Abstract 3280: ASCL2+ tumor cells modulate the response of colorectal cancer to MAPK targeting therapy

Abstract

Abstract Colorectal cancer (CRC) is commonly treated with MAPK pathway inhibitors (MAPKi) including EGFRi, BRAFi, and most recently KRAS G12C inhibitors. However, clinical trials repeatedly demonstrate that durability of benefit is short-lived in many patients. While genomic mechanisms of acquired resistance have been described, this explains a small minority of patients and further research is needed to determine the underlying biology limiting MAPKi therapy durability in CRC. Potential responsible mechanisms were explored using transcriptomic data from patients treated with MAPKi combination therapy, showing upregulation of stem-related signatures in nonresponders (FDR<0.001), therefore CRC stem markers with the greatest expression increase were identified. The top markers (ASCL2, LGR5, LRIG1) were evaluated using a metacohort of single cell RNAseq (scRNAseq) data from patients with CRC (n=72), and ASCL2 was selected for further study due to its high marker coexpression and tumor cell specificity (OR=8.27, p<0.0001). Additionally, ASCL2 expression increased in multiple patient-derived organoid and xenograft (PDX) models that were treated with a spectrum of MAPKi therapies (BRAFi, EGFRi, G12Ci, MEKi, SOSi). As such, we postulated that ASCL2+ tumor cells play an important role in the limited response durability of MAPKi therapy in CRC. To investigate, ASCL2 reporter models were treated with MAPKi therapy, resulting in the significant enrichment of ASCL2+ tumor cells, independent of driver mutation or treatment regimen used. Using Single Cell BarcodeSeq, by combining molecular barcoded PDXs with single cell transcriptomics, we found that MAPKi therapy engaged intrinsic cellular plasticity resulting in cells transitioning into the ASCL2+ phenotype. Additional patient-level data confirmed enrichment of the ASCL2+ transcriptomic signature at progression while on MAPKi therapy. Characterization of ASCL2+ cells showed higher stem cell frequency (p=9.7 × 10−62), clonogenic capacity (p<0.0001), expression of stem-related genes, and retention of phenotypic plasticity, indicating ASCL2+ tumor cells feature stem-like abilities, as previously described, which include therapy resistance. Lastly, depletion of ASCL2+ tumor cells resulted in improved initial response to MAPKi therapy (p<0.0001) and, more importantly, prolonged cell growth inhibition. Overall, we observe that MAPKi therapy enriches stemness programs in CRC and demonstrate MAPKi drives cells into a transient ASCL2+ stem-like state with limited sensitivity to MAPK inhibitors. Further studies are underway to identify vulnerabilities that will enable targeting of ASCL2+ tumor cells. Ultimately these findings will help guide future optimization of MAPKi regimens to prolong response durability and improve outcomes for patients with CRC. Citation Format: Oscar E. Villarreal, Hey Min Lee, Ha Tran, Annette Machado, Jumanah Alshenaifi, Chi Wut Wong, Olu Coker, Rosalba Minelli, Michael Peoples, Alejandro Hernandez Martinez, Natalie Fowlkes, Preeti Kanikarla, Kangyu Lin, Christopher Bristow, Andrea Viale, Joseph R. Marszalek, John Paul Shen, Scott Kopetz. ASCL2+ tumor cells modulate the response of colorectal cancer to MAPK targeting therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3280.