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Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk ALL subtype with high rates of relapse and poor patient outcome. Activating mutations affecting components of the JAK-STAT signaling pathway occur in the majority of Ph-like ALL cases. The use of JAK inhibitors represents a potential treatment option for Ph-like ALL, although we and others have shown that CRLF2-rearranged Ph-like ALL responds poorly to single-agent JAK inhibitors in the preclinical setting. Therefore, the aim of this study was to identify effective combination treatments against CRLF2-rearranged Ph-like ALL, and to elucidate the underlying mechanisms of synergy. We carried out a series of high-throughput combination drug screenings and found that ruxolitinib exerted synergy with standard-of-care drugs used in the treatment of ALL. In addition, we investigated the molecular effects of ruxolitinib on Ph-like ALL by combining mass spectrometry phosphoproteomics with gene expression analysis. Based on these findings, we conducted preclinical in vivo drug testing and demonstrated that ruxolitinib enhanced the in vivo efficacy of an induction-type regimen consisting of vincristine, dexamethasone, and L-asparaginase in 2/3 CRLF2-rearranged Ph-like ALL xenografts. Overall, our findings support evaluating the addition of ruxolitinib to conventional induction regimens for the treatment of CRLF2-rearranged Ph-like ALL.

BACKGROUND: The prognosis for high-risk childhood acute leukaemias remains dismal and established treatment protocols often cause long-term side effects in survivors. This study aims to identify more effective and safer therapeutics for these patients. METHODS: A high-throughput phenotypic screen of a library of 3707 approved drugs and pharmacologically active compounds was performed to identify compounds with selective cytotoxicity against leukaemia cells followed by further preclinical evaluation in patient-derived xenograft models. RESULTS: Auranofin, an FDA-approved agent for the treatment of rheumatoid arthritis, was identified as exerting selective anti-cancer activity against leukaemia cells, including patient-derived xenograft cells from children with high-risk ALL, versus solid tumour and non-cancerous cells. It induced apoptosis in leukaemia cells by increasing reactive oxygen species (ROS) and potentiated the activity of the chemotherapeutic cytarabine against highly aggressive models of infant MLL-rearranged ALL by enhancing DNA damage accumulation. The enhanced sensitivity of leukaemia cells towards auranofin was associated with lower basal levels of the antioxidant glutathione and higher baseline ROS levels compared to solid tumour cells. CONCLUSIONS: Our study highlights auranofin as a well-tolerated drug candidate for high-risk paediatric leukaemias that warrants further preclinical investigation for application in high-risk paediatric and adult acute leukaemias.

Ning Xu,1,2 Benjamin Tse,1,2 Lu Yang,1,2 Tiffany CY Tang,1,2 Michelle Haber,1,2 Kenneth Micklethwaite,3– 6 Alla Dolnikov1,2 1Children’s Cancer Institute, University of New South Wales, Sydney, NSW, Australia; 2School of Women’s and Children’s Health, University of New South Wales, Sydney, NSW, Australia; 3Blood Transplant and Cell Therapies Program, Department of Hematology, Westmead Hospital, Sydney, NSW, Australia; 4Sydney Cellular Therapies Laboratory, NSW Health Pathology, Sydney, NSW, Australia; 5Westmead Institute for Medical Research, Sydney, NSW, Australia; 6Sydney Medical School, The University of Sydney, Sydney, NSW, AustraliaCorrespondence: Alla DolnikovChildren’s Cancer Institute, University of New South Wales, PO Box 81, Randwick, NSW, 2031, AustraliaTel +61 449 904 174Email adolnikov@ccia.org.auPurpose: Despite the success of chimeric antigen receptor (CAR) T cells in clinical studies, a significant proportion of responding patients eventually relapsed, with the latter correlating with low CAR T cell expansion and persistence.Methods and Results: Using patient-derived xenograft (PDX) mouse models of CD19+ B cell acute lymphoblastic leukemia (B-ALL), we show that priming leukemia-bearing mice with 5-azacytidine (AZA) enhances CAR T cell therapy. AZA given 1 day prior to CAR T cell infusion delayed leukemia growth and promoted CAR T cell expansion and effector function. Priming leukemia cells with AZA increased CAR T cell/target cell conjugation and target cell killing, promoted CAR T cell divisions and expanded IFNγ+ effector T cells in co-cultures with CD19+ leukemia Nalm-6 and Raji cells. Transcriptome analysis revealed activation of diverse immune pathways in leukemia cells isolated from mice treated with AZA. We propose that epigenetic priming with AZA induces transcriptional changes that sensitize tumor cells to subsequent CAR T cell treatment. Among the candidate genes up-regulated by AZA is TNFSF4 which encodes OX40L, one of the strongest T cell co-stimulatory ligands. OX40L binds OX40, the TNF receptor superfamily member highly specific for activated T cells. TNFSF4 is heterogeneously expressed in a panel of pediatric PDXs, and high TNFSF4 expression correlated with increased CAR T cell numbers identified in co-cultures with individual PDXs. High OX40L expression in Nalm-6 cells increased their susceptibility to CAR T cell killing while OX40L blockade reduced leukemia cell killing.Conclusion: We propose that treatment with AZA activates OX40L/OX40 co-stimulatory signaling in CAR T cells. Our data suggest that the clinical use of AZA before CAR T cells could be considered.Keywords: CAR T cells, leukemia, AZA, patient-derived xenografts, gene expression

Purpose: TERT gene rearrangement with transcriptional superenhancers leads to TERT overexpression and neuroblastoma. No targeted therapy is available for clinical trials in patients with TERT-rearranged neuroblastoma. Experimental Design: Anticancer agents exerting the best synergistic anticancer effects with BET bromodomain inhibitors were identified by screening an FDA-approved oncology drug library. The synergistic effects of the BET bromodomain inhibitor OTX015 and the proteasome inhibitor carfilzomib were examined by immunoblot and flow cytometry analysis. The anticancer efficacy of OTX015 and carfilzomib combination therapy was investigated in mice xenografted with TERT-rearranged neuroblastoma cell lines or patient-derived xenograft (PDX) tumor cells, and the role of TERT reduction in the anticancer efficacy was examined through rescue experiments in mice. Results: The BET bromodomain protein BRD4 promoted TERT-rearranged neuroblastoma cell proliferation through upregulating TERT expression. Screening of an approved oncology drug library identified the proteasome inhibitor carfilzomib as the agent exerting the best synergistic anticancer effects with BET bromodomain inhibitors including OTX015. OTX015 and carfilzomib synergistically reduced TERT protein expression, induced endoplasmic reticulum stress, and induced TERT-rearranged neuroblastoma cell apoptosis which was blocked by TERT overexpression and endoplasmic reticulum stress antagonists. In mice xenografted with TERT-rearranged neuroblastoma cell lines or PDX tumor cells, OTX015 and carfilzomib synergistically blocked TERT expression, induced tumor cell apoptosis, suppressed tumor progression, and improved mouse survival, which was largely reversed by forced TERT overexpression. Conclusions: OTX015 and carfilzomib combination therapy is likely to be translated into the first clinical trial of a targeted therapy in patients with TERT-rearranged neuroblastoma.

