Methotrexate sensitizes drug-resistant metastatic melanoma cells to BRAF V600E inhibitors dabrafenib and encorafenib

Abstract

Acquired resistance of metastatic melanoma (MM) tumors to BRAF V600E inhibitors (BRAFi's) is commonplace in the clinic. Habitual relapse of patients contributes to <20% 5-year survival rates in MM. We previously identified serine synthesis as a critical detrminant of late-stage cancer cell resistance to BRAFi's. Pre-treatment with DNA damaging agent gemcitabine (a nucleoside analog) re-sensitized drug-resistant cancer cells to BRAFi's dabrafenib and vemurafenib. Importantly, the combination treatments were effective against BRAF wild type cancer cells potentially expanding the clinical reach of BRAFi's. In this study, we identify the antifolate methotrexate (MTX) as a sensitizer of acquired- and intrinsically-resistant MM cells to BRAFi's dabrafenib and encorafenib. We identify a novel, positive correlation between dabrafenib treatments and repair delay of MTX induced single-strand DNA (ssDNA) breaks. Cells arrest in G1 phase following simultaneous MTX + dabrafenib treatments and eventually die via apoptosis. Importantly, we identify RAS codon 12 activating mutations as prognostic markers for MTX + BRAFi treatment efficacy. We describe a method of killing drug-resistant MM cells that if translated has the potential to improve MM patient survival.

Keywords: dabrafenib; encorafenib; metastatic melanoma; methotrexate; pancreatic cancer.

Figure 1. Sensitization of SK-MEL-28VR1 and 501-mel cells to BRAFi’s dabrafenib and encorafenib by MTX

(A) Colony formation assays of SK-MEL-28VR1 cells following treatments with differential doses of MTX, dabrafenib, or MTX + dabrafenib (n = 3) (p < 0.0001). (B) Colony formation assays of 501-mel cells following treatments with differential doses of MTX, dabrafenib, or MTX + dabrafenib (n = 3) (p < 0.0001). (C) Spheroidal growth assays of SK-MEL-28VR1 and 501-mel cells following treatments with differential doses of MTX, dabrafenib, or MTX + dabrafenib (n = 3). (D) Spheroidal growth assays of SK-MEL-28VR1 cells following treatments with differential doses of MTX, encorafenib, or MTX + encorafenib.

Figure 2. MAPK activation and DNA damage checkpoint induction following MTX + dabrafenib combination treatments

(A) Western blot of p-ERK1/2 (Thr202/Tyr204) expression in differentially treated SK-MEL-28 and SK-MEL-28VR1 cells. β-actin used as loading control. 30 µg of protein loaded in each lane. (B) Western blot of RPA70 and p-CDK1 (Tyr15) expression in differentially treated SK-MEL-28 and SK-MEL28VR1 cells. β-actin used as loading control. 30 µg of protein loaded in each lane. (C) Western of cleaved PARP1 and cleaved caspase 3 expression in differentially treated SK-MEL-28 and SK-MEL-28VR1 cells. β-actin used as loading control. 30 µg of protein loaded in each lane.

Figure 3. 48 hour MTX + dabrafenib treatments cause G1/S cell cycle arrests in BRAFi-resistant MM cells

FACScan cell cycle assays following differential treatments of SK-MEL-28VR1 and 501-mel cells. 10,000 cells were analyzed. Top panels are histograms, and bottom panels are corresponding side scatter plots.