Abstract B052: Understanding latent resistance mechanisms and their phenotypes in pancreatic cancer organoids under KRASG12D inhibition

Abstract

Pancreatic ductal adenocarcinoma (PDAC) remains lethal due to acquired resistance despite the generation of novel targeted therapeutics. Here, we have generated resistant pancreatic cancer organoid (PCO) models to the tri-selective KRASG12D (ON) inhibitor Zoldonrasib to evaluate the rate of acquired resistance and characterize changes at phenotypic resistance. Furthermore, we assessed how dosage of epidermal growth factor (EGF) in media impact resistance mechanisms. PCOs were treated with Zoldonrasib in either standard EGF conditions (50ug/mL) or physiologic EGF conditions (0.5ug/mL) over the course of 96 hours with imaging (Cytation 5) to evaluate their growth rate. PCOs were determined resistant if they held a growth rate above 20% in normalized diameter under continuous treatment. PCO’s were then dose escalated in a log2 fold manner to resistance of 200nM. Resistant PCOs and their parent cultures were collected for protein analysis via western blot (LICOR Odyssey) for alterations in MAP kinase effector signaling. dPCR (QIAcuity) was done to evaluate shifts in copy number variants (CNVs) in key oncogenes post resistance. PCO38 achieved rapid resistance despite the logarithmic escalated doses of Zoldonrasib in 35 days independent of EGF stimulation. Similar, albeit slower (p-value < 0.0001), growth rates were seen in PCO33, with resistance to Zoldonrasib in standard and low EGF conditions in 42 days. Abnormal CNVs of MYC in PCO38 were observed in the parent culture (CNV = 35) and remained unchanged over the course of dose escalation. However, KRAS copy numbers in PCO38 show a limited increase in CNV from a baseline of 3 to 4.5 in standard EGF conditions (p-value = 0.09). Low EGF conditions show non-significant changes in mutant KRAS CNV during dose escalation (p-value > 0.99). Protein analysis suggests opposing signaling pathway activation in resistant PCOs to Zoldonrasib at 200nM dependent upon EGF stimulation. Interestingly, both PCO’s in standard EGF stimulation resulted in activation of the parallel AKT pathway without reactivation of the Ras-ERK node. Physiologic EGF stimulation resulted in no activation of AKT, but instead the re-activation of the Ras-ERK node itself. The rate of acquired resistance to Zoldonrasib was found to be rapid in PCO models and independent of EGF stimulation. dPCR revealed no changes in the abnormal CNVs of MYC, with limited increase in KRASG12D only with standard EGF conditions. Protein analysis suggests that decreased levels of EGF facilitate resistance to Zoldonrasib that is mediated through re-activation of the ERK node. Standard EGF conditions did result in parallel activation of AKT signaling, suggest of model specific off target activation. Ongoing work includes using a druggable CRISPR-Cas9 library screen in latent resistant PCOs to further characterize the diverging mechanisms of resistance dependent upon EGF stimulation. Lauryn E. Flannagan, Michela Cadarso, Audrey G. Sawyer, Md Shahadat Hossan, Jeremy D. Kratz. Understanding latent resistance mechanisms and their phenotypes in pancreatic cancer organoids under KRASG12D inhibition [abstract]. In: Proceedings of the AACR Conference on Pancreatic Cancer: New Frontiers in Biology and Therapeutic Development; 2026 Sep 25-28; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(18_Suppl_2):Abstract nr B052.