Histone deacetylase (HDAC) inhibitors are effective in MYCN-driven cancers, because of a unique need for HDAC recruitment by the MYCN oncogenic signal. However, HDAC inhibitors are much more effective in combination with other anti-cancer agents. To identify novel compounds which act synergistically with HDAC inhibitor, such as suberanoyl hydroxamic acid (SAHA), we performed a cell-based, high-throughput drug screen of 10,560 small molecule compounds from a drug-like diversity library and identified a small molecule compound (SE486-11) which synergistically enhanced the cytotoxic effects of SAHA. Effects of drug combinations on cell viability, proliferation, apoptosis and colony forming were assessed in a panel of neuroblastoma cell lines. Treatment with SAHA and SE486-11 increased MYCN ubiquitination and degradation, and markedly inhibited tumorigenesis in neuroblastoma xenografts, and, MYCN transgenic zebrafish and mice. The combination reduced ubiquitin-specific protease 5 (USP5) levels and increased unanchored polyubiquitin chains. Overexpression of USP5 rescued neuroblastoma cells from the cytopathic effects of the combination and reduced unanchored polyubiquitin, suggesting USP5 is a therapeutic target of the combination. SAHA and SE486-11 directly bound to USP5 and the drug combination exhibited a 100-fold higher binding to USP5 than individual drugs alone in microscale thermophoresis assays. MYCN bound to the USP5 promoter and induced USP5 gene expression suggesting that USP5 and MYCN expression created a forward positive feedback loop in neuroblastoma cells. Thus, USP5 acts as an oncogenic cofactor with MYCN in neuroblastoma and the novel combination of HDAC inhibitor with SE486-11 represents a novel therapeutic approach for the treatment of MYCN-driven neuroblastoma.

There has been dramatic improvement in treatment outcomes for many pediatric cancers over the last three decades. However, for the 20% of young people with relapsed or refractory cancer, the prognosis remains grim. Canada’s PRecision Oncology For Young peopLE (PROFYLE) and Australia’s Zero Childhood Cancer (ZERO) programs, leverage nation-wide scientific and clinical oncology expertise to provide personalized precision medicine to children, adolescent and young adult (CAYA) cancer patients who lack treatment options. Beyond improving the lives of young cancer patients nationally, PROFYLE and ZERO have partnered to create an international pipeline for knowledge sharing and sample acquisition. A key component of both programs is the use of mice for patient-derived xenografts (PDXs). However, long lead times for mouse PDX generation make the timely return of preclinical drug response data challenging. PROFYLE has uniquely incorporated zebrafish larval xenografts, which have the potential to provide comparable information in a clinically actionable timeframe. In a pilot study, we compared retrospective matched ZERO patient and mouse PDX therapeutic response data with prospective zebrafish larval PDX data as a proof-of-principle that drug efficacy signals were maintained across model systems. Three ZERO avatars: high-risk neuroblastoma, Ewing’s sarcoma, and anaplastic large cell lymphoma were shipped from Australia to Canada and transplanted into 48h casper zebrafish. Following dose optimization, zebrafish PDXs were treated with targeted single and combination drug treatments by immersion therapy. Strikingly, in as little time as a week, cell proliferation rates and drug responses to single agents and combinatorial therapy in zebrafish PDXs recapitulated mouse and patient data. To expand on this pilot project, we tested additional patient samples, including multiple subtypes of sarcomas, T-cell acute lymphoblastic leukemia and an embryonal tumor with multilayered rosettes. Results further validated the practical utility of the zebrafish larval PDX model and in fact provided drug response data when mouse PDX data were unavailable. This study demonstrates the robustness and feasibility of the zebrafish larval PDX model as a preclinical tool for personalized precision therapeutic decision-making and highlights the value of international collaboration in improving outcomes of rare childhood cancers. Citation Format: Nadine Azzam, Nicole Melong, Lissandra Tuzi, Lisa Pinto, Jamie I. Fletcher, Alvin Kamili, Biljana Dumevska, Loretta Lau, Jennifer A. Chan, Donna L. Senger, Stephanie A. Grover, Michelle Haber, David Malkin, Jason N. Berman. True bench-to-bedside science: An international pilot study modeling hard-to-cure pediatric cancers to prioritize therapeutic intervention. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4670.

Approximately 25% of human neuroblastoma is caused by amplification of the MYCN oncogene, which leads to overexpression of N‐Myc oncoprotein. The survival rate for this patient subtype is, The MYCN oncogene encodes an ‘undruggable’ protein N‐Myc. Here, we show that N‐Myc protein binds to the DDX21 gene promoter, leading to increased CEP55 expression, neuroblastoma cell cytoskeletal stability, and cell proliferation. Knocking down DDX21 suppressed tumor progression in neuroblastoma‐bearing mice. Our data demonstrate that DDX21 and CEP55 are valid therapeutic targets for the treatment of MYCN‐amplified neuroblastoma.

Philadelphia (Ph)-like acute lymphoblastic leukemia (ALL) is characterized by aberrant activation of signaling pathways and high risk of relapse. Approximately 50% of Ph-like ALL cases overexpress cytokine receptor-like factor 2 (CRLF2) associated with gene rearrangement. Activated by its ligand thymic stromal lymphopoietin (TSLP), CRLF2 signaling is critical for the development, proliferation, and survival of normal lymphocytes. To examine activation of tyrosine kinases regulated by TSLP/CRLF2, phosphotyrosine (P-Tyr) profiling coupled with stable isotope labeling of amino acids in cell culture (SILAC) was conducted using two CRLF2-rearranged (CRLF2r) Ph-like ALL cell lines stimulated with TSLP. As a result, increased P-Tyr was detected in previously reported TSLP-activated tyrosine kinases and substrates, including JAK1, JAK2, STAT5, and ERK1/2. Interestingly, TSLP also increased P-Tyr of insulin growth factor 1 receptor (IGF1R) and fibroblast growth factor receptor 1 (FGFR1), both of which can be targeted with small-molecule inhibitors. Fixed-ratio combination cytotoxicity assays using the tyrosine kinase inhibitors BMS-754807 and ponatinib that target IGF1R and FGFR1, respectively, revealed strong synergy against both cell line and patient-derived xenograft (PDX) models of CRLF2r Ph-like ALL. Further analyses also indicated off-target effects of ponatinib in the synergy, and novel association of the Ras-associated protein-1 (Rap1) signaling pathway with TSLP signaling in CRLF2r Ph-like ALL. When tested in vivo, the BMS-754807/ponatinib combination exerted minimal efficacy against 2 Ph-like ALL PDXs, associated with low achievable plasma drug concentrations. Although this study identified potential new targets in CRLF2r Ph-like ALL, it also highlights that in vivo validation of synergistic drug interactions is essential. Implication: Quantitative phosphotyrosine profiling identified potential therapeutic targets for high-risk CRLF2-rearranged Ph-like ALL.

