J. K. Thompson1, J. Blumberg5, O. Alkhalili5, H. Crawford2,4, M. Pasca Di Magliano1,3, F. O. Bednar1 1University Of Michigan,Surgery,Ann Arbor, MI, USA 2University Of Michigan,Internal Medicine,Ann Arbor, MI, USA 3University Of Michigan,Cell And Developmental Biology,Ann Arbor, MI, USA 4University Of Michigan,Molecular And Integrative Physiology,Ann Arbor, MI, USA 5University Of Michigan,Literature, Science, And Arts,Ann Arbor, MI, USA
Introduction: KRAS is the primary oncogenic driver in human pancreatic ductal adenocarcinoma (PDA). Pancreatic acinar cells are most susceptible to transformation by oncogenic Kras in mouse models of PDA. The earliest stage of transformation consists of conversion of the acinar cells to a duct-like progenitor phenotype in a process called acinar-ductal metaplasia (ADM). Transcription factor (TF) gene regulatory networks control ADM progression and pancreatic tissue repair. We have shown that deletion of Bmi1, a component of the Polycomb Repressor Complex 1 (PRC1), disrupts oncogenic Kras-driven ADM in mice. We hypothesize that loss of Bmi1 inhibits the Kras-driven changes in TF networks to abolish pancreatic neoplasia.
Methods: Mice with a pancreatic acinar cell-specific, tamoxifen inducible Cre recombinase (Ela-CreER) were bred with strains containing the oncogenic Kras G12D allele, the conditional knockout Bmi1 allele (Bmi1 flox) and the fluorescent protein tdTomato in the Rosa26 locus. We activated the Cre recombinase with five daily gavages of tamoxifen (4mg/day) and induced acute pancreatitis with intraperitoneal caerulein injections (x8/day for two consecutive days). We isolated acinar cell progeny from primary pancreata with fluorescence-activated cell sorting (FACS) using the tdTomato lineage marker one week after acute pancreatitis. Total cell RNA was isolated from the sorted cells using the RNeasy Micro Kit (QIAGEN) and complementary DNA (cDNA) was synthesized with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). We utilized a panel of 26 TaqMan qPCR probes specific for pancreatic developmental TFs and three acinar cell and duct markers (amylase, elastase, keratin 19 – CK19) to characterize the TF networks within the isolated cells.
Results: Tamoxifen gavage induced a high level of tdTomato expression in the pancreata from Ela-CreER expressing mice. RT-qPCR analysis after FACS identified Kras-driven loss of expression of most acinar cell-specific TFs one week after pancreatitis induction consistent with active ADM. Genetic deletion of Bmi1 partially restored amylase and elastase expression at the RNA level. Bmi1 loss also led to recovery of the acinar cell compartment from ADM. These findings correlated with increased expression of Mist1 and Nr5a2, key acinar fate-specific TFs. We did not observe recovery of other acinar TFs, including Pdx1 and Ptf1a/p48, one week after acute pancreatitis in cells with oncogenic Kras and deleted Bmi1.
Conclusion: Oncogenic Kras-driven ADM is controlled by changes in master TF gene regulatory networks. Bmi1 deletion leads to partial reprogramming of these networks to allow acinar cells to resist Kras-driven oncogenesis.