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Abstract Osteosarcoma is the most common primary bone malignancy in children and young adults, and it has few treatment options. As a result, there has been little improvement in survival outcomes in the past few decades. The need for models to test novel therapies is especially great in this disease since it is both rare and does not respond to most therapies. To address this, an NCI-funded consortium has characterized and utilized a panel of patient-derived xenograft models of osteosarcoma for drug testing. The exomes, transcriptomes, and copy number landscapes of these models have been presented previously. This study now adds whole genome sequencing and reverse-phase protein array profiling data, which can be correlated with drug testing results. In addition, four additional osteosarcoma models are described for use in the research community.

In embryonal rhabdomyosarcoma (ERMS) and generally in sarcomas, the role of wild-type and loss- or gain-of-function TP53 mutations remains largely undefined. Eliminating mutant or restoring wild-type p53 is challenging; nevertheless, understanding p53 variant effects on tumorigenesis remains central to realizing better treatment outcomes. In ERMS, >70% of patients retain wild-type TP53, yet mutations when present are associated with worse prognosis. Employing a kRASG12D-driven ERMS tumor model and tp53 null (tp53-/-) zebrafish, we define wild-type and patient-specific TP53 mutant effects on tumorigenesis. We demonstrate that tp53 is a major suppressor of tumorigenesis, where tp53 loss expands tumor initiation from 97% of animals. Characterizing three patient-specific alleles reveals that TP53C176F partially retains wild-type p53 apoptotic activity that can be exploited, whereas TP53P153Δ and TP53Y220C encode two structurally related proteins with gain-of-function effects that predispose to head musculature ERMS. TP53P153Δ unexpectedly also predisposes to hedgehog-expressing medulloblastomas in the kRASG12D-driven ERMS-model.

Rhabdomyosarcomas are tumours blocked in myogenic differentiation, which despite the expression of master muscle regulatory factors, including MYOD, are unable to differentiate. Here, the authors show that SNAI2 is upregulated by MYOD through super enhancers, binds to MYOD target enhancers, and arrests differentiation.

Abstract Background Aging is a comorbidity of breast cancer suggesting that aging-associated transcriptome changes may promote breast cancer progression. However, the mechanism underlying the age effect on breast cancer remains poorly understood. Method We analyzed transcriptomics of the matched normal breast tissues from the 82 breast cancer patients in The Cancer Genome Atlas (TCGA) dataset with linear regression for genes with age-associated expression that are not associated with menopause. We also analyzed differentially expressed genes between the paired tumor and non-tumor breast tissues in TCGA for the identification of age and breast cancer (ABC)-associated genes. A few of these genes were selected for further investigation of their malignancy-regulating activities with in vitro and in vivo assays. Results We identified 148 upregulated and 189 downregulated genes during aging. Overlapping of tumor-associated genes between normal and tumor tissues with age-dependent genes resulted in 14 upregulated and 24 downregulated genes that were both age and breast cancer associated. These genes are predictive in relapse-free survival, indicative of their potential tumor promoting or suppressive functions, respectively. Knockdown of two upregulated genes (DYNLT3 and P4HA3) or overexpression of the downregulated ALX4 significantly reduced breast cancer cell proliferation, migration, and clonogenicity. Moreover, knockdown of P4HA3 reduced growth and metastasis whereas overexpression of ALX4 inhibited the growth of xenografted breast cancer cells in mice. Conclusion Our study suggests that transcriptome alterations during aging may contribute to breast tumorigenesis. DYNLT3, P4HA3, and ALX4 play significant roles in breast cancer progression.

The radiation and chemotherapy used for treating medulloblastoma patients cause debilitating side effects. Here, the authors show that miR-584 acts as a therapeutic adjuvant as it sensitizes medulloblastoma to radiation and chemotherapy by targeting HDAC1 or eIF4E3 to enhance spindle defects and DNA damage.

ABSTRACT IMPACT: By assessing function of mutant (patient-specific) tp53 in zebrafish embryonal rhabdomyosarcoma will inform clinicians of the severity of mutant tp53 alleles. OBJECTIVES/GOALS: This study aims to define loss- and gain-of-function TP53 mutations by comparing effects in tp53-null and wild-type tumors. In addition, it aims to generate a rapid in vivo analysis platform to assign function to patient specific TP53 mutations in the clinic METHODS/STUDY POPULATION: To define tp53 function in ERMS pathogenesis, we previously generated a new tp53-null mutant (tp53-/-) in zebrafish by deleting the entire tp53 genomic locus using TALEN mutagenesis. tp53-/- zebrafish spontaneously develop a spectrum of tumors including sarcomas, leukemia and germ cell tumors (Ignatius…Baxi et. al., eLife) reminiscent of tumors observed in Trp53-null mice. Using the tp53-/- mutants to generate kRASG12D-induced ERMS, we discovered that tp53 is a potent repressor of metastases but rather surprisingly had no effect on self-renewal (Ignatius…Baxi et. al., eLife). Here, using tp53-/- zebrafish, we assessed effects of wild-type and mutant (patient specific) tp53 on tumor initiation, proliferation and apoptosis. RESULTS/ANTICIPATED RESULTS: ERMS tumor initiation in the tp53-/- background is observed in > 97% of animals whereas only

