For tumors harboring cMET amplification as a determinant to cause TKI resistance, targeting cMET receptor in combination to EGFR inhibitors are likely to predict a better response compared with individual targeting of EGFR. Antibodies targeting the cMET ligand, antibodies targeting MET itself and small molecules inhibitors against cMET are the therapeutic options for these combination therapies. The simultaneous inhibition of EGFR and cMET was shown to suppress the proliferation of cells and anti-tumor efficacy in mice in HCC827 cells which develop gefitinib resistance [15, 17]. Another study was carried out in NCI-H820 cells which naturally harbor EGFR T790M mutation as well as cMET amplification. In this study, small molecule cMET inhibition or knockdown of cMET along with EGFR inhibition suppressed the compensatory ERBB3 signaling and compromised cell viability [14]. From these studies, it was not clear whether T790M EGFR and cMET amplification co-occur in the same cell and whether the cell type is dependent on both of these factors. To address these issues, Xu et al. carried out a study, where they developed mouse model of adenocarcinoma harboring both T790M EGFR and cMET. In this study treatment with individual inhibitors of EGFR and cMET was un-affective and the combinatorial targeting of both these receptors caused significant tumor regression. Importantly, this study strengthened the notion that in tumors harboring both T790M and cMET amplifications, both these lesions are drivers for growth [70].

The rationale of combining EGFR and ERBB2 inhibitors is via various molecular interactions across their downstream signaling pathways. It is known that the ligand independent activation of EGFR can be mediated by ERBB2 amplification. Over-expression and amplification of ERBB2 decreased the degradation of EGFR and increases its recycling to the cell membrane [71]. For simultaneous targeting of EGFR and ERBB2, two classes of inhibitors have been developed: agents which bind reversibly and those that bind irreversibly (covalently) to the ATP binding site in the tyrosine kinase domain in EGFR and ERBB2. Due to the mechanisms governing the resistance to reversible TKI, irreversible inhibitors targeting both EGFR and ERBB2 are likely to be better therapeutic choices. Irreversible TKI such as BIBW 2992 is shown to inhibit the autophosphorylation of EGFR and ERBB2. This agent was more than 100-fold potent that gefitinib against cells harboring the T790M+L858R mutation [47]. Another irreversible inhibitor HKI-272 caused dramatic tumor regression in mice model of TKI resistance [72]. In a Phase I clinical trial of BIBW2992, out of 26 patients with adenocarcinoma, 2 showed partial response. Another Phase II single arm clinical trial using BIBW2992, recently reported partial response in 43 patients out of 67 in mutation positive patients. The disease control rate was 96 % and a median progression free survival was 10.2 months. A clinical trial utilizing HKI-272 showed stable disease in 42 % of 16 NSCLC patients previously treated with gefitinib.

To overcome the EMT associated with TKI resistance several mediators have been targeted [73]. Due to the role of ERK1/2 in EMT, ERK1/2 blockade by U0126 led to a suppression of TGF-Beta mediated EMT in NSCLC, restored epithelial phenotype of these cells and sensitized the TKI resistant cells in combination with gefitinib [74]. E-cadherin re-expression is also utilized to target EMT in overcoming TKI resistance. ZEB1 is known to down regulate E-cadherin and promote EMT. ZEB1 was shown to be inhibited by utilizing Histone deacetylase inhibitors. In this study, the combination of HDAC inhibitors with erlotinib led to the reversal of TKI resistance in HCC4006ER cells [19]. Since the up regulation of AXL kinase was shown to be a critical determinant of TKI resistance in NSCLC, targeting of AXL kinase sensitized HCC827ER cells. In this study, knockdown of AXL kinase in HCC827 parental cells did not affect survival, however, the knockdown decreased the survival of HCC827ER cells, suggesting the specific role of AXL kinase in mediating erlotinib resistance in these cells. Small molecule inhibitors of AXL kinase, MP-470 and XL-880 [28] in combination with erlotinib also decreased the viability of HCC827ER cells. The process of EMT is also governed by inflammatory signals [75]. The inflammatory enzyme COX-2 is frequently over-expressed in a variety of malignancies [76]. COX2 plays an important role in conferring malignant and metastatic phenotypes and its over-expression is involved in therapy resistance [77]. For instance, COX-2 over-expression in NSCLC is associated with apoptosis resistance, [78] angiogenesis, [79, 80] and metastasis [81, 82]. Most of these effects are mediated by prostaglandin E2 (PGE2). PGE2 and other inflammatory cytokines in the tumor microenvironment may contribute to TKI resistance by downregulating the levels of E-cadherin. These findings suggest a strong rationale of combining COX inhibitors with TKI to overcome TKI resistance [83]. However the clinical trials combining COX2 inhibitors with EGFR inhibitors did not show additional benefits compared with the individual treatment of EGFR inhibitor. It seems possible that in these trials sufficient dose of COX2 inhibitor was not used to inhibit maximum COX2 activity [84, 85].

The rationale for combining EGFR inhibitors with mTOR inhibitors was based on studies suggesting an important survival function of mTOR-AKT axis as downstream effector of EGFR signaling [86]. The ability of the irreversible EGFR inhibitor HKI-272 and rapamycin combination to promote more effective suppression of EGFR signaling to S6 and AKT kinases was demonstrated both in cultured NSCLC cell lines harboring double mutant EGFR alleles as well as in lung tumors in TL mice. The investigators suggest that HKI-272 may not sufficiently overcome the biochemical drug resistance conferred by T790M and that further suppression of an essential AKT-mTOR signal downstream of EGFR is required to achieve a therapeutic response [87].