Drug-tolerant Cells Research Articles

P76.71 RYK Confers Drug Tolerance to Osimertinib in Lung Cancer Cells with EGFR Mutations

Emergence of acquired resistance is almost inevitable during EGFR-tyrosine kinase inhibitor therapy for non-small-cell lung cancer (NSCLC) harboring EGFR mutations. Drug tolerance, a reversible state of drug insensitivity in the early phases of tyrosine kinase inhibitor therapy, is considered to serve as the basis of recurrent disease. Therefore, it is important to elucidate the molecular mechanisms of drug tolerance. Five EGFR-mutant NSCLC cell lines were used in this study. We established drug-tolerant cells (DTCs) via 72 h treatment with osimertinib (600 nM) or afatinib (60 nM). Acquisition of drug tolerance was evaluated by growth inhibitory assay, and the molecular mechanisms of drug tolerance were analyzed by phospho-RTK array. DTCs were successfully induced in PC9, HCC4006, and H1975 cells against osimertinib and in PC9 cells against afatinib. Next, we compared the phosphorylation levels of EGFR in HCC4006 cells after exposure to 600 nM osimertinib or 60 nM afatinib. 600 nM osimertinib inhibited the phosphorylation of EGFR in HCC4006 cells more than 60 nM afatinib. This was inconsistent with the experiments using PC9 cells (both 600 nM osimertinib and 60 nM afatinib induced drug tolerance in PC9 cells), showing that inhibition of EGFR phosphorylation was similar between 600 nM osimertinib and 60 nM afatinib.To investigate if the suppression level of EGFR phosphorylation was related to the acquisition of a drug-tolerant phenotype, we exposed HCC4006 cells to different concentrations of osimertinib or afatinib. Lower doses of osimertinib (66 nM and 200 nM) failed to induce a drug-tolerant state in HCC4006 cells, although the growth inhibition rate of 66 nM osimertinib for HCC4006 parental cells was identical to that of 600 nM osimertinib. However, higher doses of afatinib (180 nM and 540 nM) induced a drug-tolerant state in HCC4006 cells. We also observed that lower doses of osimertinib and afatinib failed to induce a drug-tolerant state in PC9 and H1975 cells. These results indicated that stronger inhibition of phosphorylated EGFR is necessary to induce DTCs. Next, in the analysis of HCC4006 DTCs against osimertinib, we observed increased receptor-like tyrosine kinase (RYK) expression, and siRNA-mediated RYK knockdown inhibited the proliferation of DTCs. This phenomenon was HCC4006 cell line-specific since we observed that PC9 parental cells and PC9-600 nM osim-DTCs were both resistant to RYK knockdown. In addition, cabozantinib, which reportedly inhibit RYK, monotherapy effectively inhibited the proliferation of HCC4006-600 nM osim-DTCs but not HCC4006 parental cells. However, cabozantinib was not active against PC9-600 nM osim-DTCs and PC9 parental cells. Our study found that the inducibility of DTCs depended on the type of cell line and the drug concentration. It is possible that the optimal dose of EGFR-TKI in each patient may reduce the emergence of DTCs in clinical practice. We also found high expression of RYK was a molecular mechanism of the drug-tolerant state in HCC4006 cells against osimertinib. Further studies are necessary to fully understand the DTCs that are essential for the appropriate primary double-strike therapy for lung cancers with EGFR mutations.

Tackling Drug Resistance in EGFR Exon 20 Insertion Mutant Lung Cancer.

Insertion mutations in exon 20 (Ex20ins) of the epidermal growth factor receptor (EGFR) gene are the largest class of EGFR mutations in non-small cell lung cancer (NSCLC) for which there are currently no approved targeted therapies. NSCLC patients with these mutations do not respond to clinically approved EGFR tyrosine kinase inhibitors (TKIs) and have poor outcomes. A number of early phase clinical trials are currently underway to evaluate the efficacy of a new generation of TKIs that are capable of binding to and blocking Ex20ins. Although these agents have shown some clinical activity, patient responses have been restricted by dose-limiting toxicity or rapid acquisition of resistance after a short response. Here we review the current understanding of the mechanisms of resistance to these compounds, which include on-target EGFR secondary mutations, compensatory bypass pathway activation and acquisition of an EMT phenotype. Taking lessons from conventional EGFR inhibitor therapy in NSCLC, we also consider other potential sources of resistance including the presence of drug-tolerant persister cells. We will discuss therapeutic strategies which have the potential to overcome different forms of drug resistance. We conclude by evaluating recent technological developments in drug discovery such as PROTACs as a means to better tackle TKI resistance in NSCLC harbouring Ex20ins mutations.