Amplification of the MYCN oncogene occurs in approximately 25% of primary neuroblastomas and is the single most powerful biological marker of poor prognosis in this disease. MYCN transcriptionally regulates a range of biological processes important for cancer, including cell metabolism. The MYCN-regulated metabolic gene SLC16A1, encoding the lactate transporter monocarboxylate transporter 1 (MCT1), is a potential therapeutic target. Treatment of neuroblastoma cells with the MCT1 inhibitor SR13800 increased intracellular lactate levels, disrupted the nicotinamide adenine dinucleotide (NADH/NAD(+)) ratio and decreased intracellular glutathione levels. Metabolite tracing with 13C-glucose and 13C-glutamine following MCT1 inhibitor treatment revealed increased quantities of tricarboxylic acid (TCA) cycle intermediates and increased oxygen consumption rate. MCT1 inhibition was highly synergistic with vincristine under cell culture conditions, but this combination was ineffective against neuroblastoma xenografts in mice. Post-treatment xenograft tumors had increased expression of the MCT1 homolog MCT4/SLC16A, a known resistance factor to MCT1 inhibition. We found that MCT4 was negatively regulated by MYCN in luciferase reporter assays and its expression in neuroblastoma cells was increased under hypoxic conditions and following hypoxia-inducible factor (HIF1) induction, suggesting that MCT4 may contribute to resistance to MCT1 inhibitor treatment in hypoxic neuroblastoma tumors. Co-treatment of neuroblastoma cells with inhibitors of MCT1 and LDHA, the enzyme responsible for lactate production, resulted in a large increase in intracellular pyruvate and was highly synergistic in decreasing neuroblastoma cell viability. These results highlight the potential of targeting MCT1 in neuroblastoma in conjunction with strategies that involve disruption of pyruvate homeostasis and indicate possible resistance mechanisms.

MRP1 (ABCC1) is a membrane transporter that confers multidrug resistance in cancer cells by exporting chemotherapeutic agents, often in a reduced glutathione (GSH)-dependent manner. This transport activity can be altered by compounds (modulators) that block drug transport while simultaneously stimulating GSH efflux by MRP1. In MRP1-expressing cells, modulator-stimulated GSH efflux can be sufficient to deplete GSH and increase sensitivity to chemotherapy, enhancing cancer cell death. Further development of clinically useful MRP1 modulators requires a better mechanistic understanding of modulator binding and its relationship to GSH binding and transport. Here, we explore the mechanism of two MRP1 small molecule modulators, 5681014 and 7914321, in relation to a bipartite substrate-binding cavity of MRP1. Binding of these modulators to MRP1 was dependent on the presence of GSH but not its reducing capacity. Accordingly, the modulators poorly inhibited organic anion transport by K332L-mutant MRP1, where GSH binding and transport is limited. However, the inhibitory activity of the modulators was also diminished by mutations that limit E

Neuroblastoma, the most common extracranial solid tumor in young children, arises from the sympathetic nervous system. Our understanding of neuroblastoma has been improved by the development of both genetically engineered and xenograft mouse models of the disease. Anatomical pathology is an essential component of the phenotyping of mouse models of cancer, characterizing the morphologic effects of genetic manipulation and drug treatment. The Th-MYCN model, the most widely used of several genetically engineered mouse models of neuroblastoma, was established by targeted expression of the human MYCN gene to murine neural crest cells under the control of the rat tyrosine hydroxylase promoter. Neuroblastoma development in Th-MYCN mice is preceded by neuroblast hyperplasia-the persistence and proliferation of neural crest-derived neuroblasts within the sympathetic autonomic ganglia. The neuroblastomas that subsequently develop morphologically resemble human neuroblastoma and carry chromosomal gains and losses in regions syntenic with those observed in human tumors. In this overview, we describe the essential pathologic features for investigators when assessing mouse models of neuroblastoma. We outline human neuroblastoma as the foundation for understanding the murine disease, followed by details of the murine sympathetic ganglia from which neuroblastoma arises. Sympathetic ganglia, both with and without neuroblast hyperplasia, are described. The macroscopic and microscopic features of murine neuroblastoma are explained, including assessment of xenografts and tumors following drug treatment. An approach to experimental design is also detailed. Increased understanding of the pathology of murine neuroblastoma should improve reproducibility and comparability of research findings and assist investigators working with mouse models of neuroblastoma. © 2021 Wiley Periodicals LLC.

Introduction Neuroblastoma is a cancer of the sympathetic nervous system that causes up to 15% of cancer-related deaths among children. Among the ~1,000 newly diagnosed cases per year in Europe, more than half are classified as high-risk, with a 5-year survival rate Areas covered The authors provide a critical review on methodological advances aimed at providing new therapeutic opportunities for neuroblastoma patients, including preclinical models of human disease, generation of omics data to discover new therapeutic targets, and artificial intelligence-based technologies to implement personalized treatments. Expert opinion While survival of childhood cancer has improved over the past decades, progress has been uneven. Still, survival is dismal for some cancers, including high-risk neuroblastoma. Embracing new technologies (e.g. molecular profiling of tumors, 3D in vitro models, etc.), international collaborative efforts and the incorporation of new therapies (e.g. RNA-based therapies, epigenetic therapies, immunotherapy) will ultimately lead to more effective and safer therapies for these subgroups of neuroblastoma patients.

Despite aggressive surgery, chemotherapy, and radiotherapy, survival of children and adolescents and young adults (AYAs) with sarcoma has not improved significantly in the past four decades. Immune checkpoint inhibitors (ICIs) are an exciting type of immunotherapy that offer new opportunities for the treatment of paediatric and AYA sarcomas. However, to date, most children do not derive a benefit from this type of treatment as a monotherapy. The immunosuppressive tumour microenvironment is a major barrier limiting their efficacy. Combinations of ICIs, such as anti-PD-1 therapy, with targeted molecular therapies that have immunomodulatory properties may be the key to breaking through immunosuppressive barriers and improving patient outcomes. Preclinical studies have indicated that several receptor tyrosine kinase inhibitors (RTKi) can alter the tumour microenvironment and boost the efficacy of anti-PD-1 therapy. A number of these combinations have entered phase-1/2 clinical trials, mostly in adults, and in most instances have shown efficacy with manageable side-effects. In this review, we discuss the status of ICI therapy in paediatric and AYA sarcomas and the rationale for co-treatment with RTKis. We highlight new opportunities for the integration of ICI therapy with RTK inhibitors, to improve outcomes for children with sarcoma.

Patients whose leukemias harbor a rearrangement of the Mixed Lineage Leukemia (MLL/KMT2A) gene have a poor prognosis, especially when the disease strikes in infants. The poor clinical outcome linked to this aggressive disease and the detrimental treatment side-effects, particularly in children, warrant the urgent development of more effective and cancer-selective therapeutics. The aim of this study was to identify novel candidate compounds that selectively target KMT2A-rearranged (KMT2A-r) leukemia cells. A library containing 3707 approved drugs and pharmacologically active compounds was screened for differential activity against KMT2A-r leukemia cell lines versus KMT2A-wild type (KMT2A-wt) leukemia cell lines, solid tumor cells and non-malignant cells by cell-based viability assays. The screen yielded SID7969543, an inhibitor of transcription factor Nuclear Receptor Subfamily 5 Group A Member 1 (NR5A1), that limited the viability of 7 out of 11 KMT2A-r leukemia cell lines including 5 out of 7 lines derived from infants, without affecting KMT2A-wt leukemia cells, solid cancer lines, non-malignant cell lines, or peripheral blood mononuclear cells from healthy controls. The compound also significantly inhibited growth of leukemia cell lines with a CALM-AF10 translocation, which defines a highly aggressive leukemia subtype that shares common underlying leukemogenic mechanisms with KMT2A-r leukemia. SID7969543 decreased KMT2A-r leukemia cell viability by inducing caspase-dependent apoptosis within hours of treatment and demonstrated synergy with established chemotherapeutics used in the treatment of high-risk leukemia. Thus, SID7969543 represents a novel candidate agent with selective activity against CALM-AF10 translocated and KMT2A-r leukemias that warrants further investigation.

