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Medulloblastoma is the most common malignant brain tumor in children. This disease is heterogeneous and is composed of four subtypes of medulloblastoma [WNT, Sonic Hedgehog (SHH), Group 3, and Group 4]. An immediate goal is to identify novel molecular targets for the most aggressive forms of medulloblastoma. Polo-like kinase 1 (PLK1) is an oncogenic kinase that controls cell cycle and proliferation, making it a strong candidate for medulloblastoma treatment. In this study, pediatric medulloblastomas were subtyped in two patient cohorts (discovery cohort, n = 63 patients; validation cohort, n = 57 patients) using NanoString nCounter analysis and PLK1 mRNA was assessed. We determined that the SHH and Group 3 subtypes were independently associated with poor outcomes in children as was PLK1 using Cox regression analyses. Furthermore, we screened a library of 129 compounds in clinical trials using a model of pediatric medulloblastoma and determined that PLK1 inhibitors were the most promising class of agents against the growth of medulloblastoma. In patient-derived primary medulloblastoma isolates, the PLK1 small-molecule inhibitor BI2536 suppressed the self-renewal of cells with high PLK1 but not low PLK1 expression. PLK1 inhibition prevented medulloblastoma cell proliferation, self-renewal, cell-cycle progression, and induced apoptosis. In contrast, the growth of normal neural stem cells was unaffected by BI2536. Finally, BI2536 extended survival in medulloblastoma-bearing mice with efficacy comparable with Headstart, a standard-of-care chemotherapy regimen. We conclude that patients with medulloblastoma expressing high levels of PLK1 are at elevated risk. These preclinical studies pave the way for improving the treatment of medulloblastoma through PLK1 inhibition. Cancer Res; 73(22); 6734–44. ©2013 AACR.

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PDF file - 685K, Supplementary Table S1. Summary of the pediatric MB patients included in the study Discovery cohort. Supplementary Table S2. Summary of the pediatric MB patients included in the study Validation cohort. Supplementary Table S3. PAM class prediction validation for MB subgroup assignment of Discovery cohort and cultured cells. Supplementary Table S4. PAM class prediction validation for MB subgroup assignment of Validation cohort. Supplementary Table S5. Univariate and multivariate analyses of clinical, pathological and biological endpoints in the Validation cohort. Supplementary Figure S1. MB survival in Validation cohort is stratified by molecular subtype. Supplementary Figure S2. PLK1 is invariably expressed between patient cohorts and between MB subgroups. Supplementary Figure S3. BI2536 sensitivity depends on PLK1 expression in primary MB cells. Supplementary Figure S4. MB cells lines expressing PLK1 experience cell cycle arrest and reduced proliferation with PLK1 inhibition. Supplementary Figure S5. MB cell PLK1 inhibition morphology using immunofluorescence. Supplementary Figure S6. MB cell lines treated with anti-cancer drugs.

Atypical teratoid rhabdoid tumors (ATRT) are rare and aggressive embryonal tumors of central nervous system that typically affect children younger than 3 years of age. Given the generally poor outcomes of patients with ATRT and the significant toxicities associated with conventional multi-modal therapies, there is an urgent need for more novel approaches to treat ATRT, one such approach being immunotherapy. The recent rise of large-scale, multicenter interdisciplinary studies has delineated several molecular and genetic characteristics unique to ATRT. This review aims to describe currently available data on the tumor immune microenvironment of ATRT and its specific subtypes and to summarize the emerging clinical and preclinical results of immunotherapy-based approaches. It will also highlight the evolving knowledge of epigenetics on immunomodulation in this epigenetically influenced tumor, which may help guide the development of effective immunotherapeutic approaches in the future.

There is experimental and clinical data to indicate the contribution of immune-escape mechanisms in relapsed/refractory pediatric leukemia. Studies have shown the accumulation of mutations that translate to peptides containing tumor-specific epitopes (neoantigens). The effectiveness of neoantigen-based vaccines has been shown in several clinical trials in adults. Though the initial results are encouraging, this knowledge must be developed to account for the uniqueness of pediatric cancer biology. We have completed the initial proof-of-concept analysis on a high-risk pediatric leukemia specimen and identified usable neoantigen sequences. We describe this approach, including the bioinformatics method and experimental model to verify their function that can be further broadened for personalized neoantigen prediction and testing for the generation of anticancer vaccines against high-risk pediatric leukemias.

PURPOSE Retinoblastoma is the most frequent intraocular cancer in children. It is also one of the most common causes for enucleation and carries a significant morbidity rate in affected individuals. Hence, studies on its pathophysiological and growth regulatory mechanisms are urgently needed to identify more effective novel therapeutics. METHODS Using the Y79 retinoblastoma cell line, we investigated the electrophysiological and functional activities of the T-type voltage-gated calcium channel Cav3.1, that is constitutively expressed in these cells. We also analyzed the Akt and MAPK signaling pathways downstream of the epidermal growth factor receptor (EGFR) to understand the mechanism responsible for the inhibition of Cav3.1. RESULTS We demonstrate that the EGFR inhibitor Afatinib significantly reduced cell viability and Cav3.1 mRNA expression and electrophysiological activity. At low concentrations (1 µM), Afatinib reduced the amplitude of Cav3.1 current density, whereas at a high concentration (10 µM), it completely abolished the voltage-gated calcium current. Our results show that inhibition of the MAPK pathway by a specific inhibitor VX-11e affected the Cav3.1 current in a dose-dependent manner. VX-11e (50 nM-1 µM) treatment reduced Cav3.1 current densities in Y79 cells, with complete abolishment of Cav3.1 current at higher concentrations (5 µM). We also demonstrate that the specific inhibition of the Akt kinase (using MK-2206) had no effect on the Cav3.1 currents. CONCLUSION Our study provides a functional relationship between the MAPK pathway and EGFR signaling and indicates that the MAPK signaling pathway mediates the control of Cav3.1 by EGFR in retinoblastoma.

Background: Most children with recurrent metastatic solid tumors have high mortality rates. Recent studies have shown that proteasome inhibition leads to effective tumor killing in cells that have acquired treatment resistance and metastatic properties. Objective: The purpose of this study was to test the potential of Carfilzomib (CFZ), a proteasome inhibitor, in refractory pediatric solid tumors which is currently unknown. Methods: A panel of pediatric solid tumor cell lines, including neuroblastoma, Ewing’s sarcoma, osteosarcoma, rhabdomyosarcoma and atypical teratoid rhabdoid tumor (ATRT), was used to evaluate the cytotoxic and proteasomal inhibitory effects of CFZ. A drug scheduling experiment was performed to determine the optimal dose and time to obtain effective cell killing. Combination studies of CFZ with chemotherapeutic drugs of different classes were performed to determine the extent of synergy. Results: CFZ showed effective cytotoxicity against all cell lines tested (mean IC50 = 7nM, range = 1-20nM) and activity in a fluorophore-tagged cell-based proteasome assay. Drug scheduling experiments showed that the minimum exposure of 4-8 hours/day is needed for effective cumulative killing. CFZ, when combined with chemotherapeutic drugs of different classes, synergistically enhanced the extent of cell death. Conclusions: CFZ showed cytotoxic activity against all the solid pediatric cancer cell lines tested. This study provides initial in vitro data on the potential of CFZ to treat pediatric solid tumors and supports further investigations into the components of drug scheduling, biological correlates and drug combinations for future early phase clinical trials in children.

Objective: The function of the Ras association domain family 1 (RASSF1)/modulator of apoptosis 1 (MOAP-1) molecular tumor suppressor pathway is often perturbed in many solid and blood cancers by epigenetic loss of RASSF1A via promoter specific methylation. However, a detailed analysis of expression and stability of MOAP-1 as well as effect on cellular proliferation and cell death in blood cancers has not been explored. Materials and Methods: Expression of MOAP-1 RASSF1A was performed by immunoblotting analysis, MOAP-1 effect on biology determined by cell proliferation, cell death and tumorigenicity assays. Lastly, proteomic analysis in MOAP-1 in form 1 and 2 expressing cells reveal new interacting partners to MOAP-1. Results: The expression of MOAP-1 appears to be quite varied and exists as two forms, a p39 and p46 form in blood cancers. The higher MOAP-1 p46 form was mainly observed in acute myeloid leukemia (AML) and some acute lymphocytic leukemia (ALL) patients and cell lines. Furthermore, MOAP-1 p46 form can be reduced to the p39 form upon calf intestinal or lambda phosphatase treatment, suggesting post-translational phosphorylation likely produces the slower migrating form. Blood cancer cell lines containing the p46 form of MOAP-1 appears to be more resistant to cell death signals and have increased growth. In addition, we document in vivo protection from tumorigenesis of cells containing MOAP-1 following tail vein injection of cancer cells. Lastly, proteomic analysis revealed several interesting components of the MOAP-1 interactome in blood cancer cells that we validated. Conclusions: MOAP-1 is a bona fide tumor suppressor in blood cancers with functions beyond apoptosis.

