light on potential therapeutic strategies to combat acquired resistance. Citation Format: Nobuyuki Kondo, Takahiro Utsumi, Ken Uchibori, Yasunari Miyazaki, Ryohei Katayama. Identification of low-dose ligand dependent drug-resistance in ALK/ROS1 positive NSCLC via CRISPR library screening [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 5839.

Abstract 2006: Deciphering the interplay of β-catenin/YAP signaling and alveolar lineage plasticity during targeted therapy in non-small cell lung cancer

Abstract Molecularly targeted therapies have significantly enhanced clinical outcomes by directly addressing oncogenic driver mutations in genes such as EGFR. However, despite the success of treatments targeting these mutations, long-term patient survival remains limited. This study investigates the molecular mechanisms underlying drug-tolerance in response to targeted therapies in non-small cell lung cancer (NSCLC). Using single-cell RNA sequencing of oncogene-driven human NSCLCs at the residual disease (RD) state during active targeted therapy, our investigation reveals an increase of an alveolar type I/II (AT1/2) mixed lineage program in surviving cancer cells, resembling an alveolar cell-like state. We observed increased WNT3A/4 ligands, FZD2 receptors, β-catenin signaling, and AT1/2 signatures at the RD stage. Consistent with clinical results, AT1/2 signatures and target genes of β-catenin and YAP are increased in drug-tolerant persister (DTP) cells in preclinical models. Additionally, we discovered that β-catenin and YAP form a stable nuclear protein complex in DTP cells. Disrupting β-catenin/YAP protein expression and stability in DTP cells by knocking down FZD2 or interrupting the destruction complex of β-catenin/YAP leads to re-sensitization to the EGFR inhibitor Osimertinib, suggesting a potential therapeutic approach. Furthermore, this therapeutic strategy decreased AT1/2 gene signatures. Our results highlight an interdependence between β-catenin and YAP, with both proteins playing crucial roles in sustaining DTP cells and promoting the alveolar cell like lineage plasticity. These findings shed light on the complex interplay between β-catenin/YAP signaling and alveolar signatures in the residual cancer state, offering mechanistic insights and potential therapeutic strategies to address drug tolerance and improve clinical outcomes. Citation Format: Yu-Ting Chou, Wei Wu, Daniel Kerr, Macey Slota, Trever Bivona. Deciphering the interplay of β-catenin/YAP signaling and alveolar lineage plasticity during targeted therapy in non-small cell lung 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 2006.

Abstract 5853: Regulatory network inference of lung adenocarcinoma drug-tolerant persister cells

Abstract Despite the effectiveness of the latest generation of cancer drugs, partial tumor response and subsequent disease relapse is commonly observed in patients. Recent evidence suggests that treatment resistance emerges from drug-tolerant persister cells (DTPC) that survive through non-genetic tumor cell adaptation. Although DTPC are mostly slow-cycling, a rare subset can re-enter the cell cycle under constitutive presence of treatment. Understanding how DTPC dynamically remodel their molecular portraits at the single-cell level may provide high-resolution insights into the molecular circuits associated with adaptive survival to therapy.Here, we use single-cell RNA sequencing (scRNA-seq) to identify potential mechanisms that promote survival of DTPC and decipher their underlying gene regulatory networks (GRNs), in a model of EGFR-mutant lung adenocarcinoma treated with osimertinib (EGFR inhibitor). Gene expression analysis revealed fewer cell states in DTP compared to treatment-naïve cells (TN). We confirmed the acquisition of DTPC hallmarks (e.g., high epithelial-mesenchymal transition, slow-cycling phenotype, deficient DNA repair). We also identified subpopulations of cells that display intermediate transcriptional states: a pre-existing DTP-like state in a bulk of TN cells, and DTPC with enriched cell cycle signatures, evoking a phenotype of cycling DTPC. To characterize the GRN that govern TN cells, DTPC and the intermediate cell states, we applied the network inference tool PYSCENIC, which associates transcription factors (TFs) to their candidate target genes (TGs) on the basis of the scRNA-seq data and enrichment of TF binding motifs in the genomic regions around TGs. The resulting (GRN) consisted of 5 TF modules clustered on the basis of correlated TF activity.This analysis revealed that, compared to TN cells, DTPC and the intermediate cell states display a marked decreased expression of TFs and TGs involved in mRNA splicing, and more specifically in the initiation of spliceosome assembly through E-complex and A-complex formation. Interestingly, the splicing-related TFs all appear to have affinity for binding to a GCCxC consensus motif or close variant in genes (i.e., SNRPB and SNRPE) that code for key effectors of these initiation complexes. This pilot study highlights that scRNA-seq analyses coupled with inference of regulatory networks might enable the identification of molecular mechanisms that potentially trigger cell state transitions during drug-tolerance. The role of altered mRNA splicing in persistence to EGFR-targeted therapies will be explored experimentally in patient-derived cell lines and 3D organoid cultures. Citation Format: Lisa Chabrier, Camille Leonce, Raphael Schneider, Cyril Degletagne, Pierre Saintigny, Anton Crombach, Sandra Ortiz-Cuaran. Regulatory network inference of lung adenocarcinoma drug-tolerant persister 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 5853.

