Drug-tolerant Persister Research Articles

Abstract 1693: Drug-tolerant persister cells contain heterogeneous subpopulations that utilize lung lineage pathways as drug resistance mechanism

Abstract Genomic discoveries of oncogenic driver alterations in lung cancer have led to development of effective target therapeutics; however, patients who initially respond to the therapy inevitably experience regrowth of the disease. Recent studies identified a reversible stage of cancer cells entering a quiescent state to ensure survival, called drug-tolerant persister (DTP) cells. The DTP stage is viewed as a major source of non-genetic drug resistance, while its epigenetic dynamic and regulatory mechanisms remain largely unknown. To determine the epigenetic activity reminiscent of a quiescent state during DTP development, we evaluated repressive epigenetic modifications during DTP progression and found that DTP cells had a markedly increased H3K27me3 level at the beginning of DTP stage. Treatment of EZH2 inhibitor together with target therapy synergistically inhibited cell growth, indicating the essential role of compressed chromatin structure for DTP survival. To further investigate the epigenomic heterogeneity of DTP cells, we performed single cell ATAC-seq on DTP cells. Unsupervised hierarchical clustering analysis revealed that the drug-induced DTP cells had two distinct populations compared with the untreated group with only one major population, suggesting an increased epigenomic heterogeneity. By analyzing the differentially accessible regions, motifs for TEADs and GATAs were specifically enriched in DTP cells, whose family members are well-established regulators of the lung development. This raised a possibility that the evolution of drug resistance shares similarities with organ regeneration, both require terminally differentiated cells regaining cell plasticity and serving as a progenitor cell to direct re-differentiation. Thus, we focused on alveolar lineage genes as a model to test our hypothesis that DTP cells hijack conserved developmental pathways for lung regeneration to ensure their survival and subsequent expansion. Our results showed that specific alveolar marker genes were transiently upregulated and exhibited translocation from cytoplasm to nuclei at early phase of DTP. Moreover, deletion of the alveolar lineage gene decreased cell re-growth under drug treatment. Our finding provides a new perspective of anti-relapse therapeutic strategies, that targeting lung lineage pathways to manipulate the activity of critical lineage factors during lung regeneration, for patients with minimal residual disease and relapse after target therapy. Citation Format: Yang Tian, Feng Jiang, Athina Jaffer, Benjamin Hopkins, Jian Jin, Hideo Watanabe. Drug-tolerant persister cells contain heterogeneous subpopulations that utilize lung lineage pathways as drug resistance mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1693.

Abstract 425: Characterization of TKI-induced drug-tolerant persister cells from a patient-derived cell line

Abstract Introduction: Targeted therapies provide clinical benefits in patients harboring oncogene-driven cancers, but relapses invariably occur. Studies on resistance mechanisms are essential to guide the development of next-generation inhibitors. An increasing number of studies focus on an earlier stage of disease evolution. Resistance may develop from the selection of preexisting resistant cells or by adaptation of drug-tolerant persister (DTP) cells. In this context, our study depicts DTP cells able to survive the initial tyrosine kinase inhibitor (TKI) exposure without harboring genetic changes, in order to identify the vulnerabilities existing in this cell state. Materials and Methods: This project focuses on a patient-derived cell line from the prospective MATCH-R clinical trial (NCT0251782). The model is a non-small cell lung cancer harboring EML4-ALK fusion and ALK C1156Y/G1269A mutation, sensitive to the 3rd generation ALK TKI lorlatinib. In vitro, fully resistant cells were generated by long-term lorlatinib exposure and the acquired resistance mechanism resulted from an epithelial-mesenchymal transition (EMT). DTP cells were generated under shorter drug exposures that eradicate sensitive carcinoma cells. Results and Discussion: Carcinoma cell plasticity drives cell transformation towards a phenotypic state, rendering them more tolerant to drugs. To better characterize this phenotype, we first focused on EMT and observed that DTP cells co-harbored epithelial and mesenchymal markers. We noticed that DTP cells exhibited an intracellular reactive oxygen species (ROS) rate making them dependent on protective oxidation-reduction mechanisms against oxidative stress-related damage. We revealed the overexpression of p21 and p27, known to bind to cyclin-CDK complexes and induce cell-cycle arrest. This dormant phenotype of DTP cells was underlined by a global repressive chromatin state evidenced by enrichment in H3K27me3, H3K36me3 and H3K9me3 marks; the latter mark also being a component of senescence-associated heterochromatin foci. In agreement, a high level of SA-β-gal and senescence-associated secretory phenotype components (CXCL10, PAI-1), evidenced that our DTP model displayed a senescence-like phenotype. We quantified an increase in γH2AX DNA damage foci in DTP cells compared to sensitive cells, in line with the concept that TKI treatment may have a role in DNA repair modulation that ultimately promotes new mutations. Together, our data depicted DTP features that might be relevant to identify drug candidates overcoming resistance (HDAC inhibitors, senolytic drugs and DNA repair inhibitors). Conclusion: We demonstrated potential targetable features of DTP cells. A combination of targeted therapies with DTP drug candidates could represent a therapeutic opportunity to improve the depth of response and delay the emergence of resistance in patients. Citation Format: Floriane Brayé, Francesco Facchinetti, Helena Gerber, Juliette Juigné, Giorgia Guaitoli, Jean-Paul Thiery, Santiago Ponce, Benjamin Besse, Ken A. Olaussen, Luc Friboulet. Characterization of TKI-induced drug-tolerant persister cells from a patient-derived cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 425.