Mutations in the CBL gene were first identified in adults with various myeloid malignancies. Some patients with juvenile myelomonocytic leukemia (JMML) were also noted to harbor mutations in CBL, but were found to have generally less aggressive disease courses compared to patients with other forms of Ras pathway-mutant JMML. Importantly, and in contrast to most reports in adults, the majority of CBL mutations in JMML patients are germline with acquired uniparental disomy occurring in affected marrow cells. Here, we systematically studied a large cohort of 33 JMML patients with CBL mutations and found that this disease is highly diverse in presentation and overall outcome. Moreover, we discovered somatically acquired CBL mutations in 15% of pediatric patients who presented with more aggressive disease. Neither clinical features nor methylation profiling were able to distinguish patients with somatic CBL mutations from those with germline CBL mutations, highlighting the need for germline testing. Overall, we demonstrate that disease courses are quite heterogeneous even among patients with germline CBL mutations. Prospective clinical trials are warranted to find ideal treatment strategies for this diverse cohort of patients.

Clinical trials completed in the last two decades have contributed significantly to the improved overall survival of children with cancer. In spite of these advancements, disease relapse still remains a significant cause of death in this patient population. Often, increasing the intensity of current protocols is not feasible because of cumulative toxicity and development of drug resistance. Therefore, the identification and clinical validation of novel targets in high-risk and refractory childhood malignancies are essential to develop effective new generation treatment protocols. A number of recent studies have shown that the hepatocyte growth factor (HGF) and its receptor Mesenchymal epithelial transition factor (c-MET) influence the growth, survival, angiogenesis, and metastasis of cancer cells. Therefore, the c-MET receptor tyrosine kinase and HGF have been identified as potential targets for cancer therapeutics and recent years have seen a race to synthesize molecules to block their expression and function. In this review we aim to summarize the literature that explores the potential and biological rationale for targeting the HGF/c-MET pathway in common and high-risk pediatric solid tumors. We also discuss selected recent and ongoing clinical trials with these agents in relapsed pediatric tumors that may provide applicable future treatments for these patients.

High-risk neuroblastoma is an aggressive pediatric tumor. Despite great advances in neuroblastoma therapy and supportive care protocols, no curative treatment is available for most patients with this disease. Here, we uncover that CBN attenuated the cell proliferation, invasion, and angiogenesis of neuroblastoma cell lines in a dose-dependent manner via the inhibition of the AKT pathway and the upregulation of miR-34a that targets E2F1. Both miR-34a and a 31-nt tRNAiMet fragment (tRiMetF31) derived from miR-34a-guided cleavage were downregulated in 4 examined neuroblastoma cell lines inversely correlated with the levels of its direct target, the PFKFB3 protein. Moreover, ectopic tRiMetF31 suppressed proliferation, migration, and angiogenesis in the studied neuroblastoma cell lines. Conversely, tRiMetF31 knockdown promoted PFKFB3 expression, resulting in enhanced angiogenesis. Our findings reveal a suppressive role of CBN in neuroblastoma tumorigenesis, highlighting a novel and crucial miR-34a tumor suppressor network in CBN’s antineuroblastoma actions.

ObjectiveTo examine the relationship between neonatal inflammatory cytokines and perinatal stroke using a systems biology approach analyzing serum and blood-spot cytokines from 47 patients.MethodsThis was a population-based, controlled cohort study with prospective and retrospective case ascertainment. Participants were recruited through the Alberta Perinatal Stroke Project. Stroke was classified as neonatal arterial ischemic stroke (NAIS), arterial presumed perinatal ischemic stroke (APPIS), or periventricular venous infarction (PVI). Biosamples were stored blood spots (retrospective) and acute serum (prospective). Controls had comparable gestational and maternal ages. Sixty-five cytokines were measured (Luminex). Hierarchical clustering analysis was performed to create heat maps. The Fisher linear discriminant analysis was used to create projection models to determine discriminatory boundaries between stroke types and controls.ResultsA total of 197 participants were analyzed (27 with NAIS, 8 with APPIS, 12 with PVI, 150 controls). Cytokines were quantifiable with quality control measures satisfied (standards testing, decay analysis). Linear discriminant analysis had high accuracy in using cytokine profiles to separate groups. Profiles in participants with PVI and controls were similar. NAIS separation was accurate (sensitivity 77%, specificity 97%). APPIS mapping was also distinguishable from NAIS (sensitivity 86%, specificity 99%). Classification tree analysis generated similar diagnostic accuracy.ConclusionsUnique inflammatory biomarker signatures are associated with specific perinatal stroke diseases. Findings support an acquired pathophysiology and suggest the possibility that at-risk pregnancies might be identified to develop prevention strategies.Classification of evidenceThis study provides Class III evidence that differences in acute neonatal serum cytokine profiles can discriminate between patients with specific perinatal stroke diseases and controls.

Central nervous system (CNS) atypical teratoid/rhabdoid tumor (AT/RT) is a rare, aggressive tumor that most often affects very young children. The common decisive molecular defect in AT/RT has been shown to be a single genetic alteration, i.e., the loss of hSNF5 gene that encodes for a subunit of the SWI/SNF complex that modulates chromatin remodeling activities. As a result, AT/RT cells display unregulated cell proliferation due to the dysfunction of an important epigenetic control. We have previously demonstrated the preclinical efficacy of the oncolytic double-deleted vaccinia virus (VVDD) against AT/RT. Here we report the establishment of a modified VVDD engineered to express wild type hSNF5 gene. We show that this reconstructed vaccinia virus retains comparable infectivity and in vitro cytotoxicity of the parent strain. However, in addition, hSNF5-arming of VVDD results in a decreased cell cycle S phase population and down-regulation of cyclin D1. These findings suggest that hSNF5-arming of VVDD may increase the efficacy in the treatment of AT/RT and validates, as a proof-of-concept, an experimental approach to enhance the effective use of novel modified oncolytic viruses in the treatment of tumors with loss of a tumor suppressor gene function.

Tumorigenesis, which involves the uncontrolled proliferation and differentiation of cells, has been observed to imitate a variety of pathways vital to embryonic development, motivating cancer researchers to explore the genetic origins of these pathways. The pluripotency gene regulatory network is an established collection of genes that induces stemness in embryonic cells. Dysregulation in the expression genes of the pluripotency gene networks including OCT4, SOX2, NANOG and REX1 have been implicated in tumor development, and have been observed to result in poorer patient outcomes. The p53 pathway is a highly important regulatory process in a multitude of cell types, including embryonic, and the tumor suppressor gene TP53 is widely regarded as being one of the most important genes involved in tumorigenesis. Dysregulations in TP53 expression, along with altered expression of developmentally originating p53 regulators such as MDM2 and MDM4 have been implicated in various cancers, leading to poorer prognosis. Epithelial–mesenchymal transition (EMT), the process allowing epithelial cells to undergo biochemical changes to mesenchymal phenotypes, also plays a vital role in the fate of both embryonic and neoplastic cells. Genes that regulate EMT such as Twist1, SOX9 and REX1 have been associated with an increased occurrence of EMT in cancer cells, leading to enhanced cell stemness, proliferation and metastasis. The class of RNA that does not encode for proteins, known as non-coding RNA, has been implicated in a variety of cellular processes and emerging research has shown that its dysregulation can lead to uncontrolled cell proliferation and differentiation. Genes that have been shown to play a role in this dysregulation include PIWIL1, LIN28A and LIN28B, and have been associated with poorer patient outcomes and more aggressive cancer subtypes. The identification of these developmentally regulated genes in tumorigenesis has proved to play an advantageous role in cancer diagnosis and prognosis, and has provided researchers with a multitude of new target mechanisms for novel chemotherapeutic research.

Background: Cancer is the leading cause of disease-related mortality in children and malignancies are more frequently observed in individuals with primary immunodeficiencies (PIDs). This study aimed to identify and highlight the molecular mechanisms, such as oncogenesis and immune evasion, by which PID-related genes may lead to the development of pediatric cancers. Method: We implemented a novel bioinformatics framework using patient data from the TARGET database and performed a comparative transcriptome analysis of PID-related genes in pediatric cancers between normal and cancer tissues, gene ontology enrichment, and protein–protein interaction analyses, and determined the prognostic impacts of commonly mutated and differentially expressed PID-related genes. Results: From the Fulgent Genetics Comprehensive Primary Immunodeficiency panel of 472 PID-related genes, 89 genes were significantly differentially expressed between normal and cancer tissues, and 20 genes were mutated in two or more patients. Enrichment analysis highlighted many immune system processes as well as additional pathways in the mutated PID-related genes related to oncogenesis. Survival outcomes for patients with altered PID-related genes were significantly different for 75 of the 89 DEGs, often resulting in a poorer prognosis. Conclusions: Overall, multiple PID-related genes demonstrated the connection between PIDs and cancer development and should be studied further, with hopes of identifying new therapeutic targets.

