Stephen X. Skapek

Correlation of terminal cell cycle arrest of skeletal muscle with induction of p21 by MyoD

Skeletal muscle differentiation entails the coordination of muscle-specific gene expression and terminal withdrawal from the cell cycle. This cell cycle arrest in the G0 phase requires the retinoblastoma tumor suppressor protein (Rb). The function of Rb is negatively regulated by cyclin-dependent kinases (Cdks), which are controlled by Cdk inhibitors. Expression of MyoD, a skeletal muscle-specific transcriptional regulator, activated the expression of the Cdk inhibitor p21 during differentiation of murine myocytes and in nonmyogenic cells. MyoD-mediated induction of p21 did not require the tumor suppressor protein p53 and correlated with cell cycle withdrawal. Thus, MyoD may induce terminal cell cycle arrest during skeletal muscle differentiation by increasing the expression of p21.

Inhibition of myogenic differentiation in proliferating myoblasts by cyclin D1-dependent kinase

Although the myogenic regulator MyoD is expressed in proliferating myoblasts, differentiation of these cells is limited to the G0 phase of the cell cycle. Forced expression of cyclin D1, but not cyclins A, B, or E, inhibited the ability of MyoD to transactivate muscle-specific genes and correlated with phosphorylation of MyoD. Transfection of myoblasts with cyclin-dependent kinase (Cdk) inhibitors p21 and p16 augmented muscle-specific gene expression in cells maintained in high concentrations of serum, suggesting that an active cyclin-Cdk complex suppresses MyoD function in proliferating cells.

Regulatory mechanisms that coordinate skeletal muscle differentiation and cell cycle withdrawal

Skeletal muscle differentiation entails the coupling of muscle-specific gene expression to terminal withdrawal from the cell cycle. Several models have recently been proposed which attempt to explain how regulated expression and function of myogenic transcription factors ensures that proliferation and differentiation of skeletal muscle cells are mutually exclusive processes.

Comprehensive Genomic Analysis of Rhabdomyosarcoma Reveals a Landscape of Alterations Affecting a Common Genetic Axis in Fusion-Positive and Fusion-Negative Tumors

UNLABELLED Despite gains in survival, outcomes for patients with metastatic or recurrent rhabdomyosarcoma remain dismal. In a collaboration between the National Cancer Institute, Childrens Oncology Group, and Broad Institute, we performed whole-genome, whole-exome, and transcriptome sequencing to characterize the landscape of somatic alterations in 147 tumor/normal pairs. Two genotypes are evident in rhabdomyosarcoma tumors: those characterized by the PAX3 or PAX7 fusion and those that lack these fusions but harbor mutations in key signaling pathways. The overall burden of somatic mutations in rhabdomyosarcoma is relatively low, especially in tumors that harbor a PAX3/7 gene fusion. In addition to previously reported mutations in NRAS, KRAS, HRAS, FGFR4, PIK3CA, and CTNNB1, we found novel recurrent mutations in FBXW7 and BCOR, providing potential new avenues for therapeutic intervention. Furthermore, alteration of the receptor tyrosine kinase/RAS/PIK3CA axis affects 93% of cases, providing a framework for genomics-directed therapies that might improve outcomes for patients with rhabdomyosarcoma. SIGNIFICANCE This is the most comprehensive genomic analysis of rhabdomyosarcoma to date. Despite a relatively low mutation rate, multiple genes were recurrently altered, including NRAS, KRAS, HRAS, FGFR4, PIK3CA, CTNNB1, FBXW7, and BCOR. In addition, a majority of rhabdomyosarcoma tumors alter the receptor tyrosine kinase/RAS/PIK3CA axis, providing an opportunity for genomics-guided intervention.

Arf tumor suppressor promoter monitors latent oncogenic signals in vivo.

Induction of the Arf tumor suppressor gene by elevated thresholds of mitogenic signals activates a p53-dependent transcriptional response that triggers either growth arrest or apoptosis, thereby countering abnormal cell proliferation. Conversely, Arf inactivation is associated with tumor development. Expression of Arf in tissues of adult mice is difficult to detect, possibly because its induction leads to the arrest or elimination of incipient tumor cells. We replaced coding sequences of exon 1β of the mouse cellular Arf gene with a cDNA encoding GFP, thereby producing Arf-null animals in which GFP expression is driven by the intact Arf promoter. The Arf promoter was induced in several biologic settings previously shown to elicit mouse p19Arf expression. Inactivation of Arf in this manner led to the outgrowth of tumor cells expressing GFP, thereby providing direct evidence that the Arf promoter monitors latent oncogenic signals in vivo.

Vinblastine and Methotrexate for Desmoid Fibromatosis in Children: Results of a Pediatric Oncology Group Phase II Trial

PURPOSE To determine the efficacy and safety of using vinblastine (Vbl) and methotrexate (Mtx) in children with desmoid-type fibromatosis that is recurrent or not amenable to treatment with radiation or surgery. PATIENTS AND METHODS A phase II study was conducted within the Pediatric Oncology Group. Patients were treated using Vbl (5 mg/m2/dose) and Mtx (30 mg/m2/dose), both administered by intravenous injection weekly for 26 weeks and every other week for an additional 26 weeks. Response was assessed by bidimensional measurements of tumor on axial imaging (magnetic resonance imaging or computed tomography). RESULTS Over 35 months, 28 patients were enrolled; 27 were eligible, and 26 were assessable for response. A measurable response was documented in eight patients (31%), and 10 patients had stable disease documented as the best response to treatment. Eighteen patients had disease progression at a median time of 9.1 months. Eight patients remain free of disease progression at a median of 43.4 months from study entry. Nine patients reported no to moderate toxicity. Neutropenia was the most common toxicity (n = 22) and the most common grade 4 toxicity (n = 5). Anemia, nausea, vomiting, and elevations in hepatic transaminases were also common and were reversible with interruption of chemotherapy. CONCLUSION Vbl and Mtx are well tolerated in children with desmoid-type fibromatosis. Furthermore, this combination can promote tumor regression or block tumor growth in most children.