6P AsiDNA targets drug-tolerant persister cells responsible of resistance to PARP inhibitors

6P AsiDNA targets drug-tolerant persister cells responsible of resistance to PARP inhibitors

Dose-dependence in acquisition of drug tolerant phenotype and high RYK expression as a mechanism of osimertinib tolerance in lung cancer

ObjectiveEmergence of acquired resistance is almost inevitable during EGFR-tyrosine kinase inhibitor therapy for non-small-cell lung cancer (NSCLC) harboring EGFR mutations. Drug tolerance, a reversible state of drug insensitivity in the early phases of tyrosine kinase inhibitor therapy, is considered to serve as the basis of recurrent disease. Therefore, it is important to elucidate the molecular mechanisms of drug tolerance. Materials and methodsFive EGFR-mutated NSCLC cell lines were used in this study. We established drug-tolerant cells (DTCs) via 72 h treatment with osimertinib (600 nM) or afatinib (60 nM). Acquisition of drug tolerance was evaluated by growth inhibitory assay, and the molecular mechanisms of drug tolerance were analyzed by phospho-RTK array. ResultsDTCs were successfully induced in PC9, HCC4006, and H1975 cells against osimertinib and in PC9 cells against afatinib. We observed that a high drug concentration was required to induce DTCs, and HCC4006 cells become tolerant when a higher dose of afatinib (>180 nM) was used. In the analysis of HCC4006 DTCs against osimertinib, we observed increased receptor-like tyrosine kinase (RYK) expression, and siRNA-mediated RYK knockdown inhibited the proliferation of DTCs. ConclusionsThese results suggest that induction of DTCs is dose-dependent, and increased RYK expression was the mechanism of drug tolerance in HCC4006 cells against osimertinib.

Heterogeneity and Clonal Evolution of Acquired PARP Inhibitor Resistance in TP53- and BRCA1-Deficient Cells.

Homologous recombination (HR)-deficient cancers are sensitive to poly-ADP ribose polymerase inhibitors (PARPi), which have shown clinical efficacy in the treatment of high-grade serous cancers (HGSC). However, the majority of patients will relapse, and acquired PARPi resistance is emerging as a pressing clinical problem. Here we generated seven single-cell clones with acquired PARPi resistance derived from a PARPi-sensitive TP53 -/- and BRCA1 -/- epithelial cell line generated using CRISPR/Cas9. These clones showed diverse resistance mechanisms, and some clones presented with multiple mechanisms of resistance at the same time. Genomic analysis of the clones revealed unique transcriptional and mutational profiles and increased genomic instability in comparison with a PARPi-sensitive cell line. Clonal evolutionary analyses suggested that acquired PARPi resistance arose via clonal selection from an intrinsically unstable and heterogenous cell population in the sensitive cell line, which contained preexisting drug-tolerant cells. Similarly, clonal and spatial heterogeneity in tumor biopsies from a clinical patient with BRCA1-mutant HGSC with acquired PARPi resistance was observed. In an imaging-based drug screening, the clones showed heterogenous responses to targeted therapeutic agents, indicating that not all PARPi-resistant clones can be targeted with just one therapy. Furthermore, PARPi-resistant clones showed mechanism-dependent vulnerabilities to the selected agents, demonstrating that a deeper understanding on the mechanisms of resistance could lead to improved targeting and biomarkers for HGSC with acquired PARPi resistance. SIGNIFICANCE: This study shows that BRCA1-deficient cells can give rise to multiple genomically and functionally heterogenous PARPi-resistant clones, which are associated with various vulnerabilities that can be targeted in a mechanism-specific manner.

Dynamic single-cell RNA sequencing identifies immunotherapy persister cells following PD-1 blockade.

Resistance to oncogene-targeted therapies involves discrete drug-tolerant persister cells, originally discovered through in vitro assays. Whether a similar phenomenon limits efficacy of programmed cell death 1 (PD-1) blockade is poorly understood. Here, we performed dynamic single-cell RNA-Seq of murine organotypic tumor spheroids undergoing PD-1 blockade, identifying a discrete subpopulation of immunotherapy persister cells (IPCs) that resisted CD8+ T cell-mediated killing. These cells expressed Snai1 and stem cell antigen 1 (Sca-1) and exhibited hybrid epithelial-mesenchymal features characteristic of a stem cell-like state. IPCs were expanded by IL-6 but were vulnerable to TNF-α-induced cytotoxicity, relying on baculoviral IAP repeat-containing protein 2 (Birc2) and Birc3 as survival factors. Combining PD-1 blockade with Birc2/3 antagonism in mice reduced IPCs and enhanced tumor cell killing in vivo, resulting in durable responsiveness that matched TNF cytotoxicity thresholds in vitro. Together, these data demonstrate the power of high-resolution functional ex vivo profiling to uncover fundamental mechanisms of immune escape from durable anti-PD-1 responses, while identifying IPCs as a cancer cell subpopulation targetable by specific therapeutic combinations.

