Phosphoproteomic Profiling Reveals ALK and MET as Novel Actionable Targets across Synovial Sarcoma Subtypes

Abstract

Despite intensive multimodal treatment of sarcomas, a heterogeneous group of malignant tumors arising from connective tissue, survival remains poor. Candidate-based targeted treatments have demonstrated limited clinical success, urging an unbiased and comprehensive analysis of oncogenic signaling networks to reveal therapeutic targets and personalized treatment strategies. Here we applied mass spectrometry-based phosphoproteomic profiling to the largest and most heterogeneous set of sarcoma cell lines characterized to date and identified novel tyrosine phosphorylation patterns, enhanced tyrosine kinases in specific subtypes, and potential driver kinases. ALK was identified as a novel driver in the Aska-SS synovial sarcoma (SS) cell line via expression of an ALK variant with a large extracellular domain deletion (ALK D2-17 ). Functional ALK dependency was confirmed in vitro and in vivo with selective inhibitors. Importantly, ALK immunopositivity was detected in 6 of 43 (14%) of SS patient specimens, one of which exhibited an ALK rearrangement. High PDGFRa phosphorylation also characterized SS cell lines, which was accompanied by enhanced MET activation in Yamato-SS cells. Although Yamato-SS cells were sensitive to crizotinib (ALK/MET-inhibitor) but not pazopanib (VEGFR/PDGFR-inhibitor) monotherapy in vitro, synergistic effects were observed upon drug combination. In vivo, both drugs were individually effective, with pazopanib efficacy likely attributable to reduced angiogenesis. MET or PDGFRa expression was detected in 58% and 84% of SS patients, respectively, with coexpression in 56%. Consequently, our integrated approach has led to the identification of ALK and MET as promising therapeutic targets in SS.

Targeted Therapy–based Combination Treatment in Rhabdomyosarcoma

“…In ARMS, the characteristic PAX3-FOXO1 protein can enhance IGF1R and ALK transcription, possibly explaining this difference in sensitivity. However, we, among others, could not find intrinsic ALK activity in rhabdomyosarcoma cells (7)(8)(9). In addition, the antitumor effects observed with the ALK inhibitor ceritinib were mostly explained by its capacity to inhibit IGF1R signaling (9).…”

“…Although the majority of patients with rhabdomyosarcoma have a low mutational burden, a recent report did reveal a number of activating mutations and epigenetic alterations that could have an effect on pathway activity (28). Also a phosphoproteomics screen in rhabdomyosarcoma cell lines, and a combined genomic and morphoproteomic screening of an ARMS sample identified and validated adequate treatment strategies (7,81). This exemplifies that molecular genomics, epigenomics, and (phospho)proteomics might have a place in rhabdomyosarcoma diagnosis to provide patients with the best, most personalized treatment available.…”

“…Amplification is more likely to occur in ARMS as a result of the PAX3-FOXO1 fusion protein regulating FGFR4 expression. Indeed, higher activated FGFR4 expression levels were observed in ARMS as compared with ERMS cell lines (7). In contrast, whole-exome/transcriptome sequencing revealed activated FGFR4 mutations in 4 of 60 patients with rhabdomyosarcoma, all of which were ERMS (28).…”

Preclinical Evaluation of Nintedanib, a Triple Angiokinase Inhibitor, in Soft-tissue Sarcoma: Potential Therapeutic Implication for Synovial Sarcoma

“…Recent evidence has suggested involvement of RTKs such as PDGFR (8,34), c-Met (11) as well as IGF1-R (35), and ErbB4 (9) among others in various sarcoma subtypes. Phosphoproteomic analysis using a mass spectrometry approach has further revealed that RTKs such as PDGFR, IGF-1R, c-Met, and ALK play a role in driving sarcoma development and progression (6,7). Previous studies from our laboratory (8) and others (20) have also elucidated the role of PDGFR and FGFR signaling pathways in driving synovial sarcoma proliferation.…”

“…Receptor tyrosine kinases (RTK) act as key mediators of signal transduction pathways that regulate cell proliferation, survival, and migration (5). Role of RTKs such as IGF-1R, PDGFR, c-Met, AXL, and FGFR as well as nonreceptor tyrosine kinases (non-RTK) such as FAK and Src acting as tumor drivers has been reported in various sarcoma subtypes over the past few years (6)(7)(8)(9)(10)(11). However, the translation of these preclinical studies into effective molecularly targeted therapies has met with only modest clinical success (12,13).…”