Preclinical therapeutics ex ovo quail eggs as a biomimetic automation-ready xenograft platform.

Preclinical cancer research ranges from in vitro studies that are inexpensive and not necessarily reflective of the tumor microenvironment to mouse studies that are better models but prohibitively expensive at scale. Chorioallantoic membrane (CAM) assays utilizing Japanese quail (Coturnix japonica) are a cost-effective screening method to precede and minimize the scope of murine studies for anti-cancer efficacy and drug toxicity. To increase the throughput of CAM assays we have built and optimized an 11-day platform for processing up to 200 quail eggs per screening to evaluate drug efficacy and drug toxicity caused by a therapeutic. We demonstrate ex ovo concordance with murine in vivo studies, even when the in vitro and in vivo studies diverge, suggesting a role for this quail shell-free CAM xenograft assay in the validation of new anti-cancer agents.

Defining the Extracellular Matrix of Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood with a propensity to metastasize. Current treatment for patients with RMS includes conventional systemic chemotherapy, radiation therapy, and surgical resection; nevertheless, little to no improvement in long term survival has been achieved in decades-underlining the need for target discovery and new therapeutic approaches to targeting tumor cells or the tumor microenvironment. To evaluate cross-species sarcoma extracellular matrix production, we have used murine models which feature knowledge of the myogenic cell-of-origin. With focus on the RMS/undifferentiated pleomorphic sarcoma (UPS) continuum, we have constructed tissue microarrays of 48 murine and four human sarcomas to analyze expression of seven different collagens, fibrillins, and collagen-modifying proteins, with cross-correlation to RNA deep sequencing. We have uncovered that RMS produces increased expression of type XVIII collagen alpha 1 (COL18A1), which is clinically associated with decreased long-term survival. We have also identified significantly increased RNA expression of COL4A1, FBN2, PLOD1, and PLOD2 in human RMS relative to normal skeletal muscle. These results complement recent studies investigating whether soft tissue sarcomas utilize collagens, fibrillins, and collagen-modifying enzymes to alter the structural integrity of surrounding host extracellular matrix/collagen quaternary structure resulting in improved ability to improve the ability to invade regionally and metastasize, for which therapeutic targeting is possible.

Refractory alveolar rhabdomyosarcoma in an 11-year-old male.

Rhabdomyosarcoma (RMS) is a mesenchymal malignancy phenocopying muscle and is among the leading causes of death from childhood cancer. Metastatic alveolar rhabdomyosarcoma is the most aggressive subtype with an 8% 5-yr disease-free survival rate when a chromosomal fusion is present and a 29% 5-yr disease-free survival rate when negative for a fusion event. The underlying biology of PAX-fusion-negative alveolar rhabdomyosarcoma remains largely unexplored and is exceedingly rare in Li-Fraumeni syndrome patients. Here, we present the case of an 11-yr-old male with fusion-negative alveolar rhabdomyosarcoma studied at end of life with a comprehensive functional genomics characterization, resulting in identification of potential therapeutic targets for broader investigation.

Probabilistic modeling of personalized drug combinations from integrated chemical screen and molecular data in sarcoma.

BACKGROUND: Cancer patients with advanced disease routinely exhaust available clinical regimens and lack actionable genomic medicine results, leaving a large patient population without effective treatments options when their disease inevitably progresses. To address the unmet clinical need for evidence-based therapy assignment when standard clinical approaches have failed, we have developed a probabilistic computational modeling approach which integrates molecular sequencing data with functional assay data to develop patient-specific combination cancer treatments. METHODS: Tissue taken from a murine model of alveolar rhabdomyosarcoma was used to perform single agent drug screening and DNA/RNA sequencing experiments; results integrated via our computational modeling approach identified a synergistic personalized two-drug combination. Cells derived from the primary murine tumor were allografted into mouse models and used to validate the personalized two-drug combination. Computational modeling of single agent drug screening and RNA sequencing of multiple heterogenous sites from a single patient's epithelioid sarcoma identified a personalized two-drug combination effective across all tumor regions. The heterogeneity-consensus combination was validated in a xenograft model derived from the patient's primary tumor. Cell cultures derived from human and canine undifferentiated pleomorphic sarcoma were assayed by drug screen; computational modeling identified a resistance-abrogating two-drug combination common to both cell cultures. This combination was validated in vitro via a cell regrowth assay. RESULTS: Our computational modeling approach addresses three major challenges in personalized cancer therapy: synergistic drug combination predictions (validated in vitro and in vivo in a genetically engineered murine cancer model), identification of unifying therapeutic targets to overcome intra-tumor heterogeneity (validated in vivo in a human cancer xenograft), and mitigation of cancer cell resistance and rewiring mechanisms (validated in vitro in a human and canine cancer model). CONCLUSIONS: These proof-of-concept studies support the use of an integrative functional approach to personalized combination therapy prediction for the population of high-risk cancer patients lacking viable clinical options and without actionable DNA sequencing-based therapy.

