azd-6244 and crenolanib

Activating mutations in the proto-oncogene KRAS are a hallmark of pancreatic ductal adenocarcinoma (PDAC), an aggressive malignancy with few effective therapeutic options. Despite efforts to develop KRAS-targeted drugs, the absolute dependence of PDAC cells on KRAS remains incompletely understood. Here we model complete KRAS inhibition using CRISPR/Cas-mediated genome editing and demonstrate that KRAS is dispensable in a subset of human and mouse PDAC cells. Remarkably, nearly all KRAS deficient cells exhibit phosphoinositide 3-kinase (PI3K)-dependent mitogen-activated protein kinase (MAPK) signaling and induced sensitivity to PI3K inhibitors. Furthermore, comparison of gene expression profiles of PDAC cells retaining or lacking KRAS reveal a role of KRAS in the suppression of metastasis-related genes. Collectively, these data underscore the potential for PDAC resistance to even the very best KRAS inhibitors and provide insights into mechanisms of response and resistance to KRAS inhibition.

Topics: Animals; Antineoplastic Agents; Benzimidazoles; Carcinoma, Pancreatic Ductal; DNA Copy Number Variations; Humans; Immunoblotting; Indazoles; Mice; Morpholines; Pancreatic Neoplasms; Phenylurea Compounds; Piperidines; Proto-Oncogene Mas; Proto-Oncogene Proteins p21(ras); Purines; Pyrimidines; Pyrimidinones; Quinazolinones; Sulfonamides; Thiazoles

The evolution of cancer therapy into complex regimens with multiple drugs requires novel approaches for the development and evaluation of companion biomarkers. Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) is a versatile platform for biomarker measurement. In this study, we describe the development and use of the LC-MRM platform to study the adaptive signaling responses of melanoma cells to inhibitors of HSP90 (XL888) and MEK (AZD6244). XL888 had good anti-tumor activity against NRAS mutant melanoma cell lines as well as BRAF mutant cells with acquired resistance to BRAF inhibitors both in vitro and in vivo. LC-MRM analysis showed HSP90 inhibition to be associated with decreased expression of multiple receptor tyrosine kinases, modules in the PI3K/AKT/mammalian target of rapamycin pathway, and the MAPK/CDK4 signaling axis in NRAS mutant melanoma cell lines and the inhibition of PI3K/AKT signaling in BRAF mutant melanoma xenografts with acquired vemurafenib resistance. The LC-MRM approach targeting more than 80 cancer signaling proteins was highly sensitive and could be applied to fine needle aspirates from xenografts and clinical melanoma specimens (using 50 μg of total protein). We further showed MEK inhibition to be associated with signaling through the NFκB and WNT signaling pathways, as well as increased receptor tyrosine kinase expression and activation. Validation studies identified PDGF receptor β signaling as a potential escape mechanism from MEK inhibition, which could be overcome through combined use of AZD6244 and the PDGF receptor inhibitor, crenolanib. Together, our studies show LC-MRM to have unique value as a platform for the systems level understanding of the molecular mechanisms of drug response and therapeutic escape. This work provides the proof-of-principle for the future development of LC-MRM assays for monitoring drug responses in the clinic.

Topics: Animals; Azabicyclo Compounds; Benzimidazoles; beta Catenin; Biomarkers, Tumor; Cell Line, Tumor; Chromatography, High Pressure Liquid; Drug Resistance, Neoplasm; GTP Phosphohydrolases; HSP90 Heat-Shock Proteins; Humans; Indoles; MAP Kinase Kinase 1; MAP Kinase Signaling System; Mass Spectrometry; Melanoma; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, SCID; Neoplasm Transplantation; NF-kappa B; Phosphoinositide-3 Kinase Inhibitors; Phthalic Acids; Piperidines; Proteomics; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Receptor, Platelet-Derived Growth Factor beta; RNA Interference; RNA, Small Interfering; Sulfonamides; Transplantation, Heterologous; Vemurafenib; Wnt Signaling Pathway