Ewing sarcoma is a small round blue cell malignancy arising from bone or soft tissue and most commonly affects adolescents and young adults. Metastatic and relapsed Ewing sarcoma have poor outcomes and recurrences remain common. Owing to the poor outcomes associated with advanced disease and the need for a clear research strategy, the Children’s Oncology Group Bone Tumor Committee formed the New Agents for Ewing Sarcoma Task Force to bring together experts in the field to evaluate and prioritize new agents for incorporation into clinical trials. This group’s mission was to evaluate scientific and clinical challenges in moving new agents forward and to recommend agents and trial designs to the Bone Tumor Committee. The task force generated a framework for vetting prospective agents that included critical evaluation of each drug by using both clinical and non-clinical parameters. Representative appraisal of agents of highest priority, including eribulin, dinutuximab, cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors, anti-angiogenic tyrosine kinase inhibitors, and poly-ADP-ribose polymerase (PARP) inhibitors, is described. The task force continues to analyze new compounds by using the paradigm established.

STAT3 is a transcription factor involved in cytokine and receptor kinase signal transduction that is aberrantly activated in a variety of sarcomas, promoting metastasis and chemotherapy resistance. The purpose of this work was to develop and test a novel putative STAT3 inhibitor, LY5.An in silico fragment-based drug design strategy was used to create LY5, a small molecule inhibitor that blocks the STAT3 SH2 domain phosphotyrosine binding site, inhibiting homodimerization. LY5 was evaluated in vitro demonstrating good biologic activity against rhabdomyosarcoma, osteosarcoma and Ewing's sarcoma cell lines at high nanomolar/low micromolar concentrations, as well as specific inhibition of STAT3 phosphorylation without effects on other STAT3 family members. LY5 exhibited excellent oral bioavailability in both mice and healthy dogs, and drug absorption was enhanced in the fasted state with tolerable dosing in mice at 40 mg/kg BID. However, RNAi-mediated knockdown of STAT3 did not phenocopy the biologic effects of LY5 in sarcoma cell lines. Moreover, concentrations needed to inhibit ex vivo metastasis growth using the PuMA assay were significantly higher than those needed to inhibit STAT3 phosphorylation in vitro. Lastly, LY5 treatment did not inhibit the growth of sarcoma xenografts or prevent pulmonary metastasis in mice.LY5 is a novel small molecule inhibitor that effectively inhibits STAT3 phosphorylation and cell proliferation at nanomolar concentrations. LY5 demonstrates good oral bioavailability in mice and dogs. However LY5 did not decrease tumor growth in xenograft mouse models and STAT3 knockdown did not induce concordant biologic effects. These data suggest that the anti-cancer effects of LY5 identified in vitro were not mediated through STAT3 inhibition.

Quaternary alkaloids are the major alkaloids isolated from Tinospora species. A previous study pointed to the necessary presence of quaternary nitrogens for strong acetylcholinesterase (AChE) inhibitory activity in such alkaloids. Repeated column chromatography of the vine of Tinospora crispa extract led to the isolation of one new protoberberine alkaloid, 4,13-dihydroxy-2,8,9-trimethoxydibenzo[a,g]quinolizinium (1), along with six known alkaloids—dihydrodiscretamine (2), columbamine (3), magnoflorine (4), N-formylannonaine (5), N-formylnornuciferine (6), and N-trans-feruloyltyramine (7). The seven compounds were isolated and structurally elucidated by spectroscopic analysis. Two known alkaloids, namely, dihydrodiscretamine and columbamine are reported for the first time for this plant. The compounds were tested for AChE inhibitory activity using Ellman’s method. In the AChE inhibition assay, only columbamine (3) showed strong activity with IC50 48.1 µM. The structure–activity relationships derived from these results suggest that the quaternary nitrogen in the skeleton has some effect, but that a high degree of methoxylation is more important for acetylcholinesterase inhibition.