Adrenomedullin-CALCRL axis controls relapse-initiating drug tolerant acute myeloid leukemia cells

Drug tolerant/resistant leukemic stem cell (LSC) subpopulations may explain frequent relapses in acute myeloid leukemia (AML), suggesting that these relapse-initiating cells (RICs) persistent after chemotherapy represent bona fide targets to prevent drug resistance and relapse. We uncover that calcitonin receptor-like receptor (CALCRL) is expressed in RICs, and that the overexpression of CALCRL and/or of its ligand adrenomedullin (ADM), and not CGRP, correlates to adverse outcome in AML. CALCRL knockdown impairs leukemic growth, decreases LSC frequency, and sensitizes to cytarabine in patient-derived xenograft models. Mechanistically, the ADM-CALCRL axis drives cell cycle, DNA repair, and mitochondrial OxPHOS function of AML blasts dependent on E2F1 and BCL2. Finally, CALCRL depletion reduces LSC frequency of RICs post-chemotherapy in vivo. In summary, our data highlight a critical role of ADM-CALCRL in post-chemotherapy persistence of these cells, and disclose a promising therapeutic target to prevent relapse in AML.

Single cell biomass tracking allows identification and isolation of rare targeted therapy-resistant DLBCL cells within a mixed population.

Adaptive resistance is a major limitation in the use of targeted therapies for cancer. Using real time biomass tracking, we demonstrate the isolation and identification of rare (1% fraction) diffuse large B cell lymphoma cells resistant to the PI3K inhibitor idelalisib, from an otherwise sensitive population. This technique allows direct study of these rare, drug tolerant cells.

Nanoengineered Disruption of Heat Shock Protein 90 Targets Drug-Induced Resistance and Relieves Natural Killer Cell Suppression in Breast Cancer.

Drug-induced resistance, or tolerance, is an emerging yet poorly understood failure of anticancer therapy. The interplay between drug-tolerant cancer cells and innate immunity within the tumor, the consequence on tumor growth, and therapeutic strategies to address these challenges remain undescribed. Here, we elucidate the role of taxane-induced resistance on natural killer (NK) cell tumor immunity in triple-negative breast cancer (TNBC) and the design of spatiotemporally controlled nanomedicines, which boost therapeutic efficacy and invigorate "disabled" NK cells. Drug tolerance limited NK cell immune surveillance via drug-induced depletion of the NK-activating ligand receptor axis, NK group 2 member D, and MHC class I polypeptide-related sequence A, B. Systems biology supported by empirical evidence revealed the heat shock protein 90 (Hsp90) simultaneously controls immune surveillance and persistence of drug-treated tumor cells. On the basis of this evidence, we engineered a "chimeric" nanotherapeutic tool comprising taxanes and a cholesterol-tethered Hsp90 inhibitor, radicicol, which targets the tumor, reduces tolerance, and optimally reprimes NK cells via prolonged induction of NK-activating ligand receptors via temporal control of drug release in vitro and in vivo. A human ex vivo TNBC model confirmed the importance of NK cells in drug-induced death under pressure of clinically approved agents. These findings highlight a convergence between drug-induced resistance, the tumor immune contexture, and engineered approaches that consider the tumor and microenvironment to improve the success of combinatorial therapy. SIGNIFICANCE: This study uncovers a molecular mechanism linking drug-induced resistance and tumor immunity and provides novel engineered solutions that target these mechanisms in the tumor and improve immunity, thus mitigating off-target effects.

Modulating proteasome inhibitor tolerance in multiple myeloma: an alternative strategy to reverse inevitable resistance

BackgroundResistance to proteasome inhibitors (PIs) is a major obstacle to the successful treatment of multiple myeloma (MM). Many mechanisms have been proposed for PI resistance; however, our mechanistic understanding of how PI resistance is inevitably acquired and reversed remains incomplete.MethodsMM patients after bortezomib relapse, MM cell lines and mouse models were used to generate matched resistant and reversed cells. RNA sequencing and bioinformatics analyses were employed to assess dysregulated epigenetic regulators. In vitro and in vivo procedures were used to characterise PI-tolerant cells and therapeutic efficacy.ResultsUpon PI treatment, MM cells enter a slow-cycling and reversible drug-tolerant state. This reversible phenotype is associated with epigenetic plasticity, which involves tolerance rather than persistence in patients with relapsed MM. Combination treatment with histone deacetylase inhibitors and high-dosage intermittent therapy, as opposed to sustained PI monotherapy, can be more effective in treating MM by preventing the emergence of PI-tolerant cells. The therapeutic basis is the reversal of dysregulated epigenetic regulators in MM patients.ConclusionsWe propose an alternative non-mutational PI resistance mechanism that explains why PI relapse is inevitable and why patients regain sensitivity after a ‘drug holiday’. Our study also suggests strategies for epigenetic elimination of drug-tolerant cells.

