Cabozantinib overcomes crizotinib resistance in ROS1 fusion-positive cancer

Abstract

Purpose: ROS1 rearrangement leads to constitutive ROS1 activation with potent transforming activity. In an ongoing phase I trial, the ALK tyrosine kinase inhibitor (TKI) crizotinib shows remarkable initial responses in patients with non-small cell lung cancer (NSCLC) harboring ROS1 fusions; however, cancers eventually develop crizotinib resistance due to acquired mutations such as G2032R in ROS1. Thus, understanding the crizotinib-resistance mechanisms in ROS1-rearranged NSCLC and identification of therapeutic strategies to overcome the resistance are required.

Experimental design: The sensitivity of CD74-ROS1-transformed Ba/F3 cells to multiple ALK inhibitors was examined. Acquired ROS1 inhibitor-resistant mutations in CD74-ROS1 fusion were screened by N-ethyl-N-nitrosourea mutagenesis with Ba/F3 cells. To overcome the resistance mutation, we performed high-throughput drug screening with small-molecular inhibitors and anticancer drugs used in clinical practice or being currently tested in clinical trials. The effect of the identified drug was assessed in the CD74-ROS1-mutant Ba/F3 cells and crizotinib-resistant patient-derived cancer cells (MGH047) harboring G2032R-mutated CD74-ROS1.

Results: We identified multiple novel crizotinib-resistance mutations in the ROS1 kinase domain, including the G2032R mutation. As the result of high-throughput drug screening, we found that the cMET/RET/VEGFR inhibitor cabozantinib (XL184) effectively inhibited the survival of CD74-ROS1 wild-type (WT) and resistant mutants harboring Ba/F3 and MGH047 cells. Furthermore, cabozantinib could overcome all the resistance by all newly identified secondary mutations.

Conclusions: We developed a comprehensive model of acquired resistance to ROS1 inhibitors in NSCLC with ROS1 rearrangement and identified cabozantinib as a therapeutic strategy to overcome the resistance.

Figure 1. Several ALK inhibitors effectively inhibit the growth of CD74-ROS1-addicted Ba/F3 cells

(A) Ba/F3 cells expressing CD74-ROS1 (clone #6) were seeded in 96 well-plates and treated with indicated concentration of crizotinib, ceritinib, AP26113, ASP3026, or alectinib for 72 h. Cell viability was analyzed using the CellTiter-Glo assay. (B) IC₅₀ values (nM) of Ba/F3 cell lines expressing CD74-ROS1 (clone #6) against various ALK inhibitors are shown. Average IC₅₀ values against crizotinib, ceritinib or AP26113 were calculated from the 3 independent experiments. IC₅₀ values against ASP3026 and alectinib were calculated from the single experiment. (C) Inhibition of phospho-ROS1 by various ALK inhibitors in Ba/F3 models. CD74-ROS1 expressing Ba/F3 cells were exposed to increasing concentrations of crizotinib, ceritinib, AP26113, or ASP3026 for 3 h. Cell lysates were immunoblotted to detect the indicated proteins.

Figure 2. Identification of crizotinib and ceritinib-resistant mutations by accelerated mutagenesis screening

(A) Number of the ROS1 kinase domain mutations found in the N-ethyl-N-nitrosourea (ENU)-treated CD74-ROS1 Ba/F3 clones isolated after growth in the presence of 100 and 200 nM of crizotinib or 200 nM of ceritinib. (B) Inhibition of phospho-ROS1 by crizotinib and ceritinib in ENU selected crizotinib- or ceritinib-resistant Ba/F3 clones. CD74-ROS1-wildtype expressing Ba/F3 cells clone 3 or ENU selected K2003I or Ba/F3 cells harboring the G2032R mutation were exposed to increasing concentrations of crizotinib or ceritinib for 2 h. Cell lysates were immunoblotted to detect the indicated proteins. (C) IC₅₀ values for CD74-ROS1 kinase domain mutant Ba/F3 cells treated with crizotinib or ceritinib. IC₅₀ values are shown in the lower table. IC₅₀ values of Ba/F3 parental cells cultured with IL-3 and CD74-ROS1-WT expressing Ba/F3 cells were shown for comparison.