Abstract 3268: Biomarkers of drug-tolerant persistent melanoma cells in human, correlation with tumor microenvironment and response to treatment

Abstract Introduction: Drug tolerant cancer persister cells can be silent for years but constitute a reservoir of genetic resistance and can eventually result in tumor relapse. We showed that targeted therapy (TT)-tolerant BRAFV600 melanoma persister cells (MPC) have a reduced global mRNA translation activity but an increased translation efficiency of a subset of mRNAs encoding proteins involved in transcription, chromatin remodeling and epigenetic changes. Here, we identify protein markers of MPC in patient biopsies treated either with TT or with immune checkpoint inhibitors (ICI). Since persister cells are not destroyed by the immune system, we explored the interactions of MPC with their immune microenvironment. Single cell expression of these candidate MPC markers, as well markers of the tumor microenvironment (31 lymphoid and myeloid markers), were studied in sequential biopsies from melanoma patients treated with TT or ICI by CyTOF. Spatial localization of MPC markers and various types of immune cells were studied by multiplex immunohistochemistry (Akoya Codex). In addition, the in vitro effect of the cell supernatants of MPC or naïve parental cells on immune cells was studied. Results: Biomarkers of persistence included 7 protein markers that are regulated at the translational level (MLL3, HDAC6, CCSER2, MT-ND5, ANK2, HIPK2, CBP) and 4 that are transcriptionally regulated (MITF, NGFR, CD36 and SLIT2). Some MPC markers were expressed at baseline in melanoma patients’ biopsies (n = 27) and their baseline expression tended to be associated with progressive disease (to TT or ICI). Paired biopsies from 18 patients showed that an increase in HDAC6, ANK2, HIPK2, CBP5 and MITF was statistically correlated with a short survival regardless of the type of treatment. Baseline lymphocytic infiltrate, especially CD4+ T cells, was associated with favorable clinical outcome. In addition, we found that the supernatants derived from naïve parental and persister cells have distinct effects on human CD4+, CD8+ T cells and macrophages. MPC supernatant had a significant inhibitory effect on the proliferation and activation of CD4+, CD8+ T cells as well as the expression of immune checkpoint markers (PD-1, PD-L1, CTLA4, LAG3, Tim3) compared to supernatant of naïve parental cells. We also found that supernatants of MPC induced M2-like polarization of macrophages. One major candidate to convey this effect is MIF (Macrophage migration inhibitory factor) that is specifically increased in MPC supernatant. Conclusion: Melanoma persister cells can be identified in patients’ biopsies during TT as well as ICI therapy. The expression of MPC protein markers is associated with poor clinical outcome regardless of the type of treatment. Their expression is inversely correlated with a hot immune environment that could result from the effect of immunosuppressive cytokines secreted by persister cells. Citation Format: Hongxu Chen, Shensi Shen, Sara Faouzi, Alexia Alfaro, Dorothee Baille, Séverine Roy, Naima Benannoune, Hugues Hermann, Samad Muhammadnejad, Stephan Vagner, Caroline Robert. Biomarkers of drug-tolerant persistent melanoma cells in human, correlation with tumor microenvironment and response to treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3268.

Abstract 3460: Targeting the MIF-CD74 axis to overcome resistance to tyrosine kinase inhibitors in non-small cell lung cancer

Abstract Development of tyrosine kinase inhibitors (TKIs) against oncogenic drivers has significantly improved survival of patients with oncogene-mutated non-small cell lung cancer (NSCLC). However, acquired resistance to TKIs emerges over time in essentially all patients who initially respond. Recent evidence suggests that drug-tolerant persister (DTP) cells, which survive and adapt to targeted therapies during an early phase of treatment, play an important role in the emergence of drug resistance. We previously reported that CD74 expression is upregulated in EGFR-mutated lung cancer after treatment with EGFR-TKIs and that CD74 can be one of the DTP cell markers. However, the mechanism underlying CD74 expression and the role of CD74 in DTP cells remain unclear. Here, we sought to identify the mechanism using cell culture systems and transgenic mouse models. We confirmed that CD74 upregulation at the mRNA level after TKIs treatments in various oncogene-mutated cell lines, including those with EGFR mutations, ROS1 fusions, and ALK fusions. We found that CIITA, upstream of CD74, and TNF-α expression were induced by TKIs treatments in tumor cells, leading to an increase in CD74 expression. In addition, we found that TKIs treatments enhanced the autocrine secretion of macrophage migration inhibitory factor (MIF), a ligand of CD74, from tumor cells, suggesting that autocrine stimulation of the CD74 signaling results in block of apoptosis and emergence of DTP cells. To examine whether CD74 plays an important role in the emergence of resistance to TKIs in vivo, we crossed lung-specific EGFR-L858R-T790M transgenic mice with Cd74 knockout mice. Complete deletion of CD74 overcame or delayed resistance to TKIs. Taken together, the MIF-CD74 axis can be a novel target to overcome resistance in driver-mutated NSCLC. Citation Format: Motohiro Izumi, Meghan Lee, Daisuke Shibahara, Ikei S. Kobayashi, David Plotnick, Susumu S. Kobayashi. Targeting the MIF-CD74 axis to overcome resistance to tyrosine kinase inhibitors in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3460.

Abstract 99: Apoptotic DNase DFFB mediates cancer persister cell mutagenesis and acquired drug resistance

Abstract Tumors frequently acquire resistance to therapy resulting in disease relapse and patient mortality. Therapeutic approaches to overcome acquired resistance are hindered by limited treatment options and the presence of multiple parallel resistance mechanisms. Rather than treating resistance after it emerges, it may possible to prevent it by inhibiting the adaptive processes which initiate resistance. However, these processes and the genes which control them are poorly understood. Here we report that drug-tolerant cancer persister cells, which constitute residual disease and seed relapse, undergo drug stress-induced sublethal apoptotic signaling resulting in activation of apoptotic DNase DFFB without cell death. DFFB induces DNA damage and mutagenesis in persister cells, revealing DFFB as a key regulator of stress-induced mutagenesis in persister cells. Furthermore, we found in multiple tumor types that DFFB is required for acquired resistance to targeted therapies. Mechanistically, DFFB induces multiple putative resistance mutations and may also promote epigenetic changes which result in resistance. These results reveal DFFB as a promising potential therapeutic target to prevent acquired resistance. In addition, this stress-sensing mutagenic mechanism may promote mutagenesis in other contexts including normal tissues because sublethal apoptotic signaling may result from a variety of physiological stresses. Citation Format: August Finley Williams, David Gervasio, Claire Turkal, Anna Stuhlfire, Michael Wang, Brandon Mauch, Ariel West, Michelle Paw, Mehrshad Hairani, Cooper Lathrop, Sophie Harris, Jennifer Page, Matthew Hangauer. Apoptotic DNase DFFB mediates cancer persister cell mutagenesis and acquired drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 99.