Resistance to chemotherapy remains one of the most significant obstacles to successful cancer treatment. While inhibiting drug efflux mediated by ATP-binding cassette (ABC) transporters is a seemingly attractive and logical approach to combat multidrug resistance (MDR), small molecule inhibition of ABC transporters has so far failed to confer clinical benefit, despite considerable efforts by medicinal chemists, biologists, and clinicians. The long-sought treatment to eradicate cancers displaying ABC transporter overexpression may therefore lie within alternative targeting strategies. When aberrantly expressed, the ABC transporter multidrug resistance-associated protein 1 (MRP1, ABCC1) confers MDR, but can also shift cellular redox balance, leaving the cell vulnerable to select agents. Here, we explore the physiological roles of MRP1, the rational for targeting this transporter in cancer, the development of small molecule MRP1 inhibitors, and the most recent developments in alternative therapeutic approaches for targeting cancers with MRP1 overexpression. We discuss approaches that extend beyond simple MRP1 inhibition by exploiting the collateral sensitivity to glutathione depletion and ferroptosis, the rationale for targeting the shared transcriptional regulators of both MRP1 and glutathione biosynthesis, advances in gene silencing, and new molecules that modulate transporter activity to the detriment of the cancer cell. These strategies illustrate promising new approaches to address multidrug resistant disease that extend beyond the simple reversal of MDR and offer exciting routes for further research.

Members of the ABC transporter family, particularly P-glycoprotein (P-gp, ABCB1), breast cancer resistance protein (BCRP, ABCG2) and multidrug resistance protein 1 (MRP1, ABCC1) are well characterized mediators of multidrug resistance, however their pharmacological inhibition has so far failed as a clinical strategy. Harnessing collateral sensitivity, a form of synthetic lethality where cells with acquired multidrug resistance exhibit hypersensitivity to unrelated agents, may be an alternative approach to targeting multidrug resistant tumour cells. We characterized a novel small molecule modulator that selectively enhanced MRP1-dependent efflux of reduced glutathione (GSH), an endogenous MRP1 substrate. Using cell lines expressing high levels of endogenous MRP1 from three difficult to treat cancer types-lung cancer, ovarian cancer and high-risk neuroblastoma-we showed that the MRP1 modulator substantially lowered intracellular GSH levels as a single agent. The effect was on-target, as MRP1 knockdown abolished GSH depletion. The MRP1 modulator was synergistic with the GSH synthesis inhibitor buthionine sulfoximine (BSO), with the combination exhausting intracellular GSH, increasing intracellular reactive oxygen species (ROS) and abolishing clonogenic capacity. Clonogenicity was rescued by the ROS scavenger N-acetylcysteine, implicating GSH depletion in the effect. The MRP1 modulator in combination with BSO also strongly sensitized cancer cells to MRP1-substrate chemotherapeutic agents, particularly arsenic trioxide, and was more effective than either the MRP1 modulator or BSO alone. GSH-depleting MRP1 modulators may therefore provide an enhanced therapeutic window to treat chemo-resistant MRP1-overexpressing pediatric and adult cancers.

Around 10% of acute leukemias harbor a rearrangement of the MLL/KMT2A gene, and the presence of this translocation results in a highly aggressive, therapy-resistant leukemia subtype with survival rates below 50%. There is a high unmet need to identify safer and more potent therapies for MLL-rearranged (MLL-r) leukemia that can be combined with established chemotherapeutics to decrease treatment-related toxicities. The curaxin, CBL0137, has demonstrated nongenotoxic anticancer and chemopotentiating effects in a number of preclinical cancer models and is currently in adult Phase I clinical trials for solid tumors and hematological malignancies. The aim of our study was to investigate whether CBL0137 has potential as a therapeutic and chemopotentiating compound in MLL-r leukemia through a comprehensive analysis of its efficacy in preclinical models of the disease. CBL0137 decreased the viability of a panel of MLL-r leukemia cell lines (n = 12) and xenograft cells derived from patients with MLL-r acute lymphoblastic leukemia (ALL, n = 3) in vitro with submicromolar IC50s. The small molecule drug was well-tolerated in vivo and significantly reduced leukemia burden in a subcutaneous MV4;11 MLL-r acute myeloid leukemia model and in patient-derived xenograft models of MLL-r ALL (n = 5). The in vivo efficacy of standard of care drugs used in remission induction for pediatric ALL was also potentiated by CBL0137. CBL0137 exerted its anticancer effect by trapping Facilitator of Chromatin Transcription (FACT) into chromatin, activating the p53 pathway and inducing an Interferon response. Our findings support further preclinical evaluation of CBL0137 as a new approach for the treatment of MLL-r leukemia.

Chromosome 17q21-ter is commonly gained in neuroblastoma, but it is unclear which gene in the region is important for tumorigenesis. The JMJD6 gene at 17q21-ter activates gene transcription. Here we show that JMJD6 forms protein complexes with N-Myc and BRD4, and is important for E2F2, N-Myc and c-Myc transcription. Knocking down JMJD6 reduces neuroblastoma cell proliferation and survival in vitro and tumor progression in mice, and high levels of JMJD6 expression in human neuroblastoma tissues independently predict poor patient prognosis. In addition, JMJD6 gene is associated with transcriptional super-enhancers. Combination therapy with the CDK7/super-enhancer inhibitor THZ1 and the histone deacetylase inhibitor panobinostat synergistically reduces JMJD6, E2F2, N-Myc, c-Myc expression, induces apoptosis in vitro and leads to neuroblastoma tumor regression in mice, which are significantly reversed by forced JMJD6 over-expression. Our findings therefore identify JMJD6 as a neuroblastoma tumorigenesis factor, and the combination therapy as a treatment strategy., Although the gain in chromosome 17q21-ter is commonly associated with neuroblastoma, it is not clear which gene of this region mediates tumorigenesis. Here, the authors are showing that JMJD6, which locates in that region, is a neuroblastoma tumorigenic factor.

Caspase-2 is a highly conserved cysteine protease with roles in apoptosis and tumor suppression. Our recent findings have also demonstrated that the tumor suppression function of caspase-2 is context specific. In particular, while caspase-2 deficiency augments lymphoma development in the EμMyc mouse model, it leads to delayed neuroblastoma development in Th-MYCN mice. However, it is unclear how caspase-2 mediates these differential outcomes. Here we utilized RNA sequencing to define the transcriptomic changes caused by caspase-2 (Casp2−/−) deficiency in tumors from EμMyc and Th-MYCN mice. We describe key changes in both lymphoma and neuroblastoma-associated genes and identified differential expression of the EGF-like domain-containing gene, Megf6, in the two tumor types that may contribute to tumor outcome following loss of Casp2. We identified a panel of genes with altered expression in Th-MYCN/Casp2−/− tumors that are strongly associated with neuroblastoma outcome, with roles in melanogenesis, Wnt and Hippo pathway signaling, that also contribute to neuronal differentiation. In contrast, we found that key changes in gene expression in the EμMyc/Casp2−/− tumors, are associated with increased immune signaling and T-cell infiltration previously associated with more aggressive lymphoma progression. In addition, Rap1 signaling pathway was uniquely enriched in Casp2 deficient EμMyc tumors. Our findings suggest that Casp2 deficiency augments immune signaling pathways that may be in turn, enhance lymphomagenesis. Overall, our study has identified new genes and pathways that contribute to the caspase-2 tumor suppressor function and highlight distinct roles for caspase-2 in different tissues.