Background Efficacy and safety data of COVID-19 vaccines among cancer populations have been limited; however, preliminary data from recent studies have emerged regarding their immunogenicity and safety in this population. In this review, we examined current peer-reviewed publications containing serological and safety data following COVID-19 vaccination of patients with cancer. Methods This analysis examined 21 studies with a total of 5,012 cancer patients, of which 2,676 (53%) had hematological malignancies, 2,309 (46%) had solid cancers, and 739 were healthy controls. Serological responses by anti-SARS-CoV-2 spike protein S1/S2 IgG (anti-S IgG) antibody levels and post-vaccination patient questionnaires regarding vaccine-related side effects following the first and second dose were collected and analyzed. Results In general, a single dose of COVID-19 vaccine yields weaker and heterogeneous serological responses in both patients with hematological and solid malignancies. Upon receiving a second dose, serological response rates indicate a substantial increase in seropositivity to the SARS-CoV-2 spike protein in all cancer cohorts, but antibody titers remain reduced in comparison to healthy controls. Furthermore, seroconversion in patients with hematological malignancies was significantly lower compared to patients with solid tumors. COVID-19 vaccines are safe and well-tolerated in cancer patients based on current data of local and systemic effects. Conclusion Together, these data support the prioritization of patients with cancer to receive their first and second doses to minimize the risk of COVID-19 infection and severe complications in this vulnerable population. Additional prophylactic measures must be considered for high-risk patients where current vaccination programs may not mount sufficient protection against SARS-CoV-2 infection.

Although treatment strategies for pediatric leukemia have improved overall survival rates in the recent past, relapse rates in certain subgroups such as infant leukemia remain unacceptably high. Despite undergoing extensive chemotherapy designed to target the rapidly proliferating leukemia cells, many of these children experience relapse. In refractory leukemia, the existence of cell populations with stemness characteristics, termed leukemia stem cells (LSCs), which remain quiescent and subsequently replenish the blast population, has been described. A significant body of evidence exists, derived largely from xenograft models of adult acute myeloid leukemia, to support the idea that LSCs may play a fundamental role in refractory disease. In addition, clinical studies have also linked LSCs with increased minimal residual disease, higher relapse rate, and decreased survival rates in these patients. Recently, a number of reports have addressed effective ways to utilize new-generation genomic sequencing and transcriptomic analyses to identify targeted therapeutic agents aimed at LSCs, while sparing normal hematopoietic stem cells. These data underscore the value of timely translation of knowledge from adult studies to the unique molecular and physiological characteristics seen in pediatric leukemia. We aim to summarize this article in the rapidly expanding field of stem cell biology in hematopoietic malignancies, focusing particularly on relevant preclinical models and novel targeted therapeutics, and their applicability to childhood leukemia.

BACKGROUND Recent studies have shown that cell cycle events are tightly controlled by complex and shared activities of a select group of kinases. Among these, polo-like kinases (Plks) are regulatory mitotic proteins that are overexpressed in several types of cancer and are associated with poor prognosis. MATERIALS AND METHODS We have evaluated, in preclinical in vitro studies, the activity of a panel of Plk inhibitors against cell lines derived from refractory pediatric leukemia, as well as primary leukemia cells, in culture. Through in vitro growth inhibition studies, Western blot analysis for the expression and activation of key regulators of cell growth and survival and gene silencing studies, we specifically examined the ability of these agents to induce cytotoxicity through the activation of apoptosis and their capacity to interact and modulate the expression and phosphorylation of Aurora kinases. RESULTS Our findings show that the various Plk-1 inhibitors in development show potential utility for the treatment of pediatric leukemia and exhibit a wide range of phosphorylation and target modulatory capabilities. Finally, we provide evidence for a complex interregulatory relationship between Plk-1 and Aurora kinases enabling the identification of synergy and biologic correlates of drug combinations targeting the 2 distinct enzyme systems. DISCUSSION This information provide the rationale for the evaluation of Plk-1 as an effective target for therapeutics in refractory pediatric leukemia and indicate compensatory activities between Plk-1 and Aurora kinases, providing insight into some of the complex mechanisms involved in the process of cell division.

High-risk, relapsed and refractory neuroblastoma are associated with poor 5-years survival rates, demonstrating the need for investigational therapeutic agents to treat this disease. Taurolidine is derived from the aminosulfoacid taurine and has known anti-microbial and anti-inflammatory properties. Taurolidine has also demonstrated anti-neoplastic effects in a range of cancers, providing the rationale to investigate the activity of taurolidine against neuroblastoma in preclinical studies. We investigated the in vitro activity of taurolidine against neuroblastoma using the alamar blue cytotoxicity assay, phase-contrast light microscopy, western blotting and analysis of global gene expression by RNA-Seq. In vivo activity of taurolidine was evaluated using mouse xenograft models. In vitro pre-clinical data show that taurolidine is cytotoxic to neuroblastoma cell lines, inducing cell death by apoptosis. Analysis of global gene expression and determination of signaling pathway activation scores using the in silico Pathway Activation Network Decomposition Analysis (iPANDA) platform indicates that taurolidine has an effect on the Notch, mitogen-activated protein kinase (MAPK) and interleukin-10 (IL-10) signaling pathways. In vivo experiments in xenograft mouse models show that taurolidine decreases tumor growth and improves survival. These results provide supportive pre-clinical data on the activity of taurolidine against neuroblastoma. The findings support the rationale for further evaluation of taurolidine for the treatment of relapsed/refractory neuroblastoma patients in an early phase clinical trial.

Background: Neuroblastoma (NB) is one of the most aggressive and common solid tumors in pediatrics. Development of effective new therapeutics for NB is in progress to help reduce mortality...

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in children. Pathogenesis of RMS is associated with aggressive growth pattern and increased risk of morbidity and mortality. There are two main subtypes or RMS: embryonal and alveolar. The embryonal type is characterized by distinct molecular aberrations, including alterations in the activity of certain protein kinases. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that plays a vital role in focal adhesion (FA) assembly to promote cytoskeleton dynamics and regulation of cell motility. It is regulated by multiple phosphorylation sites: tyrosine 397, Tyr 576/577, and Tyr 925. Tyrosine 397 is the autophosphorylation site that regulates FAK localization at the cell periphery to facilitate the assembly and formation of the FA complex. The kinase activity of FAK is mediated by the phosphorylation of Tyr 576/577 within the kinase domain activation loop. Aberrations of FAK phosphorylation have been linked to the pathogenesis of different types of cancers. In this regard, pY397 upregulation is linked to increase ERMS cell motility, invasion, and tumorigenesis. In this study, we have used an established human embryonal muscle rhabdomyosarcoma cell line RD as a model to examine FAK phosphorylation profiles to characterize its role in the pathogenies of RMS. Our findings revealed a significant increase of FAK phosphorylation at pY397 in RD cells compared to control cells (hTERT). On the other hand, Tyr 576/577 phosphorylation levels in RD cells displayed a pronounced reduction. Our data showed that Y925 residue exhibited no detectable change. The in vitro analysis showed that the FAK inhibitor, PF-562271 led to G1 cell-cycle arrest induced cell death (IC50, ~ 12 µM) compared to controls. Importantly, immunostaining analyses displayed a noticeable reduction of Y397 phosphorylation following PF-562271 treatment. Our data also showed that PF-562271 suppressed RD cell migration in a dose-dependent manner associated with a reduction in Y397 phosphorylation. The data presented herein indicate that targeting FAK phosphorylation at distinct sites is a promising strategy in future treatment approaches for defined subgroups of rhabdomyosarcoma.

Purpose Neuroblastoma is the most common extracranial cancer in children. Although the prognosis for low-risk neuroblastoma patients is good, the 5-year survival rates for high-risk and relapsed patients are low. The poor survival rates for these patients demonstrate the need for novel therapeutic approaches to treat this disease. PV-10 is a sterile 10% solution of Rose Bengal that has previously been shown to induce cell death in a range of adult cancers, providing the rationale for studying the activity of PV-10 against neuroblastoma in preclinical studies. Methods The effects of PV-10 on neuroblastoma were investigated in vitro. Cytotoxicity assays were performed using the alamar blue assay on the following cell lines: SK-N-AS, SK-N-BE(2), IMR5, LAN1, SHEP, and SK-N-SH neuroblastoma cells, SK-N-MC neuroepithelioma cells, and normal primary, BJ, and WI38 fibroblasts. Phase-contrast, fluorescence, and time-lapse video microscopy; flow cytometry; and Western blotting were used to investigate the effects of PV-10 on SK-N-AS and IMR5 cells. Synergy with commonly used anticancer drugs was determined by calculation of combination indices in SK-N-AS and IMR5 cells. Mouse xenograft models of SK-N-AS and IMR5 tumors were also used to evaluate the efficacy of PV-10 in vivo. Results In vitro preclinical data demonstrate that pharmacologically relevant concentrations of PV-10 are cytotoxic to neuroblastoma cell lines. Studies to investigate target modulation in neuroblastoma cell lines show that PV-10 disrupts lysosomes, decreases the percentage of cells in S phase, and induces apoptosis in a concentration-, time-, and cell-line-dependent manner, and we also identify agents that are synergistic with PV-10. Furthermore, experiments in xenograft mouse models show that PV-10 induces tumor regression in vivo. Conclusion Our study provides preclinical data on the efficacy of PV-10 against neuroblastoma and provides rationale for the development of an early phase clinical trial of this agent in relapsed and refractory neuroblastoma patients.