Preclinical rationale for entinostat in embryonal rhabdomyosarcoma.

BACKGROUND: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in the pediatric cancer population. Survival among metastatic RMS patients has remained dismal yet unimproved for years. We previously identified the class I-specific histone deacetylase inhibitor, entinostat (ENT), as a pharmacological agent that transcriptionally suppresses the PAX3:FOXO1 tumor-initiating fusion gene found in alveolar rhabdomyosarcoma (aRMS), and we further investigated the mechanism by which ENT suppresses PAX3:FOXO1 oncogene and demonstrated the preclinical efficacy of ENT in RMS orthotopic allograft and patient-derived xenograft (PDX) models. In this study, we investigated whether ENT also has antitumor activity in fusion-negative eRMS orthotopic allografts and PDX models either as a single agent or in combination with vincristine (VCR). METHODS: We tested the efficacy of ENT and VCR as single agents and in combination in orthotopic allograft and PDX mouse models of eRMS. We then performed CRISPR screening to identify which HDAC among the class I HDACs is responsible for tumor growth inhibition in eRMS. To analyze whether ENT treatment as a single agent or in combination with VCR induces myogenic differentiation, we performed hematoxylin and eosin (H&E) staining in tumors. RESULTS: ENT in combination with the chemotherapy VCR has synergistic antitumor activity in a subset of fusion-negative eRMS in orthotopic "allografts," although PDX mouse models were too hypersensitive to the VCR dose used to detect synergy. Mechanistic studies involving CRISPR suggest that HDAC3 inhibition is the primary mechanism of cell-autonomous cytoreduction in eRMS. Following cytoreduction in vivo, residual tumor cells in the allograft models treated with chemotherapy undergo a dramatic, entinostat-induced (70-100%) conversion to non-proliferative rhabdomyoblasts. CONCLUSION: Our results suggest that the targeting class I HDACs may provide a therapeutic benefit for selected patients with eRMS. ENT's preclinical in vivo efficacy makes ENT a rational drug candidate in a phase II clinical trial for eRMS.

The HDAC3-SMARCA4-miR-27a axis promotes expression of the PAX3:FOXO1 fusion oncogene in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood with an unmet clinical need for decades. A single oncogenic fusion gene is associated with treatment resistance and a 40 to 45% decrease in overall survival. We previously showed that expression of this PAX3:FOXO1 fusion oncogene in alveolar RMS (aRMS) mediates tolerance to chemotherapy and radiotherapy and that the class I-specific histone deacetylase (HDAC) inhibitor entinostat reduces PAX3:FOXO1 protein abundance. Here, we established the antitumor efficacy of entinostat with chemotherapy in various preclinical cell and mouse models and found that HDAC3 inhibition was the primary mechanism of entinostat-induced suppression of PAX3:FOXO1 abundance. HDAC3 inhibition by entinostat decreased the activity of the chromatin remodeling enzyme SMARCA4, which, in turn, derepressed the microRNA miR-27a. This reexpression of miR-27a led to PAX3:FOXO1 mRNA destabilization and chemotherapy sensitization in aRMS cells in culture and in vivo. Furthermore, a phase 1 clinical trial (ADVL1513) has shown that entinostat is tolerable in children with relapsed or refractory solid tumors and is planned for phase 1B cohort expansion or phase 2 clinical trials. Together, these results implicate an HDAC3-SMARCA4-miR-27a-PAX3:FOXO1 circuit as a driver of chemoresistant aRMS and suggest that targeting this pathway with entinostat may be therapeutically effective in patients.

Epirubicin and Ifosfamide with Preoperative Radiation for High-Risk Soft Tissue Sarcomas.