Minimal residual disease (MRD) measurement is a cornerstone of contemporary acute lymphoblastic leukaemia (ALL) treatment. The presence of immunoglobulin (Ig) and T cell receptor (TCR) gene recombinations in leukaemic clones allows widespread use of patient-specific, DNA-based MRD assays. In contrast, paediatric solid tumour MRD remains experimental and has focussed on generic assays targeting tumour-specific messenger RNA, methylated DNA or microRNA.We examined the feasibility of using whole-genome sequencing (WGS) data to design tumour-specific polymerase chain reaction (PCR)-based MRD tests (WGS-MRD) in 18 children with high-risk relapsed cancer, including ALL, high-risk neuroblastoma (HR-NB) and Ewing sarcoma (EWS) (n = 6 each).Sensitive WGS-MRD assays were generated for each patient and allowed quantitation of 1 tumour cell per 10WGS facilitated the development of patient-specific MRD tests in ALL, HR-NB and EWS with potential clinical utility in monitoring treatment response. WGS data could be used to design patient-specific MRD assays in a broad range of tumours.

Simple Summary Neuroblastoma is a devasting childhood cancer in which multiple copies (amplification) of the cancer-causing gene MYCN strongly predict poor outcome. Neuroblastomas are reliant on high levels of cellular components called polyamines for their growth and malignant behavior, and the gene regulating polyamine synthesis is called ODC1. ODC1 is often coamplified with MYCN, and in fact is regulated by MYCN, and like MYCN is prognostic of poor outcome. Here we studied a naturally occurring genetic variant or polymorphism that occurs in the ODC1 gene, and used gene editing to demonstrate the functional importance of this variant in terms of ODC1 levels and growth of neuroblastoma cells. We showed that this variant impacts the ability of MYCN to regulate ODC1, and that it also influences outcome in neuroblastoma, with the rarer variant associated with a better survival. This study addresses the important topic of genetic polymorphisms in cancer. Ornithine decarboxylase (ODC1), a critical regulatory enzyme in polyamine biosynthesis, is a direct transcriptional target of MYCN, amplification of which is a powerful marker of aggressive neuroblastoma. A single nucleotide polymorphism (SNP), G316A, within the first intron of ODC1, results in genotypes wildtype GG, and variants AG/AA. CRISPR-cas9 technology was used to investigate the effects of AG clones from wildtype MYCN-amplified SK-N-BE(2)-C cells and the effect of the SNP on MYCN binding, and promoter activity was investigated using EMSA and luciferase assays. AG clones exhibited decreased ODC1 expression, growth rates, and histone acetylation and increased sensitivity to ODC1 inhibition. MYCN was a stronger transcriptional regulator of the ODC1 promoter containing the G allele, and preferentially bound the G allele over the A. Two neuroblastoma cohorts were used to investigate the clinical impact of the SNP. In the study cohort, the minor AA genotype was associated with improved survival, while poor prognosis was associated with the GG genotype and AG/GG genotypes in MYCN-amplified and non-amplified patients, respectively. These effects were lost in the GWAS cohort. We have demonstrated that the ODC1 G316A polymorphism has functional significance in neuroblastoma and is subject to allele-specific regulation by the MYCN oncoprotein.

Targeting cell division by chemotherapy is a highly effective strategy to treat a wide range of cancers. However, there are limitations of many standard-of-care chemotherapies: undesirable drug toxicity, side-effects, resistance and high cost. New small molecules which kill a wide range of cancer subtypes, with good therapeutic window in vivo, have the potential to complement the current arsenal of anti-cancer agents and deliver improved safety profiles for cancer patients. We describe results with a new anti-cancer small molecule, WEHI-7326, which causes cell cycle arrest in G2/M, cell death in vitro, and displays efficacious anti-tumor activity in vivo. WEHI-7326 induces cell death in a broad range of cancer cell lines, including taxane-resistant cells, and inhibits growth of human colon, brain, lung, prostate and breast tumors in mice xenografts. Importantly, the compound elicits tumor responses as a single agent in patient-derived xenografts of clinically aggressive, treatment-refractory neuroblastoma, breast, lung and ovarian cancer. In combination with standard-of-care, WEHI-7326 induces a remarkable complete response in a mouse model of high-risk neuroblastoma. WEHI-7326 is mechanistically distinct from known microtubule-targeting agents and blocks cells early in mitosis to inhibit cell division, ultimately leading to apoptotic cell death. The compound is simple to produce and possesses favorable pharmacokinetic and toxicity profiles in rodents. It represents a novel class of anti-cancer therapeutics with excellent potential for further development due to the ease of synthesis, simple formulation, moderate side effects and potent in vivo activity. WEHI-7326 has the potential to complement current frontline anti-cancer drugs and to overcome drug resistance in a wide range of cancers.

Diffuse intrinsic pontine glioma (DIPG) is an incurable malignant childhood brain tumor, with no active systemic therapies and a 5-year survival of less than 1%. Polyamines are small organic polycations that are essential for DNA replication, translation and cell proliferation. Ornithine decarboxylase 1 (ODC1), the rate-limiting enzyme in polyamine synthesis, is irreversibly inhibited by difluoromethylornithine (DFMO). Herein we show that polyamine synthesis is upregulated in DIPG, leading to sensitivity to DFMO. DIPG cells compensate for ODC1 inhibition by upregulation of the polyamine transporter SLC3A2. Treatment with the polyamine transporter inhibitor AMXT 1501 reduces uptake of polyamines in DIPG cells, and co-administration of AMXT 1501 and DFMO leads to potent in vitro activity, and significant extension of survival in three aggressive DIPG orthotopic animal models. Collectively, these results demonstrate the potential of dual targeting of polyamine synthesis and uptake as a therapeutic strategy for incurable DIPG., Diffuse intrinsic pontine glioma (DIPG) is an almost incurable malignant childhood brain tumor. Here, the authors show that the polyamine synthetic pathway is activated in DIPG and that the dual targeting of polyamine synthesis and uptake results in prolonged survival in animal models.

Despite the success of immunotherapies in adult solid cancers and pediatric hematological malignancies, limited progress has been made towards implementing these strategies in pediatric solid tumors. These tumors exhibit a high potential to escape antitumor immunity, making them difficult to target by current immunotherapies. This review highlights the altered metabolic pathways in pediatric solid tumors that promote immune escape, and discusses current novel strategies targeting these pathways. We further explore how these strategies could be applied to potentiate immunotherapies for pediatric solid cancers and pose key questions yet to be addressed. Translational challenges to facilitate clinical application of antimetabolic strategies through personalized medicine are identified. We propose preclinical testing of antimetabolic approaches in combination with immunotherapies for pediatric solid cancers.