Abstract 3262: Elucidating the mechanism of resistance to tyrosine kinase inhibitors in ROS1-and ALK-fusion non-small cell lung cancer: The role of CD74

Abstract The therapeutic landscape in treating advanced oncogene-mutated non-small cell lung cancer (NSCLC) was revolutionized with the advent of tyrosine kinase inhibitors (TKIs). Despite the efficacious breakthrough and improved patient outcomes, disease progresses once more due to the seemingly inevitable development of drug resistance in tumor cells. This poses a considerable predicament in the clinical management of oncogene-mutated NSCLC and signifies the importance of elucidating the mechanism of resistance in tumor cells that harbor these genomic alterations which give rise to oncogenic drivers. Recent evidence suggests that drug-tolerant persister (DTP) cells, which are tolerant to initial exposure to TKIs give rise to acquired drug-resistant cells. Currently, the mechanism by which tumor cells undergo a drug-tolerant state is not well understood in NSCLCs driven by ALK or ROS1 fusion. Previously, we identified CD74 as a gene that may play a role in a drug-tolerant state in EGFR-mutated lung cancer via single-cell RNA-sequencing (scRNA-seq). Here, we sought to determine whether the CD74 signaling also leads to resistance to TKIs in NSCLCs with ALK or ROS1 fusion. We showed that CD74 expression increased at both the mRNA and protein levels after treatment with respective TKIs in ALK and ROS1 fusion cells lines. Notably, the ALK and ROS1 fusion cell lines also exhibited an increase in mRNA expression levels of upstream and downstream genes of CD74. Additionally, we generated CD74 knock-out and overexpression models in two cells lines, HCC78 (SLC34A2-ROS1 fusion) and H2228 (EML4–ALK fusion), to further examine the role of CD74 expression in DTP cells. We demonstrated that resistance to TKIs were dependent on CD74 expression. Furthermore, it was determined that anti-CD74 antibody-drug conjugates, in combination with TKIs, synergistically induced apoptosis in these cell lines. In conclusion, our data suggest that CD74 can be a therapeutic target in NSCLC with ALK or ROS1 fusion. Citation Format: Vivian Ho, Motohiro Izumi, Meghan Lee, Daisuke Shibahara, Ikei S. Kobayashi, Susumu S. Kobayashi. Elucidating the mechanism of resistance to tyrosine kinase inhibitors in ROS1-and ALK-fusion non-small cell lung cancer: The role of CD74 [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 3262.

Abstract 1221: Selective translational activation of PBK (PDZ- binding kinase) reveals a molecular vulnerability of drug-tolerant persister cells to targeted therapies in EGFR-mutant lung adenocarcinoma

Abstract In lung adenocarcinoma (LUAD), EGFR-targeted therapies often result in incomplete tumor responses followed by disease progression caused by acquired resistance. As EGFR mutations are truncal (i.e., present in every cell of a tumor), it remains unclear why a fraction of tumor cells can survive in the presence of drug treatment. It is increasingly recognized that drug-tolerant persister cells (DTPCs) can escape selective drug pressure and are important drivers of therapy failure and tumor relapse. The mechanisms underlying tumor cell adaptation and survival of DTPCs during therapy are not fully understood.Using EGFR-mutant LUAD cell lines treated with osimertinib, we unexpectedly observed that DTPCs display uncoupled mRNA and protein variations of PBK (PDZ Binding Kinase), a spindle assembly serine/threonine protein kinase. Mechanistically we discovered that although global protein synthesis is decreased upon osimertinib treatment, PBK translation is selectively maintained in DTPCs.The establishment and survival of DTPCs following EGFR inhibition is critically dependent on PBK, as genetic depletion of PBK reduces persistence to osimertinib treatment in vitro. PBK knockout leads to suppression of ERK1/2 reactivation following EGFR inhibition, and results in decreased expression of ZEB1 and Vimentin, key effectors of epithelial-to-mesenchymal transition (EMT), a hallmark of the DTPC phenotype. Consistently, combined treatment with osimertinib and OTS514 (PBK inhibitor) in vitro, reduces the DTPC fraction and decreases the enrichment of EMT-related transcriptional signatures. Similar results were obtained in LUAD patient-derived organoids, used to recapitulate the phenotype of residual disease in patients. In PDX treated with osimertinib, PBK is activated at both residual disease and acquired resistance. Concordantly, combined EGFR/PBK pharmacological inhibition was also effective in reverting osimertinib acquired resistance in vitro. Finally, in matched diagnosis and relapse samples from 7 EGFR-mutant LUAD patients treated with EGFR inhibitors, we observed increased PBK activation at the time of acquired resistance in 5 cases, including 3 EGFRT790M-positive cases, suggesting that PBK activation may co-occur with genetic resistant alterations.Our findings unveil a novel mechanism by which EGFR-mutant lung cancer DTPCs selectively translate PBK mRNA as an adaptive survival mechanism to persist during osimertinib treatment; and provide a therapeutic target of non-genetic drug resistance otherwise unlooked-for in routine transcriptomic analyses. A deeper understanding on the mRNA translational reprogramming in DTPCs is currently performed to uncover therapeutic vulnerabilities to reduce residual disease and delay or revert the emergence of acquired resistance. Citation Format: Camille Leonce, Anne-Laure Désage, Nour El-Houda Mourksi, Geneviève De Souza, Béatrice Vanbervliet, Camille Charrondière, Constance Nicq, Jérémy Néri, Aurélie Swalduz, Laurie Tonon, Eric Cumunel, Nicolas Gadot, Gabriel Ichim, Jean-Jacques Diaz, Christine Lovly, Julien Mazières, Mayeul Tabutin, Maurice Pérol, Sylvie Lantuejoul, Olivier Calvayrac, Fabien Forest, Virginie Marcel, Pierre Saintigny, Sandra Ortiz-Cuaran. Selective translational activation of PBK (PDZ- binding kinase) reveals a molecular vulnerability of drug-tolerant persister cells to targeted therapies in EGFR-mutant lung adenocarcinoma [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 1221.

