Two novel ALK mutations mediate acquired resistance to the next-generation ALK inhibitor alectinib

Abstract

Purpose: The first-generation ALK tyrosine kinase inhibitor (TKI) crizotinib is a standard therapy for patients with ALK-rearranged non-small cell lung cancer (NSCLC). Several next-generation ALK-TKIs have entered the clinic and have shown promising activity in crizotinib-resistant patients. As patients still relapse even on these next-generation ALK-TKIs, we examined mechanisms of resistance to the next-generation ALK-TKI alectinib and potential strategies to overcome this resistance.

Experimental design: We established a cell line model of alectinib resistance, and analyzed a resistant tumor specimen from a patient who had relapsed on alectinib. We developed Ba/F3 models harboring alectinib-resistant ALK mutations and evaluated the potency of other next-generation ALK-TKIs in these models. We tested the antitumor activity of the next-generation ALK-TKI ceritinib in the patient with acquired resistance to alectinib. To elucidate structure-activity relationships of ALK mutations, we performed computational thermodynamic simulation with MP-CAFEE.

Results: We identified a novel V1180L gatekeeper mutation from the cell line model and a second novel I1171T mutation from the patient who developed resistance to alectinib. Both ALK mutations conferred resistance to alectinib as well as to crizotinib, but were sensitive to ceritinib and other next-generation ALK-TKIs. Treatment of the patient with ceritinib led to a marked response. Thermodynamics simulation suggests that both mutations lead to distinct structural alterations that decrease the binding affinity with alectinib.

Conclusions: We have identified two novel ALK mutations arising after alectinib exposure that are sensitive to other next-generation ALK-TKIs. The ability of ceritinib to overcome alectinib-resistance mutations suggests a potential role for sequential therapy with multiple next-generation ALK-TKIs.

Fig. 1. H3122 CHR-A1 cells are resistant to alectinib and harbor a V1180L mutation in the ALK kinase domain

(A) Cells were seeded in 96-well black plates and treated with increasing concentrations of alectinib for 72 hrs. Cell survival was analyzed using the CellTiter-Glo assay. While H3122 cells showed high sensitivity to alectinib (red line), H3122 CHR-A1 cells (green line) were as insensitive to alectinib as non-ALK rearranged cell lines. (B) Table summarizing IC50 values (nM) of each cell line against alectinib. (C) Parental H3122 and CHR-A1 cells were treated with alectinib at the indicated concentrations for 6 hrs. Cell extracts were immunoblotted to detect the indicated proteins. (D) Two-color FISH (ALK-5′ region (red) / ALK-3′ region (green)) analysis was performed on parental H3122 and CHR-A1 cells. Most of the cells harbor isolated or split green signals (arrowhead, EML4-ALK). Shown on the right are electrophoretograms of EML4-ALK cDNA from parental H3122 and CHR-A1 cells. The g3538c mutation within exon 23 results in a V1180L substitution which corresponds to a gatekeeper mutation in ALK.

Fig. 2. Discovery of the ALK I1171T secondary mutation in a patient with acquired resistance to crizotinib and alectinib

(A) Secondary I1171T mutation was only detected in the post-alectinib treated specimen. Shown are electrophoretograms of ALK kinase domain cDNA from the post-alectinib treatment. (B) Sensitivity of MGH056-1 cells to crizotinib or alectinib. MGH056-1 cells, parental H3122 and CHR-A1 cells were treated with indicated concentrations of crizotinib or alectinib. Cell survival was analyzed with CellTiter-Glo. (C) MGH056-1 cells were exposed to increasing concentrations of crizotinib or alectinib for 6 hrs. Cell lysates were immunoblotted to detect the indicated proteins.

Fig. 3. Biochemical and structural basis of alectinib resistance mediated by ALK I1171T and V1180L mutations

(A) Ba/F3 cells expressing WT, I1171T or V1180L EML4-ALK were seeded in 96-well plates and treated with the indicated concentrations of alectinib for 72 hrs. Cell viability was analyzed using the CellTiter-Glo assay. (B) Inhibition of phospho-ALK by alectinib in Ba/F3 models. WT or mutated EML4-ALK-expressing Ba/F3 cells were exposed to increasing concentrations of alectinib for 2 hrs. Cell lysates were immunoblotted to detect the indicated proteins. (C) Estimated interaction energies of the indicated complexes (alectinib with WT ALK (green), ALK I1171T (cyan) or ALK V1180L (magenta)) by MP-CAFEE methods are shown. In the MP-CAFEE methods, the free energy value (ΔG) is estimated by summation of coulomb and van-der-Waals (vdw) potential energies. (D) Shown are the complex average structures in the equilibrated molecular dynamics simulation for wild-type ALK (green), ALK I1171T (cyan) and ALK V1180L (magenta).

Fig. 4. Ceritinib overcomes alectinib-resistance in Ba/F3 models and H3122 CHR-A1 cells

(A) Parental Ba/F3 cells and Ba/F3 cells expressing WT, I1171T or V1180L EML4-ALK were seeded in 96-well plates and treated with the indicated concentrations of TAE-684 (left), crizotinib (center), or ceritinib (right) for 72 hrs. Cell viability was analyzed using the CellTiter-Glo assay. (B) Cancer cell lines, including parental H3122 and alectinib-resistant H3122 CHR-A1 cells, were seeded in 96-well plates and treated with increasing concentrations of TAE-684 (left), crizotinib (center) or ceritinib (right) for 72 hrs. Cell viability was measured using CellTiter-Glo. Both parental H3122 cells (red line) and H3122 CHR-A1 cells (green line) showed marked sensitivity to TAE-684 and ceritinib. Non-ALK rearranged cell lines (A549, H460, HCC827 and PC-9 cells) showed minimal growth inhibition when exposed to ALK inhibitors. (C) Suppression of ALK signaling by ALK inhibitors (TAE684, crizotinib or ceritinib) in parental H3122 and CHR-A1 cells. Cells were exposed to increasing concentrations of TAE684, crizotinib or ceritinib for 6 hrs. Cell lysates were immunoblotted to detect the indicated proteins.

Fig. 5. Preclinical and clinical activity of ceritinib in alectinib-resistant cancer cells harboring the I1171T mutation

(A) MGH056-1 cells were seeded in 96-well plates and treated with the indicated concentration of crizotinib, alectinib, or ceritinib for 72 hrs. Cell viability was measured using CellTiter-Glo. (B) MGH056 cells were exposed to increasing concentrations of crizotinib or ceritinib for 6 hrs. Cell lysates were immunoblotted to detect the indicated proteins. (C) Axial CT scan images of patient MGH056. Shown on the left is an axial slice through this patient’s liver after he had relapsed on alectinib and prior to ceritinib. Shown on the right is a comparable axial image after 18 weeks of ceritinib treatment demonstrating marked improvement in his liver metastases.