Abstract LB-298: AP26113, a potent ALK inhibitor, overcomes mutations in EML4-ALK that confer resistance to PF-02341066 (PF1066)

Abstract

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.

Treatment Efficacy and Resistance Mechanisms Using the Second-Generation ALK Inhibitor AP26113 in Human NPM-ALK–Positive Anaplastic Large Cell Lymphoma

“…A tyrosine substitution was found in an NSCLC patient that relapsed after Crizotinib treatment, whereas substitutions with an arginine, an isoleucine, or a glycine were predicted in vitro as resistant to AP26113. Notably, the S1206C was the only mutation detected at AP26113 500 nmol/L, indicating that this residue has a key role in conferring high AP26113 resistance (34). All data obtained by 3 H thymidine incorporation test were validated by Western blot (Figs.…”

“…The gatekeeper L1196M was one of the first causes of Crizotinib resistance found in patients. A moderate AP26113 resistance was predicted in vitro (29,34,35); however, the IC 50 value is low enough to consider this mutation sensitive to AP26113. Because in SUP-M2AR500B cells both L1122V and L1196M were found, we hypothesize that the first is the mutation driving resistance, while the latter was selected at low doses and never counterselected, remaining as a passenger in the high-dose-resistant clone.…”

“…Mutations involving F1174 were recognized as activating in neuroblastoma and phenylalanine substitution with a leucine was found in an IMT patient that relapsed after Crizotinib treatment (24). Clones carrying cysteine, valine, or isoleucine in residue 1174 instead of phenylalanine were selected at AP26113 100 nmol/L and 200 nmol/L in a previous Ba/F3 screen (34). In our screening, Ba/F3 cells bearing the single mutation are completely sensitive to all drugs studied, including AP26113.…”

Tackling ALK in non-small cell lung cancer: the role of novel inhibitors

“…Brigatinib is a tyrosine kinase inhibitor that showed preclinical activity against ALK and 9 clinically-identified crizotinib-resistant mutants (86,87). Moreover, this compound exerts in vitro activity against both ALK and EGFR (88), making it a suitable therapeutic option for patients progressing to crizotinib with activation of EGFR pathway as the mechanism of resistance (14,15,66).…”

“…With respect to the possibility to use second-generation ALK inhibitors, like ceritinib and alectinib, in crizotinibnaïve patients, it can be argued that these novel agents generally show more than 10-fold higher potency against the ALK-rearranged protein with a wild-type ALK kinase domain (66,84,86,93) and display increased affinity for second-site mutated ALK. Potentially, second-generation ALK inhibitors, compared with crizotinib, may offer an improved duration of response with a delay in the onset of ALK resistance and a treatment option available for secondary mutation-dependent resistance.…”