Resistance and partial responses to targeted monotherapy are major obstacles in cancer treatment. Systematic approaches to identify efficacious drug combinations for cancer are not well established, especially in the context of genotype. To address this, we have tested pairwise combinations of an array of small molecule inhibitors on early passage melanoma cultures using combinatorial drug screening. Results reveal several inhibitor combinations effective for melanomas with activating RAS or BRAF mutations, including mutant BRAF melanomas with intrinsic or acquired resistance to vemurafenib. Inhibition of both EGFR and AKT sensitized treatment-resistant BRAF-mutant melanoma cultures to vemurafenib. Melanomas with RAS mutations were more resistant to combination therapies relative to BRAF mutants, but were sensitive to combinations of statins and cyclin-dependent kinase inhibitors in vitro and in vivo. These results demonstrate the utility of combinatorial drug screening for discovering unique treatment regimens that overcome resistance phenotypes of mutant BRAF and RAS driven melanomas.

The receptor tyrosine kinase ERBB4, a member of the epidermal growth factor receptor (EGFR) family, is unusual in that when phosphorylated, ERBB4 can undergo intramembrane proteolysis, releasing a soluble intracellular domain (ICD) that activates transcription in the nucleus. We found that ERBB4 activated the transcriptional coactivator YAP, which promotes organ and tissue growth and is inhibited by the tumor-suppressor Hippo pathway. Overexpressing ERBB4 in cultured mammary epithelial cells or adding the ERBB4 ligand neuregulin 1 (NRG1) to breast cancer cell cultures promoted the expression of genes regulated by YAP, such as CTGF. Knocking down YAP or ERBB4 prevented the induction of CTGF expression by NRG1, as did preventing ERBB4 cleavage by treating cells with the pan-EGFR inhibitors lapatinib or erlotinib. A PPxY motif in the ERBB4 ICD enabled its interaction with WW domains in YAP, similar to the mode of interaction between YAP and the kinase LATS1, which inhibits the transcriptional activity of YAP. The ERBB4 ICD coimmunoprecipitated with YAP and TEAD1, a YAP coactivator, suggesting that the ERBB4 ICD may functionally interact with YAP and TEAD to promote transcriptional activity. NRG1 stimulated YAP activity to an extent comparable to that of EGF or LPA (lysophosphatidic acid), known activators of YAP. NRG1 stimulated YAP-dependent cell migration in breast cancer cell lines. These observations connect the unusual nuclear function of a growth factor receptor with a mechanosensory pathway and suggest that NRG1-ERBB4-YAP signaling may underlie the aggressive behavior of tumor cells.

MET amplification as a mechanism of acquired resistance to EGFR targeted therapies in non-small cell lung carcinoma (NSCLC) led to investigation of novel combinations of EGFR and MET kinase inhibitors. However, promiscuous interactions between MET and ERBB family members have made it difficult to evaluate the effects of MET on EGFR signaling, both independent of drug treatment and in the context of drug resistance. We addressed this issue by establishing a 32D model cell system wherein ERBBs or MET are expressed alone and in combination. Using this model, we determined that EGFR signaling is sufficient to induce MET phosphorylation, although MET activation is enhanced by co-expression of ERBB3. EGFR-MET crosstalk was not direct but occurred by a combined regulation of MET levels and intermediary signaling through MAP kinases. In NSCLCs harboring either wild-type or mutant EGFR, inhibiting EGFR or MAP kinases reduced MET activation and protein levels. Furthermore, MET signaling promoted EGFR-driven migration and invasion. Lastly, EGFR-MET signaling was enhanced in a highly metastatic EGFR mutant cell subpopulation, compared to the indolent parental line, and MET attenuation decreased the incidence of brain metastasis. Overall, our results establish that EGFR-MET signaling is critical for aggressive behavior of NSCLCs and rationalize its continued investigation as a therapeutic target for tumors harboring both wild-type and mutant EGFR at early stages of progression.

We have found that ERBB4 activates sterol regulatory element binding protein-2 (SREBP-2) to enhance expression of genes essential for cholesterol metabolism including mevalonate pathway enzymes and the low-density lipoprotein receptor (LDLR). ERBB4 is unusual among receptor kinases in undergoing ligand-induced proteolytic cleavage to release a soluble intracellular domain that enters the nucleus and modifies transcription. Expression of the ERBB4 intracellular domain or activation of ERBB4 with the ligand Neuregulin 1 (NRG1) in mammary epithelial cells induced expression of SREBP target genes involved in cholesterol biosynthesis including HMGCR, HMGCS1, and LDLR beyond amounts induced through lipoprotein depletion. ERBB4 increased expression of cholesterogenic genes by enhancing abundance of the mature form of SREBP-2. NRG1 activated SREBP-2 through ERBB family kinases and PI3K, but independent from AKT or mTORC1 activity. NRG1 increased cholesterol metabolism by 1) increasing de novo biosynthesis through the mevalonate pathway, and 2) enhancing LDL binding and uptake through the LDLR. As all EGFR family receptors can appropriate ERBB4 signaling by cross-activating ERBB4, these data show that the ERBBs are linked to SREBP-regulated cholesterol metabolism, with potential impact on dyslipidemia and cancer.