Abstract 457: Persister cell state-associated lncRNA signatures in non-small cell lung cancer

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide, largely due to late-stage diagnosis and a 5-year survival rate of 9%. Targeted therapies for oncogene-dependent tumors like tyrosine kinase inhibitors (TKIs) improve outcomes yet their clinical efficacy is often limited by occult drug-tolerant persister cells (DTPCs), causing recurrence. Long non-coding RNAs (lncRNAs) are transcripts over 200 nucleotides and typically non-protein coding. They regulate cellular functions by interacting with substrates through their complex secondary structures. Their deregulation is linked to oncogenesis and therapy resistance. While their role in lung cancer has been documented, their involvement in the DTPC state remains unexplored. Our study aims to investigate the role of lncRNAs in mechanisms that drive failure to targeted therapy in NSCLC. We comprehensively analyzed single-cell RNA-seq data from 49 NSCLC biopsies, pre- and during TKI therapy (residual disease, RD). We identified several lncRNAs that were significantly, differentially expressed in RD when compared to treatment naïve (TN) tumors. We orthogonally validated these findings in a minimum of 2 lung cancer cell lines in TN or DTPCs (9-15 days upon TKI exposure in culture) [p<0.05, absolute log2 fold change > 0.3]. We found that MALAT1, HCG11, and EPB41L4A-AS1 (lncRNA-UP signature) displayed significantly increased expression in RD relative to TN tumors and parental cell lines. We also found significant downregulation of LUCAT1 and LINC00152 (lncRNA-DOWN signature) in RD samples. We further performed Kaplan-Meier survival analysis in an independent set of RD samples stratified by differential expression of our lncRNA signatures (n=18/group). The analysis showed improved overall survival in patients whose DTPCs expressed a high level of the lncRNA-UP signature (hazard ratio, HR=0.56; log-rank test p=0.0331). We did not observe significant differences in survival for the lncRNA-DOWN signature. We additionally performed survival analysis using the GEPIA database for lung adenocarcinoma (primarily TN, n= 239/group). Consistently, the analysis showed improved overall survival for high levels of the lncRNA-UP signature (HR=0.71; p=0.029), whereas high levels of the lncRNA-DOWN signature were indicative of inferior outcomes (HR=1.4, p=0.046). We systematically identified DTPC-associated lncRNA signatures from our unique DTPC cohort, indicating that deregulation of lncRNA expression may play a role in acquired drug resistance in lung cancer. DTPC-associated lncRNA expression patterns are linked to differential overall survival, suggesting a possible tumor-suppressive or promoting role and highlighting their potential as predictive biomarkers and therapeutic targets. Further investigation into the functional roles of these lncRNAs and their interplay with coding molecules is warranted. Citation Format: Jovanka Gencel-Augusto, Wei Wu, Trever G. Bivona. Persister cell state-associated lncRNA signatures in non-small cell lung 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 457.

Abstract 4653: Unraveling the vulnerabilities of targeted therapy-tolerant persister cells in NSCLC

