Abstract A012: Investigating the heterogeneity of different KRAS point mutations on DRP1 activation and its regulation of the TME in pancreatic cancer

Abstract

Despite research efforts, outcomes for patients with pancreatic ductal adenocarcinoma (PDAC) remain dismal, with a 5-year survival rate of 13%. Our group identified that DRP1 loss in KRAS-mutant PDAC tumor cells alters mitochondrial shape and reduces glycolysis and cell proliferation in murine models. However, how Ras-induced mitochondrial dynamics in human PDAC models impact the PDAC tumor microenvironment (TME) remains unclear. We hypothesize that reliance on DRP1 activation in PDAC is KRAS-mutant-specific and shapes the TME to promote survival. Four human PDAC patient-derived xenograft (PDX) cell lines with KRAS mutations (PDX 395 & 188 KRASG12Rand PDX 366 & 608 KRASG12D) were stably transduced with a lentivirus in which a tetracycline-responsive promoter drives murine Drp1 expression. Next, the cells were transduced with a lentivirus expressing CAS9 and a guide targeting the human DRP1 gene. To maintain physiological levels of murine Drp1 expression, low-dose doxycycline (dox) kept expression comparable to endogenous human DRP1. We developed two in vivo mouse models: an orthotopic PDX model and a splenic injection model of liver metastasis. In both models, the tumor cells are human, and the TME cells are mouse. To test how DRP1 loss affects primary and metastatic tumor growth, we injected DRP1-/- and control cell lines into the pancreas or spleen. Additionally, in vivo splenic injection experiments using the PDX 395 KRASG12R cell lines, with mice on dox water, controlled when DRP1 is lost during tumor growth. Injecting 395 parent cell lines into mice with no, low (0.25mg/mL), or high (0.5mg/mL) dox water will allow for the understanding of whether dox affects tumor growth in the liver. To identify additional mechanisms that may contribute to differences in in vivo growth, possibly depending on the KRAS point mutation, we performed a cytokine Luminex assay followed by confirmation ELISAs. We will test the DRP1-/- cell lines for this dependance in PDX 366 and 608 KRASG12D, compared with PDX 395 and 188 KRASG12R, by measuring activation of NF-κB, AKT, FAS, and G6PD, and enzymes in glycolysis and the MAPK pathway by immunoblot. We will also perform in vitro macrophage engulfment assays on the PDX 395 KRASG12R and 366 KRASG12D sgcontrol and sgDRP1 cell lines on and off dox. The PDX 395 KRASG12R cell line grows differently in both the pancreas and liver when DRP1 is lost compared to the two KRASG12D cell lines. DRP1 may play a role in establishing the PDX 395 cell line in the liver, and its clearance may be DRP1-dependent. Dox treatment of mice with PDX 395 tumors does not affect tumor clearance in the liver. There is an increased trend in IL-6 secretion from the PDX 395 KRASG12R cell line and a significant three- to fivefold increase in PDX 366 KRASG12D upon DRP1 loss compared to controls. These alterations in IL-6 secretion upon DRP1 loss may represent a DRP1-dependent adaptation. In human PDAC cell lines, adaptations that overcome DRP1 targeting and enable metastasis may be KRAS-mutant-specific.