Abstract 137: Epigenetic regulation of APOBEC3A mutagenesis and tumor evolution during targeted therapy 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 accumulation of chromosomal aberrations and delayed the emergence of drug resistance. Thus, we hypothesize that inhibition of A3A may represent a potential therapeutic strategy to halt the evolution of resistant clones and prevent acquired resistance to lung cancer targeted therapies. Understanding the molecular mechanism of A3A induction during targeted therapy is a crucial step to develop targeting A3A therapies.Using a targeted drug combination screen, we found that DNA methyltransferase (DNMT) inhibitors induce expression of APOBEC3A in non-small cell lung cancer cells, phenocopying the effects of targeted therapies. RNA-seq profiling revealed that both targeted therapies and DNMT inhibitors increase expression of non-coding repeat RNAs. Repeatome analysis identified distinct classes of non-codding RNAs including endogenous retrovirus elements (ERV) in drug tolerant persister cells. Activation of endogenous intracellular viral sensing pathways by exogenous nucleic acids mimicking viral infection induced APOBEC3A, and genetic perturbation of RIG-I or MAVS reduced APOBEC3A induced by targeted therapy. These findings suggest that epigenetic derepression of retroviral repeat elements may underly APOBEC3A mutagenic activity and tumor evolution during lung cancer targeted therapy. Citation Format: Hideko Isozaki, Ramin Sakhtemani, Naveed Nikpour, Susanna Monroe, Jessica Lin, Lecia Sequist, Zofia Piotrowska, Justin Gainor, Rémi Buisson, Michael Lawrence, Aaron Hata. Epigenetic regulation of APOBEC3A mutagenesis and tumor evolution during targeted therapy in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 137.

Abstract 3864: Targeting IL-6/STAT3 signaling in EGFR-mutant drug tolerant persister cells

Abstract While the majority of epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC) patients initially respond to treatment with an EGFR tyrosine kinase inhibitor (TKI), most will eventually develop acquired resistance to these agents. The evolution of tumor cells with acquired resistance to targeted agents initially involves the persistence of a drug-tolerant subpopulation of tumor cells which display a transient and flexible resistant state. These persister cells can remain quiescent for prolonged periods of time, but eventually give rise to fully drug resistant tumor cells that resume growth and metastatic spread. The signaling pathways that allow persister cells to remain viable or to re-activate expansive growth are poorly understood. We and others have reported that EGFR TKI resistance that occurs independent of secondary EGFR mutations or MET amplification is associated with an epithelial to mesenchymal transition (EMT). Our analysis of tumor cells that had undergone EMT-mediated resistance to the EGFR TKI osimertinib revealed that interleukin-6 (IL-6) was highly upregulated in resistant cells as compared to parental cells. We find that in the setting of EGFR mutant NSCLC, IL-6 suppresses the anti-tumor cell activity of T cells and NK cells. To assess the role of IL-6 in the early events of EGFR TKI resistance, we generated drug-tolerant persister cells by treating EGFR mutant HCC4006 and HCC827 NSCLC cells with the EGFR TKI osimertinib for 10 days and then assessed IL-6 expression. We observed that IL-6 was upregulated at both the RNA and protein levels in EGFR-TKI persister cells as compared to parental cells. While the cell surface expression of IL6R was not significantly increased in EGFR-TKI persister cells as compared to parental cells, we observed increased secretion of soluble IL6R in persister cells by ELISA assay. We next assessed changes in protein expression and activation in drug tolerant persister cells by reverse phase protein array (RPPA) and observed that p-STAT3, a key component of the IL-6 signaling pathway, was significantly elevated in persister cells as compared parental cells or cells treated with osimertinib for 24 hours. Using genetically engineered mouse models (GEMM) of EGFRL858R mutant lung cancer, we also observed a similar upregulation of both IL-6 and p-STAT3 expression at the RNA and protein levels in EGFR-mutant tumors treated with osimertinib for 2 or 4 weeks. Additionally, circulating levels of IL-6 also are elevated in EGFRL858R GEMMs treated with osimertinib for 2 or 4 weeks. Collectively, our data indicates that upregulation of IL-6 is an early event in the evolution of drug resistance. Given the role of IL-6 in promoting EGFR TKI resistance, further studies investigating whether targeting of IL-6 signaling may impair the survival or outgrowth of persister populations are warranted. Citation Format: Sonia A. Patel, Monique Nilsson, Yan Yang, Li Shen, Jing Wang, Alissa Poteete, Xiaoxing Yu, Xiaoyang Ren, Xiuning Le, John Heymach. Targeting IL-6/STAT3 signaling in EGFR-mutant drug tolerant persister cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3864.

