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Leptomeningeal dissemination (LMD) is the primary cause of treatment failure in children with group 3 medulloblastoma (MB). Building on our previous work on protein phosphatase 2A (PP2A) activation in MB, here we present preclinical and molecular data on the effects of two novel classes of PP2A activators on disease processes of LMD in group 3 MB. The PP2A activators used in this study are ATUX-6156 and ATUX-6954 (diarylmethylcycloamine sulfonylureas), and ATUX-1215 and ATUX-5800 (diarylmethyl-4-aminotetrahydropyran-sulfonamides). Treatment with these compounds led to suppression of the endogenous PP2A inhibitor, cancerous inhibitor of PP2A (CIP2A), enhanced phosphatase activity (10-60%), and reduced MB viability, migration, and invasion, prerequisites for MB cells to access the cerebrospinal fluid, affecting the initiation stage of LMD. PP2A activator treatment of MB cells led to apoptosis mediated via caspase 9/PARP signaling due to decreased phosphorylation of Bad, impeding the dispersal stage of LMD. Cell proliferation and LMD-driving cellular traits and molecules pertinent to the third stage, colonization, were also affected. Treatment with ATUX-1215 or ATUX-5800 prevented LMD in an intraventricular murine model of MB, possibly mediated by disruption of the CCL2-CCR2 axis by altered NF-kB phosphorylation via disrupted AKT signaling. The present investigation offers proof-of-principle data for PP2A-based reactivation therapy for Group 3 MB and provides the first indications that PP2A reactivation may challenge the current paradigm in targeting the 3-stage process of MB LMD. Further investigations of PP2A activators are warranted as these compounds may prove beneficial as therapeutics for MB., Competing Interests: Conflict of interest Patent applications WO2023/023594 and WO2021/188949, inventor MO, are assigned to Atux Iskay LLC. The other authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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.)

