Search

Ewing sarcoma (ES) is a malignant tumor of bone and soft tissue that most often occurs in children, adolescents, and young adults. Debate and controversy remain in the management of relapsed/refractory ES (RR-ES). The authors leveraged the expertise assembled by the National Ewing Sarcoma Tumor Board, a multidisciplinary virtual tumor board that meets monthly to discuss challenging cases of ES. In this review, they focus on select topics that apply to the management of patients with RR-ES. The specific topics covered include the initial approach of such patients and discussion of the goals of care, the role of molecular testing, chemotherapy regimens and novel agents to consider, the role of maintenance therapy, and the use of high-dose chemotherapy with autologous stem cell rescue. The data referenced are often limited to subgroup analyses and/or compiled from multiple sources. Although not intended to replace the clinical judgement of treating physicians, these guidelines are intended to support clinicians and provide some clarity and recommendations for the management of patients with RR-ES. PLAIN LANGUAGE SUMMARY: Ewing sarcoma (ES) is a bone and soft tissue cancer that most often occurs in teenagers and young adults. This article uses the experience of the National Ewing Sarcoma Tumor Board, a multi-institution, multidisciplinary virtual tumor board that meets monthly to discuss challenging cases of ES and to address questions related to the treatment of patients with relapsed ES. Although not intended to replace the clinical judgement of treating physicians and limited by available data, these consensus recommendations will support clinicians who treat patients with this challenging malignancy, made even more difficult when it recurs., (© 2024 The Author(s). Cancer published by Wiley Periodicals LLC on behalf of American Cancer Society.)

Clinical trials conducted by the Intergroup Rhabdomyosarcoma (RMS) Study Group and the Children's Oncology Group have been pivotal to establishing current standards for diagnosis and therapy for RMS. Recent advancements in understanding the biology and clinical behavior of RMS have led to more nuanced approaches to diagnosis, risk stratification, and treatment. The complexities introduced by these advancements, coupled with the rarity of RMS, pose challenges to conducting large-scale phase 3 clinical trials to evaluate new treatment strategies for RMS. Given these challenges, systematic planning of future clinical trials in RMS is paramount to address pertinent questions regarding the therapeutic efficacy of drugs, biomarkers of response, treatment-related toxicity, and patient quality of life. Herein, the authors outline the proposed strategic approach of the Children's Oncology Group Soft Tissue Sarcoma Committee to the next generation of RMS clinical trials, focusing on five themes: improved novel agent identification and preclinical to clinical translation, more efficient trial development and implementation, expanded opportunities for knowledge generation during trials, therapeutic toxicity reduction and quality of life, and patient engagement., (© 2024 American Cancer Society.)

Background: Pediatric gastroenteropancreatic neuroendocrine tumors are exceedingly rare, resulting in most pediatric treatment recommendations being based on data derived from adults. Trametinib is a kinase inhibitor that targets MEK1/2 and has been employed in the treatment of cancers harboring mutations in the Ras pathway., Methods: We utilized an established human pediatric gastroenteropancreatic neuroendocrine-like tumor patient-derived xenograft (PDX) with a known NRAS mutation to study the effects of MEK inhibition. We evaluated the effects of trametinib on proliferation, motility, and tumor growth in vivo. We created an intraperitoneal metastatic model of this PDX, characterized both the phenotype and the genotype of the metastatic PDX and again, investigated the effects of MEK inhibition., Results: We found target engagement with decreased ERK1/2 phosphorylation with trametinib treatment. Trametinib led to decreased in vitro cell growth and motility, and decreased tumor growth and increased animal survival in a murine flank tumor model. Finally, we demonstrated that trametinib was able to significantly decrease gastroenteropancreatic neuroendocrine intraperitoneal tumor metastasis., Conclusions: The results of these studies support the further investigation of MEK inhibition in pediatric NRAS mutated solid tumors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Invasive aspergillosis is a known complication in patients with hematologic malignancies. Tracheopleural fistulas are very rare and reported in immunocompromised adults. We present a case of invasive pulmonary aspergillosis with tracheopleural fistula in a pediatric patient with a history of rhabdomyosarcoma and macrophage activation syndrome. This case highlights the importance of recognizing life-threatening fungal infections and coordinating surgical subspecialities for patient care.