Abstract 5950: Ccr2+ monocyte derived macrophages influence trajectories of acquired therapy resistance in BRAF-mutant melanoma

Abstract Therapies targeting oncogene addiction have had a tremendous impact on patient care, but drug resistance continues to be problematic. One method to deal with this challenge entails extending the scope of anti-cancer treatment beyond tumor cells by additionally altering their tumor microenvironment. Understanding how the tumor microenvironment can contribute to the evolution of diverse resistance pathways could aid in the rational design of sequential treatments that enforce pressure towards a more predictable resistance trajectory. Tumor associated macrophages often support neoplastic growth and are frequently the most abundant immune cell found in tumors. Here, we used clinically relevant in vivo Braf-mutant melanoma models and Ccr2-RFP; Cx3cr1-GFP mice to track the long-term changes in tumor cells and macrophages under targeted therapy with Braf/Mek inhibitors. We assessed the dynamic evolution of the macrophage population generated by therapy pressure-induced stress. During the onset of a drug-tolerant persister state, Ccr2+ macrophage infiltration rises, underscoring a prominent role at this point in melanoma evolution for development of drug-resistance. Importantly, we show that a lack of melanoma infiltrating Ccr2+ macrophages delays onset of resistance. In this case, rebounded tumor cells from Ccr2 knockout recipients favor the evolution of cell-extrinsic mediated resistance mechanisms, which can be reversed when these adapted melanoma cells are relocated to a microenvironment with functional Ccr2. Conversely, Ccr2 presence promotes development of cell-intrinsic drug resistance with a stable phenotype in rebounded tumor cells. Overall, this demonstrates that the development of resistance may be manipulated and directed to improve timing and probability of relapse. Citation Format: Dahihm Kim, Luye An, Jiwon Moon, Andrew White. Ccr2+ monocyte derived macrophages influence trajectories of acquired therapy resistance in BRAF-mutant melanoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5950.

Abstract 1692: mTOR inhibition attenuates chemosensitivity through induction of a persister state

Abstract Effective cancer therapeutics often kill a large fraction of tumor cells, while sparing a small surviving population, which can resurface as future tumor recurrence. Mechanisms of resistance in these residual tumors may involve rare preexisting clones with mutations, as well as de novo development of resistance through various genetic and/or non-genetic processes. In the context of acute stress resulting from cancer therapeutics, the initial adaptation is essential for tumor cell survival and allows for subsequent development of resistance through additional genetic and non-genetic mechanisms. As an integrator of environmental signals and cellular response, the mTOR pathway is a critical driver of cellular adaptation to environmental changes. In particular, mTOR regulates embryonic diapause, a reversible dormancy state in mammalian development in response to unfavorable environments. Analogous to its role in regulating embryonic diapause, mTOR inhibition may induce a diapause-like state in cancer cells, leading to drug tolerance and persistence of residual tumors following therapy. In line with this notion, recent studies have reported markedly reduced mTOR activity in residual tumors. However, the causal relationship between mTOR inhibition and chemosensitivity has not been investigated. Through a genome-wide CRISPR knockout library screen in pancreatic cancer cells treated with chemotherapeutic agents, we have identified the mTOR pathway as a prominent determinant of chemosensitivity. Pharmacological suppression of mTOR activity in cancer cells from diverse tissue origins leads to the persistence of a reversibly resistant population, which is otherwise eliminated by chemotherapeutic agents. Conversely, activation of the mTOR pathway increases chemosensitivity and predicts better survival among various human cancers. Survival of the persisters is dependent on the regulation of autophagy, G2/M checkpoint, and cell death pathways, as revealed by a small-molecule chemical library screen. Thus, mTOR plays a causal yet paradoxical role in regulating chemotherapeutic response; inhibition of the mTOR pathway, while suppressing tumor expansion, facilitates the development of a reversible drug-tolerant persister state. Citation Format: Yuanhui Liu, Nancy Azizian, Delaney Sullivan, Yulin Li. mTOR inhibition attenuates chemosensitivity through induction of a persister state [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1692.

Abstract 4865: Mechanisms of EGFR inhibitor sensitivity and resistance in chordoma

Abstract Target discovery studies have identified EGFR inhibition as a promising therapeutic strategy in chordoma, motivating ongoing clinical trials. Yet EGFR is not mutated in chordoma, leaving the mechanisms associated with EGFR inhibitor (EGFRi) sensitivity and resistance unclear. In this study, we profiled a panel of over 10 chordoma cell lines to categorize their sensitivity to afatinib, a second-generation EGFRi with potency against the wild-type receptor. Several cell lines, including U-CHCF365, UM-Chor1, MUG-Chor1, and U-CH1, were highly sensitive to afatinib in cell viability assays, with absolute IC50 values below 50 nanomolar. In contrast, other chordoma cell lines, including UM-Chor5C and U-CHCF359B, exhibited limited response to EGFRi at concentrations exceeding 1 micromolar. Among sensitive cell lines, we found that afatinib promoted cell death in some cases (e.g., U-CH1) whereas in others it induced a profound cell cycle arrest. Moreover, in U-CH1 cells, acute afatinib treatment enriched a population of drug-tolerant persister cells with a fusiform morphology, potentially indicative of an epithelial-mesenchymal transition. Ongoing studies are focused on understanding the mechanisms of afatinib-induced cell death, drug tolerance, and acquired resistance, with a view to designing rational combination strategies capable of enhancing the magnitude and duration of therapeutic response. In cell lines with matched xenograft models, in vitro responses to afatinib were consistent in vivo. In a panel of 12 chordoma patient-derived xenograft (PDX) models treated with afatinib or cetuximab, differential sensitivity to EGFRi was recapitulated - with EGFRi promoting complete or negligent tumor growth inhibition in sensitive or resistant models, respectively. Finally, we combined drug sensitivity profiling of chordoma cell lines and PDXs with whole-exome and whole-transcriptome sequencing to identify genomic and transcriptomic features associated with sensitivity and resistance to EGFRi in chordoma. Collectively, our data identify a striking degree of differential sensitivity to EGFRi in chordoma, and begin to shed light on factors associated with primary and acquired resistance. These results provide a framework for guiding patient selection and identifying potential combination therapy regimens to improve EGFRi efficacy and address resistance. Citation Format: Nindo Punturi, Lee Dolat, Joan Levy, Josh Sommer, Daniel M. Freed. Mechanisms of EGFR inhibitor sensitivity and resistance in chordoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4865.

