PF-06463922, an ALK/ROS1 Inhibitor, Overcomes Resistance to First and Second Generation ALK Inhibitors in Preclinical Models

Abstract

We report the preclinical evaluation of PF-06463922, a potent and brain-penetrant ALK/ROS1 inhibitor. Compared with other clinically available ALK inhibitors, PF-06463922 displayed superior potency against all known clinically acquired ALK mutations, including the highly resistant G1202R mutant. Furthermore, PF-06463922 treatment led to regression of EML4-ALK-driven brain metastases, leading to prolonged mouse survival, in a superior manner. Finally, PF-06463922 demonstrated high selectivity and safety margins in a variety of preclinical studies. These results suggest that PF-06463922 will be highly effective for the treatment of patients with ALK-driven lung cancers, including those who relapsed on clinically available ALK inhibitors because of secondary ALK kinase domain mutations and/or brain metastases.

Figure 1. PF-06463922 is a potent inhibitor of wild-type ALK and crizotinib-resistant ALK mutants

A) PF-06463922 in biochemical kinase assays with the indicated recombinant ALK kinase domain constructs. Ki values are geometric means with a 95% confidence interval for n independent measurements. B) Biochemical kinase activity of recombinant human wild-type ALK kinase domain treated with the indicated concentrations of PF-06463922, crizotinib, ceritinib and alectinib. The kinase activity was assayed by a microfluidic mobility shift assay. C) IC₅₀ of PF-06463922, crizotinib, ceritinib and alectinib on ALK phosphorylation (left panel) and cell viability (right panel) across different Ba/F3 cell lines expressing wild-type or mutated EML4-ALK relative to parental IL-3–dependent Ba/F3 cells. Values are presented as mean +/− SD (n=3). See also Figure S1 and Table S1.

Figure 2. PF-06463922 potently inhibits ALK fusion wild type and mutant-mediated tumor cell survival

(A–C) Cell viability assays of H3122 EML4-ALK^WT (A), H3122 EML4- ALK^L1196M (B), H3122 EML4-ALK^G1269A (C) cells treated with the indicated doses of crizotinib or PF-06463922 for 72 hours. Cell viability was assayed by Cell-Titer-Glo. For panels A–C, values are presented as mean +/− SEM (n=3) D) IC₅₀ of PF-06463922, crizotinib, ceritinib and alectinib on ALK phosphorylation in H3122 cell lines expressing EML4-ALK^WT, EML4-ALK^L1196M and EML4-ALK^G1269A. Values are presented as mean +/− SEM (n=3–7). E) Cleaved caspase 3/7 induction by PF-06463922 treatment in the three different H3122 cell models. Values are presented as mean +/− SD (n=3). F) Cell viability assay of the alectinib-resistant H3122 cell line (EML4-ALK^V1180L) treated with the indicated doses of crizotinib, PF-06463922 or alectinib for 72 hours. Values are presented as mean +/− SEM (n=6) G) Cell survival and ALK phosphorylation IC50s of PF-06463922, crizotinib, ceritinib and alectinib on crizotinib-resistant patient-derived cell line SNU2535 (EML4-ALK^G1269A). Values are presented as mean +/− SEM (n=3–14). H) Cell viability assay of ceritinib-resistant patient-derived cell line MGH021-5 (SQSTM1-ALK^G1202R) treated with the indicated doses of crizotinib, PF-06463922 or alectinib for 7 days. I) Cell viability assay of alectinib-resistant patient-derived cell line MGH056-1 (EML4-ALK^I1171T) treated with the indicated doses of crizotinib, PF-06463922 or alectinib for 72 hours. For panels H and I values are presented as mean +/− SEM (n=3). See also Figure S2.

Figure 3. PF-06463922 ALK target inhibition PK/PD relationships in ALK fusion driven subcutaneous tumor xenograft models in mice

Mini-pump infusion study in H3122 model expressing endogenous EML4-ALK^WT(A), H3122 model expressing engineered EML4-ALK^L1196M (B), H3122 model expressing engineered human EML4-ALK^G1269(C), 3T3 model expressing engineered human EML4-ALK^G1202R (D). Tumor sizes (left) and pALK level and PF-06463922 free plasma concentration (right) of each group are indicated. ALK phosphorylation in tumors was measured at the time of sacrifice following the last tumor volume measurement. The tumors were collected and processed immediately after sacrifice. Tumor volumes, ALK phosphorylation and plasma concentration values are presented as mean +/− SEM (n=8–12). See also Figure S3, Table S2, Table S3 and Table S4.

Figure 4. PF-06463922 antitumor efficacy in ALK fusion driven subcutaneous xenograft tumor models in mice

A) Subcutaneous tumor growth in the H3122 EML4-ALK^L1196M tumor model treated with orally dosed crizotinib 75 mg/kg BID or PF-06463922 0.3–10 mg/kg BID for 13 days. Tumor volumes are presented as mean +/− SEM (n=12). B) Activated-Caspase3 positive cell numbers following 3-day of oral BID administration of PF-06463922 in the H3122-EML4-ALK^L1196M model (cf. Fig. 4A). Values = Mean +/− SEM (n=7–9). C) Long term subcutaneous tumor growth in the EML4-ALK^WT MGH051 crizotinib-resistant patient-derived model treated with crizotinib 25 mg/kg QD or PF-06463922 10 mg/kg BID. The mice treated with crizotinib were shifted to PF-06463922 after 107 days of treatment (arrow). Tumor volumes are presented as mean +/− SD (n=5–12). D) Long term subcutaneous tumor growth of the H3122-EML4-ALKG^G1269A tumors treated with PF-06463922 subcutaneous pump infusion at 11 mg/kg/day for 172 days. Tumor volumes are presented as mean +/− SEM (n=5–12). See also Figure S4.

Figure 5. PF-06463922 antitumor efficacy in ALK fusion-driven intracranial tumor models

A) Representative MRI images showing regression of large established H3122 EML4-ALK^WT intracranial tumors in mice following PF-06463922 infusion. B) Quantitation of brain tumor sizes following PF-06463922 treatment in the H3122 EML4-ALK^WT intracranial model shown in panel A. Values are presented as mean +/− SEM. C) Oral dosing of PF-06463922, crizotinib and alectinib. Long term brain orthotopic tumor growth of H3122 EML4-ALK^WT cells expressing secreted luciferase treated with crizotinib 50 mg/kg QD or alectinib 60 mg/kg QD or PF-06463922 10 mg/kg BID. The mice treated with alectinib were shifted to PF-06463922 at the indicated times (blue arrows). D) Pharmacodynamic analysis of H3122 EML4-ALK^WT brain tumors treated for 3 days and collected 3 hours after last treatment. (E,F) Brain tumor growth (E) and Kaplan-Meier survival curves (p<0.0001) (F) of MGH006 EML4-ALK^WT patient-derived cell line in mice treated orally dosed with crizotinib 100 mg/kg QD or PF-06463922 10 mg/kg/day BID for 42 days. G) Pharmacodynamic analysis of MGH006 brain tumors treated for 3 days and collected 3 hours after last treatment. Individual blood Gluc activity values are presented for each mouse (n=5–7 per group). See also Figure S5.

Figure 6. PF-06463922 preclinical pharmacology profile

A) Cell survival curves of Ba/F3 parental (left) or EML4-ALK^WT –expressing cells (right) following treatment for 48 hr with crizotinib, alectinib or PF-06463922. Cell survival was assayed using Cell-Titer-Glo. Values are presented as mean +/− SEM (n=3) B) PF-06463922 preclinical pharmacology profile and safety margin. See also Figure S6.