Abstract Genetic profiling and the development of targeted therapy has improved survival of many cancer patients, but resistance to targeted therapy remains a major barrier to long-term patient survival. To develop more pro-active strategies to improve clinical outcomes, our research focuses on drug-tolerant persister cells that constitute a reservoir of cancer cells that survive and adapt during the initial stage of therapy. These cells typically display therapy resistance in a reversible manner and set the stage for the subsequent emergence of cells with full acquired resistance. We utilized quantitative proteomics to characterize persister cells generated from EGFRmut, KRASmut, and ALKmut non-small cell lung cancer (NSCLC) cell lines after treatment with osimertinib, sotorasib, and lorlatinib, respectively. Integrating proteomics data with network analysis revealed a decrease in the signaling network associated with cell cycle regulation and DNA replication. Meanwhile, persister cells exhibited an enrichment in proteins linked to lysosomes, amino acids, and fatty acid metabolism. The data revealed a consistent metabolic shift in persister cells, with an increased dependence on oxidative phosphorylation (OXPHOS) for ATP generation. Small-scale drug screening was performed based on findings from proteomics and confirmed that persister cells are vulnerable to inhibitors of OXPHOS mitochondrial complexes. Combination treatment of OXPHOS inhibitors and each targeted therapy delayed the emergence of resistance. One complex V inhibitor, Gboxin, selectively inhibited the growth of persister cells. The therapeutic potential of complex V was further validated by CRISPR-based genetic knockdown studies. The metabolic transition in drug tolerant persister cells may serve as a critical adaptive resistance mechanism to provide a survival advantage under targeted therapy-induced stress. Understanding this metabolic shift may lead to new treatments that aim to prevent or delay the onset of acquired resistance in NSCLC. Citation Format: Donghwa Kim, Dmitry Kuchenov, Aubhishek Zaman, Neal Bennett, Emilio Ramos, Ken Nakamura, Arun P. Wiita, Sourav Bandyopadhyay, Trever Bivona. Unraveling the vulnerabilities of targeted therapy-tolerant persister cells in NSCLC [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 4653.

Abstract 5626: Essential role of Slug during the evolution of drug-tolerance in lung adenocarcinoma

Abstract A major obstacle in the efficacy of targeted therapies of oncogene-driven tumors are drug-tolerant persister cells (DTPs) that build the basis for the outgrowth of drug-resistant clones and ultimately limit patient survival. During treatment, DTPs enter a reversible senescent state to survive therapy while cell death is induced in non-DTPs. Here, using RNA-Seq and proteomic analyses, we identified drug-induced TGFβ2 secretion in DTPs derived from different oncogene-driven lung cancer cell lines. As expected, TGFβ2 induces epithelial-to-mesenchymal transition (EMT) that over time promotes the ability of cells to survive targeted therapy. Using CRISPR/Cas9-mediated loss-of-function and reconstitution experiments we show that downstream of TGFβ2, expression of the transcription factor SNAI2 (SLUG) is essential for the outgrowth of DTPs during targeted treatment. Unsupervised RNA-Seq data analyses in combination with Cut&Tag profiling revealed that EMT signaling is in part a SLUG regulated process but that is also independent of direct SLUG binding at the transcription start sites (TSS) of EMT signature genes. In contrast, we observed high occupancy of SLUG at TSS of genes involved in the regulation of cell cycle, being repressed in DTPs and at TSS of genes that regulate sphingolipid metabolism and MAPK signaling, being induced in DTPs. In line with our previous findings, a motif-based analysis of the Cut&Tag data (TOBIAS) furthermore uncovered a tight connection between DTP outgrowth and inflammatory signaling induced through IRF7, IRF4 or STAT1 activation. In vivo, EGFR-mutant SLUG deficient cells showed a significantly prolonged tumor onset and a higher response rate to osimertinib treatment. Overall, we uncover a major role of TGFβ2/SLUG-mediated EMT signaling that may be induced indirectly through SLUG-dependent reprogramming of cell cycle directed and metabolic processes in drug tolerant cells. These insights may offer unique therapeutic opportunities to limit the outgrowth of drug resistant tumors in cancer patients. Citation Format: Jenny Ostendorp, Hannah Lea Tumbrink, Pascal Hunold, Michaela Hoehne, Philipp Jurmeister, Anastasia Dekker, Felix Heisel, Johannes Brägelmann, Frederick Klauschen, Robert Haensel-Hertsch, Martin L. Sos. Essential role of Slug during the evolution of drug-tolerance in lung adenocarcinoma [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 5626.

Abstract 3289: APOBEC3A drives tumor evolution through activation of ERVs in non small cell lung cancer

Abstract Acquired drug resistance to even the most effective anti-cancer targeted therapies remains an unsolved clinical problem. Although many drivers of acquired drug resistance have been identified, the underlying molecular mechanisms shaping tumor evolution during treatment are incompletely understood. We recently demonstrated that lung cancer targeted therapies commonly used in the clinic induce the expression of cytidine deaminase APOBEC3A (A3A), leading to sustained mutagenesis in drug-tolerant cancer cells persisting during therapy. Preventing therapy-induced A3A mutagenesis by gene deletion decreased the chromosomal aberration events and delayed the emergence of drug resistance. Thus, inhibition of A3A may represent a potential therapeutic strategy to prevent acquired resistance. Understanding the regulatory mechanisms of A3A may provide alternative approaches to inhibit A3A mutagenesis. Using a panel drug screen, we found that DNA methyltransferase (DNMT) inhibitor remarkably induces A3A in non-small cell lung cancer cells. RNA-seq profiling revealed that targeted therapy as well as DNMT inhibitor treatment activate expression of distinct classes non-coding repetitive RNA elements, including endogenous retrovirus (ERVs), in drug-tolerant persister cells. Activation of intracellular viral sensing pathways by introduction of exogenous nucleic acids mimicking viral elements induced A3A expression. While the transcription factor NFkB was acutely activated by targeted therapy, TBK1-IRF3 signaling was activated at later time points. These findings suggest that reactivation of ERVs may underlie A3A mutagenic activity and tumor evolution during targeted therapy. Citation Format: Hideko Isozaki, Ramin Sakhtemani, Naveed Nikpour, Susanna Monroe, Michael Lawrence, Aaron Hata. APOBEC3A drives tumor evolution through activation of ERVs in non small cell lung 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 3289.

