Discoidin death receptor 2 (DDR2) is a receptor tyrosine kinase activated by binding of its discoidin domain to fibrillar collagen (Figure 1) (15). Apart from its important role in postnatal development and tissue repair, DDR2 also regulates primary and metastatic cancer progression. Though unclear, several downstream signaling pathways have been implicated in DDR2 signaling, including Shp-2, Src, STAT, and MAPK pathways (16). The S768R mutation results in substitution of serine with arginine at position 768 in DDR2. In addition to lung squamous cell cancers, DDR2 mutations are found in cancers of the kidney, breast, endometrium, colon, and brain (17). Hammerman et al. identified oncogenic mutations in the DDR2 kinase gene in 3.8% of the 222 Sanger sequenced lung squamous cell samples (18).

Dasatinib (Sprycel, Bristol-Myers Squibb, NY, USA) is a marketed multitargeted kinase inhibitor that blocks DDR2 (18, 19). Hammerman et al. reported selective in vitro killing of squamous cancer cell lines harboring DDR2 mutations by either knockdown of DDR2 with RNA interference or by treatment with dasatinib (18). In vivo sensitivity of DDR2 to dasatinib was demonstrated by inhibition of tumors induced into athymic nude mice. As demonstrated in other studies (19) potent inhibitory effect of imatinib on DDR2 induced oncogenic transformation was also reported in this study. However, such inhibition was most shown to be most potent with dasatinib, given concurrent Src inhibition. Hammerman et al. also independently verified the presence of S768R mutation in the pre-treatment tumor sample of a female patient with squamous cell cancer with no EGFR mutation who responded to a combination of dasatinib and erlotinib in a different study (20). Currently this combination is being studied in phase I trials recruiting patients with advanced cancer. Dasatinib monotherapy is being studied in advanced squamous cell lung cancers especially in individuals whose tumors harbor mutations in the DDR2 gene (Table 1). Dasatinib can cause pleural effusions. The Brown University Oncology Group evaluated dasatinib in a phase I study with chemoradiation in an unselected cohort of patients with stage III NSCLC. This trial needed to be terminated due to pulmonary toxicity. Therefore caution is needed when dasatinib is utilized for patients with lung cancer and prior thoracic radiation (21).

Phosphatidylinositol-3 kinases (PI3K) belong to a family of heterodimeric lipid kinases. Normally, PI3K converts phosphatidy linositol-3,4-bisphosphate [PI(4,5)P2] to phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3], which in turn activates downstream AKT/mTOR pathway to regulate growth, survival, and motility. Three classes of PI3K exist; class IA is the only protein found to have somatic mutations in human cancers (22). PI3K contains catalytic and regulatory subunits encoded by separate genes. Mutations mostly occur in the helical and kinase domains of PIK3CA, the gene that encodes the p110α catalytic subunit of PI3K. These mutations are reported in up to 9% of squamous cell lung cancers and are more common in squamous cell than in adenocarcinomas (22–24). Evidence for the oncogenic potential of PIK3CA mutations stems from the association of PI3K with various oncogenes like phosphatase and TENsin homolog (PTEN) and AKT (Figure 2) (25, 26). Amplification of PIK3CA in the 3q locus (3q26) is also a mechanism of oncogene activation in squamous cell lung cancer. Copy number gains of up to 43% in lung squamous cell cancer were demonstrated suggesting that amplifications of PIK3CA are more frequent than mutations (27, 28).

PIK3CA mutations were found to coexist with EGFR mutation in studies by Sun et al. (four of four tumors) and Kawano et al. (three of eight tumors) (24, 29). Activation of PI3K/AKT pathway promotes resistance to EGFR TKIs in EGFR-mutant NSCLC (30). PIK3CA mutations have been detected in about 5% of EGFR-mutant lung cancers with acquired resistance to EGFR-TKI therapy (31).

In a phase I dose-escalation study in advanced solid tumors using BKM120 (Novartis, Basel, Switzerland), an oral pan-Class I PI3K inhibitor, two lung cancer patients were included, and one responded with stable disease (32). PI-103, a dual PI3Kα and mTOR inhibitor showed clear antitumor activity in two gefitinib-resistant NSCLC cell lines, A549 and H460 (33). BYL719 (Novartis, Basel, Switzerland), BKM120 (Novartis, Basel, Switzerland), AZD5363 (AstraZeneca, London, UK), SAR256212 and SAR245409 (Sanofi, Paris, France) are other targeted PIK3CA inhibitors being studied in phase I trials for advanced solid tumors with an alteration of the PIK3CA gene (Table 1). Caution is needed when biologic agents that effect overlapping pathways are utilized together. For example in a Brown University Oncology Group phase I study combining the mTOR inhibitor ridaforolimus with anti-EGFR antibody, severe mucositis developed at ridaforolimus doses typically not associated with this toxicity (NCT01212627).