Background: Chemoresistance contributes to relapse in high-risk neuroblastoma. Cancer cells acquire resistance through multiple mechanisms, including drug efflux pumps. In neuroblastoma, multidrug resistance-associated protein-1 (MRP1/ABCC1) efflux pump expression correlates with worse outcomes. These pumps are regulated by PIM kinases, a family of serine-threonine kinases, overexpressed in neuroblastoma. We hypothesized PIM kinase inhibition would sensitize neuroblastoma cells by modulating MRP1., Methods: Kocak database query evaluated ABCC1, PIM1, PIM2, and PIM3 expression in neuroblastoma patients. SK-N-AS and SK-N-BE(2) cells were treated with doxorubicin or the pan-PIM kinase inhibitor, AZD1208. Flow cytometry assessed intracellular doxorubicin accumulation. AlamarBlue assay measured viability. The lethal dose 50% (LD 50 ) of each drug and combination indices (CI) were calculated and isobolograms constructed to determine synergy., Results: Kocak database query demonstrated positive correlation between PIM genes and ABCC1. PIM kinase inhibition increased intracellular doxorubicin accumulation in both cell lines, suggesting PIM kinase regulation of MRP1. Isobolograms showed synergy between AZD1208 and doxorubicin., Conclusions: The correlation between PIM and ABCC1 gene expression suggests PIM kinases may contribute to neuroblastoma chemotherapeutic resistance. PIM kinase inhibition increased intracellular doxorubicin accumulation. Combination treatment with AZD1208 and doxorubicin decreased neuroblastoma cell viability in a synergistic fashion. These findings support further investigations of PIM kinase inhibition in neuroblastoma., Type of Study: Basic Science Research., Level of Evidence: NA., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Sarcomas are a heterogenous collection of bone and soft tissue tumors. The heterogeneity of these tumors makes it difficult to standardize treatment. CDK 4/6 inhibitors are a family of targeted agents which limit cell cycle progression and have been shown to be upregulated in sarcomas. In the current preclinical study, we evaluated the effects of lerociclib, a CDK4/6 inhibitor, on pediatric sarcomas in vitro and in 3D bioprinted tumors., Methods: The effects of lerociclib on viability, proliferation, cell cycle, motility, and stemness were assessed in established sarcoma cell lines, U-2 OS and MG-63, as well as sarcoma patient-derived xenografts (PDXs). 3D printed biotumors of each of the U-2 OS, MG-63, and COA79 cells were utilized to study the effects of lerociclib on tumor growth ex vivo., Results: CDK 4/6, as well as the intermediaries retinoblastoma protein (Rb) and phosphorylated Rb were identified as targets in the four sarcoma cell lines. Lerociclib treatment induced cell cycle arrest, decreased proliferation, motility, and stemness of sarcoma cells. Treatment with lerociclib decreased sarcoma cell viability in both traditional 2D culture as well as 3D bioprinted microtumors., Conclusions: Inhibition of CDK 4/6 activity with lerociclib was efficacious in traditional 2D sarcoma cell culture as well as in 3D bioprints. Lerociclib holds promise and warrants further investigation as a novel therapeutic strategy for management of these heterogenous groups of tumors., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Neuroblastoma arises from aberrancies in neural stem cell differentiation. PIM kinases contribute to cancer formation, but their precise role in neuroblastoma tumorigenesis is poorly understood. In the current study, we evaluated the effects of PIM kinase inhibition on neuroblastoma differentiation., Methods: Versteeg database query assessed the correlation between PIM gene expression and the expression of neuronal stemness markers and relapse free survival. PIM kinases were inhibited with AZD1208. Viability, proliferation, motility were measured in established neuroblastoma cells lines and high-risk neuroblastoma patient-derived xenografts (PDXs). qPCR and flow cytometry detected changes in neuronal stemness marker expression after AZD1208 treatment., Results: Database query showed increased levels of PIM1, PIM2, or PIM3 gene expression were associated with higher risk of recurrent or progressive neuroblastoma. Increased levels of PIM1 were associated with lower relapse free survival rates. Higher levels of PIM1 correlated with lower levels of neuronal stemness markers OCT4, NANOG, and SOX2. Treatment with AZD1208 resulted in increased expression of neuronal stemness markers., Conclusions: Inhibition of PIM kinases differentiated neuroblastoma cancer cells toward a neuronal phenotype. Differentiation is a key component of preventing neuroblastoma relapse or recurrence and PIM kinase inhibition provides a potential new therapeutic strategy for this disease., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: The tumor suppressor, protein phosphatase 2A (PP2A), is downregulated in hepatoblastoma. We aimed to examine the effects of two novel compounds of the tricyclic sulfonamide class, ATUX-3364 (3364) and ATUX-8385 (8385), designed to activate PP2A without causing immunosuppression, on human hepatoblastoma., Methods: An established human hepatoblastoma cell line, HuH6, and a human hepatoblastoma patient-derived xenograft, COA67, were treated with increasing doses of 3364 or 8385, and viability, proliferation, cell cycle and motility were investigated. Cancer cell stemness was evaluated by real-time PCR and tumorsphere forming ability. Effects on tumor growth were examined using a murine model., Results: Treatment with 3364 or 8385 significantly decreased viability, proliferation, cell cycle progression and motility in HuH6 and COA67 cells. Both compounds significantly decreased stemness as demonstrated by decreased abundance of OCT4, NANOG, and SOX2 mRNA. The ability of COA67 to form tumorspheres, another sign of cancer cell stemness, was significantly diminished by 3364 and 8385. Treatment with 3364 resulted in decreased tumor growth in vivo., Conclusion: Novel PP2A activators, 3364 and 8385, decreased hepatoblastoma proliferation, viability, and cancer cell stemness in vitro. Animals treated with 3364 had decreased tumor growth. These data provide evidence for further investigation of PP2A activating compounds as hepatoblastoma therapeutics., Competing Interests: Conflicts of interest MO is the inventor of ATUX-3364 and ATUX-8385 and the CEO of Atux Iskay, LLC. The other authors declare no competing financial interests in relation to the work described., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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.)