The use of three-dimensional (3D) bioprinting has remained at the forefront of tissue engineering and has recently been employed for generating bioprinted solid tumors to be used as cancer models to test therapeutics. In pediatrics, neural crest-derived tumors are the most common type of extracranial solid tumors. There are only a few tumor-specific therapies that directly target these tumors, and the lack of new therapies remains detrimental to improving the outcomes for these patients. The absence of more efficacious therapies for pediatric solid tumors, in general, may be due to the inability of the currently employed preclinical models to recapitulate the solid tumor phenotype. In this study, we utilized 3D bioprinting to generate neural crest-derived solid tumors. The bioprinted tumors consisted of cells from established cell lines and patient-derived xenograft tumors mixed with a 6% gelatin/1% sodium alginate bioink. The viability and morphology of the bioprints were analyzed via bioluminescence and immunohisto chemistry, respectively. We compared the bioprints to traditional twodimensional (2D) cell culture under conditions such as hypoxia and therapeutics. We successfully produced viable neural crest-derived tumors that retained the histology and immunostaining characteristics of the original parent tumors. The bioprinted tumors propagated in culture and grew in orthotopic murine models. Furthermore, compared to cells grown in traditional 2D culture, the bioprinted tumors were resistant to hypoxia and chemotherapeutics, suggesting that the bioprints exhibited a phenotype that is consistent with that seen clinically in solid tumors, thus potentially making this model superior to traditional 2D culture for preclinical investigations. Future applications of this technology entail the potential to rapidly print pediatric solid tumors for use in high-throughput drug studies, expediting the identification of novel, individualized therapies., Competing Interests: The authors declare no conflict of interests., (Copyright: © 2023, Quinn CH, Beierle AM, Julson JR, et al.)

Background: Protein phosphatase 2A (PP2A) functions as an inhibitor of cancer cell proliferation, and its tumor suppressor function is attenuated in many cancers. Previous studies utilized FTY720, an immunomodulating compound known to activate PP2A, and demonstrated a decrease in the malignant phenotype in neuroblastoma. We wished to investigate the effects of two novel PP2A activators, ATUX-792 (792) and DBK-1154 (1154)., Methods: Long-term passage neuroblastoma cell lines and human neuroblastoma patient-derived xenograft (PDX) cells were used. Cells were treated with 792 or 1154, and viability, proliferation, and motility were examined. The effect on tumor growth was investigated using a murine flank tumor model., Results: Treatment with 792 or 1154 resulted in PP2A activation, decreased cell survival, proliferation, and motility in neuroblastoma cells. Immunoblotting revealed a decrease in MYCN protein expression with increasing concentrations of 792 and 1154. Treatment with 792 led to tumor necrosis and decreased tumor growth in vivo., Conclusions: PP2A activation with 792 or 1154 decreased survival, proliferation, and motility of neuroblastoma in vitro and tumor growth in vivo. Both compounds resulted in decreased expression of the oncogenic protein MYCN. These findings indicate a potential therapeutic role for these novel PP2A activators in neuroblastoma.