Survival outcomes for patients with osteosarcoma (OS) have remained stagnant over the past three decades. Insulin-like growth factor 1 receptor (IGF1R) is over-expressed in a number of malignancies, and anti-IGF1R antibodies have and are currently being studied in clinical trials. Understanding the molecular aberrations which result in increased tumor response to anti-IGF1R therapy could allow for the selection of patients most likely to benefit from IGF1R targeted therapy.IGF1R mRNA expression was assessed by RT PCR in OS patient primary tumors, cell lines, and xenograft tumors. IGF1R copy number was assessed by 3 approaches: PCR, FISH, and dot blot analysis. Exons 1-20 of IGF1R were sequenced in xenograft tumors and 87 primary OS tumors, and surface expression of IGF1R was assessed by flow cytometry. Levels of mRNA and protein expression, copy number, and mutation status were compared with tumor response to anti-IGF1R antibody therapy in 4 OS xenograft models.IGF1R mRNA is expressed in OS. Primary patient samples and xenograft samples had higher mRNA expression and copy number compared with corresponding cell lines. IGF1R mRNA expression, cell surface expression, copy number, and mutation status were not associated with tumor responsiveness to anti-IGF1R antibody therapy.IGF1R is expressed in OS, however, no clear molecular markers predict response to IGF1R antibody-mediated therapy. Additional pre-clinical studies assessing potential predictive biomarkers and investigating targetable molecular pathways critical to the proliferation of OS cells are needed.

Summary Objective  ABT-751 is a novel antimitotic agent that binds tubulin at the colchicine binding site. ABT-751 is undergoing Phase I trials in children, but has not been evaluated against a range of pediatric tumor models in vivo. Materials and methods  ABT-751 was evaluated against 27 subcutaneously implanted xenograft models of childhood cancer including neuroblastoma [4], osteosarcoma [4], Ewing sarcoma [2] rhabdomyosarcoma [8], medulloblastoma [1] and eight kidney cancer lines (six Wilms tumors, two rhabdoid). ABT-751 was administered at 100 mg/kg P.O. on a schedule of 5 days on, 5 days off, 5 days on, repeating the cycle at 21 days. Tumor diameters were measured at 7 day intervals for a period of 12 weeks. Three measures of antitumor activity were used: (1) clinical response criteria [e.g., partial response (PR), complete response (CR), etc.]; (2) treated to control (T/C) tumor volume at day 21; and (3) a time to event measure based on the median event free survival (EFS) of treated and control lines. Results  ABT-751 induced regression in 4 of 25 models (16%) including models of neuroblastoma that are refractory to vincristine and paclitaxel. Other regressions occurred in rhabdomyosarcoma and Wilms tumor models. ABT-751 significantly increased event free survival (EFS > 2.0) in eight models (33%) in addition to those with objective responses. Conclusions  ABT-751 demonstrated intermediate activity against this tumor panel. Neuroblastoma models appear somewhat more sensitive to this agent, with objective regressions also in rhabdomyosarcoma and Wilms tumor. ABT-751 was also active in several tumor lines intrinsically refractory to vincristine or paclitaxel. [ABSTRACT FROM AUTHOR]

Chemotherapy is commonly used in the treatment of children with high-grade glioma, although its usefulness is uncertain. We conducted a multi-institutional study to evaluate the efficacy of temozolomide given after radiotherapy in children with newly diagnosed high-grade glioma and unfavorable low-grade glioma (gliomatosis cerebri or bithalamic involvement). Optional window therapy of intravenous irinotecan (10 doses of 20 mg/m2 per cycle × 2) was given over 6 weeks. The 5-day schedule of temozolomide (200 mg/m2 per day) started 4 weeks after the completion of radiotherapy and continued for a total of 6 cycles. Thirty-one eligible patients (median age: 12.3 years) participated. Tumors most commonly involved cerebral hemispheres (n = 13, 42%) and thalamus (n = 14, 45%). Whereas six patients underwent radical resection, the remainder had limited surgery, including biopsy (n = 14, 45%). The predominant histologic diagnoses were glioblastoma multiforme (n = 15, 48%) and anaplastic astrocytoma (n = 10, 32%). Two patients had bithalamic grade II astrocytoma. Twenty-seven patients received radiotherapy (median dose: 59.4 Gy), including craniospinal irradiation in 3 because of leptomeningeal spread. Four patients did not receive radiotherapy in this study because of consent withdrawn (n = 2), toxicity during window therapy (n = 1), or at the physician’s discretion (n = 1). Twenty-three patients received 112 cycles of temozolomide therapy. The 2-year progression-free and overall survival estimates were 11 ± 5% and 21 ± 7%, respectively. Although the heterogeneity of prognostic factors in our patients made assessment of treatment outcome more difficult, the addition of 6 cycles of temozolomide after radiotherapy did not seem to alter the poor outcome of these patients. [ABSTRACT FROM AUTHOR]