Increasing incidence coupled with poor prognosis and treatments that are virtually unchanged over the past 20 years have made the need for the development of novel therapeutics for hepatoblastoma imperative. PIM kinases have been implicated as drivers of tumorigenesis in multiple cancers, including hepatocellular carcinoma. We hypothesized that PIM kinases, specifically PIM3, would play a role in hepatoblastoma tumorigenesis and that PIM kinase inhibition would affect hepatoblastoma in vitro and in vivo. Parameters including cell survival, proliferation, motility, and apoptosis were assessed in human hepatoblastoma cells following PIM3 knockdown with siRNA or treatment with the PIM inhibitor AZD1208. An in vivo model of human hepatoblastoma was utilized to study the effects of PIM inhibition alone and in combination with cisplatin. PIM kinases were found to be present in the human hepatoblastoma cell line, HuH6, and in a human hepatoblastoma patient-derived xenograft, COA67. PIM3 knockdown or inhibition with AZD1208 decreased cell survival, attachment independent growth, and motility. Additionally, inhibition of tumor growth was observed in a hepatoblastoma xenograft model in mice treated with AZD1208. Combination therapy with AZD1208 and cisplatin resulted in a significant increase in animal survival when compared to either treatment alone. The current studies showed that PIM kinase inhibition decreased human hepatoblastoma tumorigenicity both in vitro and in vivo, implying that PIM inhibitors may be useful as a novel therapeutic for children with hepatoblastoma.

Background Biliary rhabdomyosarcoma (RMS) is the most common biliary tumor in children. The biliary tract is classified as a favorable primary site. Therefore, patients with localized biliary RMS were included in two consecutive low‐risk studies, D9602 and ARST0331, by the Children's Oncology Group (COG). The outcome for these patients treated with low‐risk therapy has not been reported. Procedure Patients with biliary RMS enrolled on COG low‐risk trials D9602 or ARST0331 were analyzed. All patients received systemic chemotherapy and those with Group II (microscopic residual) or Group III (macroscopic residual) disease received 36‐50.4 Gy adjuvant radiotherapy (RT). Delayed primary excision (DPE) was allowed on both studies. Results Seventeen patients with biliary RMS were treated on D9602 (n = 7) or ARST0331 (n = 10). Median age was 3.5 years (range 1.7‐10.3). Ten (59%) patients had tumors >5 cm and 14 (82%) had Group III disease. Fifteen (88%) patients received RT. The 5‐year event‐free survival (EFS) and overall survival (OS) were 70.6% (95% confidence interval [CI]: 46.9‐94.3%) and 76.5% (95% CI: 54.6‐98.4%), respectively. The majority of patients (80%) who received RT did not have disease recurrence while both patients who did not receive RT had local relapse. Five (36%) of 14 patients with Group III disease underwent DPE; two experienced a local relapse. In the nine patients without DPE, two developed local relapse. Conclusions Patients with localized biliary RMS treated on low‐risk studies had suboptimal outcomes. These patients may benefit from therapy on intermediate‐risk studies.

Hepatoblastoma is the most common primary liver tumor in children, but treatment has not changed significantly in the past 20 years. We have previously demonstrated that Proviral Integration site for Moloney murine leukemia (PIM) kinases promote tumorigenesis in hepatoblastoma. Stem cell-like cancer cells (SCLCCs) are a subset of cells thought to be responsible for chemoresistance, metastasis, relapse, and recurrence. The aim of this study was to identify SCLCCs in hepatoblastoma and determine the role of PIM kinases in SCLCCs. Hepatoblastoma cells were separated into CD133-enriched and CD133-depleted populations and the frequency of SCLCCs was assessed. CD133 expression was determined in the presence or absence of the PIM inhibitor, AZD1208. The effects of AZD1208 on proliferation, apoptosis, and motility were assessed in vitro and the effect of AZD1208 on tumor growth was examined in vivo. We identified CD133 as a marker for SCLCCs in hepatoblastoma and showed that PIM kinases promote a SCLCC phenotype. PIM kinase inhibition with AZD1208 decreased proliferation, migration, and invasion and increased apoptosis in both SCLCCs and non-SCLCCs in a long-term passaged hepatoblastoma cell line and patient-derived xenograft. Additionally, tumor growth in mice implanted with hepatoblastoma SCLCCs was decreased with PIM inhibition such that 57% of the tumors regressed. These findings identify CD133 as a marker for SCLCCs in hepatoblastoma and provide evidence that inhibition of PIM kinases decreases stemness and tumorigenicity of SCLCCs in hepatoblastoma, making them potential therapeutic targets for the treatment of hepatoblastoma.