Gene Panel of Persister Cells as a Prognostic Indicator for Tumor Repopulation After Radiation.

Tumor repopulation during cycles of radiotherapy limits the radio-response in ensuing cycles and causes failure of treatment. It is thus of vital importance to unveil the mechanisms underlying tumor repopulating cells. Increasing evidence suggests that a subpopulation of drug-tolerant persister cancer cells (DTPs) could survive the cytotoxic treatment and resume to propagate. Whether these persister cells contribute to development of radio-resistance remains elusive. Based on the genetic profiling of DTPs by integrating datasets from Gene Expression Omnibus database, this study aimed to provide novel insights into tumor-repopulation mediated radio-resistance and identify predictive biomarkers for radio-response in clinic. A prognostic risk index, grounded on four persister genes (LYNX1, SYNPO, GADD45B, and PDLIM1), was constructed in non-small-cell lung cancer patients from The Cancer Genome Atlas Program (TCGA) using stepwise Cox regression analysis. Weighted gene co-expression network analysis further confirmed the interaction among persister-gene based risk score, radio-response and overall survival time. In addition, the predictive role of risk index was validated in vitro and in other types of TCGA patients. Gene set enrichment analysis was performed to decipher the possible biological signaling, which indicated that two forces behind persister cells, stress response and survival adaptation, might fuel the tumor repopulation after radiation. Targeting these persister cells may represent a new prognostic and therapeutic approach to enhance radio-response and prevent radio-resistance induced by tumor repopulation.

Abstract PO-100: Expressed molecular barcoding coupled with single cell RNAseq enables a high resolution investigation into the evolution of drug tolerance

Abstract EGFR targeted kinase inhibitors (TKIs) are the standard of care in non-small cell lung cancer (NSCLC) patients with activating mutations in the epidermal growth factor receptor (EGFR). Patients initially respond well to EGFR inhibitors, although the majority only achieve a partial response and a subset of drug-tolerant persister cells remain at minimal residual disease (MRD). These drug-tolerant persister cells represent a cell reservoir from which de novo genetic mutations, such as EGFRT790M or MET amplification, can arise to render the tumor fully drug-resistant. Previous studies suggest that drug-tolerant cells rely on an altered chromatin state to survive EGFR-inhibition. However, it is still unclear whether the drug-tolerant cell population emerges through selection for cells that pre-existed in that state or through and adaptation in response to drug. It is also unknown if drug-tolerant persister cells rely on a single survival mechanism that could be exploited to more effectively target this population or if multiple independent mechanisms are being utilized and need to be targeted to fully suppress drug tolerance. Despite the urgent clinical need to answer these questions, we have lacked the techniques capable of the dynamic resolution necessary to investigate the emergence of drug tolerance throughout the course of treatment within individual cell lineages. Here we present a strategy to investigate the clonal evolution of drug tolerance in EGFRmut NSCLC using an expressed molecular barcoding library coupled with single cell RNAseq (scRNAseq). We found that the cell lineages that are destined to become drug-tolerant are pre-defined, although the epigenetic drug-tolerant state does not pre-exist. We observed multiple distinct heterogeneous classes of drug-tolerant cells with unique gene expression signatures as well as distinct trajectories in response to EGFRi. We observed evidence of putative mechanisms of drug tolerance, such as EMT and adaptive MAPK signaling, in parallel trajectory classes across cell lines. Finally, we compared EGFRi/TKI drug combinations versus EGFRi/chemotherapy combinations to investigate which therapeutic approach was more efficacious in targeting multiple trajectory classes of drug tolerant cells. Taken together, our work presents a new technology that enables a comprehensive interrogation of drug response over time and provides greater insight into how drug-tolerant cells evolve over the course of drug treatment, which ultimately can help inform combination treatment strategies for patients in the clinic. Citation Format: Jennifer L. Cotton, Viveksagar Krisnamurthy Radhakrishna, Javier Estrada Diez, David A. Ruddy, Kathleen Sprouffske, Gaylor Boulay, Michelle Piquet, Joel Wagner, Youngchul Song, Xiaoyan Li, Katja Schumacher, Joshua Korn, Erick J. Morris, Peter S. Hammerman, Jeffrey A. Engelman, Matthew J. Niederst. Expressed molecular barcoding coupled with single cell RNAseq enables a high resolution investigation into the evolution of drug tolerance [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-100.

Inflammasome Sensor NLRP1 Confers Acquired Drug Resistance to Temozolomide in Human Melanoma.

