Abstract 4685: Mechanistic guidance of ALK inhibition for the treatment of neuroblastoma

Receptor tyrosine kinases (RTKs) are important therapeutic targets in a wide variety of human diseases, especially neoplastic disorders. Several drugs that target the tyrosine kinase domains of the epidermal growth factor receptor (EGFR) or other tyrosine kinases are now in clinical use, and it has become clear that their use requires guidance from the results of genetic, biochemical and structural studies of their targets. A similar situation is emerging with another RTK-the anaplastic lymphoma kinase (ALK)-in neuroblastoma (NB), where activating mutations are therapeutic targets. In our study, inhibition of in vitro proliferation of NB cell lines was associated with abrogation of phosphorylation of ALK by PF-02341066, an orally bioavailable, ATP-competitive inhibitor of ALK and c-Met. Cell lines expressing F1174L were observed to be resistant to PF-02341066 compared to cells expressing R1275Q in vitro. Additionally, the inhibitor caused complete and sustained regression of xenografts harboring the R1275Q mutation which accounts for ∼50% of all mutations in human NB. However, the compound displayed modest tumor growth delay against xenografts harboring F1174L mutations. Little is known about the normal mechanism of activation of this kinase, and our data indicate that, under the proper conditions, classical trans-autophosphorylation of the activation loop in the recombinantly expressed tyrosine kinase domain (TKD) results in a marked (∼80 fold) increase in catalytic efficiency. Recent crystal structures of ALK-TKD have revealed, surprisingly, that autoinhibition is achieved by a mechanism similar to that of EGFR rather than other insulin receptor family members. However, ALK relieves this autoinhibition solely by autophosphorylation whereas EGFR depends on allosteric interactions between dimerized domains. Our kinetic data indicate that the R1275Q and F1174L mutations perturb autoinhibitory features of this kinase and activate the TKD to varying degrees, with the F1174L mutant displaying more dramatic dysregulation. Furthermore, F1174L displayed a lower Km, ATP value compared to R1275Q, directly analogous to the EGFR L858R/T790M resistant double mutant compared to EGFR harboring the L858R mutation alone. Additionally, F1174L was nearly identical to wild-type and R1275Q ALK in screens using a panel of commercially available inhibitors. This suggests that the reason for the relative resistance of F1174L-expressing cell lines to PF-02341066 is due primarily to stronger ATP affinity. This study provides preclinical rationale for the use of PF-02341066 in NB as well as important insight into how ALK is regulated in normal and disease states. To effectively target F1174L, an innate resistance mechanism for NB cells expressing it, as well as an acquired resistance mechanism for ALK-translocated tumors, we propose that next-generation ALK inhibitors should bind the ATP-pocket with higher affinity or irreversibly. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4685. doi:10.1158/1538-7445.AM2011-4685