Cancer is the leading cause of death by disease in children, and over 15% of pediatric cancer-related mortalities are due to neuroblastoma. Current treatment options for neuroblastoma remain suboptimal as they often have significant toxicities, are associated with long-term side effects, and result in disease relapse in over half of children with high-risk disease. There is a dire need for new therapies, and oncolytic viruses may represent an effective solution. Oncolytic viruses attack tumor cells in two ways: direct infection of tumor cells leading to cytolysis, and production of a debris field that stimulates an anti-tumor immune response. Our group has previously shown that M002, an oncolytic herpes simplex virus (oHSV), genetically engineered to express murine interleukin-12 (mIL-12), was effective at targeting and killing long term passage tumor cell lines. In the current study, we investigated M002 in three neuroblastoma patient-derived xenografts (PDXs). PDXs better recapitulate the human condition, and these studies were designed to gather robust data for translation to a clinical trial. We found that all three PDXs expressed viral entry receptors, and that the virus actively replicated in the cells. M002 caused significant tumor cell death in 2D culture and 3D bioprinted tumor models. Finally, the PDXs displayed variable susceptibility to M002, with a more profound effect on high-risk neuroblastoma PDXs compared to low-risk PDX. These findings validate the importance of incorporating PDXs for preclinical testing of oncolytic viral therapeutics and showcase a novel technique, 3D bioprinting, to test therapies in PDXs. Collectively, our data indicate that oHSVs effectively target high-risk neuroblastoma, and support the advancement of this therapy to the clinical setting.

Patient-derived xenografts provide significant advantages over long-term passage cell lines when investigating efficacy of treatments for solid tumors. Our laboratory encountered a high-grade, metastatic, neuroendocrine-like tumor from a pediatric patient that presented with a unique genetic profile. In particular, mutations in TYRO3 and ALK were identified. We established a human patient-derived xenoline (PDX) of this tumor for use in the current study. We investigated the effect of crizotinib, a chemotherapeutic known to effectively target both TYRO3 and ALK mutations. Crizotinib effectively decreased viability, proliferation, growth, and the metastatic properties of the PDX tumor through downregulation of STAT3 signaling, but expression of PDGFRß was increased. Sunitinib is a small molecule inhibitor of PDGFRß and was studied in this PDX independently and in combination with crizotinib. Sunitinib alone decreased viability, proliferation, and growth in vitro and decreased tumor growth in vivo. In combination, sunitinib was able to overcome potential crizotinib-induced resistance through downregulation of ERK 1/2 activity and PDGFRß receptor expression; consequently, tumor growth was significantly decreased both in vitro and in vivo. Through the use of the PDX, it was possible to identify crizotinib as a less effective therapeutic for this tumor and suggest that targeting PDGFRß would be more effective. These findings may translate to other solid tumors that present with the same genetic mutations., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier Inc.)

Introduction: The poor therapeutic efficacy seen with current treatments for neuroblastoma may be attributed to stem cell-like cancer cells (SCLCCs), a subpopulation of cancer cells associated with poor prognosis and disease recurrence. Retinoic acid (RA) is a differentiating agent used as maintenance therapy for high-risk neuroblastoma but nearly half of children treated with RA relapse. We hypothesized that 6-Methyl-UAB30 (6-Me), a second-generation rexinoid recently developed with a favorable toxicity profile compared to RA, would reduce cancer cell stemness in human neuroblastoma patient-derived xenografts (PDXs)., Methods: Cells from three neuroblastoma PDXs were treated with 6-Me and proliferation, viability, motility, and cell-cycle progression were assessed. CD133 expression, sphere formation, and mRNA abundance of stemness and differentiation markers were evaluated using flow cytometry, in vitro extreme limiting dilution analysis, and real-time PCR, respectively., Results: Treatment with 6-Me decreased proliferation, viability, and motility, and induced cell-cycle arrest and differentiation in all three neuroblastoma PDXs. In addition, 6-Me treatment led to decreased CD133 expression, decreased sphere-forming ability, and decreased mRNA abundance of Oct4, Nanog, and Sox2, indicating decreased cancer cell stemness., Conclusions: 6-Me decreased oncogenicity and reduced cancer cell stemness of neuroblastoma PDXs, warranting further exploration of 6-Me as potential novel therapy for neuroblastoma., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript., (Copyright © 2021. Published by Elsevier Inc.)