Patients presenting with metastatic hepatoblastoma have limited treatment options and survival rates as low as 25%. We previously demonstrated that Proviral Integration site in Maloney murine leukemia virus 3 (PIM3) kinase promotes tumorigenesis and cancer cell stemness in hepatoblastoma. In this study, we assessed the role of PIM3 kinase in promoting hepatoblastoma metastasis. We utilized a tail vein injection model of metastasis to evaluate the effect of CRISPR/Cas9-mediated PIM3 knockout, stable overexpression of PIM3, and pharmacologic PIM inhibition on the formation of lung metastasis. In vivo studies revealed PIM3 knockout impaired the formation of lung metastasis: 5 out of 6 mice injected with wild type hepatoblastoma cells developed lung metastasis while none of the 7 mice injected with PIM3 knockout hepatoblastoma cells developed lung metastasis. PIM3 overexpression in hepatoblastoma increased the pulmonary metastatic burden in mice and mechanistically, upregulated the phosphorylation and cell surface expression of CXCR4, a key receptor in the progression of cancer cell metastasis. CXCR4 blockade with AMD3100 decreased the metastatic phenotype of PIM3 overexpressing cells, indicating that CXCR4 contributed to PIM3's promotion of hepatoblastoma metastasis. Clinically, PIM3 expression correlated positively with CXCR4 expression in primary hepatoblastoma tissues. In conclusion, we have shown PIM3 kinase promotes the metastatic phenotype of hepatoblastoma cells through upregulation of CXCR4 cell surface expression and these findings suggest that targeting PIM3 kinase may provide a novel therapeutic strategy for metastatic hepatoblastoma., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Cancer continues to be a significant cause of non-traumatic pediatric mortality. Diagnosis of pediatric solid tumors is paramount to prescribing the correct treatment regimen. Recent efforts have focused on non-invasive methods to obtain tumor tissues, but one of the challenges encountered is the ability to obtain an adequate amount of viable tissue. In this study, a wireless, inductor-capacitor (LC) sensor was employed to detect relative permittivity of pediatric tumor tissues. There is a comparison of resonant frequencies of tumor tissues between live versus dead tissues, the primary tumor tissue versus tissue from the organs of origin or metastasis, and treated versus untreated tumors. The results show significant shifts in resonant frequencies between the comparison groups. Dead tissues demonstrated a significant shift in resonant frequencies compared to alive tissues. There were significant differences between the resonant frequencies of normal tissues versus tumor tissues. Resonant frequencies were also significantly different between primary tumors compared to their respective metastases. These data indicate that there are potential clinical applications of LC technology in the detection and diagnosis of pediatric solid tumors., Competing Interests: The authors declare the following competing financial interest(s): Michael Ohlmeyer is the inventor of Atux-792 and is the founder of and vice president of chemistry at Atux-Iskay, LLC. The other authors have nothing to declare., (© 2022 The Authors. Published by American Chemical Society.)