Summary: Diffuse intrinsic pontine glioma (DIPG) is an aggressive and incurable childhood brain tumor for which new treatments are needed. CBL0137 is an anti-cancer compound developed from quinacrine that targets facilitates chromatin transcription (FACT), a chromatin remodeling complex involved in transcription, replication, and DNA repair. We show that CBL0137 displays profound cytotoxic activity against a panel of patient-derived DIPG cultures by restoring tumor suppressor TP53 and Rb activity. Moreover, in an orthotopic model of DIPG, treatment with CBL0137 significantly extends animal survival. The FACT subunit SPT16 is found to directly interact with H3.3K27M, and treatment with CBL0137 restores both histone H3 acetylation and trimethylation. Combined treatment of CBL0137 with the histone deacetylase inhibitor panobinostat leads to inhibition of the Rb/E2F1 pathway and induction of apoptosis. The combination of CBL0137 and panobinostat significantly prolongs the survival of mice bearing DIPG orthografts, suggesting a potential treatment strategy for DIPG.

Background: Epithelial ovarian cancer (EOC) is the most lethal gynaecological malignancy with over 80% of cases already disseminated at diagnosis and facing a dismal five-year survival rate of 35%. EOC cells often spread to the greater omentum where they take-up cholesterol. Excessive amounts of cholesterol can be cytocidal, suggesting that cholesterol efflux through transporters may be important to maintain homeostasis, and this may explain the observation that high expression of the ATP-binding cassette A1 (ABCA1) cholesterol transporter has been associated with poor outcome in EOC patients. Methods: ABCA1 expression was silenced in EOC cells to investigate the effect of inhibiting cholesterol efflux on EOC biology through growth and migration assays, three-dimensional spheroid culture and cholesterol quantification. Results: ABCA1 suppression significantly reduced the growth, motility and colony formation of EOC cell lines as well as the size of EOC spheroids, whilst stimulating expression of ABCA1 reversed these effects. In serous EOC cells, ABCA1 suppression induced accumulation of cholesterol. Lowering cholesterol levels using methyl-B-cyclodextrin rescued the effect of ABCA1 suppression, restoring EOC growth. Furthermore, we identified FDA-approved agents that induced cholesterol accumulation and elicited cytocidal effects in EOC cells. Conclusions: Our data demonstrate the importance of ABCA1 in maintaining cholesterol balance and malignant properties in EOC cells, highlighting its potential as a therapeutic target for this disease.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive paediatric brainstem tumour, with a median survival of less than 1 year. Polyamines are intracellular polycations that control important aspects of cell growth and are often upregulated in cancer. Difluoromethylornithine (DFMO) is an FDA-approved inhibitor of the enzyme ornithine decarboxylase (ODC1) which is a key driver of polyamine synthesis. We investigated the efficacy of polyamine pathway inhibitors as a therapeutic strategy against DIPG. We found that there were high over-expression levels of polyamine synthetic enzymes from DIPG primary patient samples and neurosphere cultures. Using alamar blue cytotoxicity and soft-agar clonogenic assays, we found that DFMO inhibited the proliferation of DIPG neurospheres. However, DIPG cells compensated for DFMO inhibition by increasing expression of the polyamine transporter SLC3A2 and subsequently uptake of polyamines. Addition of polyamine transporter inhibitor AMXT 1501 to DFMO led to synergistic inhibition of DIPG proliferation in vitro. Consistent with the in vitro results, the combination of DFMO and AMXT 1501 significantly prolonged the survival of mice bearing 3 different DIPG orthografts with at least 2/3 of the animals surviving up to 160 days. Addition of irradiation further improved the survival of mice treated with DFMO and AMXT 1501. Differential expression analysis showed that the polyamine transporter, SLC3A2, was significantly overexpressed in DIPG and other paediatric brain tumours including high grade gliomas compared with normal brain tissue. Our results suggest that DIPG tumours are exquisitely sensitive to polyamine inhibitors, and that dual blockade of polyamine synthesis and transport is a promising novel therapeutic strategy. AMXT 1501 is currently in clinical development, and following completion of an adult Phase 1 trial, a clinical trial of AMXT 1501 + DFMO for DIPG patients is planned through the CONNECT consortium.

DIPG is an aggressive and incurable childhood brain tumour for which new treatments are needed. A high throughput drug screen of 3500 pharmaceutical compounds identified anti-malarials, including quinacrine as having potent activity against DIPG neurospheres. CBL0137, a compound modelled on quinacrine, is a novel anti-cancer compound which targets Facilitates Chromatin Transcription (FACT), a chromatin remodelling complex involved in transcription, replication, and DNA repair. CBL0137 effectively crosses the blood-brain barrier and has recently completed Phase I testing in adult patients. CBL0137 induced apoptosis in DIPG neurospheres and had profound cytotoxic activity against a panel of DIPG cultures. In a DIPG orthotopic model, treatment with CBL0137 significantly improved survival. We found that treatment with CBL0137 up-regulated TP53 and increased histone H3.3 acetylation and tri-methylation in DIPG cells. We therefore examined the interaction between CBL0137 and the histone deacetylase (HDAC) inhibitor panobinostat. In vitro experiments showed that the two agents had profound synergistic activity against DIPG neurospheres in clonogenic assays and enhanced caspase activation and apoptosis. The FACT subunit SSRP1 was found to directly interact with H3.3K27M and treatment with CBL0137 targeted this epigenetic defect, restoring histone H3.3 trimethylation and leading to tumor cell death. Transcriptomic analysis and immunoblotting indicated that combination treatment activated signalling pathways controlled by Retinoblastoma (RB)/E2F1 and subsequently increased phosphorylation and enzymatic activity of enhancer of zeste homolog 2 (EZH2). Consistent with the in vitro results, the combination of CBL0137 and panobinostat significantly prolonged survival in two independent orthotopic models of DIPG, while histological analysis showed restoration of H3K27me3 and decreased Ki67 positive cells. In addition to panobinostat, CBL0137 has been found to combine synergistically in vitro and in vivo with PARP and BET inhibitors. Given these promising results, a paediatric trial of CBL0137 will open through the Children’s Oncology Group with an expansion cohort for DIPG patients.

Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Amplification of the MYCN oncogene is associated with an aggressive phenotype and poor outcome in childhood neuroblastoma. Polyamines are highly regulated essential cations that are frequently elevated in cancer cells, and the rate-limiting enzyme in polyamine synthesis, ornithine decarboxylase 1 (ODC1), is a direct transcriptional target of MYCN. Treatment of neuroblastoma cells with the ODC1 inhibitor difluoromethylornithine (DFMO), although a promising therapeutic strategy, is only partially effective at impeding neuroblastoma cell growth due to activation of compensatory mechanisms resulting in increased polyamine uptake from the surrounding microenvironment. In this study, we identified solute carrier family 3 member 2 (SLC3A2) as the key transporter involved in polyamine uptake in neuroblastoma. Knockdown of SLC3A2 in neuroblastoma cells reduced the uptake of the radiolabeled polyamine spermidine, and DFMO treatment increased SLC3A2 protein. In addition, MYCN directly increased polyamine synthesis and promoted neuroblastoma cell proliferation by regulating SLC3A2 and other regulatory components of the polyamine pathway. Inhibiting polyamine uptake with the small-molecule drug AMXT 1501, in combination with DFMO, prevented or delayed tumor development in neuroblastoma-prone mice and extended survival in rodent models of established tumors. Our findings suggest that combining AMXT 1501 and DFMO with standard chemotherapy might be an effective strategy for treating neuroblastoma.