Background: Novel therapies are needed for patients with hepatoblastoma because of an increasing incidence of disease and poor prognosis for advanced, refractory, and recurrent disease. PIM kinases promote tumorigenesis in hepatoblastoma. A novel PIM inhibitor, PIM447, has shown promise in inhibiting oncogenesis in hematologic and lymphoid malignancies. We hypothesized that PIM inhibition with PIM447 would result in decreased tumorigenesis in hepatoblastoma., Methods: The effects of PIM447 on hepatoblastoma viability, proliferation, motility, apoptosis, and tumor cell stemness were assessed in HuH6, a human hepatoblastoma cell line, and COA67, a human hepatoblastoma patient-derived xenograft., Results: PIM447 significantly decreased the viability, proliferation, and motility of HuH6 and COA67 cells. Apoptosis significantly increased following PIM447 treatment. PIM447 had a significant impact on tumor cell stemness as evidenced by decreased expression of CD133 and reduced ability of HuH6 and COA67 cells to form tumorspheres. Furthermore, combining PIM447 with cisplatin resulted in a significant decrease in cell viability compared to either treatment alone., Conclusion: We showed that PIM447 inhibits oncogenesis and potentiates the effects of cisplatin in hepatoblastoma and, therefore, warrants further investigation as a potential therapeutic agent for hepatoblastoma., (Copyright © 2021. Published by Elsevier Inc.)

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., (© 2021 Wiley Periodicals LLC.)

Despite increasing incidence, treatment for hepatoblastoma has not changed significantly over the past 20 years. Chemotherapeutic strategies continue to rely on cisplatin, as it remains the most active single agent against hepatoblastoma. However, chemoresistance remains a significant challenge with 54-80% of patients developing resistance to chemotherapy after 4-5 cycles of treatment. Stem cell-like cancer cells (SCLCCs) are a subset of cells thought to play a role in chemoresistance and disease recurrence. We have previously demonstrated that Proviral Integration site for Moloney murine leukemia virus (PIM) kinases, specifically PIM3, play a role in hepatoblastoma cell proliferation and tumor growth and maintain the SCLCC phenotype. Here, we describe the development of a cisplatin-resistant hepatoblastoma xenograft model of the human HuH6 cell line and a patient-derived xenograft, COA67. We provide evidence that these cisplatin-resistant cells are enriched for SCLCCs and express PIM3 at higher levels than cisplatin-naïve cells. We demonstrate that PIM inhibition with AZD1208 sensitizes cisplatin-resistant hepatoblastoma cells to cisplatin, enhances cisplatin-mediated apoptosis, and decreases the SCLCC phenotype seen with cisplatin resistance. Together, these findings indicate that PIM inhibition may be a promising adjunct in the treatment of hepatoblastoma to effectively target SCLCCs and potentially decrease chemoresistance and subsequent disease relapse.

Investigation of the mechanisms responsible for aggressive neuroblastoma and its poor prognosis is critical to identify novel therapeutic targets and improve survival. Enhancer of Zeste Homolog 2 (EZH2) is known to play a key role in supporting the malignant phenotype in several cancer types and knockdown of EZH2 has been shown to decrease tumorigenesis in neuroblastoma cells. We hypothesized that the EZH2 inhibitor, GSK343, would affect cell proliferation and viability in human neuroblastoma. We utilized four long-term passage neuroblastoma cell lines and two patient-derived xenolines (PDX) to investigate the effects of the EZH2 inhibitor, GSK343, on viability, motility, stemness and in vivo tumor growth. Immunoblotting confirmed target knockdown. Treatment with GSK343 led to significantly decreased neuroblastoma cell viability, migration and invasion, and stemness. GSK343 treatment of mice bearing SK-N-BE(2) neuroblastoma tumors resulted in a significant decrease in tumor growth compared to vehicle-treated animals. GSK343 decreased viability, and motility in long-term passage neuroblastoma cell lines and decreased stemness in neuroblastoma PDX cells. These data demonstrate that further investigation into the mechanisms responsible for the anti-tumor effects seen with EZH2 inhibitors in neuroblastoma cells is warranted., Competing Interests: The authors have declared that no competing interests exist.

