Supplemental figure 1: (A) RT-PCR of human genomic DNA probed for CDKN2A in Mia PaCa2, Panc-1, HS 766T, HPNE and PDXs, compared to positive control cells PA-1[54]. Subsequent RT-PCR of mouse genomic DNA probed for CDKN2A in KPC (KRAS mutant, p53 null) and KTC (KRAS mutant, HuR null) mouse organoids to validate CDKN2A status. (B) Pico green cell viability assay on PDX cell lines treated with palbociclib, abemaciclib or gemcitabine for 5 days, with IC50 tabulated below. (C) Cell titer glow cell viability assay to calculate IC50 for abemaciclib in KPC (IC50 4.9µM) and CDKN2A null organoids (IC50 4.0µM). (D) Top: Pico green assays to assess short-term abemaciclib drug response in Mia PaCa2, which has a deletion of CDKN2A gene, compared to HPNE, which has a preserved CDKN2A gene. IC50 for both cell lines are shown in the parenthesis. Bottom: Drug curves for HPNE cells treated with abemaciclib, gemcitabine or oxaliplatin are graphed with IC50 shown in parenthesis. (E) Crystal violet colony formation images for cells treated with palbociclib or abemaciclib at respective concentrations. (F) PCR primer sequences used for RT-PCR reactions.
ARTICLE ABSTRACT
Mutation or promoter hypermethylation of CDKN2A is found in over 90% of pancreatic ductal adenocarcinomas (PDAC) and leads to loss of function of cell-cycle inhibitors p16 (INK4A) and p14 (ARF) resulting in unchecked proliferation. The CDK4/6 inhibitor, abemaciclib, has nanomolar IC50s in PDAC cell lines and decreases growth through inhibition of phospho-Rb (pRb), G1 cell-cycle arrest, apoptosis, and the senescent phenotype detected with β-galactosidase staining and relevant mRNA elevations. Daily abemaciclib treatments in mouse PDAC xenograft studies were safe and demonstrated a 3.2-fold decrease in tumor volume compared with no treatment (P < 0.0001) accompanying a decrease in both pRb and Ki67. We determined that inhibitors of HuR (ELAVL1), a prosurvival mRNA stability factor that regulates cyclin D1, and an inhibitor of Yes-Associated Protein 1 (YAP1), a pro-oncogenic, transcriptional coactivator important for CDK6 and cyclin D1, were both synergistic with abemaciclib. Accordingly, siRNA oligonucleotides targeted against HuR, YAP1, and their common target cyclin D1, validated the synergy studies. In addition, we have seen increased sensitivity to abemaciclib in a PDAC cell line that harbors a loss of the ELAVL1 gene via CRISP-Cas9 technology. As an in vitro model for resistance, we investigated the effects of long-term abemaciclib exposure. PDAC cells chronically cultured with abemaciclib displayed a reduction in cellular growth rates (GR) and coresistance to gemcitabine and 5-fluorouracil (5-FU), but not to HuR or YAP1 inhibitors as compared with no treatment controls. We believe that our data provide compelling preclinical evidence for an abemaciclib combination–based clinical trial in patients with PDAC.
Our data suggest that abemaciclib may be therapeutically relevant for the treatment in PDAC, especially as part of a combination regimen inhibiting YAP1 or HuR.