Figure 3. Sensitivity of the CD74-ROS1 mutant reintroduced Ba/F3 cells to crizotinib or ceritinib

(A) Inhibition of phospho-ROS1 by crizotinib and ceritinib in each WT or mutant CD74-ROS1 introduced Ba/F3 cells. Each Ba/F3 cells were exposed to increasing concentrations of crizotinib or ceritinib for 2 h. Cell lysates were immunoblotted to detect the indicated proteins. (B, C) WT or mutant (G2032R, L1951R, or L2026M) CD74-ROS1-introduced Ba/F3 cells were seeded on 96-well plates and treated with the indicated concentration of crizotinib (B) or ceritinib (C) for 72 h. Cell viability was analyzed using the CellTiter-Glo assay. IC-50 values of each mutant Ba/F3 clone to crizotinib or ceritinib were shown in Supplementary Figure S4B. (D) Resistant mutation residues in the structural models of wild-type ROS1 kinase domain with crizotinib. Three-dimensional mapping of each identified ROS1 mutation based on the crystal structure of ROS1 with crizotinib. Each of the three ROS1 mutations is mapped on a ribbon diagram. Figures were drawn using PyMol software with the crystal structure information of PDB ID 3ZBF. Other identified mutations mapped on the whole ROS1 kinase domain is shown in Supplementary Figure S5.

Figure 4. Newly identified inhibitors effectively inhibit phospho-ROS1 of wildtype CD74-ROS1, or both WT and G2032R crizotinib-resistant mutant

(A, B) Inhibition of phospho-ROS1 by various identified ROS1 inhibitors selected from the high throughput screening. CD74-ROS1-WT-expressing (clone 6) or CD74-ROS1-G2032R-expressing Ba/F3 cells were exposed to increasing concentrations of crizotinib, CEP701, SB218078 (A), cabozantinib (XL184), TAE684, or foretinib (B) for 2 h. Following treatment, the cell lysates were immunoblotted to detect the indicated proteins.

Figure 5. Cabozantinib overcomes crizotinib-resistance caused by the mutations in CD74-ROS1

(A) Ba/F3 parental cells (with IL-3) or those expressing CD74-ROS1-wildtype (WT) or CD74-ROS1-G2032R were seeded in 96-well plates and treated with the indicated concentration of crizotinib or cabozantinib (XL184) for 72 h. Cell viability was analyzed using the CellTiter-Glo assay. (B) IC₅₀ values of Ba/F3 cells expressing WT or G2032R mutated CD74-ROS1 treated with crizotinib, ceritinib, AP26113 or cabozantinib (XL184). (C) Comparison of the inhibition of phospho-ROS1 and its downstream by crizotinib and cabozantinib in Ba/F3 cells expressing CD74-ROS1 WT or G2032R exposed to increasing concentrations of crizotinib or cabozantinib (XL-184) for 2 h. Cell lysates were immunoblotted to detect the indicated proteins. (D) Inhibition of phospho-ROS1 by cabozantinib in each mutant expressing Ba/F3 cells. CD74-ROS1 WT or mutants expressing Ba/F3 cells were exposed to increasing concentrations of cabozantinib for 2 h. Cell lysates were immunoblotted to detect the indicated proteins. (E) Average IC50 values (from the 3 independent experiments) of each Ba/F3 cells to cabozantinib (XL184) were shown.

Figure 6. Cabozantinib inhibits the growth of G2032R mutation harboring MGH047 cells and the phosphorylation of CD74-ROS1

(A) Crizotinib-resistant CD74-ROS1-positive NSCLC patient-derived MGH047 cells were seeded on 96-well plates and treated with the indicated concentration of crizotinib ceritinib or and cabozantinib (XL184) for 7 days. Cell viability was analyzed using the CellTiter-Glo assay. (B) Comparison of the inhibition of phospho-ROS1 and its downstream signaling by crizotinib, cabozantinib or ceritinib in CD74-ROS1 G2032R expressing MGH047 cells. MGH047 cells were exposed to the indicated concentrations of crizotinib cabozantinib (XL184) or ceritinib for 6 h. Cell lysates were immunoblotted to detect the indicated proteins.