Alveolar rhabdomyosarcoma: morphoproteomics and personalized tumor graft testing further define the biology of PAX3-FKHR(FOXO1) subtype and provide targeted therapeutic options

Abstract

Alveolar rhabdomyosarcoma (ARMS) represents a block in differentiation of malignant myoblasts. Genomic events implicated in the pathogenesis of ARMS involve PAX3-FKHR (FOXO1) or PAX7-FKHR (FOXO1) translocation with corresponding fusion transcripts and fusion proteins. Commonalities in ARMS include uncontrollable proliferation and failure to differentiate. The genomic-molecular correlates contributing to the etiopathogenesis of ARMS incorporate PAX3-FKHR (FOXO1) fusion protein stimulation of the IGF-1R, c-Met and GSK3-β pathways. With sequential morphoproteomic profiling on such a case in conjunction with personalized tumor graft testing, we provide an expanded definition of the biology of PAX3-FKHR (FOXO1) ARMS that integrates genomics, proteomics and pharmacogenomics. Moreover, therapies that target the genomic and molecular biology and lead to tumoral regression and/or tumoral growth inhibition in a xenograft model of ARMS are identified.

Significance: This case study could serve as a model for clinical trials using relatively low toxicity agents in both initial and maintenance therapies to induce remission and reduce the risk of recurrent disease in PAX3-FKHR (FOXO1) subtype of ARMS.

Keywords: PAX3-FKHR subtype; alveolar rhabdomyosarcoma; morphoproteomics; targeted therapy; xenograft testing.

Figure 1. The patient's ARMS, PAX3-FKHR subtype with: H&E stained section showing dedifferentiated tumor cells

A. high expression of insulin-like growth factor (IGF)-1 receptor [Tyr1165/1166] in the cytoplasm of the tumor cells B. activation of c-Met tyrosine kinase as evidenced by the moderate expression in the cytoplasm of phosphorylated (p)-c-Met (Tyr1234/1235) at up to 2+ C. constitutive activation of the mTORC2/Akt pathway with concomitant nuclear expression of p-mTOR (Ser 2448) and p-Akt (Ser473) consistent with mTORC2 pathway activation D. and E. and downstream signaling from IGF-1R. Contrast with negative control F. (DAB[3,3′-diaminobenzidine] brown chromogenic signal; original magnifications x400 for Frames A-F).

Figure 2. Dedifferentiated tumor cells in the patient's ARMS, PAX3-FKHR subtype showing correlative expression in tumoral nuclei of: Sirt1

A. Gli2 B. and EZH2 C. and D. DAB brown chromogenic signal, original magnifications x400 for Frames A, B and D and x100 for Frame C).

Figure 3. Relative growth of treatment groups at test completion

Note tumor regression with entinostat and tumor growth inhibition with valproic acid, a class I histone deacetylase inhibitor, celecoxib and metformin.

Figure 4. Tumor volume and agent activity data plotted against test days 0 to 18

Note tumor regression over time with entinostat and tumor growth inhibition over time with valproic acid, a class I histone deacetylase inhibitor, celecoxib and metformin vis-à-vis the control.

Figure 5. A schematic depicting the biology of PAX3-FKHR (FOXO1) subtype of ARMS that integrates genomics and proteomics in a pathogenetic sequence, resulting in a dedifferentiated state and promoting proliferation (see )

Moreover, therapies that can target the genomics and molecular biology and take advantage of pharmacogenomics to relieve the block in differentiation to a more benign form and result in reduced proliferation are also illustrated in the schematic (see ) and covered in detail in the Discussion. A proof of concept is provided in part by the results of xenograft testing of the patient's ARMS, PAX3-FKHR (FOXO1) subtype with the application of entinostat showing tumor regression and a combination of valproic acid, a class I histone deacetylase inhibitor, celecoxib and metformin showing tumor growth inhibition (see Figures 3 and 4, Table 1 and Discussion).