Simple SummaryAcquired drug resistance remains a challenge in the management of cancer patients. Strategies to overcome drug resistance and enhance the response include the combination therapy with agents that target known resistance mechanisms. Inflammasomes are mediators of inflammation. We previously reported the involvement of NACHT, LRR and PYD domains-containing protein (NLRP) 1/3 inflammasomes in the melanoma microenvironment and tumor growth. The aim of this study was to further determine the role of NLRP1 in acquired drug resistance in melanoma. We, for the first time, demonstrate that NLRP1 inflammasome is involved in the development of acquired drug resistance of melanoma. Because drug-tolerant cancer cells become cross-tolerant to other classes of cancer drugs, shared mechanisms could be involved in acquired drug resistance to other drugs.Cancer cells gain drug resistance through a complex mechanism, in which nuclear factor-κB (NF-κB) and interleukin-1β (IL-1β) are critical contributors. Because NACHT, LRR and PYD domains-containing protein (NLRP) inflammasomes mediate IL-1β maturation and NF-κB activation, we investigated the role of inflammasome sensor NLRP1 in acquired drug resistance to temozolomide (TMZ) in melanoma. The sensitivity of melanoma cells to TMZ was negatively correlated with the expression levels of O6-methylguanine-DNA methyltransferase (MGMT), the enzyme to repair TMZ-induced DNA lesions. When MGMT-low human melanoma cells (1205Lu and HS294T) were treated with TMZ for over two months, MGMT was upregulated, and cells became resistant. However, the resistance mechanism was independent of MGMT, and the cells that acquired TMZ resistance showed increased NLRP1 expression, NLRP inflammasome activation, IL-1β secretion, and NF-κB activity, which contributed to the acquired resistance to TMZ. Finally, blocking IL-1 receptor (IL-1R) signaling with IL-1R antagonist decreased TMZ-resistant 1205Lu tumor growth in vivo. Although inflammation has been associated with drug resistance in various cancers, our paper is the first to demonstrate the involvement of NLRP in the development of acquired drug resistance. Because drug-tolerant cancer cells become cross-tolerant to other classes of cancer drugs, NLRP1 might be a suitable therapeutic target in drug-resistant melanoma, as well as in other cancers.

Beneficial effect of erlotinib and trastuzumab emtansine combination in lung tumors harboring EGFR mutations

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) is the standard therapy for non-small cell lung cancer (NSCLC) harboring EGFR mutations, but the resistance is inevitable. The drug-tolerant persister cancer cells are thought to be involved in the resistance. We recently reported that HER2 expression had a negative impact on time-to-treatment-failure in patients with EGFR mutant NSCLC. In this study, we hypothesized that HER2 might be a potential target for alternative combination therapy in NSCLC harboring EGFR mutations. In vitro study showed that the level of HER2 expression had no correlation with the sensitivity to EGFR-TKI, erlotinib but showed some correlation with HER2-inhibitor, ado-trastuzumab emtansine (T-DM1) in multiple EGFR-mutant lung cancer cell lines. In addition, HER2 expression was increased in persister cancer cells in 11–18 cell line harboring EGFR L858R or HCC827 cell line harboring EGFR exon 19 deletion after the exposure to erlotinib in vitro and in vivo. The combination of erlotinib and T-DM1 showed a superior inhibitory effect on cell proliferation compared with those of the erlotinib or T-DM1 alone in either 11–18 or HCC827 cells in vitro. The combination therapy also induced a significantly greater inhibitory effect on tumor growth in xenograft model in mice transplanted with either 11–18 or HCC827 cells compared with erlotinib alone or T-DM1 alone. No body weight loss was observed in these mice. These results suggested that the combination therapy with EGFR-TKI and T-DM1 might be a potentially promising strategy for treating lung cancer harboring EGFR mutations.

Drug-Tolerant Idling Melanoma Cells Exhibit Theory-Predicted Metabolic Low-Low Phenotype.

Cancer cells adjust their metabolic profiles to evade treatment. Metabolic adaptation is complex and hence better understood by an integrated theoretical-experimental approach. Using a minimal kinetic model, we predicted a previously undescribed Low/Low (L/L) phenotype, characterized by low oxidative phosphorylation (OXPHOS) and low glycolysis. Here, we report that L/L metabolism is observed in BRAF-mutated melanoma cells that enter a drug-tolerant “idling state” upon long-term MAPK inhibition (MAPKi). Consistently, using publicly available RNA-sequencing data of both cell lines and patient samples, we show that melanoma cells decrease their glycolysis and/or OXPHOS activity upon MAPKi and converge toward the L/L phenotype. L/L metabolism is unfavorable for tumor growth, yet supports successful cell division at ~50% rate. Thus, L/L drug-tolerant idling cells are a reservoir for accumulating mutations responsible for relapse, and it should be considered as a target subpopulation for improving MAPKi outcomes in melanoma treatment.