Background/purpose: Metastatic hepatoblastoma continues to pose a significant treatment challenge, primarily because the precise mechanisms involved in metastasis are not fully understood, making cell lines and preclinical models that depict the progression of disease and metastasis-related biology paramount. We aimed to generate and characterize a metastatic hepatoblastoma cell line to create a model for investigation of the molecular mechanisms associated with metastasis., Materials/methods: Using a murine model of serial tail vein injections of the human hepatoblastoma HuH6 cell line, non-invasive bioluminescence imaging, and dissociation of metastatic pulmonary lesions, we successfully established and characterized the metastatic human hepatoblastoma cell line, HLM_3., Results: The HLM_3 cells exhibited enhanced tumorigenicity and invasiveness, both in vitro and in vivo compared to the parent HuH6 cell line. Moreover, HLM_3 metastatic hepatoblastoma cells exhibited a stem cell-like phenotype and were more resistant to the standard chemotherapeutic cisplatin., Conclusion: This newly described metastatic hepatoblastoma cell line offers a novel tool to study mechanisms of tumor metastasis and evaluate new therapeutic strategies for metastatic hepatoblastoma., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Serine-threonine kinase receptor associated protein (STRAP), a scaffolding protein, is upregulated in many solid tumors. As such, we hypothesized that STRAP may be overexpressed in neuroblastoma tumors and may play a role in neuroblastoma tumor progression., Methods: We examined two publicly available neuroblastoma patient databases, GSE49710 (n = 498) and GSE49711 (n = 498), to investigate STRAP expression in human specimens. SK-N-AS and SK-N-BE(2) human neuroblastoma cell lines were stably transfected with STRAP overexpression (OE) plasmid, and their resulting phenotype studied. PamChip® kinomic peptide microarray evaluated the effects of STRAP overexpression on kinase activation., Results: In human specimens, higher STRAP expression correlated with high-risk disease, unfavorable histology, and decreased overall neuroblastoma patient survival. STRAP OE in neuroblastoma cell lines led to increased proliferation, growth, supported a stem-like phenotype and activated downstream FAK targets. When FAK was targeted with the small molecule FAK inhibitor, PF-573,228, STRAP OE neuroblastoma cells had significantly decreased growth compared to control empty vector cells., Conclusion: Increased STRAP expression in neuroblastoma was associated with unfavorable tumor characteristics. STRAP OE resulted in increased kinomic activity of FAK. These findings suggest that the poorer outcomes in neuroblastoma tumors associated with STRAP overexpression may be secondary to FAK activation., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Hepatoblastoma remains one of the most difficult childhood tumors to treat and is alarmingly understudied. We previously demonstrated that Proviral Insertion site in Maloney murine leukemia virus (PIM) kinases, specifically PIM3, are overexpressed in human hepatoblastoma cells and function to promote tumorigenesis. We aimed to use CRISPR/Cas9 gene editing with dual gRNAs to introduce large inactivating deletions in the PIM3 gene and achieve stable PIM3 knockout in the human hepatoblastoma cell line, HuH6. PIM3 knockout of hepatoblastoma cells led to significantly decreased proliferation, viability, and motility, inhibited cell-cycle progression, decreased tumor growth in a xenograft murine model, and increased animal survival. Analysis of RNA sequencing data revealed that PIM3 knockout downregulated expression of pro-migratory and pro-invasive genes and upregulated expression of genes involved in apoptosis and differentiation. Furthermore, PIM3 knockout decreased hepatoblastoma cancer cell stemness as evidenced by decreased tumorsphere formation, decreased mRNA abundance of stemness markers, and decreased cell surface expression of CD133, a marker of hepatoblastoma stem cell-like cancer cells. Reintroduction of PIM3 into PIM3 knockout cells rescued the malignant phenotype. Successful CRISPR/Cas9 knockout of PIM3 kinase in human hepatoblastoma cells confirmed the role of PIM3 in promoting hepatoblastoma tumorigenesis and cancer cell stemness., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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.)

Background: Serine-threonine kinase receptor-associated protein (STRAP) plays an important role in neural development but also in tumor growth. Neuroblastoma, a tumor of neural crest origin, is the most common extracranial solid malignancy of childhood and it continues to carry a poor prognosis. The recent discovery of the role of STRAP in another pediatric solid tumor, osteosarcoma, and the known function of STRAP in neural development, led us to investigate the role of STRAP in neuroblastoma tumorigenesis. Methods: STRAP protein expression was abrogated in two human neuroblastoma cell lines, SK-N-AS and SK-N-BE(2), using transient knockdown with siRNA, stable knockdown with shRNA lentiviral transfection, and CRISPR-Cas9 genetic knockout. STRAP knockdown and knockout cells were examined for phenotypic alterations in vitro and tumor growth in vivo. Results: Cell proliferation, motility, and growth were significantly decreased in STRAP knockout compared to wild-type cells. Indicators of stemness, including mRNA abundance of common stem cell markers Oct4, Nanog, and Nestin, the percentage of cells expressing CD133 on their surface, and the ability to form tumorspheres were significantly decreased in the STRAP KO cells. In vivo, STRAP knockout cells formed tumors less readily than wild-type tumor cells. Conclusion: These novel findings demonstrated that STRAP plays a role in tumorigenesis and maintenance of neuroblastoma stemness.