Introduction: PV-10 (10% Rose Bengal) is a small molecule agent previously shown to have potent immunotherapeutic and anti-tumor activities against a number of tumors including metastatic melanoma and refractory neuroblastoma, and is currently undergoing clinical testing as a single-agent for refractory metastatic neuroendocrine cancer (NCT02693067) and in combination with checkpoint inhibitors for metastatic melanoma (NCT02557321) and metastatic uveal melanoma (NCT00986661). We have previously determined that PV-10 induces cell death at pharmacologically relevant concentrations in a panel of phenotypically diverse adult solid tumor cell lines. However, the molecular consequences of this phenomenon have not yet been fully elucidated. In this study, we investigated the target validation and modulation of PV-10 on protein kinase signalling and their associated impact on specific oncogenic pathways in these tumor cells. Methods: A panel of human tumor cell lines derived from breast (MCF-7, T-47D, MDA-MB-231), colorectal (LoVo, T-84), head and neck (CAL-27, Detroit-562, FaDu, UM-SCC-1), and testicular (NCC-IT, NTERA-2, TCAM-2) tissues were treated with PV-10 and their cytotoxic and pro-apoptotic effects were described. Protein kinase profiling was performed using the human phospho-kinase antibody array (#ARY003C, R&D Systems). Western blotting was used to investigate autophagic markers and protein kinase activity. Cell migration inhibition was determined by wound healing assay following treatment with a sublethal dose of PV-10 or pan-WNK inhibitor WNK463. Tumor xenograft studies were carried out according to established protocols. Results: Treatment with PV-10 leads to consistent inhibition of WNK lysine deficient protein kinase 1 (WNK1) phosphorylation based on protein kinase profiling of drug-treated cancer cells when compared with vehicle-treated cells. WNK1 has been implicated as an inhibitor of autophagy and a promoter of cell migration and invasion in several cancers. Western blot analysis showed that PV-10 leads to the downregulation of SQSTM1/p62, upregulation of Beclin-1, and conversion of LC3B-I into LC3B-II, indicating activation of autophagy. PV-10 was also found to significantly inhibit the migration of cancer cells similar to the inhibitor WNK463. Significant tumor regression and target modulation was noted in tumor-bearing animals treated with PV-10. Conclusion: In addition to the known activity of PV-10 to mediate tumor-specific immune responses and cytotoxic effects in neoplasms, we have identified novel therapeutic targets for PV-10, such as WNK1, and provide new insights into its effect on autophagy and metastasis. Our data provide essential mechanism-based evidence and biomarkers of activity to formulate effective PV-10 backbone clinical studies in the future. Citation Format: Son Tran, Satbir Thakur, Mohit Jain, Chunfen Zhang, Aru Narendran. Identification and in vivo validation of unique anti-oncogenic properties and mechanisms involving protein kinase signalling and autophagy mediated by the investigational new agent PV-10 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1121.

10024 Background: KMT2a rearrangements are a hallmark of infant (less than 1 year of age) leukemia and are associated with poor prognosis. Menin is a ubiquitous protein that binds to the N-terminal of the KMT2a-fusion protein to mediate the oncogenic activity of KMT2a-rearranged leukemia cells. In this study, we evaluated the effect of multiple menin inhibitors and effective drug combinations for the treatment of KMT2A-rearranged infant leukemia. Methods: FISH analysis and immunoblotting confirmed the presence of KMT2a-fusion in the primary patient samples and cell lines studied. Lymphocytes from healthy donors were used as controls. Cells were treated with various concentration of multiple menin inhibitors for 72 hours and growth inhibition was measured using alamar blue assay. Infant leukemia cells were treated with a panel of FDA-approved agents (n = 221) to identify potential synergy, additive- and antagonistic-effects with specific menin inhibitors. Therapeutic drug interaction properties were established by calculating combination indices (CI) using the Chou-Talalay method. Mode of cell death and cellular target modulations were determined by western blot analysis. The effect of targeting menin in the context of the leukemogenic bone marrow microenvironment was determined through stromal cell co-cultures. Results: A comprehensive analysis of menin inhibitors on infant leukemia cell lines and primary patient cells exhibited significant and quantitatively diverse responses in cells with distinct molecular properties. Within the panel of menin inhibitors, infant B-ALL cells were found to be more sensitive to MI-463, MI-503 and MI-136 with a mean IC50 of 5.9µM, 6.1µM and 10.2µM, respectively. On the other hand, MI-3 exhibited an IC50 of 35.6µM. The cytotoxic effect of menin inhibitors on normal lymphocytes was minimal, suggesting a favourable therapeutic window (p < 0.0001). High-throughput screening with a library of > 200 FDA-approved drugs revealed significant sensitivity of distinct infant leukemia cells to proteasome, HDAC and CDK9 inhibitors. Among these, substantial drug synergy was observed between menin and proteasome inhibitors. For example, carfilzomib synergised with menin inhibitors over a broad range of concentrations with a CI value of 0.7. Conclusions: In this study, we present and discuss the initial proof-of-concept preclinical data for the effective anti-leukemic activity of menin inhibition against KMT2A-rearranged infant leukemia cells. Furthermore, the comprehensive drug screen and drug combination studies identified a spectrum of mechanistically-validated synergies, providing usable data for the formulation of multi-agent clinical studies for this currently unmet need in pediatric oncology.

Background: Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase and a catalytic subunit of the polycomb repressive complex 2 (PRC2) that catalyzes the mono-, di-, and tri-methylation of histone H3 at Lys 27 (H3K27me3) to facilitate chromatin remodeling and gene silencing functions. The overexpression of EZH2 is associated with high levels of H3K27me3 in various types of cancers. Previous reports showed a significant association of EZH2 aberrations in pediatric cancers such as soft tissue sarcomas and glioblastoma. Recent reports in human subjects and animal models have also suggested a central role of EZH2 in the induction and progression of acute myeloid leukemia. Methods: In this study, we aimed to investigate the molecular status of EZH in cell lines derived from distinct pediatric leukemia to assess the efficacy of targeting EZH2 to suppress cancer cell survival and proliferation. Cell cytotoxicity and the half maximal inhibitory concentration (IC50) were measured by Alamar blue cytotoxicity assay. The molecular status of EZH2 was profiled by DNA sequencing and western blot analysis of EZH2 protein levels in THP-1, SEM, and MV4:11. In addition, Methylation of histone H3 was evaluated by immunoblotting and immunostaining analyses. Results: Our results showed that EZH2 protein is overexpressed in the pediatric monocytic cell line THP-1 , but not in other leukemia derived cell lines MV4;11 and SEM. Screening a panel of methyltransferase inhibitors revealed that three inhibitors; GSK126, UNC1999 and EPZ-5687 are the most potent inhibitors that suppressed EZH2 activity selectively on lysine 27 which resulted in increased apoptosis and inhibition of AKT and ERK protein phosphorylation in THP-1 cells. Our data demonstrated a significant increase in apoptosis in cells treated with drug combination (EZH2i and selinexor) compared to EZH2i inhibitors alone. Conclusions: Taken together, our data provide initial evidence that targeting EZH2 is a promising therapeutic strategy for the treatment of subtypes of pediatric AML. Also, combining EZH2 inhibitors with selinexor may increase the treatment efficacy in these patients. Findings from this study indicate further evaluation and consideration of these compounds for early phase clinical studies in selected pediatric malignancies in the future.

Mutations in the CBL gene were first identified in adults with various myeloid malignancies. Some patients with juvenile myelomonocytic leukemia (JMML) were also noted to harbor mutations in CBL, but were found to have generally less aggressive disease courses compared to patients with other forms of Ras pathway-mutant JMML. Importantly, and in contrast to most reports in adults, the majority of CBL mutations in JMML patients are germline with acquired uniparental disomy occurring in affected marrow cells. Here, we systematically studied a large cohort of 33 JMML patients with CBL mutations and found that this disease is highly diverse in presentation and overall outcome. Moreover, we discovered somatically acquired CBL mutations in 15% of pediatric patients who presented with more aggressive disease. Neither clinical features nor methylation profiling were able to distinguish patients with somatic CBL mutations from those with germline CBL mutations, highlighting the need for germline testing. Overall, we demonstrate that disease courses are quite heterogeneous even among patients with germline CBL mutations. Prospective clinical trials are warranted to find ideal treatment strategies for this diverse cohort of patients.

Background: The function of the Ras association domain family 1 (RASSF1)/modulator of apoptosis 1 (MOAP-1) molecular tumor suppressor pathway is often perturbed in many solid and blood cancers by epigenetic loss of RASSF1A via promoter specific methylation. However, a detailed analysis of expression and stability of MOAP-1 as well as effect on cellular proliferation and cell death in blood cancers has not been explored. Methods: Expression of MOAP-1 RASSF1A was performed by immunoblotting analysis, MOAP-1 effect on biology determined by cell proliferation, cell death and tumorigenicity assays. Lastly, proteomic analysis in MOAP-1 in form 1 and 2 expressing cells reveal new interacting partners to MOAP-1. Results: The expression of MOAP-1 appears to be quite varied and exists as two forms, a p39 and p46 form in blood cancers. The higher MOAP-1 p46 form was mainly observed in acute myeloid leukemia (AML) and some acute lymphocytic leukemia (ALL) patients and cell lines. Furthermore, MOAP-1 p46 form can be reduced to the p39 form upon calf intestinal or lambda phosphatase treatment, suggesting post-translational phosphorylation likely produces the slower migrating form. Blood cancer cell lines containing the p46 form of MOAP-1 appears to be more resistant to cell death signals and have increased growth. In addition, we document in vivo protection from tumorigenesis of cells containing MOAP-1 following tail vein injection of cancer cells. Lastly, proteomic analysis revealed several interesting components of the MOAP-1 interactome in blood cancer cells that we validated. Conclusions: MOAP-1 is a bona fide tumor suppressor in blood cancers with functions beyond apoptosis.

