Interrogating KRAS, ERK, and MYC Signaling in Pancreatic Cancer with Endogenous PET Imaging

Objectives: Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly cancers with a 5-year survival rate of less than 10%. Currently, there is a critical need for specific, clinically translatable, and non-invasive tools to assess metabolic effects, predict treatment response, and demarcate biology within pancreatic cancer. KRAS mutations have been identified in >90% of PDAC and are connected to a number of signaling pathways relevant to cancer, including mitogen-activated protein kinase (MAPK) and extracellular regulated kinase (ERK). A non-invasive biomarker to study protein signaling downstream of KRAS would greatly enhance our ability to stratify patients that will benefit from targeted therapies. There is recent literature that suggests a link between KRAS and downstream MYC signaling, but the relationship is not well understood. A key downstream event of MYC dysregulation is overexpression of the transferrin receptor (TfR), which has been targeted for cancer therapeutics and imaging. In this study, we aimed to test if zirconium-89 transferrin (⁸⁹Zr[Zr]-Tf), an endogenous PET agent developed in our lab, could measure changes in MYC, depending on KRAS status of pancreatic cancer tumors. We also sought to test if anti-MYC and anti-ERK therapies could be measured with ⁸⁹Zr[Zr]-Tf PET, biodistribution studies, and ex vivo immunohistochemistry.

Methods: Doxycycline-inducible KRAS mutant murine cells (iKras[asterisk]p53[asterisk]) and human PDAC cell lines (Capan-2, Suit-2, MIA PaCa-2, and BxPC-3) were treated with BRD4 inhibitors JQ1 and OTX015, and ERK inhibitor SCH772984. Mice were inoculated were iKras[asterisk]p53[asterisk] tumors and human PDAC xenografts. Mice bearing iKras[asterisk]p53[asterisk] tumors were imaged with ⁸⁹Zr[Zr]-Tf pre- and post-withdrawal of doxycycline dependence to observe differential uptake based on status of oncogenic KRAS. Human PDAC xenografts were administered JQ1 or SCH772984 (50 mg/kg twice daily), and PET imaging studies with ⁸⁹Zr[Zr]-Tf were performed to monitor tumoral uptake and response to drug treatment. Biodistribution studies 48 h post-radiotracer administration quantified tumor and tissue uptake in each cohort.

Results: A statistically significant decrease in cell proliferation and protein expression (phospho-ERK, MYC, and TfR) was observed via western blot and flow cytometry in multiple PDAC cell lines (P < 0.05-0.001) upon drug treatment. Female athymic nude were inoculated with iKras[asterisk]p53[asterisk] tumors and showed differential uptake of ⁸⁹Zr[Zr]-Tf depending upon KRAS dependency, with significantly higher (P < 0.01-0.05) uptake in mice that were withdrawn from inducible oncogenic KRAS, which was confirmed by an increase in MYC and TfR protein expression via ex vivo immunohistochemistry. A therapy study with BRD4 inhibitor JQ1 showed a statistically significant decrease (P < 0.05) in ⁸⁹Zr[Zr]-Tf uptake in drug vs. vehicle-treated animals bearing Capan-2 and Suit-2 (KRAS mutant) xenografts. JQ1 therapy showed no statistical difference in BxPC-3 (KRAS wild-type) xenografts, but therapy with ERK inhibitor SCH772984 (same dosing and schedule) in BxPC-3 mice showed a statistically significant decrease in ⁸⁹Zr[Zr]-Tf uptake (P < 0.05) in drug vs. vehicle-treated animals. Ex vivo immunohistochemical analysis of resected human PDAC tumors reflects the data observed via PET imaging and radiotracer biodistribution.

Conclusions: Our radiotracer shows promise as a tool for interrogating proteins downstream of oncogenic KRAS such as ERK and MYC via transferrin receptor in PDAC. This strategy can pave the way to assess oncogene status and predict early therapy response to targeted inhibitors in pancreatic cancer. Research Support. This research was supported by the National Institutes of Health (1R01CA17661-01), the Center for Molecular Imaging and Nanotechnology Tow Fellowship (K.E.H.), MSKCC CCSG Grant (P30 CA008748), the American Cancer Society Research Scholar Grant (130635-RSG-17-005-01-CCE), and the UCSF CCSG (P30CA082103).

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