BACKGROUND: The optimal treatment of high-risk soft tissue sarcomas (STS) of the extremities remains controversial. We report follow-up from a phase II study of dose-intense chemotherapy with preoperative hypofractionated radiation in this population supplemented with subsequent data from an extensive institutional experience using this regimen. METHODS: Patients with localized, intermediate- or high-grade STS of the extremity or body wall measuring > 5 cm were treated with epirubicin 30 mg/m2/day and ifosfamide 2.5 g/m2/day on days 1-4 every 21 days for 3 preoperative and 3 postoperative cycles. During cycle 2 of preoperative therapy, epirubicin was omitted, and a total of 28 Gy of radiation (8 fractions) was delivered. Twenty-five patients were treated on the phase II study (2002-2005). Fifty-one additional patients were identified from a retrospective chart review (2005-2014). RESULTS: The 5-year rates for overall survival, distant disease-free survival, and freedom from local regional failure were 70.4% (95% CI 59.2-83.7%), 55.9% (95% CI 44.5-70.2%), and 87.2% (95% CI 77.9-96.5%) respectively. Thirty-eight percent of tumors (29/76) demonstrated ≥ 90% pathologic response. Wound complications occurred in 32% (24/76) of patients. DISCUSSION: Treatment with preoperative radiation and pre- and post-operative epirubicin and ifosfamide was associated with favorable clinical outcomes. Survival and recurrence rates were comparable to those reported with other preoperative chemotherapy regimens in high-risk extremity sarcomas. Use of trimodality therapy should be considered for appropriate high-risk STS patients.

Integration of genomic, transcriptomic and functional profiles of aggressive osteosarcomas across multiple species.

In complex, highly unstable genomes such as in osteosarcoma, targeting aberrant checkpoint processes (metabolic, cell cycle or immune) may prove more successful than targeting specific kinase or growth factor signaling pathways. Here, we establish a comparative oncology approach characterizing the most lethal osteosarcomas identified in a biorepository of tumors from three different species: human, mouse and canine. We describe the development of a genetically-engineered mouse model of osteosarcoma, establishment of primary cell cultures from fatal human tumors, and a biorepository of osteosarcoma surgical specimens from pet dogs. We analyzed the DNA mutations, differential RNA expression and in vitro drug sensitivity from two phenotypically-distinct cohorts: tumors with a highly aggressive biology resulting in death from rapidly progressive, refractory metastatic disease, and tumors with a non-aggressive, curable phenotype. We identified ARK5 (AMPK-Related Protein Kinase 5, also referred to as NUAK Family Kinase 1) as a novel metabolic target present in all species, and independent analyses confirmed glucose metabolism as the most significantly aberrant cellular signaling pathway in a model system for highly metastatic tumors. Pathway integration analysis identified Polo Like Kinase 1 (PLK1)-mediated checkpoint adaptation as critical to the survival of a distinctly aggressive osteosarcoma. The tumor-associated macrophage cytokine CCL18 (C-C Motif Chemokine Ligand 18) was significantly over-expressed in aggressive human osteosarcomas, and a clustering of mutations in the BAGE (B Melanoma Antigen) tumor antigen gene family was found. The theme of these features of high risk osteosarcoma is checkpoint adaptations, which may prove both prognostic and targetable.

Preclinical testing of the glycogen synthase kinase-3ß inhibitor tideglusib for rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma. RMS often arise from myogenic precursors and displays a poorly differentiated skeletal muscle phenotype most closely resembling regenerating muscle. GSK3ß is a ubiquitously expressed serine-threonine kinase capable of repressing the terminal myogenic differentiation program in cardiac and skeletal muscle. Recent unbiased chemical screening efforts have prioritized GSK3ß inhibitors as inducers of myodifferentiation in RMS, suggesting efficacy as single agents in suppressing growth and promoting self-renewal in zebrafish transgenic embryonal RMS (eRMS) models in vivo. In this study, we tested the irreversible GSK3ß-inhibitor, tideglusib for in vivo efficacy in patient-derived xenograft models of both alveolar rhabdomyosarcoma (aRMS) and eRMS. Tideglusib had effective on-target pharmacodynamic efficacy, but as a single agent had no effect on tumor progression or myodifferentiation. These results suggest that as monotherapy, GSK3ß inhibitors may not be a viable treatment for aRMS or eRMS.

NFκB signaling in alveolar rhabdomyosarcoma.