Alternatives to conventional cancer treatments are highly sought after for high-risk malignancies that have a poor response to established treatment modalities. With research advancing rapidly in the past decade, neoantigen-based immunotherapeutic approaches represent an effective and highly tolerable therapeutic option. Neoantigens are tumor-specific antigens that are not expressed in normal cells and possess significant immunogenic potential. Several recent studies have described the conceptual framework and methodologies to generate neoantigen-based vaccines as well as the formulation of appropriate clinical trials to advance this approach for patient care. This review aims to describe some of the key studies in the recent literature in this rapidly evolving field and summarize the current advances in neoantigen identification and selection, vaccine generation and delivery, and the optimization of neoantigen-based therapeutic strategies, including the early data from pivotal clinical studies.

BackgroundPopulation-based examination of comorbidity is an emerging field of study.AimsThe purpose of the present population level study is to expand our understanding of how cancer and mental illness are temporally associated.MethodA sample of 83 648 056 physician billing records for 664 838 (56% female) unique individuals over the age of 18 was stratified on ages 19–49 years and 50+ years, with temporal order of mental disorder and cancer forming the basis of comparison.ResultsMental disorders preceded cancers for both genders within each age strata. The full range of cancers and mental disorders preceding or following each pivot ICD class are described in terms of frequency of diagnosis and duration in days, with specific examples illustrated.ConclusionsThe temporal comorbidity between specific cancers and mental disorders may be useful in screening or clinical planning and may represent indicators of disease mechanism that warrant further screening or investigation.Declaration of interestNone.

Introduction: Cancer is one of the most common causes of death in patients with primary immunodeficiencies (PIDs). The immune surveillance concept postulates that PIDs are an important contributor to cancer growth and outcomes. Although several immunodeficiency syndromes are known to be associated with malignancies in children and adults, currently, the molecular mechanisms that link immune functions to cancers are poorly understood. Here we describe distinct molecular characteristics in adult cancers with respect to PID-associated genes that impact survival outcomes in affected patients. Methods: In this study, we integrated transcriptome data of 28 adult cancers from the public database The Cancer Genome Atlas (TCGA) with respective healthy tissues as control from the Genotype-Tissue Expression (GTEx) project. Unified datasets integrating GTEx healthy samples and TCGA cancer data were provided by the Recount2 protocol and differential expression analyses (DEA) were carried out using the integrated limma-voom and edgeR pipelines in the TCGAbiolinks R package. Gene ontology (GO) enrichment analyses were performed using the pathfindR R package. Mutational and clinical data of adult cancer patient samples in TCGA and the AACR Project GENIE were consolidated using cBioPortal and the maftools R package. PID-associated genes were curated from the latest registry by the European Society for Immunodeficiencies (ESID). Results: Of 307 PID-associated genes, 151 (32.0%) up-regulated and 88 (18.6%) down-regulated genes were identified across the 28 unified TCGA-GTEx datasets. From the identified differentially expressed gene sets, GO analysis revealed enrichment in immunological pathways related to complement and coagulation cascades (27/28 datasets), systemic lupus erythematosus (25/28), in addition to Fanconi anemia (22/28). The greatest frequency of mutations in PID-associated genes from the consolidated TCGA and GENIE datasets were KRAS (14.83%), KMT2D (10.03%), TERT (8.99%), PRKDC (7.59%) and PTEN (6.18%) across all cancer types, with a total of 259 affected PID-associated genes. Survival analyses using Cox proportional-hazards and Kaplan-Meier models will assess the clinical implications of the identified molecular alterations in affected patients. Conclusion: Our integrative approach aids in the elucidation of molecular mechanisms that bridge PIDs to tumour biology. These data may help identify important biomarkers and potential future targeted therapeutics. Citation Format: Son Tran, Luis Murguia-Favela, Aru Narendran. The impact of primary immunodeficiency informed molecular landscape on the biology and prognosis of refractory malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2255.

Enzastaurin is an orally administered drug that was intended for the treatment of solid and haematological cancers. It was initially developed as an isozyme specific inhibitor of protein kinase Cβ (PKCβ), which is involved in both the AKT and MAPK signalling pathways that are active in many cancers. Enzastaurin had shown encouraging preclinical results for the prevention of angiogenesis, inhibition of proliferation and induction of apoptosis as well as showing limited cytotoxicity within phase I clinical trials. However, during its assessment in phase II and III clinical trials the efficacy of enzastaurin was poor both in combination with other drugs and as a single agent. In this review, we will discuss the development of enzastaurin from drug design to clinical testing, exploring target identification, validation and preclinical assessment. Finally, we will consider the clinical evaluation of enzastaurin as an example of the challenges associated with drug development. In particular, we discuss the poor translation of drug efficacy from preclinical animal models, inappropriate end point analysis, limited standards in phase I clinical trials, insufficient use of biomarker analysis and also patient stratification, all of which contributed to the failure to achieve approval of enzastaurin as an anticancer therapeutic.

Background: Atypical teratoid rhabdoid tumor of the central nervous system (CNS ATRT) is a malignancy that commonly affects young children. The biological mechanisms contributing to tumor aggressiveness and resistance to conventional therapies in ATRT are unknown. Previous studies have shown the activity of insulin like growth factor-I receptor (IGF-1R) in ATRT tumor specimens and cell lines. IGF-1R has been shown to cross-talk with other receptor tyrosine kinases (RTKs) in a number of cancer types, leading to enhanced cell proliferation. Objective: This study aims to evaluate the role of IGF-1 receptor cross-talk in ATRT biology and the potential for therapeutic targeting. Methods: Cell lines derived from CNS ATRT specimens were analyzed for IGF-1 mediated cell proliferation. A comprehensive receptor tyrosine kinase (RTK) screen was conducted following IGF-1 stimulation. Bioinformatic analysis of publicly available cancer growth inhibition data to identify correlation between IC50 of a VEGFR inhibitor and IGF-1R expression. Results: Comprehensive RTK screen identified VEGFR-2 cross-activation following IGF-1 stimulation. Bioinformatics analysis demonstrated a positive correlation between IC50 values of VEGFR inhibitor Axitinib and IGF-1R expression, supporting the critical influence of IGF-1R in modulating response to anti-angiogenic therapies. Conclusion: Overall, our data present a novel experimental framework to evaluate and utilize receptor cross-talk mechanisms to select effective drugs and combinations for future therapeutic trials in ATRT.

10035 Background: Neuroblastoma (NB) is the most common extracranial solid tumor in children. Despite the development of risk stratification tools to improve prognostication, prediction of patient survival outcomes in NB remains poor. In this study we used an unbiased machine-learning algorithm to develop and validate a transcriptomic signature capable of predicting 5-year overall (OS) and event-free survival (EFS) in these patients. Methods: The TARGET-Neuroblastoma dataset (n = 243) was used as the training set. Two independent NB cohorts, E-MTAB-179 (n = 478) and GSE85047 (n = 266) were used as validation sets. Elastic net regression was employed to identify transcripts associated with EFS. Association of the developed signature with EFS and OS was evaluated using univariate Cox proportional hazards (CoxPH), Kaplan-Meier, and 5-year receiver-operator characteristic curves in validation cohorts. Further, the independent prognostic value of the signature was assessed using multivariate CoxPH models with relevant clinicopathologic variables including age, INSS stage, and N-Myc amplification status in both validation sets. Finally, a nomogram was developed to integrate the signature with prognostic clinicopathologic variables to evaluate their combined efficacy for prediction of 5-year EFS and OS. Results: We identified a 21-gene signature that demonstrates significant association with EFS and OS in both E-MTAB-178 and GSE49710 validation cohorts. Moreover, the signature is independent of clinicopathological variables and can be effectively incorporated into a risk model, improving the prognostic performance. Several genes within the signature have been previously implicated in NB, including ECEL1, HOXC9 and ARAF1. Conclusions: To the best of our knowledge, we are the first to use an unbiased machine learning approach to generate a transcriptomic gene signature for neuroblastoma prognosis externally validated in multiple cohorts across platforms. This 21-gene transcriptomic signature significantly associated with EFS and OS in this disease. Combining this signature with current prognostic clinicopathologic variables will improve risk stratification of affected patients and may inform effective clinical decision-making.[Table: see text]

