Abstract
Cutaneous malignant melanoma (CMM) is the deadliest form of skin cancer and clinically challenging due to its propensity to develop therapy resistance. Reactive oxygen species (ROS) can induce DNA damage and play a significant role in CMM. MTH1 protein protects from ROS damage and is often overexpressed in different cancer types including CMM. Herein, we report that MTH1 inhibitor TH1579 induced ROS levels, increased DNA damage responses, caused mitotic arrest and suppressed CMM proliferation leading to cell death both in vitro and in an in vivo xenograft CMM zebrafish disease model. TH1579 was more potent in abrogating cell proliferation and inducing cell death in a heterogeneous co-culture setting when compared with CMM standard treatments, vemurafenib or trametinib, showing its broad anticancer activity. Silencing MTH1 alone exhibited similar cytotoxic effects with concomitant induction of mitotic arrest and ROS induction culminating in cell death in most CMM cell lines tested, further emphasizing the importance of MTH1 in CMM cells. Furthermore, overexpression of receptor tyrosine kinase AXL, previously demonstrated to contribute to BRAF inhibitor resistance, sensitized BRAF mutant and BRAF/NRAS wildtype CMM cells to TH1579. AXL overexpression culminated in increased ROS levels in CMM cells. Moreover, silencing of a protein that has shown opposing effects on cell proliferation, CAV-1, decreased sensitivity to TH1579 in a BRAF inhibitor resistant cell line. AXL-MTH1 and CAV-1-MTH1 mRNA expressions were correlated as seen in CMM clinical samples. Finally, TH1579 in combination with BRAF inhibitor exhibited a more potent cell killing effect in BRAF mutant cells both in vitro and in vivo. In summary, we show that TH1579-mediated efficacy is independent of BRAF/NRAS mutational status but dependent on the expression of AXL and CAV-1.
Highlights
Sensitivity to TH1579 is independent of BRAF/NRAS mutational background followed by trypsinization and resuspension in media
Using CETSA, we observed no direct target engagement of TH1579 to CAV-1 (Supplementary Fig. S8a), indicating that the observed reduction of CAV-1 levels is a downstream effect of the treatment
Knocking down MTH1 alone in A375VR4 did not downregulate CAV-1 (Fig. 5j) while silencing of CAV-1 led to downregulation of both AXL and MTH1, which may explain the loss of effect by TH1579. (Supplementary Fig. S8b)
Summary
Treatment efficacy to MAPK pathway targeting therapy of advanced BRAF-mutated CMM is high, but often not long-lasting due to resistance development [2]. ROS can promote cancer development and cancer cell survival [8,9,10], but interestingly suppress tumor growth through induction of apoptosis and senescence via DNA damage [11]. We and others [16, 17] hypothesized that the upregulated level of MTH1 is a salvage pathway for the cancer cell to survive and escape the elevated ROS-induced oxidative damage and apoptosis/ senescence. Resistance to BRAFi has been associated with reactivation of the MAPK pathway stemming from upregulation of RTKs such as AXL [20,21,22,23], which has been associated with resistance to DNA damaging therapies [24].
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