As we continue to learn about the unique biology of pediatric and childhood cancers and as new therapies are being developed, we asked some experts to highlight the most notable advances in our understanding of these diseases and in the development of treatments in the past decade, and to point out current challenges that still need to be addressed.

Anticancer agents exerting the best synergistic anticancer effects with BET bromodomain inhibitors were identified by screening an FDA-approved oncology drug library. The synergistic effects of the BET bromodomain inhibitor OTX015 and the proteasome inhibitor carfilzomib were examined by immunoblot and flow cytometry analysis. The anticancer efficacy of OTX015 and carfilzomib combination therapy was investigated in mice xenografted withThe BET bromodomain protein BRD4 promotedOTX015 and carfilzomib combination therapy is likely to be translated into the first clinical trial of a targeted therapy in patients with

The identification of rearrangements driving expression of neurotrophic receptor tyrosine kinase (NTRK) family kinases in tumors has become critically important because of the availability of effective, specific inhibitor drugs. Whole-genome sequencing (WGS) combined with RNA sequencing (RNA-seq) can identify novel and recurrent expressed fusions. Here we describe three SPECC1L–NTRK fusions identified in two pediatric central nervous system cancers and an extracranial solid tumor using WGS and RNA-seq. These fusions arose either through a simple balanced rearrangement or in the context of a complex chromoplexy event. We cloned the SPECC1L–NTRK2 fusion directly from a patient sample and showed that enforced expression of this fusion is sufficient to promote cytokine-independent survival and proliferation. Cells transformed by SPECC1L–NTRK2 expression are sensitive to a TRK inhibitor drug. We report here that SPECC1L–NTRK fusions can arise in a range of pediatric cancers. Although WGS and RNA-seq are not required to detect NTRK fusions, these techniques may be of benefit when NTRK fusions are not suspected on clinical grounds or not identified by other methods.

Pediatric cancers, particularly high-risk solid tumors, urgently need effective and specific therapies. Their outlook has not appreciably improved in decades. Immunotherapies such as immune checkpoint inhibitors offer much promise, but most are only approved for use in adults. Though several hundred clinical trials have tested immune-based approaches in childhood cancers, few have been guided by biomarkers or clinical-grade assays developed to predict patient response and, ultimately, to help select those most likely to benefit. There is extensive evidence in adults to show that immune profiling has substantial predictive value, but few studies focus on childhood tumors, because of the relatively small disease population and restricted use of immune-based therapies. For instance, only one published study has retrospectively examined the immune profiles of pediatric brain tumors after immunotherapy. Furthermore, application and integration of advanced multiplex techniques has been extremely limited. Here, we review the current status of immune profiling of pediatric solid tumors, with emphasis on tumor types that represent enormous unmet clinical need, primarily in the context of immune checkpoint inhibitor therapy. Translating optimized and informative immune profiling into standard practice and access to personalized combination therapy will be critical if childhood cancers are to be treated effectively and affordably.

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk ALL subtype with high rates of relapse and poor patient outcome. Activating mutations affecting components of the JAK-STAT signaling pathway occur in the majority of Ph-like ALL cases. The use of JAK inhibitors represents a potential treatment option for Ph-like ALL, although we and others have shown that CRLF2-rearranged Ph-like ALL responds poorly to single-agent JAK inhibitors in the preclinical setting. Therefore, the aim of this study was to identify effective combination treatments against CRLF2-rearranged Ph-like ALL, and to elucidate the underlying mechanisms of synergy. We carried out a series of high-throughput combination drug screenings and found that ruxolitinib exerted synergy with standard-of-care drugs used in the treatment of ALL. In addition, we investigated the molecular effects of ruxolitinib on Ph-like ALL by combining mass spectrometry phosphoproteomics with gene expression analysis. Based on these findings, we conducted preclinical in vivo drug testing and demonstrated that ruxolitinib enhanced the in vivo efficacy of an induction-type regimen consisting of vincristine, dexamethasone, and L-asparaginase in 2/3 CRLF2-rearranged Ph-like ALL xenografts. Overall, our findings support evaluating the addition of ruxolitinib to conventional induction regimens for the treatment of CRLF2-rearranged Ph-like ALL.

The Zero Childhood Cancer Program is a precision medicine program to benefit children with poor-outcome, rare, relapsed or refractory cancer. Using tumor and germline whole genome sequencing (WGS) and RNA sequencing (RNAseq) across 252 tumors from high-risk pediatric patients with cancer, we identified 968 reportable molecular aberrations (39.9% in WGS and RNAseq, 35.1% in WGS only and 25.0% in RNAseq only). Of these patients, 93.7% had at least one germline or somatic aberration, 71.4% had therapeutic targets and 5.2% had a change in diagnosis. WGS identified pathogenic cancer-predisposing variants in 16.2% of patients. In 76 central nervous system tumors, methylome analysis confirmed diagnosis in 71.1% of patients and contributed to a change of diagnosis in two patients (2.6%). To date, 43 patients have received a recommended therapy, 38 of whom could be evaluated, with 31% showing objective evidence of clinical benefit. Comprehensive molecular profiling resolved the molecular basis of virtually all high-risk cancers, leading to clinical benefit in some patients.

Epithelial ovarian cancer (EOC) is a complex disease comprising discrete histological and molecular subtypes, for which survival rates remain unacceptably low. Tailored approaches for this deadly heterogeneous disease are urgently needed. Efflux pumps belonging to the ATP-binding cassette (ABC) family of transporters are known for roles in both drug resistance and cancer biology and are also highly targetable. Here we have investigated the association of ABCC4/MRP4 expression to clinical outcome and its biological function in endometrioid and serous tumors, common histological subtypes of EOC. We found high expression of ABCC4/MRP4, previously shown to be directly regulated by c-Myc/N-Myc, was associated with poor prognosis in endometrioid EOC (P = .001) as well as in a subset of serous EOC with a "high-MYCN" profile (C5/proliferative; P = .019). Transient siRNA-mediated suppression of MRP4 in EOC cells led to reduced growth, migration and invasion, with the effects being most pronounced in endometrioid and C5-like serous cells compared to non-C5 serous EOC cells. Sustained knockdown of MRP4 also sensitized endometrioid cells to MRP4 substrate drugs. Furthermore, suppression of MRP4 decreased the growth of patient-derived EOC cells in vivo. Together, our findings provide the first evidence that MRP4 plays an important role in the biology of Myc-associated ovarian tumors and highlight this transporter as a potential therapeutic target for EOC.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive and incurable childhood brain tumor for which new treatments are needed. A high throughput drug screen of 3600 pharmaceutical compounds found that anti-malarials, including quinacrine had potent activity against DIPG neurospheres. CBL0137 is a novel anti-cancer compound developed from quinacrine, which targets Facilitates Chromatin Transcription (FACT), a chromatin remodelling complex involved in transcription, replication, and DNA repair. We have found that CBL0137 displays profound cytotoxic activity against a panel of patient derived DIPG cultures, inhibiting cell proliferation and clonogenic potential, restoring tumor suppressor TP53 and Rb activity and inducing cell death through induction of apoptosis. Moreover, in an orthotopic model of DIPG, treatment with CBL0137 significantly extended animal survival. Histone mutations leading to the loss of histone trimethylation result in epigenetic dysregulation driving DIPG tumorigenesis. Treatment with CBL0137 targets this epigenetic defect, restoring both histone H3.3 acetylation and trimethylation and leading to tumor cell death. Combined epigenetic treatment with the histone deacetylase (HDAC) inhibitor panobinostat led to inhibition of the Rb/E2F1 pathway, and increased the enzymatic activity of enhancer of zeste homolog 2 (EZH2), leading to the restoration of H3K27 trimethylation. This combination therapy had synergistic activity against DIPG neurospheres with induction of apoptosis. Consistent with the in vitro results, the combination of CBL0137 and panobinostat significantly prolonged the survival of mice bearing DIPG orthografts suggesting a potential treatment strategy for DIPG.