10033 Background: Although survival rates have improved in the recent past, relapse and refractory disease remain a significant cause of death in children with leukemia. This calls for an urgent need for the development of novel therapies that could effectively treat leukemias in children. The E26 transformation specific (ETS) family of transcription factors regulate various normal cellular functions but are abnormally expressed in various cancers, including leukemia. TK216 is an ETS inhibitor, that has shown pre-clinical activity and clinical efficacy in solid tumors. In this study, we explore the feasibility of using TK216 as a therapeutic agent for the treatment of high risk refractory pediatric leukemia. Methods: A panel of pediatric leukemia derived cell lines and primary blast cells representing a spectrum of molecular abnormalities seen in pediatric leukemia were treated in vitro with TK216 to determine cytotoxicity. Normal lymphocytes were used as controls and cell viability was determined 72 hours post-treatment by Alamar blue assay. The induction of tumor cell apoptosis and target modulation were detected by Western blotting. Alterations in the cell cycle were assessed by FACS analysis with PI staining. Drug combination studies were carried out with established anti-leukemic agents to identify synergy for greater therapeutic efficiency. Results: TK216 decreased cell viability in leukemia cells compared to normal lymphocyte controls in a dose-dependent manner with variations in sensitivity noted with inherent molecular abnormalities. The IC50 values observed ranged from 0.22 µM for the most sensitive cell line, MV4-11 to 0.95 µM for least sensitive cell line, SUP-B15. Apoptosis induction upon TK216 treatment was confirmed by PARP cleavage and caspase 3 activation. Cell cycle analysis demonstrated increased sub-G1 population of cells after TK216 treatment. A strong correlation between sub-G1 population and sensitivity of the cell line towards TK216 (47% in MV4-11 vs 3.72% in SUP-B15) was observed. Screening of a panel of 200 FDA approved anti-cancer agents in drug combination studies identified potential agents for drug synergy. Significant drug synergy was noted with TK216 in combination with the epigenetic modifier 5-azacytidine and the Bcl-2 inhibitor, Venetoclax. [Combination Index for Venetoclax and TK216, mean = 0.65 for MV4-11 and 0.33 for SUP-B15]. Conclusions: Data from our study demonstrate that the ETS inhibitor TK216 induces apoptosis and cell cycle arrest in pediatric leukemia cells at physiologically relevant concentrations. Our combination studies identified distinct anti-cancer agents that could be used for developing effective drug combination regimens with TK216. Overall, our findings provide essential preclinical data for the consideration of TK216 in early phase clinical trials for the treatment of selected high-risk and refractory childhood leukemia.

e14544 Background: PV-10 (10% rose bengal disodium; 4,5,6,7-tetrachloro-2’,4’,5’,7’-tetraiodofluorescein) is a novel therapeutic agent previously shown to have potent anti-tumor activity following intratumoral injection in melanoma and refractory neuroblastoma, and currently is undergoing clinical testing as a single-agent for refractory metastatic neuroendocrine cancer (NCT02693067) and in combination with checkpoint inhibitors for metastatic melanoma (NCT02557321) and metastatic uveal melanoma (NCT00986661). Given the established clinical efficacy of PV-10 in adult melanoma and hepatic cancers via intratumoral injection, there is a need to evaluate the therapeutic potential of PV-10 in high-risk and refractory adult solid tumors via systemic administration. Our study aims to identify the clinical potential of systemically-delivered PV-10 by first generating prerequisite in vitro data for adult malignancies. Methods: Cytotoxicity assays were performed using the Alamar Blue assay to study the effects of PV-10 in vitro 96-hours post-treatment against a panel of adult solid tumor cell lines derived from breast (MCF-7, T-47D, MDA-MB-231), colorectal (HCT-116, LoVo, T-84), head and neck (CAL-27, Detroit-562, FaDu, UM-SCC-1), and testicular (NCC-IT, NTERA-2, TCAM-2) tissues. Light microscopy and Western blotting were used to investigate apoptosis induction and target modulation in tumor cells after PV-10 treatment. Results: In vitro results from our study demonstrate that PV-10 is cytotoxic at pharmacologically relevant concentrations across the indicated cell lines. Specifically, tumor cell lines originating from testicular tissues were highly sensitive to PV-10 treatment (Mean ± SD IC50: 37.5 ± 16.4 µM; n = 3) compared to breast (117.5 ± 71.0 µM; n = 3), colorectal (64.79 µM; n = 3), and head and neck (106.6 ± 29.2 µM; n = 4) cell lines. Western blot analyses showed dose- and time-dependent activation of pro-apoptotic protein markers in caspase-3 and PARP cleavage, indicating drug-induced apoptosis. Conclusions: This study provides the first pre-clinical results of PV-10 as a novel systemically-delivered therapeutic agent for a range of high-risk and refractory adult solid tumors. Data obtained from our in vitro experiments using a broad repertoire of cell lines that represent diverse molecular and phenotypic subtypes of solid tumors in adults can serve as prerequisite pre-clinical data to establish clinical testing in these populations.

Y-box-binding protein 1 (YB-1) is a regulatory protein that is associated with drug resistance and relapse in solid tumors. As YB-1 mediates some of its activity through growth factor receptor signaling dysregulation, the present study compared the expression of YB-1 and interleukin 7 (IL-7) receptor α (IL-7Rα) in pediatric B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) and normal BCP cells. The expression levels of IL-7Rα and YB-1 were higher in relapsed vs. diagnostic samples of primary BCP ALL; however, co-expression was also observed in a minor BCP cell population in samples from healthy donors. Functional crosstalk between YB-1 and IL-7R was detected: Overexpression of YB-1 increased surface levels of IL-7R in B cells, and the stimulation of BCP ALL cell lines and primary samples by IL-7 activated YB-1 by phosphorylation at S102 in a phosphatidylinositol 3-kinase-independent and MEK1/2-dependent manner. Targeted knockdown of YB-1 reduced IL-7-mediated protection against rapamycin, and an inhibitor of MEK1/2 potentiated rapamycin-mediated killing in the presence of IL-7. These data establish a novel link between two well-characterized pro-survival factors in acute leukemia, and suggest that YB-1 inhibition may represent a novel therapeutic strategy for increasing sensitivity to chemotherapy in patients with refractory acute B-cell leukemia.

A metallomics approach can be used to probe the mechanisms by which the co-administration of sulfur-containing ‘chemoprotective agents’ can modulate the metabolism of cisplatin in the bloodstream., Numerous in vivo studies have shown that the severe toxic side-effects of intravenously administered cisplatin can be significantly reduced by the co-administration of sulfur-containing ‘chemoprotective agents’. Using a metallomics approach, a likely biochemical basis for these potentially useful observations was only recently uncovered and appears to involve the reaction of chemoprotective agents with cisplatin-derived Pt-species in human plasma to form novel platinum–sulfur complexes (PSC's). We here reveal aspects of the structure of two PSC's and establish the identification of an optimal chemoprotective agent to ameliorate the toxic side-effects of cisplatin, while leaving its antineoplastic activity largely intact, as a feasible research strategy to transform cisplatin into a safer and more effective anticancer drug.

Introduction: There is strong experimental and clinical data to indicate the critical involvement of immune evasion in relapsed leukemia in children. A well-defined characteristic of refractory leukemia is the accumulation of genetic aberrations and mutations that may act as drivers or passengers in the process of tumor recurrence. Many of these mutations get translated into proteins that contain tumor-specific immune-stimulatory epitopes (neoantigens) that can elicit host antitumor immune responses. Although, in general, the mutation rate is lower in pediatric tumors, recent studies have shown that almost 90% of pediatric leukemias carry potentially actionable neoepitopes. In this study, we describe the results from a comprehensive experimental approach of neoantigen prediction coupled with antigen processing and HLA-binding prediction algorithms with in vitro validation assays for the generation of neoantigen vaccines against high-risk leukemias in children. Methods: DNA and RNA from leukemia cells and matched fibroblasts were obtained. Raw reads were aligned to human reference genome and somatic variants (SNVs) were called using Strelka v1.0.1441. RNA-seq data from leukemic cells were used to predict neoantigen expression levels resulting from SNVs using STAR (2.4.1)12 and Cufflinks v2.2.1. Normalized expression data were then cross-referenced with the list of SNVs to identify leukemia-specific mutant proteins. HLA typing for each sample was carried out from RNA-seq data using seq2HLA v2.2. Using the patient's HLA phenotype, we then used NetMHCons v1.1 to predict short peptides derived from leukemia-specific mutant proteins that will bind to autologous HLA Class I molecules. These 8/9-mers were filtered to predict a high likelihood of proteasomal or immune-proteasomal processing and transporter associated with antigen processing (TAP) using NetChop v3.1 and the immune epitope database (IEDB), respectively. The peptides identified were rank-ordered based on the composite immunogenicity score derived from MHC class I binding affinities, proteasomal processing and TCR binding predictions and synthesized accordingly. Peripheral blood derived dendritic cells (DCs) and CD8+ T-cells were isolated and expanded in culture with relevant cytokines. The DCs were pulsed with peptides and then co-cultured with CD8+ T-cells. After five days, the primed CD8+ T-Cells were separated, washed and exposed to the patient's leukemic cells at varying ratios and the leukemia specific CD8+ T-cell activation was quantified by IFN gamma secretion using ELISpot assays. Results: In the leukemia specimen studied, approximately 5% of all on-target germline mutations were found only in leukemic cells. Tumor mutational burden was, on average, 0.34 mut/Mb. Analysis of the highest ranking synthetic peptides (approximately 10 per leukemia sample) showed leukemia-specific activation of patient's T-cells as measured by the mean number of spots observed in ELISpot assays. For example, in patient one (15 year old male, high-risk ALL, one year off therapy), 14 individual short sequences were identified and corresponding peptides were synthesized. Among these, three peptides were not soluble and three peptides showed significant activity above controls. Maximum leukemia specific T-cell activation was noted with peptide #7 QQSALVLL (mean 135 ELISpots compared to 72 in controls, p Discussion: Completed data, including the vaccine peptide sequences and corresponding activities showed the feasibility of identifying pediatric leukemia neoantigen sequences in personalized mutational landscapes of these patients. In addition, we have provided an in vitro experimental approach to validate the potential of such vaccines in future clinical studies and this methodology can also be used to identify agents for effective combinations such as immune checkpoint inhibitors. A clinical trial using these strategies is in development for the treatment of high-risk leukemia in children. Disclosures No relevant conflicts of interest to declare.