Abstract 4078: Acquired resistance to PARP inhibitors evolves from drug-tolerant cells vulnerable to AsiDNA

Abstract Purpose: PARP inhibitors (PARPi) resistance remains a major clinical hurdle as virtually all patients treated with PARPi will eventually relapse. We already demonstrated that co treatment with AsiDNA abrogates and reverses PARPi-acquired resistance. However, the mechanisms by which AsiDNA prevents genetic adaptations driving resistance and allows long-term efficacy of PARPi remain unknown. Here, we describe that PARPi resistance occurs through emergence of drug-tolerant cells (DTC) preventing PARPi from achieving long-term efficacy. Emerging evidence implicates a survival of residual DTC that constitutes a reservoir from which drug-resistant proliferative cells may emerge. We demonstrate in this study that AsiDNA is able to target specifically DTC and therefore represents a therapeutic opportunity to impede tumor relapse under PARPi treatment. Experimental design: AsiDNA is a double stranded (DS) DNA molecule (decoy oligonucleotide) that mimics DS DNA breaks to interfere with DNA repair, by over-activating a false DNA damage signaling through DNA-PK and PARP enzymes. We used different treatment protocols, cyclic or continuous, to select resistance to the PARPi Olaparib and Talazoparib and assessed the impact of AsiDNA addition on resistance abrogation, durability and irreversibility. We also addressed the mechanisms underlying the survival and evolution of the residual DTC under PARPi treatment, and how AsiDNA could impede their outgrowth. Results: Long-term treatment of BRCA2-mutated cells initially highly sensitive to PARPi leads to acquired resistance in all independently treated populations. Addition of AsiDNA to PARPi completely and irreversibly abolishes resistance emergence. This AsiDNA-dependent resistance abrogation is maintained for at least twelve weeks after AsiDNA removal (nine weeks after treatment start), indicating that AsiDNA maintains PARPi efficacy. PARPi-acquired resistance does not occur through selection of pre-existing BRCA2-wt and/or HR proficient sub-clones, but rather via (epi)-genetic evolution toward an active HR pathway and high genetic instability. We hypothesize that PARPi resistance occurs through a DTC stage as it is described for other targeted therapies like tyrosine kinase inhibitors (TKi). DTC are characterized by a transient senescent phenotype and evolve progressively into proliferative state. We showed that repeated treatments with PARPi lead to the apparition of a DTC-like population displaying a senescent phenotype evolving over time into proliferative cells. The co-treatment with AsiDNA and PARPi prevents DTC regrowth. Conclusion: Several groups have demonstrated that resistance to TKi can emerge from DTC. Our findings provide for the first time the evidence that PARPi-resistant cells can evolve from DTC, and point to the therapeutic opportunity of combining AsiDNA and PARPi to prevent or overcome resistance in clinical situation. Citation Format: Wael Jdey, Olivier Calvayrac, Orlane Maloudi, Florent Ubelmann, Gilles Favre, Françoise Bono. Acquired resistance to PARP inhibitors evolves from drug-tolerant cells vulnerable to AsiDNA [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4078.

Abstract 2994: Inhibition of the NRP2b:GSK3β binding interaction with peptides and macrocycles exerts anticancer effects in lung cancer