Alveolar rhabdomyosarcoma (aRMS) is a pediatric soft tissue cancer commonly associated with a chromosomal translocation that leads to the expression of a Pax3:Foxo1 or Pax7:Foxo1 fusion protein, the developmental underpinnings of which may give clues to its therapeutic approaches. In aRMS, the NFκB-YY1-miR-29 regulatory circuit is dysregulated, resulting in repression of miR-29 and loss of the associated tumor suppressor activity. To further elucidate the role of NFκB in aRMS, we first tested 55 unique sarcoma cell lines and primary cell cultures in a large-scale chemical screen targeting diverse molecular pathways. We found that pharmacological inhibition of NFκB activity resulted in decreased cell proliferation of many of the aRMS tumor cultures. Surprisingly, mice that were orthotopically allografted with aRMS tumor cells exhibited no difference in tumor growth when administered an NFκB inhibitor, compared to control. Furthermore, inhibition of NFκB by genetically ablating its activating kinase inhibitor, IKKß, by conditional deletion in a mouse model harboring the Pax3:Foxo1 chimeric oncogene failed to abrogate spontaneous tumor growth. Genetically engineered mice with conditionally deleted IKKß exhibited a paradoxical decrease in tumor latency compared with those with active NFκB. However, using a synthetic-lethal approach, primary cell cultures derived from tumors with inactivated NFκB showed sensitivity to the BCL-2 inhibitor navitoclax. When used in combination with an NFκB inhibitor, navitoclax was synergistic in decreasing the growth of both human and IKKß wild-type mouse aRMS cells, indicating that inactivation of NFκB alone may not be sufficient for reducing tumor growth, but, when combined with another targeted therapeutic, may be clinically beneficial.

EphB4/EphrinB2 therapeutics in Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma affecting children and is often diagnosed with concurrent metastases. Unfortunately, few effective therapies have been discovered that improve the long-term survival rate for children with metastatic disease. Here we determined effectiveness of targeting the receptor tyrosine kinase, EphB4, in both alveolar and embryonal RMS either directly through the inhibitory antibody, VasG3, or indirectly by blocking both forward and reverse signaling of EphB4 binding to EphrinB2, cognate ligand of EphB4. Clinically, EphB4 expression in eRMS was correlated with longer survival. Experimentally, inhibition of EphB4 with VasG3 in both aRMS and eRMS orthotopic xenograft and allograft models failed to alter tumor progression. Inhibition of EphB4 forward signaling using soluble EphB4 protein fused with murine serum albumin failed to affect eRMS model tumor progression, but did moderately slow progression in murine aRMS. We conclude that inhibition of EphB4 signaling with these agents is not a viable monotherapy for rhabdomyosarcoma.

IGF1R as a Key Target in High Risk, Metastatic Medulloblastoma.

Risk or presence of metastasis in medulloblastoma causes substantial treatment-related morbidity and overall mortality. Through the comparison of cytokines and growth factors in the cerebrospinal fluid (CSF) of metastatic medulloblastoma patients with factors also in conditioned media of metastatic MYC amplified medulloblastoma or leptomeningeal cells, we were led to explore the bioactivity of IGF1 in medulloblastoma by elevated CSF levels of IGF1, IGF-sequestering IGFBP3, IGFBP3-cleaving proteases (MMP and tPA), and protease modulators (TIMP1 and PAI-1). IGF1 led not only to receptor phosphorylation but also accelerated migration/adhesion in MYC amplified medulloblastoma cells in the context of appropriate matrix or meningothelial cells. Clinical correlation suggests a peri-/sub-meningothelial source of IGF-liberating proteases that could facilitate leptomeningeal metastasis. In parallel, studies of key factors responsible for cell autonomous growth in MYC amplified medulloblastoma prioritized IGF1R inhibitors. Together, our studies identify IGF1R as a high value target for clinical trials in high risk medulloblastoma.

Evaluation of Soft Tissue Sarcoma Response to Preoperative Chemoradiotherapy Using Dynamic Contrast-Enhanced Magnetic Resonance Imaging.