Introduction: The activation of stimulator of interferon (IFN) genes (STING), an intracellular receptor system located in the endoplasmic reticulum, has been shown to augment antitumor immunity through the induction of pro-inflammatory cytokines. Currently, a number of STING agonists have been developed to treat refractory malignancies. Our previous studies have identified PV-10 (4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein) as a novel therapeutic agent with potent activity following intra-tumoral injection. Here we describe a previously unidentified mechanism by which PV-10 may facilitate sustained immune activation and therapeutic antitumor activity. Methods: The well-established acute monocytic leukemia (AML) cell line THP-1 was used as a model to study STING activation in vitro. Cells were treated with PV-10 and the induction of STING was evaluated by Western blot analysis using cGAMP as a positive control. Proteins that associate with STING in the presence of PV-10 were purified by immunoprecipitation and analyzed by mass spectrometry (LC-MS/MS, Mascot database). The culture supernatants from PV-10 treated cells were probed for a panel of 42 immune cytokines using the Bio-Plex multiplex bead-based assay system. Results: We show that the exposure of THP-1 cells to PV-10 leads to the appearance of a new 70-KD STING dimer band detected by specific antibodies. Compared to cGAMP controls no induction of PDL-1 was noted. Mass spectrometric analysis of immuno-precipitates of STING in these cells showed the presence of Heat Shock Proteins (HSP) 60, 70 and 90 as well as Polyadenylate Binding Protein 1 (PABP1) to the dimerized STING complex. The chemokine assays showed specific upregulation of a distinct set of pro-inflammatory and cytotoxic T-cell recruitment cytokines. A peak in the induction of MCP-3 and IFN gamma was seen at 24 hours (2 fold) and an approximately 10-fold increase in IL-6, IL-8 and IP-10 was seen in the 24 hours following exposure to PV-10. A significant increase in MCP-1 levels was also noted. Discussion: The compound PV-10 has been shown to induce tumor necrosis following intra-tumoral injection. Our present data show a possible mechanism of this agent through STING dimerization and HSP association leading to a sustained pro-inflammatory and immune response. We provide experimental data, for the first time, for a role of HSPs in STING-mediated immune activation pathways and the description of an agent that may play a role in effective single-agent immunotherapy or drug combination therapy approaches in future clinical studies. Citation Format: Satbir Thakur, Chunfen Zhang, Laurent Brechenmacher, Luis Murgia-Favela, Aru Narendran. Association of heat shock proteins as chaperone for STING: A potential link in a key immune activation mechanism revealed by the novel anti-cancer agent PV-10 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5393.

Leukemia accounts for 30% of all childhood cancers and although the survival rate for pediatric leukemia has greatly improved, relapse is a major cause of treatment failure. Therefore, the development and introduction of novel therapeutics to treat relapsed pediatric leukemia is urgently needed. The proteasome inhibitor bortezomib has been shown to be effective against adult hematological malignancies such as multiple myeloma and lymphoma, but is frequently associated with the development of resistance. Carfilzomib is a next-generation proteasome inhibitor that has shown promising results against refractory adult hematological malignancies. Carfilzomib has been extensively studied in adult hematological malignancies, providing the rationale for evaluating proof-of-concept activity of carfilzomib in pediatric leukemia. The effects of carfilzomib on pediatric leukemia cell lines and primary pediatric leukemia patient samples were investigated in vitro using the alamar blue cytotoxicity assay, western blotting, and a proteasome activity assay. Synergy with commonly used anticancer drugs was determined by calculation of combination indices. In vitro preclinical data show pharmacologically relevant concentrations of carfilzomib are cytotoxic to pediatric leukemia cell lines and primary pediatric leukemia cells. Target modulation studies validate the effective inhibition of the proteasome and induction of apoptosis. We also identify agents that have effective synergy with carfilzomib in these cells. Our data provide pre-clinical information that can be incorporated into future early-phase clinical trials for the assessment of carfilzomib as a treatment for children with refractory hematological malignancies.

Background Melatonin is a natural health product used for sleep disturbances. In preliminary studies of adults with advanced cancer, 20 mg of melatonin daily was associated with reduction in anorexia and weight loss-symptoms that also impact pediatric oncology patients. High doses of melatonin have not been studied in pediatrics. Methods This was a multicenter single-arm phase I dose-escalation study utilizing a 3 + 3 design to determine the safety and tolerability of escalating doses of melatonin in pediatric oncology patients with relapsed solid tumors. Melatonin was given for 8 weeks at three dose levels-0.075 mg/kg (maximum 5 mg), 0.15 mg/kg (maximum 10 mg), and 0.3 mg/kg (maximum 20 mg). Results Melatonin was well tolerated at all three dose levels with no significant adverse events or dose-limiting toxicities. The only grade 3/4 toxicities were myelosuppression, which was attributed to the concomitant chemotherapy and occurred at all dose levels. Weight gain occurred in seven of nine patients, with a median increase of 1.1 kg (range -3.3 to 4.5) or 3.4% (range -10.2 to 8.7), with two patients losing weight (one in dose level 1 and one level 3). Conclusions Melatonin is well tolerated at a dose of 0.3 mg/kg (maximum 20 mg), in the pediatric population. This study provides the background for further study of high-dose melatonin in pediatric oncology patients.

Solid tumors represent one of the most widespread causes of death in children across the world. Neuroblastoma (NB) constitutes about 8% of all childhood tumors, yet accounts for more than 15% of death, with an unacceptable overall survival rate. Despite the current multimodal therapeutic approaches involving surgery, radiation, chemotherapy with myeloablative therapy and hematopoietic stem cell rescue, there is growing realization of the limitations of conventional agents to improve the outcome in high risk metastatic disease. Hence, efforts have intensified to identify new targets and novel therapeutic approaches to improve cure rates in these children. Among the significant number of new therapeutics that are being evaluated for cancer each year, the agents that have been developed for common adult malignancies have the added advantage of having usable toxicity data already available for consideration. To identify potential therapeutic targets, we screened a small molecule library of 151 small kinase inhibitors against NB cell lines. Based on our initial screening data, we further examined the potential of Bcr-Abl targeting small molecule inhibitors to affect the growth and survival of NB cells. Our findings confirm the diversity in activity among the currently available Bcr-Abl inhibitors, possibly reflecting the molecular heterogeneity and off-target activity in each combination. In depth analyses of ponatinib, an orally bioavailable multi-target kinase inhibitor and an effective agent in the treatment of refractory Philadelphia chromosome (Ph) positive leukemia, show growth inhibition at sub-micromolar concentrations. In addition, we also identified the potential of this agent to interfere with insulin-like growth factor-1 receptor (IGF-1R) signaling pathways and Src activity. Ponatinib also induced apoptosis, indicated by caspase-9 and PARP cleavage. Furthermore, at sub-lethal conditions ponatinib significantly inhibited the ability of these cells to migrate. Our findings provide initial data on the potential of ponatinib to target key growth regulatory pathways and provide the rationale for further studies and its evaluation in future early phase clinical trials for the treatment of refractory NB.

Background Diffuse intrinsic pontine gliomas (DIPGs) and high-grade astrocytomas (HGA) continue to have dismal prognoses. The combination of cetuximab and irinotecan was demonstrated to be safe and tolerable in a previous pediatric phase 1 combination study. We developed this phase 2 trial to investigate the safety and efficacy of cetuximab given with radiation therapy followed by adjuvant cetuximab and irinotecan. Methods Eligible patients of age 3–21 years had newly diagnosed DIPG or HGA. Patients received radiation therapy (5,940 cGy) with concurrent cetuximab. Following radiation, patients received cetuximab weekly and irinotecan daily for 5 days per week for 2 weeks every 21 days for 30 weeks. Correlative studies were performed. The regimen was considered to be promising if the number of patients with 1-year progression-free survival (PFS) for DIPG and HGA was at least six of 25 and 14 of 26, respectively. Results Forty-five evaluable patients were enrolled (25 DIPG and 20 HGA). Six patients with DIPG and five with HGA were progression free at 1 year from the start of therapy with 1-year PFS of 29.6% and 18%, respectively. Fatigue, gastrointestinal complaints, electrolyte abnormalities, and rash were the most common adverse events and generally of grade 1 and 2. Increased epidermal growth factor receptor copy number but no K-ras mutations were identified in available samples. Conclusions The trial did not meet the predetermined endpoint to deem this regimen successful for HGA. While the trial met the predetermined endpoint for DIPG, overall survival was not markedly improved from historical controls, therefore does not merit further study in this population.