Abstract Introduction: Neuropilin-2b (NRP2b) expression is associated with pro-tumorigenic properties of NSCLCs. We previously reported that NRP2b promotes metastasis and drug resistance, while the canonical NRP2a isoform is inhibitory. Our recent work indicates that the NRP2b cytoplasmic domain recruits GSK3β to phosphorylate and promote degradation of co-recruited PTEN, thereby enhancing AKT activity leading to enhanced survival, migration and drug resistance. We identified a 15-amino acid motif near the NRP2b C-terminus required for interaction with GSK3β. Herein, we describe the anticancer effect of peptides and drug-like macrocycles designed to disrupt this interaction. Methods: We used the cytoplasmic sequence of NRP2b for decoy peptides designed to disrupt the interaction with GSK3β. Control peptides were synthesized with alanine replacements for three amino acids suggested as crucial for GSK3β recruitment. All peptides were N-terminally myristoylated to promote association with and flipping to the inner leaflet of the plasma membrane. A library of over 42,000 drug-like macrocycles was screened in silico for compounds predicted to fit and compete with the NRP2b:GSK3β binding interface. Decoy peptides and macrocycles were assessed for anti-NRP2b activity using assays for migration and drug-tolerant persister cells. Transwell migration assays were performed using a Neuro Probe Reusable Multiwell Chemotaxis Chamber. Persister cell assays were performed with EGFR mutant PC9 and HCC827 cells in the presence of EGFR inhibitors (gefitinib or osimertinib), followed by drug withdrawal and colony formation assays. Results: As previously reported, knockdown of NRP2b inhibited, while knockdown of NRP2a enhanced, migration of lung cancer cell lines. Importantly, emergence of drug-tolerant persister cells was similarly inhibited by knockdown of NRP2b and enhanced by knockdown of NRP2a. Wild type, but not control, peptides abrogated the pro-tumorigenic effects of NRP2a knockdown on both migration and persister cell formation with IC50s of ~250 nM. The most effective macrocycles, M3 and M7, inhibited NRP2b-dependent migration with IC50s of 1-3 µM. Both macrocycles also inhibited generation of persister cell colonies from shNRP2a-PC-9 cells. However, only M3 was effective at blocking persisters from shNRP2a-HCC827 cells. Conclusions: NRP2b:GSK3β interaction is a therapeutic target in NSCLC affecting migration/invasion and persister colony formation associated with the emergence of resistance to EGFR inhibitors. This work justifies further development of these peptides and drug-like macrocycles as therapeutic modalities in lung cancer. Citation Format: Cecile Nasarre, Yuri K. Peterson, Patrick Nasarre, Anastasios Dimou, Kent E. Armeson, Harry A. Drabkin, Nancy Demore, Chadrick E. Denlinger, Robert M. Gemmill. Inhibition of the NRP2b:GSK3β binding interaction with peptides and macrocycles exerts anticancer effects in lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2994.

Abstract 2986: NR2F1 underlies persistence of residual disease in melanoma

Abstract Despite the clinical success of targeted therapy and checkpoint inhibitors in melanoma, therapeutic responses are transient, followed by relapse that may be driven by a small subpopulation of residual or drug-tolerant cells. Understanding residual disease and metastasis mechanisms can provide clues both for developing improved versions of a drug and for guiding the selection of appropriate drug combinations for melanoma therapy. Here, we found that the well-known marker of tumor dormancy, Nuclear Receptor Subfamily 2 Group F Member 1 (NR2F1), was overexpressed in minimal disease residual cells following CDK4/6 and MEK inhibitors (CDK4/6i+MEKi) treatment in vivo. Furthermore, melanoma cells overexpressing NR2F1 were less sensitive to (CDK4/6i+MEKi) or BRAF and MEK inhibitors (BRAFi+MEKi) treatment in vitro and in vivo models, inhibiting apoptosis. Surprisingly, we did not find any evidence of decreased cell growth in our model. Using a three-dimensional tumor spheroid assay in vitro, we found the NR2F1 expression enhanced melanoma invasion following CDK4/6i+MEKi or BRAFi+MEKi treatments. The use of published RNA Seq data sets that were gathered from the GEO database and Single Cell Seq data sets from PDX melanoma samples showed that high expression of NR2F1 is enriched in the undifferentiated cell state and invasive cells, respectively. Furthermore, BRAF mutant patient sample with an acquired mutation in NRAS Q61R following BRAFi+MEKi+CDKi presented a high expression of NR2F1. Altogether, these findings suggest that NR2F1 may play a role in residual disease persistence besides known features of tumor dormancy, especially important in determining responses to dramatic changes in the environment, such as changes induced by anti-cancer therapy. Citation Format: Manoela Tiago, Jessica L. Teh, Timothy J. Purwin, Weijia Cai, Connor Hollingworth, Melisa Lopez-Anton, Julio A. Aguirre-Ghiso, Andrew E. Aplin. NR2F1 underlies persistence of residual disease in melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2986.

Abstract 3081: SHOC2 is a critical modulator of the sensitivity to EGFR-TKI in non-small cell lung cancer cells