This study aims to assess the utility of quantitative dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) parameters in comparison with imaging tumor size for early prediction and evaluation of soft tissue sarcoma response to preoperative chemoradiotherapy. In total, 20 patients with intermediate- to high-grade soft tissue sarcomas received either a phase I trial regimen of sorafenib + chemoradiotherapy (n = 8) or chemoradiotherapy only (n = 12), and underwent DCE-MRI at baseline, after 2 weeks of treatment with sorafenib or after the first chemotherapy cycle, and after therapy completion. MRI tumor size in the longest diameter (LD) was measured according to the RECIST (Response Evaluation Criteria In Solid Tumors) guidelines. Pharmacokinetic analyses of DCE-MRI data were performed using the Shutter-Speed model. After only 2 weeks of treatment with sorafenib or after 1 chemotherapy cycle, Ktrans (rate constant for plasma/interstitium contrast agent transfer) and its percent change were good early predictors of optimal versus suboptimal pathological response with univariate logistic regression C statistics values of 0.90 and 0.80, respectively, whereas RECIST LD percent change was only a fair predictor (C = 0.72). Post-therapy Ktrans, ve (extravascular and extracellular volume fraction), and kep (intravasation rate constant), not RECIST LD, were excellent (C > 0.90) markers of therapy response. Several DCE-MRI parameters before, during, and after therapy showed significant (P < .05) correlations with percent necrosis of resected tumor specimens. In conclusion, absolute values and percent changes of quantitative DCE-MRI parameters provide better early prediction and evaluation of the pathological response of soft tissue sarcoma to preoperative chemoradiotherapy than the conventional measurement of imaging tumor size change.

Proptosis Caused by Rhabdomyomatous Mesenchymal Hamartomata Occurring in the Orbit.

Two infants were referred for progressive orbital proptosis. MRI in both cases demonstrated a homogenous mass in the orbit adherent to and isointense with a rectus muscle. Histopathology in both cases demonstrated a bland proliferation of spindle cells with entrapped skeletal muscle. Immunochemistry demonstrated that the abnormal tissue was of skeletal muscle origin, consistent with rhabdomyomatous mesenchymal hamartoma (RMH). Observation was elected due to the reported benign nature of RMH. In contrast to RMH of the cutaneous tissues that typically follows a benign course, RMH of the orbit may present with rapid growth.

Murine model of hepatic breast cancer.

BACKGROUND AND AIMS: Breast cancer is the most common cancer in women and the second leading cause of cancer-related deaths in this population. Breast cancer related deaths have declined due to screening and adjuvant therapies, yet a driving clinical need exists to better understand the cause of the deadliest aspect of breast cancer, metastatic disease. Breast cancer metastasizes to several distant organs, the liver being the third most common site. To date, very few murine models of hepatic breast cancer exist. METHODS: In this study, a novel murine model of liver breast cancer using the MDA-MB-231 cell line is introduced as an experimental (preclinical) model. RESULTS: Histological typing revealed consistent hepatic breast cancer tumor foci. Common features of the murine model were vascular invasion, lung metastasis and peritoneal seeding. CONCLUSIONS: The novel murine model of hepatic breast cancer established in this study provides a tool to be used to investigate mechanisms of hepatic metastasis and to test potential therapeutic interventions.

Giant Aneurysmal Bone Cyst of the Anterior Cranial Fossa and Paranasal Sinuses Presenting in Pregnancy: Case Report and Literature Review.

Background and Purpose Aneurysmal bone cysts (ABCs) rarely involve the cranium and have seldom been reported in pregnancy. Clinical Presentation We describe a case of a 28-year-old woman who presented at 37 weeks of gestation with 3 months of gradually worsening vision, 10 months of proptosis, and restricted ocular motility on the left. Brain imaging revealed a multicystic enhancing mass measuring 5.9 × 5.3 × 3.7 cm, centered on the cribriform plate on the left, extending into the anterior cranial fossa superiorly as well as the left nasal cavity, maxillary, sphenoid, and frontal sinuses. Her clinical course is described in detail; 3-month postoperative imaging demonstrated no residual mass. Conclusion A literature review revealed five previous cases of ABCs associated with pregnancy. We report a rare case of a giant ABC of fibrous dysplasia involving the paranasal sinuses and anterior cranial fossa. We postulate on the possible influence of pregnancy on the clinical course.

Inflammatory myofibroblastic tumor of the orbit: a case report.