Purpose-Hepatoblastoma is the most common primary liver cancer of childhood and has few prognostic indicators. We have previously shown that Proviral Integration site for Moloney murine leukemia virus (PIM3) kinase decreased hepatoblastoma tumorigenicity. We sought to determine the effect of PIM3 overexpression on hepatoblastoma cells and whether expression of PIM3 correlated with patient/tumor characteristics or survival. Methods-The hepatoblastoma cell line, HuH6, and patient-derived xenograft, COA67, were utilized. Viability, proliferation, migration, sphere formation, and tumor growth in mice were assessed in PIM3-overexpressing cells. Immunohistochemistry was performed for PIM3 on patient samples. Correlation between stain score and clinical/pathologic characteristics was assessed. Results-PIM3 overexpression rescued the anti-proliferative effect observed with PIM3 knockdown. Sphere formation was increased in PIM3 overexpressing cells. Cells with PIM3 overexpression yielded larger tumors than those with empty vector. Seventy-four percent of samples expressed PIM3. There was no statistical difference in patient characteristics between subjects with strong versus weak PIM3 staining, but patients with strong PIM3 staining had decreased survival. Conclusions-PIM3 expression plays a role in hepatoblastoma tumorigenesis. PIM3 was present in the majority of hepatoblastomas and higher PIM3 expression correlated with decreased

It has become increasingly clear that agents that disrupt calcium homeostasis may also be toxic to developing neurons. Using isolated primary neurons, we sought to understand the neurotoxicity of agents such as MK801 (which blocks ligand-gated calcium entry), BAPTA (which chelates intracellular calcium), and thapsigargin (TG; which inhibits the endoplasmic reticulum Ca 2+ -ATPase pump). Thus, E18 at cotical neuons wee grown for 1 day in vitro (DIV) and then exposed to vehicle (0.1% DMSO), MK801 (0.01-20 μM), BAPTA (0.1-20 μM), or TG (0.001-1 μM) for 24 h. We found that all three agents could profoundly influence early neuronal maturation (growth cone expansion, neurite length, neurite complexity), with the order of potency being MK801 < BAPTA < TG. We next asked if cultures exposed to these agents were able to re-establish their developmental program once the agent was removed. When we examined network maturity at 4 and 7 DIV, the order of recovery was MK801 > BAPTA > TG. Thus, mechanistically distinct ways of disrupting calcium homeostasis differentially influenced both short-term and long-term neuronal maturation. These observations suggest that agents that act by altering intracellular calcium and are used in obstetrics or neonatology may be quite harmful to the still-developing human brain.

Patient-derived xenografts (PDXs) provide an opportunity to evaluate the effects of therapies in an environment that more closely resembles the human condition than that seen with long-term passage cell lines. In the current studies, we investigated the effects of FAK inhibition on two neuroblastoma PDXs in vitro. Cells were treated with two small molecule inhibitors of FAK, PF-573,228 (PF) and 1,2,4,5-benzentetraamine tetrahydrochloride (Y15). Following FAK inhibition, cell survival and proliferation decreased significantly and cell cycle arrest was seen in both cell lines. Migration and invasion assays were used to determine the effect of FAK inhibition on cell motility, which decreased significantly in both cell lines in the presence of either inhibitor. Finally, tumor cell stemness following FAK inhibition was evaluated with extreme limiting dilution assays as well as with immunoblotting and quantitative real-time PCR for the expression of stem cell markers. FAK inhibition decreased formation of tumorspheres and resulted in a corresponding decrease in established stem cell markers. FAK inhibition decreased many characteristics of the malignant phenotype, including cancer stem cell like features in neuroblastoma PDXs, making FAK a candidate for further investigation as a potential target for neuroblastoma therapy.

Highlights Rare, high grade neuroendocrine-like pediatric tumor has TYRO3 and ALK mutations. Crizotinib downregulates STAT3 and ERK1/2 signaling in tumor. In vivo analysis demonstrated crizotinib resistance. Crizotinib resistant cells have upregulation of PDGFRß signaling. PDGFRß inhibition overrides resistance.

Despite an increase in incidence, treatments for hepatoblastoma remain virtually unchanged for the past 20 years, emphasizing the need for novel therapeutics. FTY720 (fingolimod) is an immunomodulator approved for use in multiple sclerosis in children that has been demonstrated to have anti-cancer properties in multiple cancer types. We have demonstrated that FTY720 activates PP2A in hepatoblastoma, but does not do so via inhibition of the endogenous inhibitors, CIP2A and I2PP2A, as previously observed in other cancers. PP2A activation in hepatoblastoma decreased cell viability, proliferation, and motility and induced apoptosis. In a subcutaneous xenograft model, FTY720 decreased tumor growth. FTY720 in combination with the standard chemotherapeutic, cisplatin, decreased proliferation in a synergistic manner. Finally, animals bearing subcutaneous hepatoblastoma xenografts treated with FTY720 and cisplatin in combination had significantly decreased tumor growth compared to those treated with either drug alone. These findings show that targeting PP2A with FTY70 shows promise in the treatment of hepatoblastoma and that combining FTY720 with cisplatin may be a novel and effective strategy to better treat this devastating pediatric liver tumor.