The Influential Role of BCL2 Family Members in Synovial Sarcomagenesis

Abstract

Synovial sarcomas are deadly soft tissue malignancies associated with t(X;18) balanced chromosomal translocations. Expression of the apoptotic regulator BCL2 is prominent in synovial sarcomas and has prompted the hypothesis that synovial sarcomagenesis may depend on it. Herein, it is demonstrated that Bcl2 overexpression enhances synovial sarcomagenesis in an animal model. Furthermore, we determined increased familial clustering of human synovial sarcoma patients with victims of other BCL2-associated malignancies in the Utah Population Database. Conditional genetic disruption of Bcl2 in mice also led to reduced sarcomagenesis. Pharmacologic inhibition specific to BCL2 had no demonstrable efficacy against human synovial sarcoma cell lines or mouse tumors. However, targeting BCLxL in human and mouse synovial sarcoma with the small molecule BH3 domain inhibitor, BXI-72, achieved significant cytoreduction and increased apoptotic signaling. Thus, the contributory role of BCL2 in synovial sarcomagenesis does not appear to render it as a therapeutic target, but mitochondrial antiapoptotic BCL2 family members may be.Implications: The association of BCL2 expression with synovial sarcoma is found to fit with a subtle, but significant, impact of its enhanced presence or absence during early tumorigenesis. However, specific pharmacologic inhibition of BCL2 does not demonstrate a persistent dependence in fully developed tumors. Conversely, inhibition of the BCL2 family member BCLxL resulted in nanomolar potency against human synovial sarcoma cell lines and 50% tumor reduction in a genetically engineered mouse model. Mol Cancer Res; 15(12); 1733-40. ©2017 AACR.

Figure 1. Bcl2 overexpression in synovial sarcoma enhances sarcomagenesis

A. Schematic of alleles for SS18-SSX2 and Bcl2 overexpression with their respective recombination products (IRES, internal ribosomal entry site). B. Schematic of TATCre injection technique resulting in the combinatorial expression of hSS2 and Bcl2. C. Radiograph image of hSS2;Bcl2^OE mouse 8 months post TATCre injection in the right forearm and right hindlimb (black arrow). D. Photomicrographs of H&E histology examples of monophasic, biphasic, and poorly differentiated synovial sarcomas from hSS2;Bcl2^OE mice induced with TATCre. E. Schematic of Myf5Cre+ distribution in E11.5 embryo resulting in the expression of hSS2 or hSS2;Bcl2^OE during embryogenesis. F. Kaplan-Meier plot of the nonmorbid fraction of hSS2;Bcl2^OE (gray) and hSS2;Bcl2^WT (blue) induced in Myf5Cre+ mice. Statistical difference (Log-rank test) between hSS2;Bcl2^OE and hSS2;Bcl2^WT, p value = 0.023. G. Photomicrographs of H&E histology examples of monophasic, biphasic, and poorly differentiated synovial sarcomas from hSS2;Bcl2^WT and hSS2;Bcl2^OE, mice induced in Myf5Cre+ cells. H. Fraction of monophasic, biphasic, and poorly differentiated histologies in Myf5Cre;hSS2;Bcl2^WT and Myf5Cre;hSS2;Bcl2^OE, mice. (All magnification bars = 10 µm)

Figure 2. Bcl2 conditional deletion in synovial sarcoma mildly disrupts sarcomagenesis

A. Schematic of alleles for Bcl2^fl/fl with its respective recombination product. Also demonstrating the location of primers for detection of the recombined allele (dPCR) and the primers for normalization (nPCR). B. Schematic of Myf5Cre+ distribution in E11.5 embryo resulting in the expression of hSS2 or hSS2;Bcl2^fl/fl during embryogenesis. C. Kaplan-Meier plot of the nonmorbid fraction of hSS2;Bcl2^fl/fl (pink), hSS2;Bcl2^fl/wt (purple) and hSS2;Bcl2^WT (blue) induced in Myf5Cre+ mice. Statistical difference (Log-rank test) between hSS2;Bcl2^fl/fl and hSS2;Bcl2^WT, p value = 0.03. D. Graph depicting the average number of tumors per mouse between hSS2;Bcl2^fl/fl and hSS2;Bcl2^WT, genotypes (p value = 0.34). E. PCR detection of the recombined Bcl2^fl/fl allele compared to wildtype. F. Quantification of the recombined Bcl2^fl/fl allele normalized to adjacent primers, demonstrating that not all Bcl2^fl/fl tumors recombined to delete Bcl2. G. Photomicrographs of H&E histology examples of monophasic, biphasic, and poorly differentiated synovial sarcomas from hSS2;Bcl2^WThSS2;Bcl2^fl/wt and hSS2;Bcl2^fl/fl mice induced in Myf5Cre+ cells. H. Fraction of monophasic, biphasic, and poorly differentiated histologies in Myf5Cre;hSS2;Bcl2^WT and Myf5Cre;hSS2;Bcl2^fl/fl mice. I. Immunoblots detecting the absence of BCL2 in fully recombined tumors following TATCre injection from hSS2;Bcl2^fl/fl mice compared to tumors from hSS2;Bcl2^WT mice. (All magnification bars = 10 µm)

Figure 3. Targeting BCL2 and BCLxL in synovial sarcoma

A. Cell viability curves for ABT-199, BXI-72 and doxorubicin in SYO-1 (blue), SS1A (red) and FUJI (green) after 48 hours of treatment. B. IC₅₀ data for ABT-199, BXI-72 and doxorubicin in SYO-1, SS1A and doxorubicin (± SEM). C. Mac Synergy plot between doxorubicin and ABT-199 demonstrating minimal synergy (< 50% synergy) after 72 hours of treatment. D. FDG-PET/CT image of Pten^fl/fl;hSS2 mice treated with control, ABT-199 or doxorubicin. E. Graph of percent change in total lesion glycolysis (TLG) for synovial sarcomas treated with control, ABT-199 or doxorubicin. F. Immunoblotting for BCLxL across five human synovial sarcoma cell lines with GAPDH as a loading control. G. Graph depicting percent volume changes after 7 days of 20 mg/kg BXI-72 in Pten^fl/fl;hSS2 mice induced with local injection of TATCre and example gross photographs of Pten^fl/fl;hSS2 mice with synovial sarcomas on the left hindlimb treated with control DMSO or 20 mg/kg BXI-72. H. Graph depicting percent necrosis in synovial sarcomas treated with control DMSO or BXI-72. Example H&E photomicrographs of necrosis identified in tumors treated with control DMSO or BXI-72: top left is viable tumor from control; top right is replacement of tumor with fibrosis in BXI-72 treated mice; bottom left is necrosis by ghost cells (anucleate); bottom right is necrosis/apoptosis by fragmentation. I. Immunoblotting for BID (pro-apoptosis), BCLxL and tubulin in extracted synovial sarcomas treated with DMSO or BXI-72. (All magnification bars = 10 µm)