Abstract B032: KRAS copy number variation and mutant allele fractions predict in vitro response of PDX-derived human pancreatic cancer cell lines to KRASG12D inhibitor MRTX1133
Authors: Sarcar Bhaswati , Tassielli Alexandra , Wang Liang , Dai Ruifan , Read Meagan D. , Beato Francisca , Kim Dae W. , Hodul Pamela J. , Permuth Jennifer B. , Haura Eric B. , Malafa Mokenge P. , Fleming Jason B. 
Published: May 2023

Abstract
Introduction: KRASG12D is the most common KRAS mutation in pancreatic ductal adenocarcinoma (PDAC). It is well known that activating point mutations in one allele are sufficient for tumor growth in patients harboring KRASG12D mutations and it is challenging to target this mutation with effective KRASG12D inhibitors. Driver gene amplification is a known mechanism of acquired resistance to therapy in pancreatic cancers. Although other genetic features, such as KRAS gene copy number variations (CNV) and mutant allele fractions (MAF), have been observed, their influence on response to treatment remains unclear. Current discovery revealed that MRTX1133 is a potent, non-covalent and high-affinity small molecule that selectively targets KRASG12D. Therefore, with the efforts underway to develop effective therapy response using the KRASG12D-targeted drug, MRTX1133, it is paramount to improve our understanding regarding the role of KRAS CNV/MAF in PDAC. Methods: Using quantitative digital PCR (DPCR), we first measured the absolute copy number of KRAS wild type (WT), and mutant type (MT) and we then calculated MAF by formula: MAF = MT allele numbers/(MT+WT allele numbers) in DNA from various KRASG12D mutant PDAC cell lines and PDX-derived primary cell lines. 3D viability and cell signaling assays determined sensitivity to MRTX1133 in established and PDX-derived primary cell lines in vitro. We also assessed the representative KRAS gene CNV of those cell lines by TaqMan real time PCR. DPCR was also utilized to determine the MAF in PDX-derived tissues. Results: We found a trend in response to MRTX1133 in KRASG12D cell lines based on their MAF and representative KRAS gene CNV. Resistant, and sensitive KRASG12D cell lines have average MAF of 75.23%, and 52.81% respectively. Most of the resistant cells have higher KRAS gene copy number gain (6-8) compared to sensitive cells (3-4). Interestingly, our data suggests that higher MAF (&gt;53%) and gene copy numbers (&gt;4) may predict a resistant phenotype in KRASG12D cells to MRTX1133. Mechanistically, MRTX1133 inhibits ERK1/2 signaling and increases apoptosis in sensitive cells. Representative sensitive and resistant cell lines are further evaluated for any changes in baseline and MRTX1133-induced MAF and copy number alterations by DPCR and TaqMan real time PCR. However, the results indicated that there are no significant differences in MAF and KRAS CNV in the sensitive and resistant cells on pre and post treatment of MRTX1133. We further extended our study by evaluating the MAF targeting KRASG12D in various PDX-derived tissues by quantitative DPCR. With exception to a few cases, most of the analyzed PDX tissues contained balanced MAF (~50%), which may show sensitivity to MRTX1133 according to our findings. Conclusions: Our current observations indicate 1) KRAS CN gain and MAF increase may appear to play major roles in the progression of a resistant phenotype 2) KRAS CNV and MAF status may contribute to criteria to select PDAC patients who are most likely to benefit from therapy with MRTX1133.
Citation Format: Bhaswati Sarcar, Alexandra Tassielli, Liang Wang, Ruifan Dai, Meagan D. Read, Francisca Beato, Dae W. Kim, Pamela J. Hodul, Jennifer B. Permuth, Eric B. Haura, Mokenge P. Malafa, Jason B. Fleming. KRAS copy number variation and mutant allele fractions predict in vitro response of PDX-derived human pancreatic cancer cell lines to KRASG12D inhibitor MRTX1133 [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B032.