Retinoic acid (RA) therapy has been utilized as maintenance therapy for high-risk neuroblastoma, but over half of patients treated with RA relapse. Neuroblastoma stem cell-like cancer cells (SCLCCs) are a subpopulation of cells characterized by the expression of the cell surface marker CD133 and are hypothesized to contribute to drug resistance and disease relapse. A novel rexinoid compound, 9-cis-UAB30 (UAB30), was developed having the same anti-tumor effects as RA but a more favorable toxicity profile. In the current study, we investigated the efficacy of UAB30 in neuroblastoma patient-derived xenografts (PDX). Two PDXs, COA3 and COA6, were utilized and alterations in the malignant phenotype were assessed following treatment with RA or UAB30. UAB30 significantly decreased proliferation, viability, and motility of both PDXs. UAB30 induced cell-cycle arrest as demonstrated by the significant increase in percentage of cells in G1 (COA6: 33.7 ± 0.7 vs. 43.3 ± 0.7%, control vs. UAB30) and decrease in percentage of cells in S phase (COA6: 44.7 ± 1.2 vs. 38.6 ± 1%, control vs. UAB30). UAB30 led to differentiation of PDX cells, as evidenced by the increase in neurite outgrowth and mRNA abundance of differentiation markers. CD133 expression was decreased by 40% in COA6 cells after UAB30. The ability to form tumorspheres and mRNA abundance of known stemness markers were also significantly decreased following treatment with UAB30, further indicating decreased cancer cell stemness. These results provide evidence that UAB30 decreased tumorigenicity and cancer cell stemness in neuroblastoma PDXs, warranting further exploration as therapy for high-risk neuroblastoma., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The role of adjuvant radiotherapy (RT) remains unclear in patients with localized, completely resected (group I) alveolar rhabdomyosarcoma (ARMS)., Procedure: Patients with group I ARMS enrolled on any one of three prior Children's Oncology Group (COG) clinical trials (D9602, D9803, or ARST0531) were analyzed. All patients received systemic chemotherapy and 36 Gy adjuvant RT (if given) to the primary site at week 12 or week 4 for D9602/D9803 and ARST0531, respectively., Results: Thirty-six patients with group I ARMS were treated on D9602 (n = 6), D9803 (n = 17), or ARST0531 (n = 13), of whom 24 (67%) were male. The median age was 4.1 years (range, 0.8-45.8). Twenty (56%) patients had an unfavorable primary site, and 10 (28%) had tumors > 5 cm. FOXO1-fusion status was negative, positive, and unknown in 10 (28%), 15 (42%), and 11 (30%) tumors, respectively. Twenty-two (61%) patients received RT. Overall, the four-year event-free survival (EFS) and overall survival (OS) were 70.8% and 88.3%, respectively. Patients with FOXO1 positivity who received RT had superior EFS compared with those who did not (77.8% vs 16.7%; P = 0.03). Among 10 patients who were FOXO1 negative, the outcome was similar with or without RT., Conclusions: Although limited by a small sample size, data from this study support the routine use of adjuvant RT in patients with FOXO1-positive disease even after complete resection. Additionally, omitting adjuvant RT is rational for patients with FOXO1-negative ARMS and will be prospectively investigated in the current COG trial ARST1431., (© 2020 Wiley Periodicals, Inc.)

