Combination of HDAC and FYN inhibitors in synovial sarcoma treatment

Abstract

The SS18-SSX fusion protein is an oncogenic driver in synovial sarcoma. At the molecular level, SS18-SSX functions as both an activator and a repressor to coordinate transcription of different genes responsible for tumorigenesis. Here, we identify the proto-oncogene FYN as a new SS18-SSX target gene and examine its relation to synovial sarcoma therapy. FYN is a tyrosine kinase that promotes cancer growth, metastasis and therapeutic resistance, but SS18-SSX appears to negatively regulate FYN expression in synovial sarcoma cells. Using both genetic and histone deacetylase inhibitor (HDACi)-based pharmacologic approaches, we show that suppression of SS18-SSX leads to FYN reactivation. In support of this notion, we find that blockade of FYN activity synergistically enhances HDACi action to reduce synovial sarcoma cell proliferation and migration. Our results support a role for FYN in attenuation of anti-cancer activity upon inhibition of SS18-SSX function and demonstrate the feasibility of targeting FYN to improve the effectiveness of HDACi treatment against synovial sarcoma.

Keywords: SS18-SSX; drug synergism; fyn; histone deacetylase inhibitor (HDACi); synovial sarcoma.

FIGURE 1

Genomic analysis of FLAG-SS18-SSX2 in CRISPR-modified synovial sarcoma cells. (A) Enrichment of FLAG-SS18-SSX2 binding sites around the transcription start site (TSS) in CRISPR-modified (SSX2-FLAG) SYO-1 cells. (B) Examples of IGV views of FLAG ChIP-seq in parental and SSX2-FLAG cells. (C) Gene ontology (GO) analysis of biological processes for FLAG ChIP-seq data.

FIGURE 2

SS18-SSX2 represses FYN gene expression in synovial sarcoma cells. (A) Comparison of the SS18-SSX2 binding genes (detected by FLAG ChIP-seq) and the genes differentially expressed upon SS18-SSX2 knockdown (RNA-seq) in SYO-1 cells. (B) FLAG ChIP-qPCR analysis of SS18-SSX2 occupancy at the FYN gene locus in SYO-1 cells. (C) qPCR analysis of FYN mRNA levels in control and SS18-SSX2-knockdown SYO-1 cells. (D) FYN mRNA expression in soft tissue sarcoma samples from the TCGA database. SS, synovial sarcoma (n = 10); LMS, leiomyosarcoma (n = 99); DAF, desmoid/aggressive fibromatosis (n = 2); MPNST, malignant peripheral nerve sheath tumor (n = 9); UPS, undifferentiated pleomorphic sarcoma (n = 50); MFS, myxofibrosarcoma (n = 25); DDLPS, dedifferentiated liposarcoma (n = 58). ChIP-qPCR (B) and qPCR (C) data represent mean ± SD of three independent experiments. *p < 0.05; **p < 0.01; ****p < 0.0001 (two-tailed Student’s t-test).

FIGURE 3

Suppression of SS18-SSX2 by FK228 induces FYN expression. (A) FLAG ChIP-qPCR analysis of SS18-SSX2 occupancy at the FYN gene locus before and after FK228 treatment. (B) qPCR analysis of FYN mRNA levels in DMSO- and FK228-treated SYO-1 cells. (C) Western blot analysis of SS18-SSX2 (anti-FLAG) and FYN protein levels in DMSO- and FK228-treated cells. GAPDH serves as the loading control. Data represent mean ± SD of three independent experiments. *p < 0.05; ****p < 0.0001 (two-tailed Student’s t-test).

FIGURE 4

The FYN inhibitor PP2 synergistically enhances the efficacy of FK228 in synovial sarcoma cells. (A) 3D plot depicting a synergistic response between FK228 and PP2 in SYO-1 cells. (B) Changes in the migration of SYO-1 cells following exposure to either FK228 (0.01 μM) or PP2 (3 μM) alone or their combination. Migrated cells were stained with crystal violet (20× magnification). (C) Effects of FK228 and PP2 treatment alone or in combination on SYO-1 spheroid growth. Representative images of SYO-1 spheroids at day 7 in 3D culture (scale bars, 100 μm). (D) A model for synergy between HDAC and FYN inhibitors in synovial sarcoma treatment. Data represent mean ± SD of 3-4 independent experiments. *p < 0.05; **p < 0.01; ****p < 0.0001; ns, not significant (two-tailed Student’s t-test).