Abstract 5864: YAP1 defines a highly plastic, persister cell population in small cell lung cancer (SCLC) following relapse

Abstract SCLC is an aggressive, neuroendocrine malignancy notable for rapid, albeit transient, responses to therapy. We have previously shown that transcriptional heterogeneity among treatment-naïve SCLC tumors underlies four distinct transcriptional subtypes, each with distinct clinical vulnerabilities. Though previously hypothesized to delineate a distinct subtype, expression of YAP1 is largely absent from treatment-naïve SCLC. Less clearly defined is the transcriptional landscape of relapsed SCLC, the evolution of which may underlie SCLC’s capacity for insurmountable resistance. To better define the biology of relapsed SCLC, we performed mass cytometry analysis of patient circulating tumor cells (CTCs), immunohistochemistry for ASCL1, NEUROD1, POU2F3, MYC and YAP1, and single-cell (sc)RNAseq of core needle biopsies from SCLC patients and preclinical models following resistance to standard-of-care therapies. In contrast to treatment-naïve SCLC, YAP1 mRNA and protein are detectable in patient CTC populations and tumors from relapsed patients and PDXs, albeit at levels lower than subtype-defining transcription factors (e.g. ASCL1, NEUROD1, or POU2F3). Notably, however, scRNAseq reveals these YAP1 expressing cells to be mutually exclusive with ASCL1 and NEUROD1, though not necessarily POU2F3. YAP1-expressing cells possess unique mesenchymal, inflamed features and exhibit high levels of transcriptional plasticity. Consistent with the upregulation of YAP1 following chemotherapy, YAP1-positive cell populations express senescence associated secretory phenotype genes and cancer stem cell genes, suggesting that these populations represent drug tolerant persister cells that may serve as a source for renewal of classical neuroendocrine populations within the tumor. Targeting these resistant populations may be critical to re-establish response in relapsed SCLC, and these analyses identified high expression of several surface proteins that are currently under investigation as targets for antibody-drug conjugates in SCLC clinical trials, including B7H3, TROP2 and HER2 antibody drug conjugates. These data suggest that the acquisition of YAP1 expression delineates a particularly recalcitrant tumor cell population observed almost exclusively in relapsed SCLC that is capable of driving tumor persistence and treatment resistance. Clinically, these data underscore the importance of constraining transcriptional plasticity in the frontline management of SCLC, but also of developing therapies aimed specifically at emergent populations capable of driving resistance, as with the YAP1-high cells identified here. The emergence of YAP1 following resistance in SCLC and the observation of YAP1 as a common feature of other high-grade neuroendocrine carcinomas exclusive of SCLC, likely explains the initial mischaracterization of YAP1 as a subtype-defining feature of SCLC. Citation Format: C. Allison Stewart, Sarah M. Groves, Runsheng Wang, Kavya Ramkumar, Yuanxin Xi, Lixia Diao, Alejandra G. Serrano, Azusa Tanimoto, Ashley M. Victorian, Sayantan Bhattacharyya, Alexa J. Halliday, Neda Kalhor, Luisa Maren Soto, Pedro Filipe Da Rocha, Natalie Vokes, Loukia G. Karacosta, Jing Wang, John V. Heymach, Vito Quaranta, Bonnie Glisson, Lauren Averett Byers, Carl M. Gay. YAP1 defines a highly plastic, persister cell population in small cell lung cancer (SCLC) following relapse [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 5864.

Abstract 2096: A novel, orally available ferroptosis inducer targeting Fe-S assembly with significant in vivo anti-tumor efficacy