Background: Patient derived xenografts (PDXs) provide a unique opportunity for investigators to study tumor cell activity, response to therapeutics, and resistance patterns without exposing the human patient to experimental compounds, and thereby play a crucial role in pre-clinical evaluation of new therapies. It has been reported that PDXs may undergo a transformation to lymphoma, most commonly associated with Epstein Barr virus (EBV). If the character of a xenograft becomes compromised and remains undetected, it could have a detrimental impact on the research community as a whole. Our lab has established a number of pediatric solid tumor PDXs which accurately recapitulate the human tumors following several passages. One particular neuroblastoma PDX was noted to grow quickly and with an unusual phenotype, leading us to hypothesize that this PDX had undergone a transformation., Methods: The PDX in question was investigated with histology, immunohistochemistry (IHC), EBER in situ hybridization, and PCR to determine its identity., Results: Histology on the tumor revealed a small, round blue cell tumor similar to the original neuroblastoma from which it was derived. IHC staining showed that the tumor was composed of lymphocytes that were CD3 positive, <5% CD4 positive, and CD20 negative. The cells were Epstein Barr virus negative. PCR demonstrated that the tumor was human and not murine in origin., Conclusion: These findings indicate that a human T Cell lymphoma developed in place of this neuroblastoma PDX. Changes in PDX identity such as this one will significantly impact studies utilizing pediatric PDXs and the mechanism by which this occurred warrants further investigation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Purpose: In spite of advances in therapy for some subtypes, group 3 medulloblastoma continues to portend a poor prognosis. A subpopulation of medulloblastoma cells expressing the cell surface marker CD133 have been posited as possible stem cell like cancer cells (SCLCC), a potential source of drug resistance and relapse. Retinoids have been shown to affect SCLCC in other brain tumors. Based on these findings, we hypothesized that the CD133-enriched cell population group 3 medulloblastoma cells would be sensitive to the novel rexinoid, UAB30., Methods: Human medulloblastoma cell lines were studied. Cell sorting based on CD133 expression was performed. Both in vitro and in vivo extreme limiting dilution assays were completed to establish CD133 as a SCLCC marker in these cell lines. Cells were treated with either retinoic acid (RA) or UAB30 and sphere forming capacity and CD133 expression were assessed. Immunoblotting was used to assess changes in stem cell markers. Finally, mice injected with CD133-enriched or CD133-depleted cells were treated with UAB30., Results: CD133-enriched cells more readily formed tumorspheres in vitro at lower cell concentrations and formed tumors in vivo at low cell numbers. Treatment with RA or UAB30 decreased CD133 expression, decreased tumorsphere formation, and decreased expression of cancer stem cell markers. In vivo studies demonstrated that tumors from both CD133-enriched and CD133-depleted cells were sensitive to treatment with UAB30., Conclusions: CD133 is a marker for medulloblastoma SCLCCs. Both CD133-enriched and CD133-depleted medulloblastoma cell populations demonstrated sensitivity to UAB30, indicating its potential as a therapeutic option for group 3 medulloblastoma., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Aggressive therapies for patients with metastatic Wilms tumor (WT) with subsequent severe late effects warrant the search for novel therapies. The role of focal adhesion kinase (FAK), a non-receptor tyrosine kinase important in pediatric solid tumor development and progression, has not been examined in metastatic WT. Using a novel patient-derived xenograft (PDX) of a primary and matched, isogenic, metastatic WT, the hypothesis of the current study was that FAK would contribute to metastatic WT and small molecule inhibition would decrease tumor growth. Immunohistochemical staining, immunoblotting, cell viability and proliferation assays, cell cycle analysis, and cellular motility and attachment-independent growth assays were performed. FAK was present and phosphorylated in both WT PDXs and in the human samples from which they were derived. FAK inhibition decreased cellular survival, proliferation, and cell cycle progression in both PDXs but only significantly decreased migration, invasion, and attachment-independent growth in the primary WT PDX. Kinomic profiling revealed that platelet-derived growth factor receptor beta (PDGFRβ) may be affected by FAK inhibition in WT. Pharmacologic inhibition of FAK and PDGFRβ was synergistic in primary WT PDX cells. These findings broaden the knowledge of metastatic WT and support further investigations on the potential use of FAK and PDGFRβ inhibitors., Competing Interests: CONFLICTS OF INTEREST The authors declare no potential conflicts of interest., (Copyright © 2019 Aye et al.)

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.

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 survival. PIM3 warrants investigation as a therapeutic target and prognostic marker for hepatoblastoma., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist.

