Functional impact and targetability of PI3KCA, GNAS, and PTEN mutations in a spindle cell rhabdomyosarcoma with MYOD1 L122R mutation

Abstract

Spindle cell/sclerosing rhabdomyosarcoma (ssRMS) is a rare subtype of rhabdomyosarcoma, commonly harboring a gain-of-function L122R mutation in the muscle-specific master transcription factor MYOD1. MYOD1-mutated ssRMS is almost invariably fatal, and development of novel therapeutic approaches based on the biology of the disease is urgently needed. MYOD1 L122R affects the DNA-binding domain and is believed to confer MYC-like properties to MYOD1, driving oncogenesis. Moreover, the majority of the MYOD1-mutated ssRMS harbor additional alterations activating the PI3K/AKT pathway. It is postulated that the PI3K/AKT pathway cooperates with MYOD1 L122R. To address this biological entity, we established and characterized a new patient-derived ssRMS cell line OHSU-SARC001, harboring MYOD1 L122R as well as alterations in PTEN, PIK3CA, and GNAS We explored the functional impact of these aberrations on oncogenic signaling with gain-of-function experiments in C2C12 murine muscle lineage cells. These data reveal that PIK3CA^I459_T462del, the novel PIK3CA variant discovered in this patient specimen, is a constitutively active kinase, albeit to a lesser extent than PI3KCA^E545K, a hotspot oncogenic mutation. Furthermore, we examined the effectiveness of molecularly targeted PI3K/AKT/mTOR and RAS/MAPK inhibitors to block oncogenic signaling and suppress the growth of OHSU-SARC001 cells. Dual PI3K/mTOR (LY3023414, bimiralisib) and AKT inhibitors (ipatasertib, afuresertib) induced dose-dependent reductions in cell growth. However, mTOR-selective inhibitors (everolimus, rapamycin) alone did not exert cytotoxic effects. The MEK1/2 inhibitor trametinib did not impact proliferation even at the highest doses tested. Our data suggest that molecularly targeted strategies may be effective in PI3K/AKT/mTOR-activated ssRMS. Taken together, these data highlight the importance of utilizing patient-derived models to assess molecularly targetable treatments and their potential as future treatment options.

Keywords: rhabdomyosarcoma.

Figure 1.

Patient presentation, clinical course, and pathologic evaluation. (A) Clinical course from initial presentation, refractory disease, and resection of the tumor. The patient underwent initial workup involving imaging, biopsy, and next-generation sequencing (NGS) via GeneTrails Comprehensive Solid Tumor Panel. He was treated on Children's Oncology Group (COG) ARST1431 until he had refractory disease. At the time of resection, the patient enrolled in the National Cancer Institute (NCI)-COG MATCH trial. (B) Initial diagnostic maxillofacial computed tomography (CT) identified a 2.3 × 4.1 × 3.0-cm enhancing soft-tissue mass (as noted with *) with bony remodeling without destructive changes. (C) Diagnostic biopsy showed intersecting spindle cells with elongated, tapered nuclei and prominent rhabdomyoblasts and strap cells on hematoxylin and eosin (H&E). (D) Desmin staining showed patchy cytoplasmic expression. (E) Myogenin exhibited punctate nuclear expression. (F) MYOD1 nuclear expression was strong and diffuse.

Figure 2.

In vitro, in vivo, and pathologic characterization of OHSU-SARC001. (A) Phase contrast imaging (10×) demonstrates spindle-shaped morphology and a focal collection of tumor cells. (B) Patient-derived xenograft (PDX) tumors were grown in NOD scid gamma (NSG) mice (P0). An increase in tumor volume (mm³) was initially measured starting at day 76 and was monitored over time until the tumor was transplanted at day 146. (C) PDX hematoxylin and eosin (H&E) staining revealed elongated spindle cells in fascicular arrangement. (D) Desmin was strongly diffuse. (E) Strong nuclear expression of mygoenin was present. (F) MYOD1 expression showed patchy nuclear expression.

Figure 3.

Crystal structure rendering of PIK3CA^I459_T462del and expression of mTOR/AKT and MAPK signaling pathways in transduced C2C12 cell lines, OHSU-SARC001, and after molecularly targeted kinase inhibitors. (A) PIK3CA^I459_T462del interfaces with the regulatory PIK3CA subunit and may disrupt the interaction between PI3K catalytic and regulatory subunits to induce catalytic activation. (B) C2C12 stable cell lines harboring wild-type and mutant cDNA suggest that PIK3CA^I459_T462del modestly activates pAkt^T308, pAkt^S473, pTsc^T1462, p70s6k^T389, pS6^S235/236, p4ebp-1^T37/46, and pEerk^T202/Y204 compared to WT PIK3CA. Of note, 4EBP1 undergoes sequential hyperphosphorylation on Thr37 and Thr46, followed by up to six residues in the carboxyl terminus. This increase in 4EBP1 phosphorylation decreases its electrophoretic mobility. GNAS^R201C does not appear to activate the mTOR/Akt or MAPK pathway compared to WT GNAS. (C) OHSU-SARC001 has PTEN loss and expresses RAP1B, B-Raf, and C-Raf. (D) In OHSU-SARC001, phosphorylation of downstream mTOR/AKT signal effectors p70S6K^T389 and pS6^S235/236 was decreased when exposed to LY3023414, everolimus, and rapamycin. Effectors p70S6K^T421/S424 were decreased in trametinib-treated cells. Trametinib treatment resulted in decreased expression of pERK^T202/Y204.

Figure 4.

Cell viability assays reveal cytotoxic effect of select molecularly targeted inhibitors in OHSU-SARC001 cells. (A–D) Dose–response cell viability assay data from the testing of (A) mTOR inhibitors, everolimus, and rapamycin, (B) dual PI3K/mTOR inhibitors, LY3023414 and bimiralisib, (C) pan-AKT inhibitors, ipatasertib and afuresertib, and (D) MEK1/2 inhibitor, trametinib. A nonlinear regression curve fit algorithm was unstable and for everolimus, R² = 0; rapamycin, R² = 0.16; and trametinib, R² = −0.22. Therefore, these data are not included. (E,F) Cell viability data from static dose testing of indicated inhibitors at 50 and 250 nM concentration, respectively. Statistical analysis with two-way ANOVA was performed using GraphPad Prism. Multiple comparison testing of dimethylsulfoxide (DMSO)-treated cells to indicated inhibitor reveals statistically significant suppression of cell growth as indicated by asterisks: ****, P < 0.0001; **, P < 0.01. (ns) Not significant.