Mutations in the DDR2 kinase gene identify a novel therapeutic target in squamous cell lung cancer

Abstract

While genomically targeted therapies have improved outcomes for patients with lung adenocarcinoma, little is known about the genomic alterations which drive squamous cell lung cancer. Sanger sequencing of the tyrosine kinome identified mutations in the DDR2 kinase gene in 3.8% of squamous cell lung cancers and cell lines. Squamous lung cancer cell lines harboring DDR2 mutations were selectively killed by knock-down of DDR2 by RNAi or by treatment with the multi-targeted kinase inhibitor dasatinib. Tumors established from a DDR2 mutant cell line were sensitive to dasatinib in xenograft models. Expression of mutated DDR2 led to cellular transformation which was blocked by dasatinib. A squamous cell lung cancer patient with a response to dasatinib and erlotinib treatment harbored a DDR2 kinase domain mutation. These data suggest that gain-of-function mutations in DDR2 are important oncogenic events and are amenable to therapy with dasatinib. As dasatinib is already approved for use, these findings could be rapidly translated into clinical trials.

Significance: DDR2 mutations are present in 4% of lung SCCs, and DDR2 mutations are associated with sensitivity to dasatinib. These findings provide a rationale for designing clinical trials with the FDA-approved drug dasatinib in patients with lung SCCs.

Keywords: DDR2; Squamous cell lung cancer; dasatinib; lung cancer genomics; tyrosine kinase inhibitors.

Conflict of interest statement

Conflicts of Interest: M.M. is a consultant to Novartis and receives research support from Novartis, receives research support from Genentech, is a founding advisor and consultant to, and an equity holder in, Foundation Medicine and patent holder for EGFR mutation testing, licensed to Genzyme Genetics. N.S.G. receives research support from Novartis. E B.H. was the principal investigator of an industry-sponsored clinical trial of dasatinib and erlotinib in lung cancer funded in part by Bristol-Myers-Squibb and the American Society for Clinical Oncology. R.K.T. reports consulting and lecture fees (Sequenom, Sanofi-Aventis, Merck, Roche, Infinity, Boehringer, Astra-Zeneca, Johnson&Johnson, Atlas-Biolabs) and research support (Novartis, AstraZeneca).

Figures

Figure 1. Sequencing of squamous lung cancer samples identifies recurrent mutations in DDR2

(a) Schema depicted for the primary, secondary and validation screens for DDR2 mutations in squamous lung cancer samples. (b) Amino acid sequence of DDR2 with the positions of the identified mutations shown in the context of the known domain structure of DDR2.

Figure 2. Lung cancer cell lines with DDR2 mutations are sensitive to drugs and RNAi targeting DDR2

(a) Proliferation of A549, NCI-H2286, HCC-366 and NCI-H1703 grown for six days in the presence of various concentrations of dasatinib. Proliferation shown relative to untreated cells at the same time point. Standard errors are shown for triplicate samples. (b) Proliferation shown of NCI-H2286 and HCC-366 cell lines ectopically expressing the T654M gatekeeper mutation in DDR2, labeled as DDR2*. Six day proliferation in the presence of dasatinib is shown as above. For NCI-H2286 and HCC-366 the gatekeeper mutation is expressed in cis with the DDR2 mutation found in the cell line. (c) Proliferation measured as above for NCI-H2286, HCC-366 and NCI-H1703 cells stably expressing sh-RNA vectors targeting either GFP or the 3′ UTR of DDR2 (DDR2 sh-RNA-2) or the coding sequence of DDR2 (DDR2 sh-RNA-5). Proliferation is measured after four days in culture as compared to day 1. Standard errors are shown for triplicate samples. Immunoblot showing relative levels of DDR2 in the cell lines used in the experiment is shown in the inset. “G” indicates cells expressing shGFP, and “2” and “5” the numbered DDR2 targeted hairpins. (d) Four-day proliferation of the DDR2 mutant NCI-H2286 and HCC-366 cell lines stably expressing ectopic DDR2 following knock-down of DDR2 by a sh-RNA targeting the 3′ UTR of DDR2 (sh-RNA-2). Proliferation of triplicate samples is presented as above relative to cells transduced with a sh-RNA targeting GFP. Protein levels of DDR2 are shown in the immunoblot below, “G” indicates sh-GFP, “2” indicates expression of sh-RNA 2 and “D” indicates expression of sh-RNA2 and DDR2.

Figure 3. Xenografts of squamous lung cancer cell lines demonstrate anti-tumor effects of dasatinib in vivo

Athymic nu/nu mice were injected subcutaneously with A549, NCI-H1703, HCC-366 and NCI-H2286 cells (n=10) and treated with dasatinib or vehicle for two weeks following tumor formation. Depicted are representative images of mice from each cohort as well as measurements of tumor size. Tumors did not form in the mice injected with HCC-366 and these mice could not be analyzed further.

Figure 4. Ectopic expression of DDR2 mutants leads to cellular transformation which can be blocked by dasatinib or combination tyrosine kinase inhibitor treatment

(a) Results from soft agar assay in which 3T3 fibroblasts expressing the L63V DDR2 mutation, the L858R EGFR mutation or the KRAS G12V mutation were plated in soft agar in the presence of various concentrations of dasatinib. Colony number of six independent samples with standard errors is shown. (b) Proliferation at four days of Ba/F3 cells expressing vector only or one of six DDR2 mutations shown in cells grown in the presence of dasatinib. For the vector control cells are grown in the presence of IL-3 to maintain viability and in the case of the DDR2 mutants all cells are IL-3 independent and cultured in the absence of IL-3. Proliferation is shown relative to untreated cells at the same time point for triplicate samples with standard errors. (c) Proliferation of Ba/F3 cells expressing DDR2 L63V co-cultured with 50 nM of nilotinib, AP24534 or dasatinib with or without 500 nM AZD0530. Proliferation is relative to untreated cells grown in parallel. (d) Immunoblots of DDR2 L63V transformed Ba/F3 cells treated for two days with the depicted concentrations of AZD0530 (AZD) in addition to 50 nM nilotinib (N), AP24534 (AP) or dasatinib (D). The first lane is an untreated sample. Shown are immunoblots probed with antibodies against phospho-Src Y416, phospho-STAT5 Y694, FLAG-DDR2 and actin.

Figure 5. Radiographic response of a patient with a S768R DDR2 mutation treated with dasatinib plus erlotinib

(a) CT scan images shown from a lung SCC patient who was treated with chemotherapy and later with dasatinib plus erlotinib. Serial CT scans are shown at the time of initiation of chemotherapy, initiation of study treatment with dasatinib and erlotinib and following two months of treatment with dasatinib plus erlotinib. (b) Top panel: Tumor dimension measurements from the subject above starting four months prior to chemotherapy treatment and extending to the time at which combination therapy with dasatinib and erlotinib was discontinued. Bottom panel: Bar graph depicting measured tumor volume by RECIST criteria of the subject prior to chemotherapy, following chemotherapy and following two months of dasatinib plus erlotinib therapy.