Abstract 3867: Chromatin modification driving sub-clonal resistance to KRAS G12C combination therapies in KRAS mutant non-small cell lung cancer

Abstract The FDA approval of the KRAS G12C inhibitor (G12Ci) sotorasib and the advancement of similar drugs into clinical trials marks a major milestone in treating KRAS G12C non-small cell lung cancer (NSCLC). However, not all patients respond (sotorasib - ORR = 37.1%, adagrasib - 43%, JDQ443 - 35%), motivating preclinical and clinical investigation into mechanisms of intrinsic and acquired resistance. For instance, clinical studies have reported on-target KRAS mutations and preclinical studies have demonstrated mitogen-activated protein kinase (MAPK) feedback reactivation including EGFP, SHP2, and WT RAS signaling. In response to targeted therapies, sub-populations of cells can enter quiescence or specific epigenetic-driven states that confer drug tolerance. However, epigenetic states defining drug-tolerant persister populations and contributing to adaptive resistance to KRAS G12Ci have not been reported. Using a lineage tracing barcoded system, we identify distinct and reversible subpopulations defined by specific chromatin and transcriptional states in KRAS NSCLC cell lines that contribute to KRAS G12Ci resistance in vitro, even prior to drug treatment. We observed that specific states, including activation of histone demethylation and SWI/SNF complex, may contribute to MAPK reactivation-driven resistance. These results suggest potential epigenetic vulnerabilities that can be exploited to improve the response to KRAS G12Ci. Moreover, we observed distinct persister subpopulations with resistance to KRAS G12Ci combination co-targeting orthogonal pathways (SHP2, CDK4/6, PI3K, and MCL-1), raising the possibility that distinct epigenetic-transcriptional states contribute to differential drug response and clonal evolution of persisters. Collectively, these results suggest that more complete tumor regression may be achieved by orthogonal strategies that target different resistant populations within the same tumor. Citation Format: Chendi Li, Qian Qin, Mohammed Usman Syed, Anahita Nimbalkar, Barbara Karakyriakou, Sarah E. Clark, Anne Y. Saiki, Paul E. Hughes, Chris Ott, Luca Pinello, Aaron N. Hata. Chromatin modification driving sub-clonal resistance to KRAS G12C combination therapies in KRAS mutant non-small cell lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3867.

Abstract 433: Mechanisms of resistance to BAY 2927088, the first reversible inhibitor targeting EGFR exon 20 insertion mutations in non-small cell lung cancer

Abstract Molecular therapies targeting EGFR-mutant non-small cell lung cancer (NSCLC) have dramatically improved prognosis for patients with the classical activating mutations, L858R and exon 19 deletion. Tumors harboring EGFR exon 20 insertions do not respond to inhibitors of classical activating mutations. Agents such as amivantamab and mobocertinib have been approved for treatment of lung cancer patients with exon 20 insertions, but agents with an improved selectivity profile versus wild-type EGFR are still needed. BAY 2927088, currently in phase I clinical trials, is a novel reversible inhibitor that targets EGFR exon 20 insertion mutations with decreased activity towards wild-type EGFR. Although most patients with advanced EGFR-mutant NSCLC eventually progress due to acquired resistance to EGFR inhibitors, the mechanisms of resistance to BAY 2927088 are currently unknown. Using two different methods of in vitro resistance generation, we identified mechanisms of intrinsic and acquired resistance to BAY 2927088 and other EGFR inhibitors. With the first method, involving initial incubation with a high concentration of inhibitor, we isolated drug-tolerant persister cells that underwent a transcriptional state transition resembling EMT. With the second method, involving gradual escalation of inhibitor concentrations, we identified NRAS hotspot mutations as a mechanism of resistance to BAY 2927088 and other EGFR inhibitors. As a parallel approach, we are developing a deep-scanning mutagenesis assay of the EGFR kinase domain to identify mutations that could cause resistance. Understanding these resistance mechanisms will help to identify second-line treatments for exon 20 insertion patients who develop resistance to EGFR inhibition. Citation Format: Gizem Karsli Uzunbas, Quinn McVeigh, Akansha Gupta, Stephanie Hoyt, Andrew Cherniack, Franziska Siegel, Stephan Siegel, Matthew Meyerson, Heidi Greulich. Mechanisms of resistance to BAY 2927088, the first reversible inhibitor targeting EGFR exon 20 insertion mutations in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 433.

Abstract 3852: IK-930, a paralog-selective novel TEAD-inhibitor, effectively attenuates drug-tolerant persister cell proliferation

Abstract The Hippo pathway is commonly mutated or altered in human cancers. In addition to the primary genetic driving mutations, the downstream effectors of the Hippo pathway comprising the transcriptional coactivators YAP1 and TAZ and the TEAD transcription factors can be activated upon prolonged inhibition of other oncogenic pathways such as EGFR and MAPK. We showed that combining the novel, paralog selective TEAD inhibitor, IK-930, with osimertinib or trametinib in EGFR or KRAS mutated tumors, led to enhanced apoptosis in vitro and beneficial antitumor activity in vivo. Experimental models demonstrated that upon inhibition of EGFR with osimertinib, a subpopulation of drug tolerant cells defined as ‘persisters’ can survive treatment allowing for the accumulation of additional genetic and epigenetic alterations. These alterations in the persister cells can give rise to resistant tumors and cancer relapse. Previous studies have implicated YAP/TEAD signaling in the emergence and survival of the persister cells. To investigate the role of YAP/TEAD signaling in persister cell emergence, EGFR and KRAS mutant NSCLC cell lines were engineered to express a TEAD luciferase reporter. Treatment with EGFR or MEK inhibitors, at concentrations that do not elicit significant cell death, induced TEAD-mediated transcription after several days of treatment, indicative of an adaptive response to chronic target inhibition. Concurrent treatment with the TEAD inhibitor, IK-930, abolished TEAD-mediated transcription. Using time lapse microscopy, the cell cycle and proliferation of EGFR mutant cultured cells, engineered to express a fluorescence-based cell-cycle indicator (FUCCI), were quantified when treated with osimertinib alone or combined with IK-930. Treatment with osimertinib led to a rapid induction of cell death. However, after several days, a drug tolerant population emerged and reentered the cell cycle even with chronic osimertinib treatment. Interestingly, the emergence of these persister cells coincided with maximum TEAD activity. Concurrent addition of IK-930, as well as addition of IK-930 after the emergence of the osimertinib-tolerant persister cells, attenuated cell proliferation and expansion, demonstrating the potential for IK-930 to prevent resistance to EGFR inhibitors and even reverse the effect when given after resistance has already emerged. Current efforts are focused on the characterization of the persister population and on evaluating the role of YAP/TEAD signaling in resistance to other targeted therapies. Citation Format: Daniel Hidalgo, Marta Sanchez-Martin, Mihir Rajurkar, George Punkosdy, Jeffrey Ecsedy, Lan Xu. IK-930, a paralog-selective novel TEAD-inhibitor, effectively attenuates drug-tolerant persister cell proliferation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3852.

