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. 2015 Mar 10;6(7):5182-94.
doi: 10.18632/oncotarget.3311.

Activation of RAS family members confers resistance to ROS1 targeting drugs

Marilisa Cargnelutti  1 , Simona Corso  1   2 , Margherita Pergolizzi  1 , Laurence Mévellec  3 , Dara L Aisner  4 , Rafal Dziadziuszko  5 , Marileila Varella-Garcia  4   6 , Paolo M Comoglio  1   2 , Robert C Doebele  6 , Jorge Vialard  7 , Silvia Giordano  1   2
Affiliations

Activation of RAS family members confers resistance to ROS1 targeting drugs

Marilisa Cargnelutti et al. Oncotarget. .

Abstract

The ROS1 tyrosine kinase is activated in lung cancer as a consequence of chromosomal rearrangement. Although high response rates and disease control have been observed in lung cancer patients bearing rearranged ROS1 tumors (ROS1+) treated with the kinase inhibitor crizotinib, many of these patients eventually relapse.To identify mechanisms of resistance to ROS1 inhibitors we generated resistant cells from HCC78 lung cancer cells bearing the SLC34A2-ROS1 rearrangement. We found that activation of the RAS pathway in the HCC78 cell model, due to either KRAS/NRAS mutations or to KRAS amplification, rendered the cells resistant to ROS1 inhibition. These cells were cross-resistant to different ROS1 inhibitors, but sensitive to inhibitors of the RAS signaling pathway. Interestingly, we identified focal KRAS amplification in a biopsy of a tumor from a patient that had become resistant to crizotinib treatment.Altogether our data suggest that the activation of members of the RAS family can confer resistance to ROS1 inhibitors. This has important clinical implications as: (i) RAS genetic alterations in ROS1+ primary tumors are likely negative predictors of efficacy for targeted drugs and (ii) this kind of resistance is unlikely to be overcome by the use of more specific or more potent ROS1 targeting drugs.

Keywords: RAS; ROS1; drug resistance; lung cancer; targeted therapy.

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Conflict of interest statement

Conflict of interest

J. Vialard and L. Mévellec are employees of Janssen Pharmaceutica NV. R. Doebele has a consulting and research grant from Pfizer; consulting, travel reimbursement and research grant from Eli Lilly/ImClone; consulting from Boehringer Ingelheim, consulting from Loxo Oncology; consulting from OxOnc to RCD, research grant from Mirati Therapeutics. D.L.Aisner has an honorarium from Illumina, Inc and from Abbott Molecular; consulting grants from Oxford Oncology and from Boehringer Ingelheim.