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., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

High-risk neuroblastoma continues to carry a poor prognosis. Nearly 50% of these tumors relapse following extensive treatment regimens. Protein phosphatase 2A (PP2A), a tumor suppressor, has been shown to be downregulated in many human cancers via multiple mechanisms including upregulation of its endogenous inhibitors, I2PP2A or CIP2A. We hypothesized that inhibition of the endogenous PP2A inhibitors or activation of PP2A would decrease tumorigenicity in human neuroblastoma cells. Four human neuroblastoma cell lines were utilized. Expression of PP2A and its endogenous inhibitors I2PP2A and CIP2A was confirmed by immunoblotting. PP2A activation was measured via phosphatase activation assay. Multiple parallel methods including siRNA inhibition of the endogenous PP2A inhibitors and pharmacologic activation of PP2A were utilized. Cell viability, proliferation, migration, and invasion assays were performed. In vivo studies were utilized to determine the effects of PP2A activation on neuroblastoma tumor growth. Inhibition of the endogenous inhibitors of PP2A or pharmacologic activation of PP2A with the PP2A activator FTY720 led to decreased neuroblastoma cell viability, proliferation, migration, and invasion. Treatment of mice bearing SK-N-AS or SK-N-BE(2) neuroblastoma tumors with FTY720 resulted in a significant decrease in tumor growth compared to vehicle-treated animals. In conclusion, activation of PP2A may provide a novel therapeutic target for neuroblastoma., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Group 3 tumors account for approximately 25-30% of medulloblastomas and have the worst prognosis. UAB30 is a novel synthetic rexinoid shown to have limited toxicities in humans and significant efficacy in the pediatric neuroectodermal tumor, neuroblastoma. We hypothesized that treatment with UAB30 would decrease tumorigenicity in medulloblastoma patient-derived xenografts (PDXs)., Methods: Three group 3 medulloblastoma PDXs (D341, D384 and D425) were utilized. Cell viability, proliferation, migration and invasion assays were performed after treatment with UAB30 or 13-cis-retinoic acid (RA). Cell cycle analysis was completed using flow cytometry. A flank model, a cerebellar model, and a model of leptomeningeal metastasis using human medulloblastoma PDX cells was used to assess the in vivo effects of UAB30 and RA., Results: UAB30 treatment led to cell differentiation and decreased medulloblastoma PDX cell viability, proliferation, migration and invasion and G1 cell cycle arrest in all three PDXs similar to RA. UAB30 and RA treatment of mice bearing medulloblastoma PDX tumors resulted in a significant decrease in tumor growth and metastasis compared to vehicle treated animals., Conclusions: UAB30 decreased viability, proliferation, and motility in group 3 medulloblastoma PDX cells and significantly decreased tumor growth in vivo in a fashion similar to RA, suggesting that further investigations into the potential therapeutic application of UAB30 for medulloblastoma are warranted.

Group 3 tumors account for 28% of medulloblastomas and have the worst prognosis. FTY720, an immunosuppressant currently approved for treatment of multiple sclerosis, has shown antitumor effects in several human cancer cell lines. We hypothesized that treatment with FTY720 (fingolimod) would decrease tumorigenicity in medulloblastoma patient-derived xenografts (PDXs). Three Group 3 medulloblastoma PDXs (D341, D384 and D425) were utilized. Expression of PP2A and its endogenous inhibitors I2PP2A and CIP2A was detected by immunohistochemistry and immunoblotting. PP2A activation was measured via phosphatase activation kit. Cell viability, proliferation, migration and invasion assays were performed after treatment with FTY720. Cell cycle analysis was completed using flow cytometry. A flank model using D425 human medulloblastoma PDX cells was used to assess the in vivo effects of FTY720. FTY720 activated PP2A and led to decreased medulloblastoma PDX cell viability, proliferation, migration and invasion and G1 cell cycle arrest in all three PDXs. FTY720 treatment of mice bearing D425 medulloblastoma PDX tumors resulted in a significant decrease in tumor growth compared to vehicle treated animals. FTY720 decreased viability, proliferation, and motility in Group 3 medulloblastoma PDX cells and significantly decreased tumor growth in vivo. These results suggest that FTY720 should be investigated further as a potential therapeutic agent for medulloblastoma.

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., Competing Interests: CONFLICTS OF INTEREST The authors have no conflicts of interest to disclose.