Inflammatory myofibroblastic tumor (IMT) is a neoplasm most commonly found in the abdominal-pelvic region, lung, and retroperitoneum. The tumor tends to affect soft tissues of children and young adults and can locally recur but rarely metastasizes. Histologically, the appearance is one of bland spindle cell proliferation with a prominent, chronic inflammatory infiltrate. This article describes 1 case of IMT found in the orbit that is presented with rapidly progressive painless proptosis. In the authors' review of the literature, they have only found 2 other case reports involving the orbit.

Lineage of origin in rhabdomyosarcoma informs pharmacological response.

Lineage or cell of origin of cancers is often unknown and thus is not a consideration in therapeutic approaches. Alveolar rhabdomyosarcoma (aRMS) is an aggressive childhood cancer for which the cell of origin remains debated. We used conditional genetic mouse models of aRMS to activate the pathognomonic Pax3:Foxo1 fusion oncogene and inactivate p53 in several stages of prenatal and postnatal muscle development. We reveal that lineage of origin significantly influences tumor histomorphology and sensitivity to targeted therapeutics. Furthermore, we uncovered differential transcriptional regulation of the Pax3:Foxo1 locus by tumor lineage of origin, which led us to identify the histone deacetylase inhibitor entinostat as a pharmacological agent for the potential conversion of Pax3:Foxo1-positive aRMS to a state akin to fusion-negative RMS through direct transcriptional suppression of Pax3:Foxo1.

PDGFRß reverses EphB4 signaling in alveolar rhabdomyosarcoma.

Alveolar rhabdomyosarcoma (aRMS) is an aggressive myogenic childhood malignancy, not infrequently presenting as incurable metastatic disease. To identify therapeutic targets, we performed an unbiased tyrosine kinome RNA interference screen in primary cell cultures from a genetically engineered, conditional mouse model of aRMS. We identified ephrin receptor B4 (EphB4) as a target that is widely expressed in human aRMS and that portends a poor clinical outcome in an expression level-dependent manner. We also uncovered cross-talk of this ephrin receptor with another receptor tyrosine kinase, PDGFRß, which facilitates PDGF ligand-dependent, ephrin ligand-independent activation of EphB4 converging on the Akt and Erk1/2 pathways. Conversely, EphB4 activation by its cognate ligand, EphrinB2, did not stimulate PDGFRß; instead, apoptosis was paradoxically induced. Finally, we showed that small-molecule inhibition of both PDGFRß and EphB4 by dasatinib resulted in a significant decrease in tumor cell viability in vitro, as well as decreased tumor growth rate and significantly prolonged survival in vivo. To our knowledge, these results are the first to identify EphB4 and its cross-talk with PDGFRß as unexpected vital determinants of tumor cell survival in aRMS, with EphB4 at the crux of a bivalent signaling node that is either mitogenic or proapoptotic.

Rb1 loss modifies but does not initiate alveolar rhabdomyosarcoma.

BACKGROUND: Alveolar rhabdomyosarcoma (aRMS) is a myogenic childhood sarcoma frequently associated with a translocation-mediated fusion gene, Pax3:Foxo1a. METHODS: We investigated the complementary role of Rb1 loss in aRMS tumor initiation and progression using conditional mouse models. RESULTS: Rb1 loss was not a necessary and sufficient mutational event for rhabdomyosarcomagenesis, nor a strong cooperative initiating mutation. Instead, Rb1 loss was a modifier of progression and increased anaplasia and pleomorphism. Whereas Pax3:Foxo1a expression was unaltered, biomarkers of aRMS versus embryonal rhabdomyosarcoma were both increased, questioning whether these diagnostic markers are reliable in the context of Rb1 loss. Genome-wide gene expression in Pax3:Foxo1a,Rb1 tumors more closely approximated aRMS than embryonal rhabdomyosarcoma. Intrinsic loss of pRb function in aRMS was evidenced by insensitivity to a Cdk4/6 inhibitor regardless of whether Rb1 was intact or null. This loss of function could be attributed to low baseline Rb1, pRb and phospho-pRb expression in aRMS tumors for which the Rb1 locus was intact. Pax3:Foxo1a RNA interference did not increase pRb or improve Cdk inhibitor sensitivity. Human aRMS shared the feature of low and/or heterogeneous tumor cell pRb expression. CONCLUSIONS: Rb1 loss from an already low pRb baseline is a significant disease modifier, raising the possibility that some cases of pleomorphic rhabdomyosarcoma may in fact be Pax3:Foxo1a-expressing aRMS with Rb1 or pRb loss of function.