Figures

FIG.1
FIG.1. HCC78 cells resistant to the ROS1 inhibitor JNJ-ROS1i-A are not dependent on ROS1 for growth
Parental cells were treated with the indicated concentrations of the inhibitor for 7 days. Resistant cells were kept in the presence of the drug concentrations to which they were made resistant. ns: not significant; **: significantly different from control (p< 0.01). B: Western blot analysis of HCC78 parental (WT) cells treated for 4 hours with JNJ-ROS1i-A with 0, 1, 2 or 4 μM and cells resistant to increasing concentrations of JNJ-ROS1i-A (ROS1i-A-RES 1, ROS1i-A-RES 2, ROS1i-A-RES 4). The blot was probed with the indicated antibodies. Vinculin was used as loading control. To evaluate ROS1 phosphorylation, cell lysates were immunoprecipitated with anti-ROS1 antibodies and the blot was probed with anti-phosphotyrosine antibodies. C: Growth rate of HCC78 cells (WT and ROS1i-A-RES 4) transfected with control or ROS1 specific siRNAs. Growth was evaluated after 96 hours of treatment. *: significantly different from control (p< 0.05).
FIG.2
FIG.2. HCC78 cells resistant to the ROS1 inhibitor JNJ-ROS1i-A are KRAS addicted and cross-resistant to other ROS1 inhibitors
A: DNA sequence analysis of HCC78 parental cells (WT) and cells resistant to the ROS1 inhibitor JNJ-ROS1i-A (ROS1i-A-RES 4). KRAS codon 12 of WT (upper panel) and ROS1i-A-RES 4 cells (lower panel) is indicated. B: Cell growth of HCC78 cells transduced with either an empty vector (MOCK) or constructs for expression of KRAS G12V (left panel) or KRAS G12C (right panel). The cells were untreated (NT) or treated with 4μM JNJ-ROS1i-A (6 days of treatment). *: significantly different from control (* p< 0.05; ** p< 0.01). C: Cell growth of ROS1i-A-RES 4 cells transfected with the indicated siRNAs and grown in the presence of JNJ-ROS1i-A 4μM. ROS1 and NRAS siRNAs were used as additional controls. D: Growth of parental (WT) or resistant (ROS1i-A-RES 4) HCC78 cells, treated with the indicated concentrations of JNJ-ROS1i-A, crizotinib or foretinib; ns: not significant; **: significantly different from control (p< 0.01). E: Growth of HCC78 WT cells transduced with empty vector (MOCK) or a KRAS G12C expression construct, treated with either 4 μM JNJ-ROS1i-A, 1.4 μM crizotinib or 50nM foretinib. Cell growth was measured after 6 days of treatment; ns: not significant; **: significantly different from control (p< 0.01). F: Growth of HCC78 cells (WT or ROS1i-A-RES 4), treated for 6 days with the MAPK inhibitor U0126 (10 μM), the AKT inhibitor MK2206 (3 μM), alone or in combination. *: significantly different from control (* p< 0.05; ** p< 0.01).
FIG.3
FIG.3. HCC78 cells resistant to crizotinib are addicted to NRAS
A: Growth of parental (WT) and crizotinib resistant (CRIZ-RES 0.72 and CRIZ-RES 1.4) cells treated with the indicated concentration of crizotinib for 6 days. Resistant cells were kept in the presence of the drug concentration to which they were made resistant. ns: not significant; *: significantly different from control (p< 0.05). B: DNA sequence analysis of parental (WT) and crizotinib resistant (CRIZ-RES 1.4) HCC78 cells. NRAS codon 61 is highlighted. C: Cell growth of crizotinib resistant cells (CRIZ-RES 1.4) transfected with the indicated siRNAs and grown in the presence of crizotinib 1.4μM. ROS1 and KRAS siRNAs were used as additional controls. **: significantly different from control (p< 0.01). D: Growth of HCC78 cells transduced with empty vector (MOCK) or NRAS Q61K and subsequently untreated (NT) or treated for 6 days with 4 μM JNJ-ROS1i-A, 1.4 μM crizotinib or 50 nM foretinib. ns: nonsignificant; ***: significantly different from control (p< 0.001).
FIG.4
FIG.4. KRAS/NRAS activating mutations or KRAS overexpression render HCC78 cells resistant to ROS1 inhibitors
A: Left panel: Growth of parental and crizotinib resistant HCC78 cells untreated or treated for 6 days with the indicated concentrations of JNJ-ROS1i-A. Right panel: Growth of parental and JNJ-ROS1i-A resistant HCC78 cells untreated or treated for 6 days with the indicated doses of crizotinib. ns: not significant; *: significantly different from control (p< 0.05); ***: significantly different from control (p< 0.001). B: Growth of parental (WT), JNJ-ROS1i-A resistant (ROS1i-A-RES 4) and crizotinib resistant (CRIZ-RES 1.4) HCC78 cells untreated (NT) or treated for 5 days with 10 and 50 nM foretinib. ns: not significant; *: significantly different from control (p< 0.05). C: Growth of HCC78 cells transduced with either an empty vector (MOCK) or a KRAS WT expression construct, untreated (NT) or treated with JNJ-ROS1i-A (4 μM), crizotinib (1.4 μM) or foretinib (50 nM). ns: not significant; *: significantly different from control (p< 0.05).
FIG.5
FIG.5. RAS pathway activation induces ROS1 down-regulation
A: ROS1 mRNA levels in HCC78 cells transduced with empty vector (MOCK), KRAS G12C, KRAS or NRAS Q61K expression constructs. B: ROS1 protein (left panel) and mRNA (right panel) levels in HCC78 WT or ROS1 inhibitor resistant cells (ROS1i-A-RES 4 and CRIZ-RES 4), treated for 3 days with the MAPK inhibitor UO126. The left panel shows a WB of the treated cells, probed with the indicated antibodies. Vinculin was used as a loading control. The right panel shows ROS1 mRNA expression evaluated by qRT-PCT. ns: not significant; **: significantly different from control (p< 0.01). C: ROS1 protein (left panel) and mRNA (right panel) levels in HCC78 WT or ROS1 inhibitor resistant cells, treated for 3 days with the AKT inhibitor MK2206; significantly different from control (* p< 0.05; ** p< 0.01).
FIG.6
FIG.6. KRAS FISH analysis in ROS1+ tumor samples
ROS1+ crizotinib-resistant patient tumor samples analyzed by FISH using KRAS (yellow) and CEP12 probes (green). Yellow arrows in panel A indicate mini-clusters corresponding to focal KRAS gene amplification in patient 1. Patient samples (2-4) in B-D were negative for KRAS gene amplification.
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