Application of Integrated Drug Screening/Kinome Analysis to Identify Inhibitors of Gemcitabine-Resistant Pancreatic Cancer Cell Growth

Abstract

Continuous exposure of a pancreatic cancer cell line MIA PaCa-2 (Mia) to gemcitabine resulted in the formation of a gemcitabine-resistant subline (Mia). In an effort to discover kinase inhibitors that inhibited Mia growth, Mia cells were exposed to kinase inhibitors (PKIS-1 library) in a 384-well screening format. Three compounds (UNC10112721A, UNC10112652A, and UNC10112793A) were identified that inhibited the growth of Mia cells by more than 50% (at 50 nM). Two compounds (UNC10112721A and UNC10112652A) were classified as cyclin-dependent kinase (CDK) inhibitors, whereas UNC10112793A was reported to be a PLK inhibitor. Dose-response experiments supported the efficacy of these compounds to inhibit growth and increase apoptosis in 2D cultures of these cells. However, only UNC10112721A significantly inhibited the growth of 3D spheroids composed of Mia cells and GFP-tagged cancer-associated fibroblasts. Multiplexed inhibitor bead (MIB)-mass spectrometry (MS) kinome competition experiments identified CDK9, CLK1-4, DYRK1A, and CSNK1 as major kinase targets for UNC10112721A in Mia cells. Another CDK9 inhibitor (CDK-IN-2) replicated the growth inhibitory effects of UNC10112721A, whereas inhibitors against the CLK, DYRK, or CSNK1 kinases had no effect. In summary, these studies describe a coordinated approach to discover novel kinase inhibitors, evaluate their efficacy in 3D models, and define their specificity against the kinome.

Application of a MYC degradation screen identifies sensitivity to CDK9 inhibitors in KRAS-mutant pancreatic cancer

“…S2A). This assay is based on kinase inhibitor competition assays to assess kinome-wide inhibitor specificity, similar to that described previously (35, 37).…”

Irreversible JNK1-JUN inhibition by JNK-IN-8 sensitizes pancreatic cancer to 5-FU/FOLFOX chemotherapy

“…Therefore, at many centers, it is currently only recommended to healthier, younger patients, which may exclude as many as 75% of metastatic PDAC patients (12). While many drug uptake and metabolic mechanisms for resistance to 5-FU and GEM are known (13,14), there is much less understanding of the signaling pathway changes, especially in the global set of protein kinases (i.e., the "kinome") that mediate cell response and survival after treatment with these widely used chemotherapy regimens in PDAC (13,15). Here, we describe an unbiased approach to assessing potential kinase targets for inhibition in combination with FOLFOX.…”

In vitro assays and techniques utilized in anticancer drug discovery

“…A study reported by Kant et al (2016) used HTS to identify natural products with antihepatocarcinogenic effects. Krulikas et al (2017) have used HTS to screen kinase targets in gemcitabine-resistant pancreatic cancer cells.…”