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Supplementary Data 6 from Abemaciclib Is Effective Against Pancreatic Cancer Cells and Synergizes with HuR and YAP1 Inhibition

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posted on 2023-04-03, 17:28 authored by Teena Dhir, Christopher W. Schultz, Aditi Jain, Samantha Z. Brown, Alex Haber, Austin Goetz, Chunhua Xi, Gloria H. Su, Liang Xu, James Posey, Wei Jiang, Charles J. Yeo, Talia Golan, Michael J. Pishvaian, Jonathan R. Brody

Supplemental figure 6: (A) Pico Green assay assessing cell viability in Mia PaCa2 HuR CRISPR knock out cells treated with abemaciclib. Assay was performed for 5 days prior to collection. (B) Western blot of Mia PaCa2 HuR CRISPR knock out cells treated with abemaciclib for 3 days and probed for pRb and HuR. Samples were quantified and normalized to untreated parental cell type. (C) Mia PaCa2 HuR CRISPR KO clone validation, both by western demonstrating no HuR product (top) and by Sanger sequencing. Clone 8 has a 476 base pair insertion, and clone 10 has a single base pair deletion in one allele and a two base pair deletion in the other allele, both leading to a frameshift mutation. (D) Pico Green assays in Mia PaCa2 cells assessing cell viability in si negative control, siHuR or siYAP1 transfected cells treated with abemaciclib. Assay was performed for 5 days prior to collection. (E) Western blot in Mia PaCa2 cells of si negative control, siHuR or siYAP1 transfected cells treated with abemaciclib for 3 days. Samples were probed for pRb, cyclin D1, HuR, YAP1 and α-tubulin. Samples were quantified and normalized to untreated si negative control. (F) qPCR results from RNA immunoprecipitation (RIP) and probed for HuR targets cyclin D1, p27 and PIM1 (positive control). Cytoplasmic extraction samples normalized to IgG. Significance is denoted as ***p<0.0001. (G) Pico Green assays in Mia PaCa2 cells assessing growth rate inhibition in si negative control or si cyclin D1 transfected cells treated with abemaciclib. Assay was performed for 5 days prior to collection. (H) Western blot in Mia PaCa2 cells of si negative control or si cyclin D1 transfected cells treated with abemaciclib for 3 days. Samples were probed for pRb, cyclin D1 and α-tubulin. Samples were quantified and normalized to untreated si negative control.

Funding

Newell Devalpine Foundation

NIGMS

National Institutes of Health

History

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.