Abstract Background: Ferroptosis is a novel form of cell death driven by iron-dependent lipid peroxidation, which disrupts cellular membranes resulting in immune-activating death. A variety of tumor types, particularly those associated with enhanced lipid and/or iron content, or which experience increased oxidative stress (including drug tolerant persister cells), are sensitive to ferroptosis, rendering ferroptosis a promising therapeutic strategy for cancer. However, currently available ferroptosis inducers show limited activity in vivo, suggesting a need for novel molecular targets to validate the clinical utility of ferroptosis induction. Clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, is typically initiated by inactivation of VHL resulting in constitutive activation of HIF-1α and HIF-2α, which contribute to disease progression, including by promoting lipid accumulation that sensitizes ccRCC cells to ferroptosis. Methods: We performed a high-throughput screen to identify small molecule inhibitors of HIF-2α followed by structure-activity-relationship studies for validation. The molecular target of these inhibitors was identified using the Drug Affinity Responsive Target Stability assay, and target validation was confirmed using siRNA and NMR. In vitro efficacy was determined in a panel of cancer cell lines and in vivo anti-tumor efficacy was verified using mouse models of cancer. Results: We identified a novel molecule, KD061, which decreases HIF-1/2α and induces ferroptosis in vivo through oral administration by targeting Iron Sulfur Cluster Assembly 2 (ISCA2). ISCA2 is a component of the late mitochondrial Iron Sulfur Cluster (L-ISC) assembly complex and ISCA2 inhibition either pharmacologically or using siRNA triggers the iron starvation response, resulting in iron/metals overload and death via ferroptosis. ISCA2 inhibition also decreases HIF-2α protein levels by blocking iron-responsive element (IRE)-dependent translation, and at higher concentrations, also decreases HIF-1α translation. These studies reveal a novel function of ISCA2 in extra-mitochondrial Fe-S assembly that may be limiting in cancer due to the increased need for these proteins in DNA replication and maintenance. Outside of ccRCC, a 92-cell line screen reveals a variety of tumor types sensitive to ISCA2 inhibition including melanoma and clear cell ovarian cancer. Treatment of tumor-bearing mice with KD061 resulted in > 65% tumor growth inhibition, associated with significantly increased lipid peroxidation, thus confirming ferroptosis in vivo, and with decreased HIF-1/2α. The anti-tumor efficacy of KD061 exceeded that of α-PD1 and was well tolerated at the therapeutic dose. Conclusions: We describe the first orally available inducer of ferroptosis with significant anti-tumor efficacy in ccRCC and other tumor types. Citation Format: Yangsook Song Green, Maria Ferreira dos Santos, Simone Ciofi-Baffoni, Xiaohui Liu, Mei Yee Koh. A novel, orally available ferroptosis inducer targeting Fe-S assembly with significant in vivo anti-tumor efficacy [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 2096.

Abstract 4759: Targeting slow-cycling persisters in EGFR mutant non-small cell lung cancer

Abstract Activating mutations in the epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer (NSCLC) patients are associated with clinical benefit in the metastatic and adjuvant settings when treated with EGFR inhibitors, such as osimertinib. Despite its dramatic efficacy, most patients are partial responders and refractive disease leave limited treatment options. The objective was to dissect the mechanism of osimertinib resistance in EGFR mutant NSCLC models to identify a means of re-sensitization. Imaging mass cytometric analysis of 76 NSCLC patients demonstrated EGFR expression was inversely correlated to CD105 membrane expression. CD105 is a transforming growth factor beta (TGF-β) family co-receptor that promotes bone morphogenic protein (BMP) signaling and inhibits TGF-β signaling. Significant elevation in cell surface CD105 by osimertinib was furthered by EGFR knockdown in two NSCLC lines with EGFR activating mutations. These lines were subjected to a program of increasing osimertinib concentrations to generate isogenic resistant lines. Heterogeneous drug-tolerant persister cells had significantly higher mutational load, increased CD105 cell surface expression as well as greater expression of bypass signaling factors such as ERK, PI3K, and BMP ligands compared to their parental counterparts. Single cell RNA-seq analysis of parental NSCLC revealed a small population of cells that shared elevated endothelial and pyrimidine metabolism, with a slow-cycling signature that was enriched in the osimertinib-resistant cells. Combining osimertinib with a CD105 neutralizing antibody, carotuximab, reduced the slow-cycling population and restored osimertinib sensitivity in resistant cell lines. The knockdown of CD105 had a more pronounced decrease in cell viability when combined with osimertinib. The osimertinib-resistant lines demonstrated more condensed chromatin and glycolytic state compared to the respective parental lines by ATAC-seq and Single Cell ENergetIc metabolism profiling, respectively. The addition of carotuximab reversed the chromatin and metabolic reprograming of osimertinib resistance. Carotuximab was discovered to initiate CD105 interaction with EGFR and its dominant active variant, EGFRv3. Ultimately, combination therapy of carotuximab and osimertinib resulted in significantly reduced tumor expansion compared to mice treated with either drug alone. Paradoxically, the small tumors from combination therapy had greater mitosis compared to the larger tumors of the osimertinib single agent treated mice. Inhibition of CD105 with carotuximab can overcome resistance to osimertinib and inhibit NSCLC tumor progression. This is a first-in-class pre-clinical foundation for a novel synthetic lethality treatment strategy resulting from the observation of global changes in EGFR-antagonist resistance as opposed to individual mutations identified in tumor subpopulations. Citation Format: Manish Thiruvalluvan, Sandrine Billet, Zhenqiu Liu, Joseph Lownik, Gabrielle Gonzales, Hyoyoung Kim, Anton L. Villamejor, Larry Milshteyn, Kamya Sankar, Edwin M. Posadas, Jean Lopategui, Sungyong You, Karen Reckamp, Neil A. Bhowmick. Targeting slow-cycling persisters in EGFR mutant non-small cell lung 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 4759.

