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TPS762 Background: Overcoming resistance to chemotherapy and immune checkpoint inhibitors remains an unmet need for PDAC. In this context, TGF-β plays a key role in PDAC by promoting the activation of fibroblasts and extracellular matrix deposition, facilitating epithelial-to-mesenchymal transition, immune evasion, and resistance to chemotherapy. Combination of FOLFIRINOX with losartan, an indirect TGF-β inhibitor with antifibrotic properties, has shown an increased R0 resection rate in pts with BR/LA PDAC, providing the rationale to explore the combination of other TGF-β targeting agents with FOLFIRINOX in the neoadjuvant setting (PMID: 34884782; PMID: 31145418). NIS793 is a first-in-class monoclonal antibody that directly binds TGF-β and reduces intratumoral fibrosis in preclinical models (PMID: 33298926). NIS793 also has an acceptable safety profile and preliminary clinical activity. This study investigates whether NIS793 modulation of the tumor fibrotic network improves clinical outcomes of FOLFIRINOX neoadjuvant therapy in BR/LA PDAC. Our aim is to demonstrate how TGF-β inhibition and stroma remodeling contribute to clinical outcomes. Methods: This is a single institution, open label, 2-part, 2-arm, non-comparator, Phase Ib study (NCT05417386) of FOLFIRINOX + NIS793 as neoadjuvant therapy for pts with untreated BR/LA PDAC. Eligible pts are adults with measurable disease, adequate organ and bone marrow function, and ECOG PS ≤1. In Part 1 (safety run-in), 6-18 pts with histologically confirmed untreated metastatic PDAC receive FOLFIRINOX + NIS793 at different dose levels. Dose escalation is primarily guided by the number of dose-limiting toxicities. Primary objectives are assessing safety, defining the recommended Phase II dose (RP2D), and providing initial evidence of clinical benefit of the combination. In Part 2, pts with untreated BR/LA PDAC are randomized 4:1 and stratified by BR vs LA. Pts in Arm 1 (n=5) receive 8 cycles of FOLFIRINOX followed by chemoradiation therapy (CRT) and surgery. Pts in Arm 2 (n=23) receive 8 cycles of FOLFIRINOX + NIS793 at RP2D followed by CRT + NIS793, surgery, and 12 cycles of adjuvant NIS793. The primary endpoint is R0 resection rate. Secondary endpoints are disease-free survival, progression-free survival, overall survival, and pathologic complete response. The effect of neoadjuvant therapy will be assessed by ferumoxytol-MRI, circulating cell-free DNA dynamics analysis, single-cell RNA sequencing, immunohistochemistry, and digital spatial analysis. Together, these correlative analyses may enable development of a mechanistic model for TGF-β inhibition in PDAC. This study is ongoing. The first pt was treated on Aug 2, 2022. Clinical trial information: NCT05417386 .

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.

Patients with non-small cell lung cancer (NSCLC) that is driven by an activating mutation in the epidermal growth factor receptor (EGFR) are routinely treated with tyrosine kinase inhibitors (TKI) to specifically target the activated EGFR signaling pathway. EGFR-mutant NSCLC tumors initially respond well to EGFR inhibitors, however a subset of drug-tolerant persister cells remain at minimal residual disease (MRD) and represent a cell reservoir from which acquired genetic mutations, such as EGFRT790M or MET amplification, can emerge to render the tumor fully drug-resistant. Prior to the emergence of genetic mutations, little is known about how drug-tolerant persister cells are able to survive EGFR targeted therapy at MRD. To better understand this cell population, we investigated drug-tolerance using single cell cloning and scRNAseq in NSCLC cell lines. Using ClonTracer barcoding, we found that the same barcodes emerged after EGFR-inhibitor treatment across multiple replicates, indicating that drug-tolerance is both pre-defined and stable over many generations. Within each individual cell line, we observed multiple distinct heterogeneous subpopulations of drug-tolerant persister cells with unique gene expression signatures and proliferation rates. Additionally, we observed evidence of putative mechanisms of drug tolerance that were shared by persister cells across cells lines and used a drug combination treatment approach to target these distinct subpopulations of drug-tolerant persister cells. Taken together, our findings provide evidence that drug-tolerant persister cell subpopulations are both predefined and heterogeneous, as well as suggesting that drug-combination treatment approaches in the clinic would be more effective at targeting multiple persister cell survival mechanisms. Citation Format: Jennifer L. Cotton, Viveksagar KrishnamurthyRadhakrishna, Julie Chen, Michelle Piquet, Joel Wagner, Gaylor Boulay, Kathleen Sprouffske, Youngchul Song, Xiaoyan Li, Katja Schumacher, Raphael Thierry, Nathaniel D. Kirkpatrick, David A. Ruddy, Joshua Korn, Erick J. Morris, Peter S. Hammerman, Jeffrey A. Engelman, Matthew J. Niederst. Molecular barcoding and single cell approaches to investigate drug tolerance in EGFRmut NSCLC [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A122. doi:10.1158/1535-7163.TARG-19-A122