Combination chemotherapy using vinblastine and methotrexate for the treatment of progressive desmoid tumor in children.

PURPOSE We report the treatment of 10 children for progressive desmoid tumor not amenable to standard surgical or radiation therapy with the use of vinblastine (VBL) and methotrexate (MTX). PATIENTS AND METHODS Ten patients aged 6.4 to 18 years with primary (two patients) or recurrent (eight patients) desmoid tumor were treated with VBL and MTX for 2 to 35 months. Patients with recurrent tumors had been previously treated with surgical resection with (two patients) or without (five patients) radiation therapy or with radiation therapy alone (one patient). No patient had previously received cytotoxic chemotherapy. The tumor response was assessed at routine intervals by physical examination and magnetic resonance imaging (MRI). RESULTS Five patients had clinical evidence of response to therapy with complete resolution (three patients) or partial resolution (two patients) of physical examination and radiographic abnormalities. Three patients had stable disease during 10 to 35 months of treatment. Two of these patients had progressive disease 9 and 37 months after treatment stopped; one patient had no progression 16 months after therapy. Two additional patients with stable disease had chemotherapy discontinued after 2 and 3 months. Common side effects included mild alopecia and myelosuppression and moderate nausea and vomiting. In patients with responding tumors, MRI showed decreased tumor size and, in two patients, changes consistent with fibrosis and decreased cellularity of the tumor. CONCLUSION Combination chemotherapy with VBL and MTX appears to control desmoid tumor without significant acute or long-term morbidity in most children. This may allow for further growth and development in these patients, which may decrease the morbidity of subsequent definitive therapy.

Cyclin-mediated inhibition of muscle gene expression via a mechanism that is independent of pRB hyperphosphorylation.

It was recently demonstrated that ectopic expression of cyclin D1 inhibits skeletal muscle differentiation and, conversely, that expression of cyclin-dependent kinase (cdk) inhibitors facilitates activation of this differentiation program (S. S. Rao, C. Chu, and D. S. Kohtz, Mol. Cell. Biol. 14:5259-5267, 1994; S. S. Rao and D. S. Kohtz, J. Biol. Chem. 270:4093-4100, 1995; S. X. Skapek, J. Rhee, D. B. Spicer, and A. B. Lassar, Science 267:1022-1024, 1995). Here we demonstrate that cyclin D1 inhibits muscle gene expression without affecting MyoD DNA binding activity. Ectopic expression of cyclin D1 inhibits muscle gene activation by both MyoD and myogenin, including a mutated form of myogenin in which two potential inhibitory cdk phosphorylation sites are absent. Because the retinoblastoma gene product, pRB, is a known target for cyclin D1-cdk phosphorylation, we determined whether cyclin D1-mediated inhibition of myogenesis was due to hyperphosphorylation of pRB. In pRB-deficient fibroblasts, the ability of MyoD to activate the expression of muscle-specific genes requires coexpression of ectopic pRB (B. G. Novitch, G. J. Mulligan, T. Jacks, and A. B. Lassar, J. Cell Biol., 135:441-456, 1996). In these cells, the expression of cyclins A and E can lead to pRB hyperphosphorylation and can inhibit muscle gene expression. The negative effects of cyclins A or E on muscle gene expression are, however, reversed by the presence of a mutated form of pRB which cannot be hyperphosphorylated. In contrast, cyclin D1 can inhibit muscle gene expression in the presence of the nonhyperphosphorylatable form of pRB. On the basis of these results we propose that G1 cyclin-cdk activity blocks the initiation of skeletal muscle differentiation by two distinct mechanisms: one that is dependent on pRB hyperphosphorylation and one that is independent of pRB hyperphosphorylation.

The Arf tumor suppressor gene promotes hyaloid vascular regression during mouse eye development

A key tumor suppressor mechanism that is disrupted frequently in human cancer involves the ARF and p53 genes. In mouse fibroblasts, the Arf gene product responds to abnormal mitogenic signals to activate p53 and trigger either cell cycle arrest or apoptosis. Recent evidence indicates that Arf also has p53-independent functions that may contribute to its tumor suppressor activity. Using Arf−/− and p53−/− mice, we have discovered a p53-independent requirement for Arf in the developmental regression of the hyaloid vascular system (HVS) in the mouse eye. Arf is expressed in the vitreous of the eye and is induced before HVS regression in the first postnatal week. In the absence of Arf, failed HVS regression causes a pathological process that resembles persistent hyperplastic primary vitreous, a developmental human eye disease thought to have a genetic basis. These findings demonstrate an essential and unexpected role for Arf during mouse eye development, provide insights into the potential genetic basis for persistent hyperplastic primary vitreous, and indicate that Arf regulates vascular regression in a p53-independent manner. The latter finding raises the possibility that Arf may function as a tumor suppressor at least in part by regulating tumor angiogenesis.

PAX-FOXO1 fusion status drives unfavorable outcome for children with rhabdomyosarcoma: a children's oncology group report.

Rhabdomyosarcoma (RMS) is divided into two major histological subtypes: alveolar (ARMS) and embryonal (ERMS), with most ARMS expressing one of two oncogenic genes fusing PAX3 or PAX7 with FOXO1 (P3F and P7F, respectively). The Childrens Oncology Group (COG) carried out a multi‐institutional clinical trial to evaluate the prognostic value of PAX‐FOXO1 fusion status.