Abstract LB-298: AP26113, a potent ALK inhibitor, overcomes mutations in EML4-ALK that confer resistance to PF-02341066 (PF1066)
Authors: Zhang Sen , Wang Frank , Keats Jeffrey , Ning Yaoyu , Wardwell Scott D. , Moran Lauren , Mohemmad Qurish K. , Anjum Rana , Wang Yihan , Zhu Xiaotian , Miret Juan J. , Dalgarno David , Narasimhan Narayana I. , Clackson Tim , Shakespeare William C. , Rivera Victor M. 
Published: February 2011

AP26113 is a potent and selective inhibitor of anaplastic lymphoma kinase (ALK) (AACR 2010; #3623). Activating gene rearrangements of ALK, such as EML4-ALK, have been identified as driver mutations in NSCLC and other cancers. There is strong precedence for the development of resistance to targeted therapies that inhibit driver mutations. Kinase domain mutations that confer resistance in patients have been successfully predicted by in vitro mutagenesis screens in BaF3 cells (e.g. BCR-ABL in CML). Here, the BaF3 system was used to identify mutations in ALK that confer resistance to PF1066, a clinically validated dual Met/ALK inhibitor (ASCO 2009; #3509), or AP26113. PF1066-resistant mutations were identified at all concentrations tested (up to 2000 nM). In contrast, 1000 nM AP26113 completely suppressed emergence of resistance. Six mutations, all in the kinase domain, were identified that confer some degree of resistance to 1 or both compounds (Table). AP26113 inhibited viability of BaF3 cells expressing these mutants with IC50s of 23 - 269 nM. PF1066 inhibited viability with IC50s of 311 -1419 nM, with 3 mutants having sensitivity indistinguishable from parental BaF3 cells, which lack EML4-ALK. The 2 mutations that confer the greatest resistance to PF1066 were examined in a BaF3 xenograft model in which compounds were administered daily by oral dosing. A 200 mg/kg dose of PF1066 induced regression of tumors expressing native EML4-ALK but was completely inactive against G1269S or L1196M (gatekeeper) mutants. In contrast, AP26113 induced regression of tumors expressing native EML4-ALK and the G1269S and L1196M mutants at 25, 50 and 50 mg/kg, respectively. Analysis of ALK phosphorylation in tumors demonstrated strong inhibition of the mutants by 50 mg/kg AP26113 but not 200 mg/kg PF1066. These results identify several mutations that may confer resistance to PF1066 in patients and suggest that more potent compounds such as AP26113 may be required to overcome such resistance.Sensitivity of BaF3 cells expressing native and mutant EML4-ALK to PF-02341066 and AP26113BaF3 cell linePF-02341066 IC50 ± SD AP26113 IC50 ± SD BaF3 cell line(nM)TI**(nM)TI**Parental*1116 ±436—2169 ±655—Native EML4-ALK153 ± 29714 ±2151EML4-ALK mutants:    F1174C497 ± 202104 ± 1921L1196M1157 ± 401166 ±733S1206R838 ± 2521269 ± 688E1210K311 ± 80488 ± 1225F1245C366 ±8373 ± 1130G1269S1419 ±618123 ± 594*Parental BaF3 cells lack EML4-ALK and therefore require IL-3 for growth**Therapeutic Index = Parental IC50 / EML4-ALK IC50

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-298.