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Mechanisms of Resistance to BRAF and MEK inhibitors in BRAF-mutant melanoma

Patel, Hima Milan
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1659529864596062

Abstract Details

2022, PhD, University of Cincinnati, Pharmacy: Pharmaceutical Sciences.
The use of BRAF and MEK inhibitors for patients with BRAF-mutant melanoma is limited as patients relapse on treatment as quickly as 6 months due to resistance. We used two approaches to treat resistant melanomas based on known and novel mechanisms of resistance. For the first approach, we generated trametinib and dabrafenib resistant (TDR) melanoma cell lines to trametinib, a MEK and dabrafenib, a BRAF inhibitor. TDR cells exhibited increased viability and maintenance of downstream p-ERK and p-Akt as compared to parental cells. Receptor tyrosine kinase arrays revealed an increase in phosphorylated insulin like growth factor 1 receptor (IGF1R) and phosphorylated insulin receptor (IR) in the drug resistant cells versus drug sensitive cells. RNA-sequencing analysis identified IGF1R and INSR upregulated in resistant cell lines compared to parental cells. Analysis of TCGA PanCancer Atlas (skin cutaneous melanoma) showed that patients with a BRAF mutation and high levels of IGF1R and INSR had a worse overall survival. BMS-754807, an IGF1R/IR inhibitor, suppressed cell proliferation along with inhibition of intracellular p-Akt in TDR cells. Dual inhibition of IGF1R and INSR using siRNA reduced cell proliferation. The combination of dabrafenib (BRAF inhibitor), trametinib (MEK inhibitor), and BMS-754807 (IGF1R/IR inhibitor) treatment reduced in vivo xenograft tumor growth. Examining the role of IGF1R and IR in mediating resistance to BRAF and MEK inhibitors will expand possible treatment options to aid in long-term success for BRAF-mutant melanoma patients. Reactive oxygen species (ROS) levels are elevated after acquisition of resistance to BRAF and MEK inhibitors. For the second approach we utilized a ROS-induced drug release (RIDR)-PI-103, a novel prodrug with a self-cyclizing moiety linked to PI-103, a PI3K inhibitor. Under high ROS conditions, RIDR-PI-103 releases PI-103, which inhibits conversion of PIP2 to PIP3. Studies in our lab demonstrate that TDR cells maintain p-Akt levels compared to parental counterparts and have significantly higher ROS. This is a rationale to explore the efficacy RIDR-PI-103 in TDR cells. We tested the effect of RIDR-PI-103 on cell viability for melanocytes, A375 TDR, WM983B TDR, and WM115 TDR using an MTT proliferation assay. RIDR-PI-103 exhibited less toxicity as compared to PI-103 at 5 µM in normal melanocytes. RIDR-PI-103 significantly inhibited TDR cell proliferation at 5 and 10 µM. This effect was consistent when TDR cells were treated with RIDR-PI-103 in long-term crystal violet cell growth and matrigel assays. 24 hour treatment with RIDR-PI-103 inhibited p-Akt (S473) along with downstream p-S6 (S240/244) and p-S6 (235/236). To examine the mechanism of action of RIDR-PI-103, we assessed the effect of glutathione (GSH) or t-butyl hydrogen peroxide (TBHP) on the TDR cells in the presence or absence of RIDR-PI-103. Addition of GSH to RIDR-PI-103 significantly rescued the cell proliferation in all three TDR cell lines while addition of TBHP and RIDR-PI-103 inhibited cell proliferation in WM115 and WM983B TDR cell lines. Examining the efficacy of RIDR-PI-103 on BRAF and MEK inhibitor resistant cells will expand possible treatment options and open up avenues for the development of new ROS based treatment therapies for BRAF-mutant melanoma patients.
Joan Garrett, Ph.D. (Committee Member)
Zalfa Abdel-Malek, Ph.D. (Committee Member)
Yuhang Zhang, Ph.D. (Committee Member)
Lisa Vinnedge, Ph.D. (Committee Member)
Timothy Phoenix, Ph.D. (Committee Member)
178 p.
Oncology
melanoma; resistance; BRAF

Recommended Citations

Citations

  • Patel, H. M. (2022). Mechanisms of Resistance to BRAF and MEK inhibitors in BRAF-mutant melanoma [Doctoral dissertation, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1659529864596062

    APA Style (7th edition)

  • Patel, Hima Milan. Mechanisms of Resistance to BRAF and MEK inhibitors in BRAF-mutant melanoma. 2022. University of Cincinnati, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1659529864596062.

    MLA Style (8th edition)

  • Patel, Hima Milan. "Mechanisms of Resistance to BRAF and MEK inhibitors in BRAF-mutant melanoma." Doctoral dissertation, University of Cincinnati, 2022. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1659529864596062

    Chicago Manual of Style (17th edition)

ucin1659529864596062
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This open access ETD is published by University of Cincinnati and OhioLINK.