Abstract 891: CD70-targeting CAR-T and CAR-NK cells demonstrate potent activity against NSCLC drug-tolerant persister cells

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths in the US. Targeted agents, such as EGFR tyrosine kinase inhibitors (TKIs) and KRAS G12C inhibitors, have provided clinical benefits for patients and can yield significant tumor shrinkage. Inevitably, however, there remain residual tumors that harbor drug-tolerant persister cells (DTPCs) not killed by initial treatment, leading to emerging of drug-resistant cells (DRCs) and tumor recurrence. The identification of effective treatment strategies to target DTPCs is a critical and unmet need. We hypothesized that DTPCs can be targeted using chimeric antigen receptor (CAR)-based cellular therapy approaches directed at cell surface proteins expressed on DTPCs and that the combination of such approaches with targeted agents will more effectively delay the emergence of fully drug-resistant cells than targeted agents alone. To this end, we first performed luciferase-based killing assays and observed that EGFR-targeting CAR-T and CAR-NK cells displayed potent cytotoxicity against DTPCs of osimertinib-treated EGFR mutant NSCLC cells. Moreover, osimertinib DTPCs showed increased sensitivity to EGFR CAR-T and CAR-NK cells compared to their parental cells, likely due to the upregulation of EGFR on the cell surface following osimertinib treatment. In a patient-derived xenograft model of HER2YVMA mutant lung cancer, we observed that HER2 CAR-NK cells showed enhanced antitumor activity in mice treated with the HER2 inhibitor, poziotinib, for two weeks, compared to CAR-NK cells only, poziotinib only, or mice treated upfront with HER2 CAR-NK cells plus poziotinib. These in vitro and in vivo data support that CAR-based cellular therapy can effectively eliminate DTPCs. To identify additional candidate cell surface proteins expressed on DTPCs that could be targeted by CAR-based cell therapy approaches, we interrogated RNA expression from our panel of DTPCs as well as public datasets containing DTPCs of targeted agents. Likewise, we investigated the expression of cell surface proteins using datasets of parental and DRCs to identify the common candidate targets that could be exploited on both DTPCs and DRCs. Our analysis identified CD70 as being consistently upregulated in both EGFR TKI-resistant cells and DTPCs of osimertinib-treated EGFR mutant NSCLC. Moreover, we observed CD70 upregulation in DTPCs and DRCs of other targeted agents, including KRAS, ALK, and RET inhibitors, suggesting that CD70 may be a common target in DTPCs across oncogenic drivers. Furthermore, we determined that CD70-targeting CAR-T and CAR-NK cells showed promising in vitro activity against the DTPCs of osimertinib-treated EGFR mutant NSCLC. These results demonstrate CAR-based cellular therapy as an effective approach to target DTPCs and identify CD70 as a novel therapeutic target for combatting DTPCs in NSCLC. Citation Format: Yan Yang, Monique Nilsson, Sonia Patel, Xiaoxing Yu, Alissa Poteete, Qian Huang, Simon Heeke, John Heymach. CD70-targeting CAR-T and CAR-NK cells demonstrate potent activity against NSCLC drug-tolerant persister cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 891.

Single-Cell Analysis Reveals Transcriptomic Features of Drug-Tolerant Persisters and Stromal Adaptation in a Patient-Derived EGFR-Mutated Lung Adenocarcinoma Xenograft Model.

Targeted therapies require life-long treatment, as drug discontinuation invariably leads to tumor recurrence. Recurrence is mainly driven by minor subpopulations of drug-tolerant persister (DTP) cells that survive the cytotoxic drug effect. In lung cancer, DTP studies have mainly been conducted with cell line models. We conducted an invivo DTP study using a lung adenocarcinoma patient-derived xenograft tumor driven by an EGFR mutation. Daily treatment of tumor-bearing mice for 5 to 6 weeks with the EGFR inhibitor erlotinib markedly shrunk tumors and generated DTPs, which were analyzed by whole exome, bulk population transcriptome, and single-cell RNA sequencing. The DTP tumors maintained the genomic clonal architecture of untreated baseline (BL) tumors but had reduced proliferation. Single-cell RNA sequencing identified a rare (approximately 4%) subpopulation of BL cells (DTP-like) with transcriptomic similarity to DTP cells and intermediate activity of pathways that are up-regulated in DTPs. Furthermore, the predominant transforming growth factor-β activated cancer-associated fibroblast (CAF) population in BL tumors was replaced by a CAF population enriched for IL6 production. Invitro experiments indicate that these populations interconvert depending on the levels of transforming growth factor-β versus NF-κB signaling, which is modulated by tyrosine kinase inhibitor presence. The DTPs had signs of increased NF-κB and STAT3 signaling, which may promote their survival. The DTPs may arise from a specific preexisting subpopulation of cancer cells with partial activation of specific drug resistance pathways. Tyrosine kinase inhibitor treatment induces DTPs revealing greater activation of these pathways while converting the major preexisting CAF population into a new state that may further promote DTP survival.