Phosphatase and TENsin homolog encodes a lipid-protein phosphatase. By dephosphorylation of PIP3, PTEN negatively regulates PI3K/AKT leading to tumor suppression (34). The R233 mutation in exon 7 results in introduction of a premature stop codon into the PTEN gene resulting in inactivation of PTEN (35). PTEN inactivation therefore increases activity of the PI3K-AKT-mTOR pathway which promotes survival. Incidence of PTEN mutation is about 4–8% in NSCLC (35, 36). Jin et al. analyzed 176 surgically resected NSCLCs. PTEN mutations were only found in smokers and were significantly more frequent in squamous cell lung cancers than in adenocarcinomas (10.2 vs. 1.7%) (35). Similar pattern was observed in the analysis of 173 NSCLC tumors by Lee et al. (36) PTEN inactivation decreases sensitivity of EGFR-mutant lung tumors to EGFR through partial uncoupling of mutant EGFR from downstream signaling leading to EGFR activation and through activation of AKT (37). PTEN loss on the contrary increases sensitivity to PI3K-AKT and FKBP12-rapamycin associated protein (mTOR) inhibitors via increased S6 kinase activity and phosphorylation of ribosomal S6 protein (38) In addition to several studies involving unselected patient population treated with PI3K/AKT inhibitors and mTOR inhibitors, a selected population with PTEN deficiency is being studied with the experimental drug GSK2636771 (GlaxoSmithKline, London, UK; Table 1).

v-AKT murine thymoma viral oncogene homolog 1 (AKT1 oncogene) encodes a class of serine-threonine protein kinases that are involved in growth, proliferation, angiogenesis, and survival. AKT1 is a downstream regulator of PI3K pathway and is activated by either loss of function of PTEN gene or mutations/amplifications of PIK3CA gene. AKT1 activation requires both plasma membrane translocation and phosphorylation at Thr308 and Ser473 (atleast at Thr308) (39–41) and involves binding of its pleckstrin homology domain (PHD) to PI(3,4,5)P3 followed by Thr308/Set473 phosphorylation or phosphorylation of Thr308 by 3-phosphoinositide-dependent protein kinase-1 (PDK1) (42, 43). By analysis of clinical tumor specimens, Carpten et al. described the E17K mutation (point mutation at nucleotide 49 that results in a lysine substitution for glutamic acid at amino acid 17) in the PHD of AKT1 which constitutively activates the kinase (44). AKT1 activation is oncogenic as evidenced by transformation of cells in culture and induction of leukemia in mice (44). E17K mutations occur in NSCLC at a rate of 1–2% and were identified in 1 of 14 lung squamous cell samples analyzed by Do et al. (45) and 2 of 36 lung squamous cell samples analyzed by Malanga et al. (46).

An open-label, multicenter, Phase Ib dose-escalation combination study using a 3 + 3 design of GDC-0068 (Genentech Inc., South San Francisco, CA, USA), a novel oral selective ATP-competitive AKT inhibitor targeting P13k-Akt-mTOR pathway in metastatic cancer refractory to standard therapy enrolled 23 patients in the GDC-0068 + docetaxel (Taxotere, Sanofi-Aventis, Paris, France) arm and is ongoing. One patient with NSCLC in this arm experienced partial response as evidenced by 32% tumor reduction by modified Response Evaluation Criteria in Solid Tumors criteria (47). Another ongoing phase I study combining the AKT inhibitor MK-2206 (Merck, NJ, USA) with gefitinib in NSCLC patient population enriched for EGFR mutations is recruiting participants (Table 1). The PHD of PDK1 also provides another potential target for drug development.

The erythropoietin-producing hepatocellular (Eph) family of receptor tyrosine kinases regulates a multitude of physiological and pathological processes. Eph receptors are tyrosine kinases involved in embryonic development (48). Eph A2 is over expressed in NSCLC (49). Mutations in EphA2 although over all rare, are more common in lung squamous cell cancer histology. Both over expression and mutation increase the risk of invasive disease. The mutation G391R in EphA2 confers susceptibility to treatment with rapamycin (Rapamune, Pfizer, NY, USA), an mTOR inhibitor and was seen in 7% of squamous cell lung cancers vs. 0% of other histologies in one report (50). Overexpression of EphA2 also leads to increased phosphorylation of Src.

Dasatinib a multitargeted TKI has activity against EphA2 receptors (51, 52). Therefore EphA2 is a potential target for specific drug development and clinical trials are indicated.

Liver Kinase B1 (LKB1, also called STK11) is frequently mutated in NSCLC and is thought to act as a tumor-suppressor gene. LKB1 acts as a tumor suppressor by activating AMPK (5′ AMP-activated protein kinase). Loss of LKB1 by point mutation or deletion suppresses AMPK, leading to increased mTOR signaling. LKB1 is thought to function in early tumorigenesis, subsequent differentiation, and the development of metastases (53). Low levels of LKB1 protein were associated with high grades of dysplasia in atypical adenomatous hyperplasia lesions, suggesting that LKB1 has an early role in the development of premalignant lesions of the lung (54). LKB1 mutations (including point mutations and deletions) were found in 34% of adenocarcinomas and 19% of squamous cell lung cancers from 144 human specimens of NSCLC (55). Mutations are more common in Whites as compared to Asians. LKB1 inactivation and KRAS mutation enhanced tumor growth and metastatic potential in mouse models.

Recently researchers at The University of Texas MD Anderson Cancer Center, investigated the effects of LKB1 mutation and mTOR inhibition on cell signaling pathways, measuring protein expression in NSCLC cell lines by reverse phase protein array. LKB1 mutant cell lines have increased insulin-like growth factor receptor (IGFR) activity with higher baseline IGFR1, insulin-like growth factor-binding protein 2 (IGFBP2), and nuclear receptor coactivator 3 (AIB1), suggesting that IGFR may be a potential therapeutic target in LKB1 mutant tumors. In addition, inhibition of the mTOR pathway was shown to upregulate the IGFR pathway, possibly as a feedback mechanism. These results support the investigation of IGFR inhibitors in combination with drugs targeting the mTOR pathway, particularly for tumors bearing LKB1 mutations (56). Currently there are no trials looking at this combination.