Re-Evaluating Multi-Kinase Inhibitors Targeting FGFR and VEGFR Pathways for Combined Therapy in Colorectal Cancer: A Preclinical Study

Abstract

Background: Overexpression and increased activity of ATP-binding cassette (ABC) transporters, such as P-glycoprotein (ABCB1/P-gp), breast cancer resistance protein (ABCG2/BCRP), and multidrug resistance-associated proteins (ABCC/MRPs), are known to be among the major contributors to multidrug resistance (MDR) in tumors to structurally similar or even different chemotherapeutic agents, thereby decreasing treatment efficacy. Thus, there is an urgent need to develop novel, effective therapies that overcome MDR and increase chemotherapy response rates. Results: We showed here that receptor tyrosine kinase inhibitors (RTKIs) designed to inhibit the overactivated FGFR and/or VEGFR signaling pathways exert an “off-target” effect on MDR proteins in colorectal cancer (CRC), thereby sensitizing CRC to certain chemotherapies. In particular, Pemigatinib and Infigratinib ranked among the top eight tested selective and non-selective FGFR/VEGFR inhibitors that effectively resensitized all types of CRCs to the DNA topoisomerase II inhibitor Doxorubicin and, in fewer experimental settings, to the antimetabolite 5-Fluorouracil. This was evidenced by a significant increase in apoptosis in CRCs treated with the aforementioned chemotherapeutic drugs in combination with RTKIs. Of note, despite the CRCs exhibiting signs of FGFR and VEGFR activation, RTKIs alone exhibited only minor pro-apoptotic and anti-proliferative activities. The enhancement of pro-apoptotic activity of Doxorubicin when co-treated with Pemigatinib, Lucitanib, and Regorafenib in HCT116 cells was consistent with a decrease in proliferative activity in CRCs treated with these RTKIs alone. This was also revealed by high synergy scores (SC) between several RTKIs (e.g., Pemigatinib, Lucitanib, and Regorafenib) and Doxorubicin in CRCs. The aforementioned effects of RTKIs were due to the inhibition of MDR-related ABC transporter activity, particularly ABCB1 and ABCG2. Indeed, in HCT116 cells, Lucitanib and, to a lesser extent, Pemigatinib impaired the efflux of the intrinsically fluorescent Doxorubicin and Mitoxantrone. Similarly, the efflux of Calcein AM, an ABCB1 substrate, was significantly impaired in Lucitanib- and Pemigatinib-treated CRCs, thereby revealing ABCB1 as a molecular target for these RTKIs. Consistent with SC data, Infigratinib and Cabozatinib effectively impaired the efflux of Doxorubicin from Colon-26 cells. Similarly, ABCB1 protein was found to be a primary target for Infigratinib in Colon-26 cancer cells, which was evidenced by competitive assay data based on the combined use of selective ABCB1 and ABCG2 inhibitors (e.g., Tariquidar and Ko-143, respectively). Importantly, ABC-inhibitory effects of RTKIs in CRCs were mediated solely by the impaired efflux of chemotherapeutic agents from CRCs and were not due to the decreased expression of ABC transporters or their subcellular distribution. Lastly, in the small-cohort allograft study, we found Infigratinib to be an effective chemosensitizing agent for the Colon-26 murine colon carcinoma in vivo. This was evidenced by the reduced growth of Colon-26 allografts in the experimental group receiving the combined therapy of Infigratinib and Doxorubicin compared with the single-treated groups. Consistent with these findings, immunohistochemical (IHC) staining revealed a substantial increase in apoptotic cells (e.g., cleaved caspase-3-positive) and a decrease in proliferative cells (e.g., Ki-67-positive) in allografts from animals treated with a combination of Infigratinib and Doxorubicin. Conclusions: We show here for the first time that some FGFR inhibitors (e.g., Pemigatinib and Infigratinib) can effectively potentiate cytotoxic and anti-proliferative activity against CRCs overexpressing MDR-related ABC transporters, thereby suggesting potential therapeutic avenues for treating CRC that progresses despite standard conventional therapies.