Abstract 1633: Chemical proteomics identifies druggable proteins in ALK-driven neuroblastomas
Authors: Smita Matkar, Renata Sano, Colleen Larmour, Kateryna Krytska, Gabriela M. Witek, Mark Gerelus, Tim J. Stuhlmiller, Gary L. Johnson, Yael P. Mosse
Journal: Cancer Research (2018)

Abstract The discovery of mutations in the ALK oncogene as the genetic etiology of familial neuroblastoma (NB) and as somatically acquired in 14% of patients with the most aggressive form of the disease has positioned ALK as the major tractable oncogene product for targeted therapy in NB. ALK tyrosine kinase domain mutations are found mostly at three hotspots (F1174, F1245 and R1275), with the F1174* and F1245* mutations conferring broad resistance to crizotinib. We have recently demonstrated that lorlatinib, a novel ATP-competitive macrocyclic ALK inhibitor, overcomes de novo resistance and exerts unprecedented activity as a single agent in patient-derived xenografts (PDXs) harboring F1174L or F1245C mutations, while also inducing durable responses in R1275Q xenografts. The objective of our work was to identify differences in reprogramming of the kinome upon ALK inhibition with crizotinib and lorlatinib. We hypothesize that identification of the targets through which lorlatinib mediates its superior anti-tumor effects will provide insights into the mechanisms by which full-length ALK inhibition abrogates tumorigenesis, activates compensatory pathways, and uncover novel targets to overcome resistance to ALK inhibition. We used Multiplexed Inhibitor Beads coupled with Mass Spectrometry (MIB/MS) to quantitatively measure kinase activity dynamics on a proteomic scale. Three PDX models harboring F1174L (COG-N-453x) or the other two most-common mutations, R1275Q (NB1643) and R1245C (Felix) were treated with either crizotinib (100 mg/kg/day) or lorlatinib (10 mg/kg/day) for 2.5 days or 6.5 days prior to MIB/MS analysis. Lorlatinib more potently inhibited ALK and preferentially inhibited a series of other tyrosine kinases, correlating with the superior efficacy of lorlatinib in pre-clinical studies. Moreover, a number of kinases involved in G2/M cell cycle transition including PLK1, CHEK1 and aurora kinases were significantly inhibited by lorlatinib, proposing that this multi-kinase inhibition is responsible for the robust anti-tumor activity observed in vivo with lorlatinib. We interrogated the role of SHP2, a ubiquitously expressed SH2 domain-containing tyrosine phosphatase recently implicated in the proliferation of receptor-tyrosine kinase driven cancers and shown to have a direct role in the G2/M checkpoint. Inhibition of ALK by treatment with lorlatinib as well as shRNA knockdown of ALK in SY5Y (ALK F1174L, PTPN11 T507K) and NB1643 NB cell lines showed decreased levels of pSHP2. Depletion of SHP2 using CRISPR constructs significantly inhibited cell proliferation in SY5Y, suggesting that SHP2 plays a crucial role in ALK driven NB. Additionally, knockdown of ALK in SY5Y downregulated PLK1 expression and arrested cells in G2/M phase. Whether SHP2 is regulated primarily downstream of ALK or jointly through alternative or compensatory signaling networks warrants further investigation. Citation Format: Smita Matkar, Renata Sano, Colleen Larmour, Kateryna Krytska, Gabriela M. Witek, Mark Gerelus, Tim J. Stuhlmiller, Gary L. Johnson, Yael P. Mosse. Chemical proteomics identifies druggable proteins in ALK-driven neuroblastomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1633.