Introduction: In children diagnosed with leukemia, relapse and its associated morbidity and mortality remain the most dreaded consequences of the disease. Therefore, the discovery and implementation of novel and broadly applicable therapeutic strategies for these patients are urgently needed. Currently, a number of precision therapeutic approaches have been formulated where molecular analyses of the malignant cells have been used to inform, often multiple, probable targets and potential therapeutic agents. However, a common drawback of this approach has been the uncertainty involved in selecting the drug with the most and clinically relevant cytotoxic potential. In vitro xenograft approaches, although can provide key information on drug activity and side effects, are time consuming and impractical and cumbersome in most cases. We have recently demonstrated the ability of a bone marrow stromal derived cell line to sustain the growth and survival of patient leukemic cells in culture that has allowed in vitro evaluations of drug response.This methodology was combined with a previously validated molecular pathway analysis program to identify effective agents or combinations for a subsequent informed precision clinical trial. Methods: Gene expression profiles from refractory pediatric leukemic cells were analyzed against similar data from more than 12, 000 tumors and outlier analyses were carried out to generate a list of overexpressed genes. This information was computed to identify hypothetically activated pathways, druggable targets and potential agents from a panel of FDA approved drugs. A bone marrow stromal cell line was established and characterized that has been shown to support leukemia cell proliferation in vitro. Briefly, stromal cells were co-cultured with leukemic cells at pre-determined ratios with and without the drugs identified in the genomic analysis. After four days in culture, leukemic cells were re-suspended and analyzed for proliferation. Target modulation and activated cell death pathways were queried by Western blot analyses. Results: Multiple targets and potential agents for effective therapeutics were identified against an initial set of relapsed leukemia specimens. For example, in patient # P700491 (pre B-ALL) gene expression data sets revealed clustering within the area of ALL and AML in the reference cancer genomics data. Comparative tumor RNA seq outlier analysis showed molecular abnormalities in BTK, JAK3 and PIK3CD, corresponding to molecular categories of RTK, JAK-SAT and PI3K-AKT-mTOR pathways targetable by the drugs Ibrutinib, WHI-P131 and Idelalisib, respectively. However, in vitro studies showed significant cell killing with Idelalisib and not with the other two agents. Target modulation assays showed effective induction of apoptosis including PARP cleavage in Idelalisib treated leukemia cells compared to controls, indicating the feasibility of this approach to effectively identify potentially applicable agents for this individual patient. Conclusions: We demonstrate the ability of a newly cloned bone marrow stromal derived cell line to sustain the growth and survival of patient leukemic cells in culture that has allowed in vitro target modulation and target validation analyses for cytotoxicity. This methodology was combined with a previously demonstrated molecular pathway interrogation program to ascertain effective agents or combinations for an experimentally informed precision clinical trial. Importantly, our data showed that genomically identified actionable targets are not universally predictive of tumor response and an in vitro cytotoxicity analysis step may enhance the accuracy of this approach. We describe the practical advantages and versatility of this work-flow to inform the selection of agents in future umbrella trials. It is anticipated that the information obtained will lead to an applicable clinical trial the near future. Disclosures Narendran: Bayer: Honoraria, Other: CANTRK Advisory Board .

e21502 Background: Neuroblastoma (NB) is the most common extracranial solid tumor and one of the most complex and difficult to treat diseases in pediatrics. Currently, even with highly aggressive treatment protocols, the prognosis for patients with high-risk and relapsed NB remains poor. Hence, there is a clear need to identify new agents and novel therapeutic strategies for the treatment of these children. Taurolidine (TRD) is derived from the aminosulfoacid taurine and has known anti-microbial and anti-inflammatory properties. TRD has demonstrated anti-neoplastic activity against a range of aggressive human tumors. We present mechanistic evidence and supportive preclinical data from in vitro and animal models of refractory NB for the development of an early phase clinical trial incorporating TRD. Methods: For in vitro activity studies, a panel of cell lines derived from patients with relapsed NB (n = 6) and normal control cells were treated with increasing concentrations of TRD and cell viability was measured by alamar blue assay. Phase-contrast light microscopy, western blotting, time-lapse video microscopy and analysis of global gene expression by RNA-Seq were used to evaluate target modulation and induction of cell death pathways. Bioluminescence imaging of mice bearing NB xenografts treated with TRD was used to investigate the efficacy of TRD in vivo. Results: Cell survival data showed that TRD is cytotoxic against NB cell lines in vitro (mean IC50 value 100 µM, range 65-135 µM). Phase-contrast and time-lapse video microscopy confirmed the antitumor effects of TRD. Western blot analyses identified that TRD induced target modulation and an effective apoptotic cascade, resulting in PARP cleavage. Gene expression analyses and signaling pathway activation scores indicated alterations in the Notch, MAPK and IL-10 signaling pathways. Xenograft studies further validated the in vivo activity of TRD with decreased tumor burden in treated mice and a measurable improvement in survival. Conclusions: Our study provides key pre-clinical data on the activity and mechanism of action of TRD against NB. The findings support the rationale for further evaluation of TRD for the treatment of relapsed/refractory NB patients in an early phase clinical trial.

INTRODUCTION: Elevated levels of LIN28A expression in AT/RT samples provide the rationale to search for agents that may interfere with cellular pathways regulated by this molecule. Ornithine decarboxylase (ODC) modulates eIF-5A through polyamines production and has been shown to directly influence the LIN28/Let-7 axis. The anti-protozoan drug α-difluoromethylornithine (DFMO) irreversibly inhibits ODC and its activity is potentiated by GC7 and Aurothioglucose (AuTG). METHODS: Bioinformatics: ATRT Pediatric Patient Dataset from Children’s Brain Tumour Tissue Consortium was evaluated (n=32). In-vitro studies: Expression levels of LIN28 and ODC in three CNS ATRT cell lines, BT12, BT16 and KCCF1, were examined by western blotting. Cells were treated with increasing concentrations of DFMO, GC7 and AuTG and viability was measured by alamar blue assay. RESULTS: mRNA expression profile of patients suggest strong evidence of co-expression between three proteins: LIN28A – ODC1: 0.37 Pearson Coefficient (p = 0.037), ODC1 – EIF5A: 0.35 Pearson Coefficient (p = 0.049) and LIN28A – EIF5A: 0.54 Pearson Coefficient (p = 0.001). Drug sensitivity studies indicated DFMO, GC7 and AuTG were cytotoxic in-vitro. IC50 values of DFMO for BT12, BT16 and KCCF1 were 80uM, 50uM and 80uM respectively. GC-7 was cytotoxic at IC50 of 115uM, 25uM and 25uM and AuTG displayed IC50 of 15uM, 42 uM and 50uM. Markers of apoptosis, including PARP cleavage, were present at 48 hours although kinetics varied between cell lines. Drug combination studies showed significant synergy between DMFO and GC7. DISCUSSION: Data from this study identified alterations in expression and activity of components in LIN28 mediated pathways in ATRT. Interference in the LIN28 pathway by the tested drugs leads to apoptosis at physiologically attainable concentrations. Therapeutic efficacy can be further enhanced by mechanistically validated drug combinations. We discuss in detail the implications of these findings with respect to the biology and novel therapeutics development for ATRT.

Embryonal tumor with multilayered rosettes (ETMR) is an aggressive central nervous system primitive neuroectodermal tumor (CNS-PNET) variant. ETMRs have distinctive histology, amplification of the chromosome 19 microRNA cluster (C19MC) at chr19q13.41-42, expression of the RNA binding protein Lin28, and dismal prognosis. Functional and therapeutic studies of ETMR have been limited by a lack of model systems.We have established a first cell line, BT183, from a case of ETMR and characterized its molecular and cellular features. LIN28 knockdown was performed in BT183 to examine the potential role of Lin28 in regulating signaling pathway gene expression in ETMR. Cell line findings were corroborated with immunohistochemical studies in ETMR tissues. A drug screen of 73 compounds was performed to identify potential therapeutic targets.The BT183 line maintains C19MC amplification, expresses C19MC-encoded microRNAs, and is tumor initiating. ETMRs, including BT183, have high LIN28 expression and low let-7 miRNA expression, and show evidence of mTOR pathway activation. LIN28 knockdown increases let-7 expression and decreases expression of IGF/PI3K/mTOR pathway components. Pharmacologic inhibition of the mTOR pathway reduces BT183 cell viability.BT183 retains key genetic and histologic features of ETMR. In ETMR, Lin28 is not only a diagnostic marker but also a regulator of genes involved in growth and metabolism. Our findings indicate that inhibitors of the IGF/PI3K/mTOR pathway may be promising novel therapies for these fatal embryonal tumors. As the first patient-derived cell line of these rare tumors, BT183 is an important, unique reagent for investigating ETMR biology and therapeutics.