Fasting-mimicking diet synergizes with ferroptosis against quiescent, chemotherapy-resistant cells.

More than ten randomized clinical trials are being tested to evaluate the efficacy, effectiveness and safety of a fasting-mimicking diet (FMD) combined with different antitumor agents. UMI-mRNA sequencing, Cell-cycle analysis, Label retention, metabolomics, Multilabeling etal. were used to explore mechanisms. A tandem mRFP-GFP-tagged LC3B, Annexin-V-FITC Apoptosis, TUNEL, H&E, Ki-67 and animal model was used to search for synergistic drugs. Here we showed that fasting or FMD retards tumor growth more effectively but does not increase 5-fluorouracil/oxaliplatin (5-FU/OXA) sensitivity to apoptosis invitro and invivo. Mechanistically, we demonstrated that CRC cells would switch from an active proliferative to a slow-cycling state during fasting. Furthermore, metabolomics shows cell proliferation was decreased to survive nutrient stress invivo, as evidenced by a low level of adenosine and deoxyadenosine monophosphate. CRC cells would decrease proliferation to achieve increased survival and relapse after chemotherapy. In addition, these fasting-induced quiescent cells were more prone to develop drug-tolerant persister (DTP) tumor cells postulated to be responsible for cancer relapse and metastasis. Then, UMI-mRNA sequencing uncovered the ferroptosis pathway as the pathway most influenced by fasting. Combining fasting with ferroptosis inducer treatment leads to tumor inhibition and eradication of quiescent cells by boosting autophagy. Our results suggest that ferroptosis could improve the antitumor activity of FMD+chemotherapy and highlight a potential therapeutic opportunity to avoid DTP cells-driven tumor relapse and therapy failure. A full list of funding bodies can be found in the Acknowledgements section.

KDM5D Histone Demethylase Identifies Platinum-Tolerant Head and Neck Cancer Cells Vulnerable to Mitotic Catastrophe

Head and neck squamous cell carcinoma (HNSCC) is a major contributor to cancer incidence globally and is currently managed by surgical resection followed by adjuvant chemoradiotherapy. However, local recurrence is the major cause of mortality, indicating the emergence of drug-tolerant persister cells. A specific histone demethylase, namely lysine-specific demethylase 5D (KDM5D), is overexpressed in diverse types of cancers and involved in cancer cell cycle regulation. However, the role of KDM5D in the development of cisplatin-tolerant persister cells remains unexplored. Here, we demonstrated that KDM5D contributes to the development of persister cells. Aurora Kinase B (AURKB) disruption affected the vulnerability of persister cells in a mitotic catastrophe-dependent manner. Comprehensive in silico, in vitro, and in vivo experiments were performed. KDM5D expression was upregulated in HNSCC tumor cells, cancer stem cells, and cisplatin-resistant cells with biologically distinct signaling alterations. In an HNSCC cohort, high KDM5D expression was associated with a poor response to platinum treatment and early disease recurrence. KDM5D knockdown reduced the tolerance of persister cells to platinum agents and caused marked cell cycle deregulation, including the loss of DNA damage prevention, and abnormal mitosis-enhanced cell cycle arrest. By modulating mRNA levels of AURKB, KDM5D promoted the generation of platinum-tolerant persister cells in vitro, leading to the identification of the KDM5D/AURKB axis, which regulates cancer stemness and drug tolerance of HNSCC. Treatment with an AURKB inhibitor, namely barasertib, resulted in a lethal consequence of mitotic catastrophe in HNSCC persister cells. The cotreatment of cisplatin and barasertib suppressed tumor growth in the tumor mouse model. Thus, KDM5D might be involved in the development of persister cells, and AURKB disruption can overcome tolerance to platinum treatment in HNSCC.

Abstract PD4-09: PD4-09 Single cell RNA-sequencing identifies intra-tumoral cellular heterogeneity and drug-induced subpopulation shifts in Triple Negative Breast Cancer mouse models

Abstract Introduction: Triple Negative Breast Cancer (TNBC) is an aggressive disease with a poor prognosis that accounts for 10-20% of breast cancer cases worldwide. Intra-tumoral heterogeneity and tumor cell plasticity are thought to contribute to drug resistance in TNBCs. Our work aims to: 1) precisely identify the intra-tumoral heterogeneity in cellular states present within TNBC, and 2) test whether drugs can block or initiate plasticity between subpopulations to improve drug sensitivity. We hypothesize that treatment(s) induce cellular plasticity, thus causing cells to shift into resistant states that persist until treatment is removed. We further hypothesize that these resistant subpopulations give rise to new tumor outgrowths once the treatment stops. Methods: To test this, we are treating TP53-/- Genetically Engineered Mouse Model (GEMM) syngeneic transplant tumors of the basal-like TNBC phenotype with the chemotherapeutic doublet of carboplatin/paclitaxel, and targeted agents implicated in plasticity including the MEK inhibitor trametinib, a chromatin remodeling inhibitor I-BET151, and the dihydroorotate dehydrogenase inhibitor brequinar. To identify cellular subpopulations and examine their response to treatment, we performed both in vivo and in vitro drug sensitivity testing, as well as gene expression profiling using single cell RNA-sequencing (scRNAseq). Results: We have identified clear intra-tumoral heterogeneity with at least 6 distinct cell states present, including basal, mesenchymal/claudin-low, and proliferative subpopulations in vivo in most TNBC GEMM models. We performed 18 individual scRNAseq experiments on the TP53-/- 2225L GEMM transplant line with the aforementioned treatments and untreated controls in triplicate and compared subpopulation frequencies in treated versus untreated tumors. Notably, treatment with trametinib and brequinar caused the rise of two rare subpopulations (i.e. 1% to 3-4% of total tumor cells) that express genes consistent with previously described drug-tolerant persisters (DTP), which we have called “Epithelial-DTP” (Tacstd2, Krt6a, and Cryab enriched) and “Mesenchymal-DTP” (Snai2 and Sca-1 enriched). A gene signature generated from the Epithelial-DTP subpopulation predicted poor patient outcomes in neoadjuvant chemotherapy treated TNBC patients. Further, in TNBC patient-derived xenografts (PDX), these two DTP subpopulations are also present and induced by treatment to an even greater frequency. Ongoing experiments include the use of fluorescence-activated cell sorting to isolate and functionally test the tumor-initiating capabilities of these two rare cell subpopulations. In addition, many experiments are underway to identify means to therapeutically target these DTP cells, with these results to be presented. Ultimately, identifying these rare drug resistant subpopulations, and identifying means to eradicate them, could vastly improve therapeutic regimens and outcomes for patients with TNBCs. Citation Format: Cherise R. Glodowski, Kevin R. Mott, Denis Okumu, Michael P. East, Timothy C. Elston, Gary L. Johnson, Charles M. Perou. PD4-09 Single cell RNA-sequencing identifies intra-tumoral cellular heterogeneity and drug-induced subpopulation shifts in Triple Negative Breast Cancer mouse models [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD4-09.

