Applying Radiolabeled Transferrin to Quantify Therapeutic Response of Triple Negative Breast Cancer to Bromodomain Inhibitors

Objectives Bromodomain and extra terminal (BET) inhibitors (e.g. JQ1 and OTX015) are transcriptional regulators that impede the proliferation of MYC-overexpressing cancers of diverse tissue origins including prostate, glioblastoma, and lymphoma. It has been well established that MYC overexpression has been found to be a key driver in multiple cancer types, and increased surface expression of transferrin receptor (TfR) is a downstream event of MYC upregulation. Transferrin labeled with 89Zr has been validated to detect MYC status in these cancer types preclinically, has recently been shown to predict treatment response from BET inhibitors in lymphoma, and is to be translated into prostate cancer patients this year. We hypothesize that 89Zr-transferrin PET will detect overexpression of MYC and TfR, predict treatment response from BET inhibitors, and improve upon the current standard of 18F-FDG PET for MYC-overexpressing triple negative breast cancer (TNBC). In this research, 89Zr-transferrin and 131I-transferrin were used to monitor treatment response to BET inhibitors and to target TNBC.

Methods TNBC cell lines (MDA-MB-231, MDA-MB-157, and Hs578T) were treated with JQ1 and OTX015 (500-1000 nM). Anti-proliferative effects via JQ1 and OTX015 in TNBC were assessed using viability assays measuring ATP synthesis. Analysis of cellular arrest was performed using flow cytometry with EdU incorporation and DAPI. Relative gene expression (MYC and TfR) was detected using quantitative RT-PCR. Western blot was used to validate MYC and TfR expression in TNBC cells and relative protein levels was quantitated post drug treatment using MYC and TfR ELISA. PET imaging studies in orthotopic MDA-MB-231 and MDA-MB-157 xenografts with 89Zr-transferrin are in progress to monitor tumoral uptake and response to drug treatment.

Results TNBC cells show a response to JQ1 and OTX015 at a nanomolar range through a variety of in vitro methods. Anti-proliferative effects and cell cycle arrest at the G0/G1 phase were observed, consistent with the mechanism of action of these agents. A drug-dependent decrease in MYC and TfR gene expression was observed via quantitative RT-PCR. Radiolabeled (131I) transferrin showed a 5-10% decrease in internalization post-drug treatment when compared with vehicle-treated TNBC cells. Time-course internalization of 89Zr-transferrin (at 37 °C vs. 4 °C) confirmed receptor-mediated endocytosis via TfR.

Conclusions Establishing biomarkers within heterogeneous cancers such as TNBC fulfills a clinical unmet need to reliably annotate tumor uptake and/or treatment response. 89Zr-transferrin is a useful tool to interrogate MYC status via TfR. Preclinical studies are warranted to further these in vitro observations, explore the mechanism of tumor response via BET inhibitors, and compare uptake of 89Zr-transferrin with 18F-FDG in TNBC tumor models. Research Support This research was supported by the National Institutes of Health (1R01CA17661-01).