The development of more effective therapies for advanced melanoma is currently attracting great interest, mainly due on the increasing incidence and high mortality rates of this aggressive form of skin tumor. In recent years, advances in targeted therapy and immunotherapy have changed the treatment algorithm of BRAF-V600E mutated metastatic melanoma patients [39]. The target therapy, mainly based on the combination of BRAF and MEK inhibitors, is in clinical practice the standard of care for BRAF-mutated advanced melanoma patients. Vemurafenib plus cobimetinib, dabrafenib plus trametinib, and encorafenib plus binimetinib are the three drug combinations so far approved for the treatment of metastatic melanoma patients harboring the BRAF-V600E mutation [18–26]. However, although these combinations are highly active, the duration of response is limited due to the onset of adaptive resistance mechanisms responsible for the progression of the disease in most of the patients [27]. Different mechanisms of resistance enable the expansion of distinct cell subpopulations in the presence of BRAF and MEK inhibitors, supporting survival and proliferation of the cancer cells that develop an adaptive response by activating feedback loops that can affect cell phenotype and melanoma progression [40–41]. Consistent evidence indicates that a multicellular heterogenous ecosystem of melanoma cells can change in a stepwise manner during the development of resistance, and many alterations accompanying this process contributes to a high plasticity of melanoma cells [42–43]. In this scenario, a better understanding of mechanisms of drug resistance and the development of new strategies able to prevent and/or overcome it, represents a challenge that needs to be pursued. Several mechanisms, including secondary mutations, bypass signaling and activation of other compensatory downstream effectors, are known to be responsible for the development of acquired resistance [44]. TKRs overexpression or activation have been shown to be able to bypass the BRAF and MEK blockade as a mechanism of resistance [45]. TKRs may in fact act as upstream activators of MAPK/AKT signaling pathways, and their increased expression in BRAF-resistant cells has been described in multiple studies [46]. Based on these data, to better understand mechanisms underlying BRAF and MEK inhibitors resistance, we generated two cell lines (SAN-VRCR and A375-VRCR) with acquired resistance to vemurafenib and cobimetinib, alone and in combination. These lines were obtained from parental SAN and A375 lines, both harboring BRAFV600E mutation and sensitive to treatment with these drugs. Moving forward vemurafenib and cobimetinib resistance, we assisted to morphological changes in microtubule organization, increased cell invasion, mobility capacity and high expression of EMT markers. All these aspects could be associated with acquired drug resistance after long-term exposure to BRAF and MEK inhibitor treatment [47]. Interestingly, in both SAN-VRCR and A375-VRCR lines the MAPK/AKT pathways were also upregulated, suggesting that the reactivation of these signaling cascades might be involved in the development of acquired resistance. Several TKRs whose activation has generally been associated with invasive behavior -such as EhpA2, DDR1 and DDR2- were highly expressed and activated in our resistant cell lines compared to parental ones. Both DDRs are known to modulate cell invasion and migration; in fact, DDRs are RTKs can target fibrillar collagens, specifically types II and III, that are the major components of extracellular matrix (ECM) [48]. The role of DDRs in promoting cell invasion is also due to their ability to modulate the expression and activity of metalloproteinases (MMPs), a group of the enzymes that degrades ECM components. Specifically, DDR1 has been shown to control melanoma cell invasion and survival and its high expression has been correlated with poor prognosis in melanoma lesions [49]. Other studies have reported that DDR2 depletion in melanoma cell lines reduced the invasive and metastatic abilities [50–51]. To note, the emerging general role of DDR family receptors as attractive targets in anti-cancer therapies has been highlighted by several studies [28–36], and EphA2 activation has been shown to be directly involved in BRAF inhibitor resistant melanoma cell lines [52]. In particular, the inhibition of DDR1 or DDR2 with nilotinib, dasatinib, or other still not-approved inhibitors, has been shown to decrease invasion and metastatic capability of several types of carcinomas [53–55].
Based on these data, we treated SAN-VRCR and A375-VRCR resistant cell lines with ALW-II-27-41, a multikinases inhibitor able to inhibit several targets, including DDR1, DDR2 and several EPH members. Strikingly, once resistant cell lines were exposed to this inhibitor, a significant reduction of expression and activation of EphA2, DDR1 and DDR2 was observed. Moreover, the ALW-II-27-41 treatment of both SAN-VRCR and A375-VRCR resistant cell lines substantially inhibited phosphorylation of AKT, MEK and MAPK downstream pathways. Importantly, the multikinase inhibitor treatment was also able to reduce the invasion and migration abilities of vemurafenib and cobimetinib-resistant cells compared to the parental ones. Collectively, these data demonstrated that inhibition of multiple TKRs with ALW-II-27-41 treatment might represent a promising therapeutic approach to overcome the limitations of targeting individual growth pathways. We suggest that the ALW-II-27-41 inhibitor, by simultaneous targeting multiple TRKs, might provide an effective therapy to overcome acquired resistance to BRAF and MEK inhibitors, possibly improving both prognosis and survival of BRAFV600E melanoma patients.