Abstract P1-13-02: The Aberrant Activity of Retrotransposon Elements Mediates the Chemo-tolerant Persister Cells Relapse in TNBC

Abstract The emergence of acquired drug resistance through therapeutic treatment remains a critical threat to efficient chemotherapy, target therapy, or immune therapy. These resistant cancer cells most often lead to relapse or metastasis. The development of drug resistance is a multi-step evolutionary adaptation for cancer cells. Tumor heterogeneity, cancer cells’ plasticity, and microenvironment contribute to the resistant clone’s formation. Therefore, a time-lapse adaptation model is critical to define the mechanism of drug resistance evolution. Recently, several studies have revealed that the initial acquired drug resistance might be conferred by transient events, such as drug-tolerant persisters (DTP) that might occur in a subpopulation of the cancer cells at the early stage of the treatment, which were then followed by the transcriptomic reprogramming and secondary-wave genetic mutations in the progression of resistance development. In the clinic, chemotherapy is still the mainstream treatment for TNBC, and one of the primary chemo agents is doxorubicin. Although the initial responsive rate of doxorubicin-based chemotherapy is up to 70%, it is well recognized that TNBC cells usually generate an evolutionary adaptive response that can result in the acquired drug-resistance and multi-drug resistant phenotypes. To date, numerous different mechanisms of acquired chemo-resistance have been reported, but the vast majority of these results have been derived from the continuous-high-dose-exposure acquired resistant cell line models. Since the chemo-treatment dosage in these artificial models is well above what is physiologically achievable in patients, few of them can mimic the actual situation of resistance development or improve the clinical trial outcomes. Moreover, most of these studies only characterized the terminal resistant cells, which are challenging to be resensitized because of their dominant genetic mutations. In this study, we hypothesize that the TNBC chemo-resistant cells may derive from the early-stage reversible chemo-tolerant “DTP-like” (CTP) cells, and early-stage epigenetic landscape perturbation might determine the progression of chemo-resistance development. To test the hypothesis and overcome the previous model limitations, based on the clinical drug exposure kinetics for doxorubicin, we developed an in vitro “pulsing-treatment CTPs regrowth” model (referred to as CTP model), which could mimic the clinical treatment and provide therapeutically relevant insights into the initial drug-induced stress response and resistance development. Leveraging this CTP model, we are able to define the early event for drug response, in which the doxorubicin-treated cells showed a senescence-like phenotype, and the interferon alpha (type I) pathway was activated. Furthermore, unexpectedly, we found that the expression of HERVs was significantly activated but LINE1s not. To further explore the TEs reactivation, we did the single cell RNA-seq for 0h, 2h, and 4 days samples. With a novel bioinformatic workflow, we integrated the TE expression information with coding genes mRNA profiling from the same single cell RNA-seq dataset and identified the IFN-enriched cluster had higher expression of HERVs. Herein, a subpopulation of HERVhigh cells with IFN activation was identified as a “hot-cluster” which might be the early determinant in the resistance evolution. Citation Format: Zijian Zhang, Yiyang Wang, Xinluo Luo, Xuwen Li, Xiaomei Zhan, Yumin Zheng, Jun Ding, Tao Wu. The Aberrant Activity of Retrotransposon Elements Mediates the Chemo-tolerant Persister Cells Relapse in TNBC [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-02.

Redox signaling in drug-tolerant persister cells as an emerging therapeutic target.

Drug-tolerant persister (DTP) cells have attracted significant interest, given their predominant role in treatment failure. In this respect, DTP cells reportedly survive after anticancer drug exposure, and their DNA repair mechanisms are altered to enhance adaptive mutation, accounting for the emergence of drug-resistant mutations. DTP cells resume proliferation upon treatment withdrawal and are responsible for cancer relapse. Current evidence suggests that DTP cells mediate redox signaling-mediated cellular homeostasis by developing various adaptive mechanisms, especially metabolic reprogramming that promotes mitochondrial oxidative respiration and a robust antioxidant process. There is an increasing consensus that disrupting redox homeostasis by intervening with redox signaling is theoretically a promising therapeutic strategy for targeting these sinister cells. In this review, we provide a comprehensive overview of the characteristics of DTP cells and the underlying mechanisms involved in redox signaling, aiming to provide a unique perspective on potential therapeutic applications based on their vulnerabilities to redox regulation.