Abstract Epidermal Growth Factor Receptor tyrosine kinase inhibitors (EGFR-TKI) have become the first line treatment for patients with EGFR mutated non-small cell lung cancer (NSCLC) as a result of multiple clinical trials demonstrating superior response and less toxicity as compared to conventional cytotoxic agents. However, despite the clinical experience among nearly two decades, it is well known that EGFR-TKI monotherapy cannot cure EGFR mutation-positive lung cancer patients due to the occurrence of drug resistance. While dramatic initial tumor shrinkage is shown in most patients with EGFR-TKI treatment, therapeutic resistance is often observed in 1-2 years. This is likely caused by the outgrowth of rare pre-existing resistant clones and/or small fraction of drug tolerant persister cells (DTPs) which survive and acquire resistance to EGFR-TKIs. Several previous studies have clarified the acquired resistance mechanisms to EGFR-TKIs, but little is known how the DTPs survive during the initial phase of drug exposure and rapidly adapt to EGFR-TKIs. Utilizing the genome wide CRISPR/Cas9 screening, we have previously reported that DTPs were capable to exploit endoplasmic reticulum stress to survive EGFR-TKI exposure (Terai, et al, Cancer Research 2018). Further analysis of this data identified multiple genes which could affect the initial drug response to EGFR-TKIs, including the scaffold protein coding gene, SHOC2. In our present study we focused on this unique protein and revealed its critical function as a modulator of drug response to EGFR-TKIs in lung cancer cells. We confirmed that depletion/overexpression of SHOC2 in PC9 cells, an EGFR-dependent NSCLC cell line, renders the cells sensitive/resistant to all 1st to 3rd generation EGFR-TKIs. We have also confirmed similar findings with another EGFR dependent NSCLC cell line named H1975. Reduction of phosphorylation levels of ERK1/2 induced by EGFR-TKIs were stronger in PC9 or H1975 cells with SHOC2 knockout, indicating that SHOC2 can facilitate ERK1/2 reactivation during EGFR-TKI treatment. The above data indicate that SHOC2 is essential for DTPs and its function is correlated with MAPK-ERK pathway activity. Celastrol, a candidate SHOC2 inhibitor, inhibited the emergence of DTPs and synergized with EGFR-TKIs. Additionally, we showed that SHOC2 depletion enhanced the growth inhibitory effect of osimertinib in vivo. Lastly, we confirmed the increased expression of SHOC2 in clinical human samples which experienced EGFR-TKI failure. Our findings could offer a new treatment strategy for NSCLC patients with EGFR mutation leading to the increase of the degree and duration of EGFR TKIs response. Citation Format: Hideki Terai, Junko Hamamoto, Tadashi Manabe, Katsura Emoto, Takeshi Masuda, Satoshi Kuronuma, Keigo Kobayashi, Keita Masuzawa, Shinnosuke Ikemura, Sohei Nakayama, Ichiro Kawada, Yusuke Suzuki, Osamu Takeuchi, Yukio Suzuki, Sumio Ohtsuki, Hiroyuki Yasuda, Kenzo Soejima, Koichi Fukunaga. SHOC2 is a critical modulator of the sensitivity to EGFR-TKI in non-small cell lung cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3081.

Abstract PR11: Active YAP as a functional marker of drug-tolerant persister cells in EGFR-mutant and ALK fusion-positive NSCLC

Abstract Targeted therapies against clinically actionable oncogenic drivers in lung adenocarcinoma have significantly improved survival of cancer patients, but durable responses are limited due to the emergence of drug resistance. Resistance development is often characterized by the retention of a small subpopulation of cancer cells under drug treatment and their evolution from non-/low-proliferative residual disease to an aggressively growing resistant tumor. Most importantly, drug-tolerant persister cells have been identified as a reservoir for a multitude of drug resistance mechanisms and thus, their characterization and the development of rational combinatorial treatment may delay or prevent resistance development and improve treatment outcome for cancer patients. Using a multitude of in vitro models such as cell culture models and patient-derived organoids, we characterized signaling and transcriptional changes in drug-tolerant persisters. We identified YAP nuclear relocalization and its increased transcriptional activity as a key marker of persisters derived from EGFR-mutant and EML4-ALK fusion-positive specimen under third-generation TKI treatment. Image analysis of cells genetically engineered via CRISPR-Cas9 to express endogenously labeled YAP-mNeonGreen validated these results. Moreover, we were able to prove the functional relevance of YAP activation in drug persistence by overexpressing active mutants of YAP that are lacking inhibitory Hippo phosphorylation sites. The latter resulted in increased nuclear levels and transcriptional activity of YAP and mediated significantly reduced cell death under high-dose drug treatment in different cell line models. Using RNA sequencing, we show a clear evolutionary path from drug-sensitive parental cells to drug-tolerant persisters and long-term derived drug-acquired resistant cells. We are currently profiling vulnerabilities of drug-tolerant EGFR-mutant and EML4-ALK fusion persisters using genetic and pharmacologic approaches. In conclusion, YAP activation is a functional marker of EGFR-mutant and EML4-ALK fusion persisters derived under high-dose drug treatment with third-generation TKIs. Targeting YAP activation either on the level of upstream signaling input, its relocalization between cytoplasm and nucleus, or its action as transcriptional coactivator may represent a promising combinatorial treatment approach to limit resistance development and improve patient survival in lung adenocarcinoma. Citation Format: Franziska Haderk, David Allegakoen, Juan Guan, Wei Wu, Trever Bivona. Active YAP as a functional marker of drug-tolerant persister cells in EGFR-mutant and ALK fusion-positive NSCLC [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr PR11.