Abstract 5882: Targeting vulnerabilities of drug-tolerant persister cells in gastric cancer

Abstract Cytotoxic drugs often fail to eradicate cancers due to the presence of treatment-persistent tumor cells that represent a reservoir for relapse. These “residual” cancer cells escape from chemotherapy- induced death by entering a reversible slow proliferation state, known as drug tolerant persister (DTP) state. Although improvement of treatment options achieved a survival benefit, Gastric Cancer (GC) is still endowed with poor prognosis. Surgery and neo/adjuvant chemotherapy remain the keystone of treatment. However, most patients with advanced cancer relapse even after complete surgical resection, suggesting a critical role for DTP cells. We obtained GC DTP cells from primary cells upon first- and second-line chemotherapy regimens (FLOT and FOLFIRI). Cells were treated with doses corresponding to the drug maximal plasmatic concentration. At the end of the treatment, cells were left without drugs (washout). During this window of time, when cells had regrown, they were re-challenged with chemotherapy and resulted still sensitive to the treatments, thus fulfilling the criteria for bona fide DTP cells. GC DTPs showed increased expression of markers associated with gastric stem cells (i.e. LGR5), downregulation of proliferative markers (i.e. Ki67) and decreased levels of PS6, evaluated as a readout of mTOR activation. Interestingly, DTPs resulted positive to X-gal-based β-galactosidase staining, suggesting a chemotherapy-induced senescence-like status. RNA-seq analysis and gene expression arrays allowed the identification of genes specifically down/upregulated in DTP cells. Using the intuitive enrichment analysis web-based tool Enrichr, we discovered that 54/173 of the downregulated genes characterizing the persister state were targets of a single miRNA. By quantitative real-time PCR, we validated the expression of this miR in all the models of FOLFIRI- and FLOT-obtained persister cells and we found it strongly upregulated (up to 8-fold), highlighting its possible contribute in the establishment of the persister state. Since among the target genes of this miR there are genes involved in the Homolougus Recombination (HR) system, we performed TaqMan™ Array assay for Human DNA repair gene expression on FOLFIRI-obtained DTPs. We identified a significant downregulation of genes strictly correlated to the HR machinery, suggesting that the FOLFIRI-induced persister cells can temporarily enter in a “BRCAness” status that may be used for a synthetic lethality approach using PARP inhibitors. Targeting the drug tolerant persister state is an essential approach to diminish cancer cells when they are potentially in their most vulnerable state. Citation Format: Elisabetta Puliga, Lisa Negro, Martina Olivero, Claudia Orrù, Fabrizio Maina, Daniela Conticelli, Simona Corso, Silvia Giordano. Targeting vulnerabilities of drug-tolerant persister cells in gastric 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 5882.

Targeting Mycobacterium tuberculosis Persistence through Inhibition of the Trehalose Catalytic Shift.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death worldwide by infectious disease. Treatment of Mtb infection requires a six-month course of multiple antibiotics, an extremely challenging regimen necessitated by Mtb's ability to form drug-tolerant persister cells. Mtb persister formation is dependent on the trehalose catalytic shift, a stress-responsive metabolic remodeling mechanism in which the disaccharide trehalose is liberated from cell surface glycolipids and repurposed as an internal carbon source to meet energy and redox demands. Here, using a biofilm-persister model, metabolomics, and cryo-electron microscopy (EM), we found that azidodeoxy- and aminodeoxy-d-trehalose analogues block the Mtb trehalose catalytic shift through inhibition of trehalose synthase TreS (Rv0126), which catalyzes the isomerization of trehalose to maltose. Out of a focused eight-member compound panel constructed by chemoenzymatic synthesis, the natural product 2-trehalosamine exhibited the highest potency and significantly potentiated first- and second-line TB drugs in broth culture and macrophage infection assays. We also report the first structure of TreS bound to a substrate analogue inhibitor, obtained via cryo-EM, which revealed conformational changes likely essential for catalysis and inhibitor binding that can potentially be exploited for future therapeutic development. Our results demonstrate that inhibition of the trehalose catalytic shift is a viable strategy to target Mtb persisters and advance trehalose analogues as tools and potential adjunctive therapeutics for investigating and targeting mycobacterial persistence.

Abstract B067: Identification of Gemcitabine-resistant Populations using scRNA-Sequencing in Triple Negative Breast Cancer Patient-Derived Xenografts