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.)

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.)

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.)

Primary pulmonary malignancies are rare in childhood. The most common, pleuropulmonary blastoma (PPB), has an incidence of 25-50 cases per year in the United States (Knight and et al., 2019) [1]. The majority of children are diagnosed with PPB before the age of four years. PPB is divided into subtypes I, Ir (type I-regressed), II, and III, which correlates to the age of diagnosis and patient prognosis [2,3]. Here we report an unusual presentation of PPB in a teen-aged female who presented with a one month history of a non-productive cough., 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.

Introduction: Retinoic acid (RA) is a differentiating agent utilized as maintenance therapy for high-risk neuroblastoma (NB), but associated toxicities limit its use. We have previously shown that a non-toxic, novel rexinoid, 9-cis-UAB30 (UAB30), decreased NB cell proliferation and in vivo tumor growth. A second generation, mono-methylated compound, 6-Methyl-UAB30 (6-Me), has been recently designed having greater potency compared with UAB30. In the current study, we hypothesized that 6-Me would inhibit NB cell proliferation and survival and induce differentiation and cell-cycle arrest., Methods: Proliferation and viability were measured in four human NB cell lines following treatment with UAB30 or 6-Me. Cell-cycle was analyzed and tumor cell stemness was evaluated with extreme limiting dilution assays and immunoblotting for expression of stem cell markers. A xenograft murine model was utilized to study the effects of 6-Me in vivo., Results: Treatment with 6-Me led to decreased proliferation and viability, induced cell cycle arrest, and increased neurite outgrowth, indicating differentiation of surviving cells. Furthermore, treatment with 6-Me decreased tumorsphere formation and expression of stem cell markers. Finally, inhibition of tumor growth and increased animal survival was observed in vivo following treatment with 6-Me., Conclusion: These results indicate a potential therapeutic role for this novel rexinoid in neuroblastoma treatment., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Malignant ovarian germ cell tumors (MOGCTs) are a rare form of ovarian malignancy. Socioeconomic status (SES) has been shown to affect survival in several gynecologic cancers. We examined whether SES impacted survival in adolescent and young adults (AYAs) with MOGCT., Materials and Methods: The National Cancer Data Base was used to identify AYAs (aged 15-39 years) with MOGCT from 1998 to 2012. Three SES surrogate variables identified were as follows: insurance type, income quartile, and education quartile. Pooled variance t-tests and chi-square tests were used to compare tumor characteristics, the time from diagnosis to staging/treatment, and clinical outcome variables for each SES surrogate variable, while controlling for age and race/ethnicity in a multivariate model. Kaplan-Meier survival estimates were calculated using the log-rank test., Results: A total of 3125 AYAs with MOGCT were identified. Subjects with lower SES measures had higher overall stage and T-stage MOGCTs at presentation. There was no significant difference in the time to staging/treatment, extent of surgery, or use of chemotherapy by SES. Subjects from a lower education background, from a lower income quartile, and without insurance had decreased survival (P ≤ 0.02 for all). Controlling for overall stage and T-stage, the difference in survival was no longer significant., Conclusions: AYAs with MOGCT from lower SES backgrounds presented with more advanced stage disease. Further studies that focus on the underlying reasons for this difference are needed to address these disparities., (Copyright © 2017 Elsevier Inc. All rights reserved.)