Combining ERBB family and MET inhibitors is an effective therapeutic strategy in cutaneous malignant melanoma independent of BRAF/NRAS mutation status

Abstract

Current treatment modalities for disseminated cutaneous malignant melanoma (CMM) improve survival; however, relapses are common. A number of receptor tyrosine kinases (RTKs) including EGFR and MET have been reported to be involved in CMM metastasis and in the development of resistance to therapy, targeting the mitogen-activated protein kinase (MAPK pathway). IHC analysis showed that patients with higher MET protein expression had a significantly shorter overall survival. In addition, silencing of MET caused an upregulation of EGFR and p-AKT, which was abrogated by concomitant silencing of MET and EGFR in CMM cells resistant to MAPK-targeting drugs. We therefore explored novel treatment strategies using clinically approved drugs afatinib (ERBB family inhibitor) and crizotinib (MET inhibitor), to simultaneously block MET and ERBB family RTKs. The effects of the combination were assessed in cell culture and spheroid models using established CMM and patient-derived short-term cell lines, and an in vivo xenograft mouse model. The combination had a synergistic effect, promoting cell death, concomitant with a potent downregulation of migratory and invasive capacity independent of their BRAF/NRAS mutational status. Furthermore, the combination attenuated tumor growth rate, as ascertained by the significant reduction of Ki67 expression and induced DNA damage in vivo. Importantly, this combination therapy had minimal therapy-related toxicity in mice. Lastly, the cell cycle G2 checkpoint kinase WEE1 and the RTK IGF1R, non-canonical targets, were altered upon exposure to the combination. Knockdown of WEE1 abrogated the combination-mediated effects on cell migration and proliferation in BRAF mutant BRAF inhibitor-sensitive cells, whereas WEE1 silencing alone inhibited cell migration in NRAS mutant cells. In summary, our results show that afatinib and crizotinib in combination is a promising alternative targeted therapy option for CMM patients, irrespective of BRAF/NRAS mutational status, as well as for cases where resistance has developed towards BRAF inhibitors.

Silencing of CEBPB-AS1 modulates CEBPB expression and resensitizes BRAF-inhibitor resistant melanoma cells to vemurafenib

“…In order to determine whether CEBPB is involved in the survival or the treatment outcome of CMM patients, we have analyzed the mRNA expression data in two data sets: TCGA database and our own AmpliSeq data obtained from a cohort of CMM patients treated with BRAFi [ 13 , 18 ]. Analysis of the TCGA database revealed a positive correlation between higher CEBPB mRNA expression and longer OS (Fig.…”

“…CEBPB mRNA expression data was extracted from previously obtained targeted sequencing of RNA from CMM fine needle aspiration samples using Ion AmpliSeq as described [ 13 , 18 ].…”

Inhibiting insulin and mTOR signaling by afatinib and crizotinib combination fosters broad cytotoxic effects in cutaneous malignant melanoma

“…We have recently shown that knockdown of MET caused an up-regulation of EGFR and induction of pAKT in BRAF inhibitor resistant CMM cells, which was blocked by simultaneous knockdown of EGFR and MET. Furthermore, we have shown that combining afatinib and crizotinib achieves an additive/synergistic effect in CMM cells and xenograft, independent of BRAF/NRAS mutation status 8 .…”

“…One of them is over-expression of receptor tyrosine kinases (RTKs) including AXL, MET, EGFR, ERBB3, IGF1R that lead to reactivation of the MAPK and/or PI3K-AKT pathways 6 . However, it has been demonstrated that resistance can be overcome by inhibiting RTKs in MAPKi resistant CMM cells [7][8][9] .…”

AXL and CAV-1 play a role for MTH1 inhibitor TH1579 sensitivity in cutaneous malignant melanoma

“…n CAV-1 expression correlates to MTH1 expression in CMM patients both in responders to targeted and immunotherapy (n = 25). Sample set previously used in[44] (*p < 0.05, Student's t test).…”