Abstract Despite major advances in the treatment of breast cancer (BC), it remains the most diagnosed and second deadliest cancer among American women. BC consists of distinct clinical subtypes, including estrogen receptor or HER2 receptor-positive and triple-negative breast cancer (TNBC) that lacks these receptors preventing the use of established precision therapies and the standard of care remains neoadjuvant chemotherapy (NACT), followed by surgery, radiation and emerging combinations with immunotherapy. While NACT is effective in some patients, up to 50% of patients are intrinsically unresponsive or acquire resistance. These patients demonstrate the highest rates of recurrence and distant metastasis. It is known that chemotherapies can effectively eliminate proliferating tumor cells, but a small population of drug-tolerant persister (DTPs) cells can remain and are thought to play a key role in relapse and resistance. We hypothesized that chemotherapy influences TNBC tumor plasticity by exerting selective pressures leading to the outgrowth of resistant subpopulations with the greatest survival advantage. To evaluate this hypothesis, we generated in vivo models of disease progression and chemotherapy resistance and investigated the plasticity of tumor cell subpopulations by single-cell RNA sequencing. We developed PDX models from a multi-drug resistant primary TNBC treated with standard-of-care chemotherapy and its patient-matched lung metastasis. To model response and resistance at the metastatic stage, we challenged this metastasis PDX with several cycles of Gemcitabine. We obtained residual disease that relapsed and eventually developed resistance, mirroring the patient response. We performed single-cell RNA sequencing (scRNAseq) of these models using a droplet-based technology from 10X Genomics. This scRNAseq data was used to compare the changes in the proportions of cellular subpopulations in each model. Our data show that the rebound model presents greater similarity to the untreated metastasis, while the resistant model significantly differs in cell population expression profiles. Interestingly, the residual disease demonstrates an intermediated state with similarities to both the untreated metastasis and the resistant model. We identified populations with hypoxic signatures in the primary tumor, its matched metastasis and the residual, rebound and resistant models. Immunohistochemistry and Nanostring GeoMx Digital Spatial Profiler validated these populations in these models. In addition, we have identified other varying populations and are currently investigating their cellular mechanisms and gene expression patterns. Using scRNA-sequencing to understand the clonal expansion of resistant subpopulations following chemotherapy reveals distinctive resistant cell features enabling the identification of the vulnerabilities of these tumors. Citation Format: Sandrine Busque, Constanza Martinez Ramirez, Ariel Madrigal Aguirre, Paul Savage, Anie Monast, Anne-Marie Fortier, Gerardo Zapata, Atilla Omeroglu, Jamil Asselah, Nathaniel Bouganim, Sarkis Meterissian, Francine Tremblay, Ari Meguerditchian, Yasser Riaz Alhosseini, Hamed S. Najafabadi, Hellen Kuasne, Morag Park. Identification of Gemcitabine-resistant Populations using scRNA-Sequencing in Triple Negative Breast Cancer Patient-Derived Xenografts [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 B067.

Abstract B089: MUC1-C is a common driver of acquired osimertinb resistance in NSCLC

Abstract Background: Osimertinib is an irreversible EGFR tyrosine kinase inhibitor approved for the first-line treatment of patients with metastatic NSCLC harboring EGFR exon 19 deletions or L858R mutations. Patients treated with osimertinib invariably develop acquired resistance by mechanisms involving additional EGFR mutations, MET amplification and other pathways. There is no known involvement of the oncogenic MUC1-C protein in acquired osimertinib resistance. Methods: H1975/EGFR(L858R/T790M) and patient-derived NSCLC cells with acquired osimertinib resistance (EMT, MET amplified, C797S mutation, or PCBP2-BRAF fusion) were investigated for MUC1-C dependence in studies of EGFR pathway activation, clonogenicity and self-renewal capacity. Results: We demonstrate that MUC1-C is upregulated in H1975 osimertinib drug tolerant persister (DTP) cells and is necessary for activation of the EGFR pathway. H1975 cells selected for stable osimertinib resistance (H1975-OR) and MGH700-2D cells isolated from a patient with acquired osimertinib resistance are shown to be dependent on MUC1-C for induction of (i) p-EGFR, p-ERK and p-AKT, (ii) EMT, and (iii) the resistant phenotype. We report that MUC1-C is also required for p-EGFR, p-ERK and p-AKT activation and self-renewal capacity in acquired osimertinib-resistant (i) MET amplified MGH170-1D #2 cells, (ii) MGH121 Res#2/EGFR(T790M/C797S) cells, and (iii) MGH845-1R5/EGFR(19del)/PCBP2-BRAF fusion cells. Importantly, targeting MUC1-C in these diverse models reverses osimertinib resistance. To extend these results, we established MET amplified MGH170-1D #2 tumors in mice and found that treatment with GO-203 inhibitor, which is a cell-penetrating peptide that blocks MUC1-C homodimerization, nuclear localization, and function, results in more pronounced inhibition and that the GO-203+osimertinib combination is more effective than either agent alone. In support of these results, high MUC1 expression is associated with poor prognosis for patients with EGFR mutant NSCLCs and those treated with osimertinib. Conclusions: Our findings demonstrate that MUC1-C is a common effector of osimertinib resistance and is a potential target for the treatment of osimertinib resistant NSCLCs. Citation Format: Naoki Haratake, Hiroki Ozawa, Yoshihiro Morimoto, Nami Yamashita, Tatsuaki Daimon, Atrayee Bhattacharya, Keyi Wang, Ayako Nakashoji, Hideko Isozaki, Aaron Hata, Donald Kufe. MUC1-C is a common driver of acquired osimertinb resistance in NSCLC [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 B089.