Therapeutic checkpoint antibodies blocking programmed death receptor 1/programmed death ligand 1 (PD-L1) signaling have radically improved clinical outcomes in cancer. However, the regulation of PD-L1 expression on tumor cells is still poorly understood. Here we show that intratumoral copper levels influence PD-L1 expression in cancer cells. Deep analysis of the The Cancer Genome Atlas database and tissue microarrays showed strong correlation between the major copper influx transporter copper transporter 1 (CTR-1) and PD-L1 expression across many cancers but not in corresponding normal tissues. Copper supplementation enhanced PD-L1 expression at mRNA and protein levels in cancer cells and RNA sequencing revealed that copper regulates key signaling pathways mediating PD-L1–driven cancer immune evasion. Conversely, copper chelators inhibited phosphorylation of STAT3 and EGFR and promoted ubiquitin-mediated degradation of PD-L1. Copper-chelating drugs also significantly increased the number of tumor-infiltrating CD8+ T and natural killer cells, slowed tumor growth, and improved mouse survival. Overall, this study reveals an important role for copper in regulating PD-L1 and suggests that anticancer immunotherapy might be enhanced by pharmacologically reducing intratumor copper levels. Significance: These findings characterize the role of copper in modulating PD-L1 expression and contributing to cancer immune evasion, highlighting the potential for repurposing copper chelators as enhancers of antitumor immunity.

Genes encoding TRK are oncogenic drivers in multiple tumour types including infantile fibrosarcoma, papillary thyroid cancer and high-grade gliomas (HGG). TRK fusions have a critical role in tumourigenesis in 40% of infant HGG. Here we report the first case of a TRK fusion-driven HGG treated with larotrectinib—the first selective pan-TRK inhibitor in clinical development. This 3-year-old girl had failed multiple therapies including chemotherapy and radiotherapy. Tumour profiling confirmed an ETV6–NTRK3 fusion. Treatment with larotrectinib led to rapid clinical improvement with near total resolution of primary and metastatic lesions on MRI imaging. This is the first report of a TRK fusion glioma successfully treated with a TRK inhibitor.

The ability of the N-MYC transcription factor to drive cancer progression is well demonstrated in neuroblastoma, the most common extracranial pediatric solid tumor, where MYCN amplification heralds a poor prognosis, with only 11% of high-risk patients surviving past 5 years. However, decades of attempts of direct inhibition of N-MYC or its paralogues has led to the conclusion that this protein is “undruggable.” Therefore, targeting pathways upregulated by N-MYC signaling presents an alternative therapeutic approach. Here, we show that MYCN-amplified neuroblastomas are characterized by elevated rates of protein synthesis and that high expression of ABCE1, a translation factor directly upregulated by N-MYC, is itself a strong predictor of poor clinical outcome. Despite the potent ability of N-MYC in heightening protein synthesis and malignant characteristics in cancer cells, suppression of ABCE1 alone selectively negated this effect, returning the rate of translation to baseline levels and significantly reducing the growth, motility, and invasiveness of MYCN-amplified neuroblastoma cells and patient-derived xenograft tumors in vivo. The growth of nonmalignant cells or MYCN-nonamplified neuroblastoma cells remained unaffected by reduced ABCE1, supporting a therapeutic window associated with targeting ABCE1. Neuroblastoma cells with c-MYC overexpression also required ABCE1 to maintain cell proliferation and translation. Taken together, ABCE1-mediated translation constitutes a critical process in the progression of N-MYC–driven and c-MYC–driven cancers that warrants investigations into methods of its therapeutic inhibition. Significance: These findings demonstrate that N-MYC–driven cancers are reliant on elevated rates of protein synthesis driven by heightened expression of ABCE1, a vulnerability that can be exploited through suppression of ABCE1.

To prevent relapse, high risk paediatric acute lymphoblastic leukaemia (ALL) is treated very intensively. However, most patients who eventually relapse have standard or medium risk ALL with low minimal residual disease (MRD) levels. We analysed recurrent microdeletions and other clinical prognostic factors in a cohort of 475 uniformly treated non-high risk precursor B-cell ALL patients with the aim of better predicting relapse and refining risk stratification. Lower relapse-free survival at 7 years (RFS) was associated with IKZF1 intragenic deletions (P 5 × 10-5 (P

Simple Summary This review explores the current trials using cellular immunotherapies in pediatric sarcoma and describes examples of promising new CAR T targets in sarcoma that are in preclinical development. We provide insights into the ways in which the immunosuppressive tumor immune microenvironment can impact on CAR T cell therapy, highlighting specific mechanisms by which the tumor microenvironment may limit CAR T efficacy. Appreciation of these mechanisms may lead to rational combinations of immunotherapies, for example, the combination of CAR T cells with checkpoint inhibitor drugs. We also describe innovations in CAR T cell generation and combination therapies that may pave the way to better clinical outcomes for these patients. Abstract Sarcomas are a diverse group of bone and soft tissue tumors that account for over 10% of childhood cancers. Outcomes are particularly poor for children with refractory, relapsed, or metastatic disease. Chimeric antigen receptor T (CAR T) cells are an exciting form of adoptive cell therapy that potentially offers new hope for these children. In early trials, promising outcomes have been achieved in some pediatric patients with sarcoma. However, many children do not derive benefit despite significant expression of the targeted tumor antigen. The success of CAR T cell therapy in sarcomas and other solid tumors is limited by the immunosuppressive tumor microenvironment (TME). In this review, we provide an update of the CAR T cell therapies that are currently being tested in pediatric sarcoma clinical trials, including those targeting tumors that express HER2, NY-ESO, GD2, EGFR, GPC3, B7-H3, and MAGE-A4. We also outline promising new CAR T cells that are in pre-clinical development. Finally, we discuss strategies that are being used to overcome tumor-mediated immunosuppression in solid tumors; these strategies have the potential to improve clinical outcomes of CAR T cell therapy for children with sarcoma.

DIPG is an aggressive paediatric brainstem tumour, with a median survival of less than 1 year. Polyamines are intracellular polycations that control important aspects of cell growth and are often upregulated in cancer. Difluoromethylornithine (DFMO) is an FDA-approved inhibitor of the enzyme ornithine decarboxylase (ODC1) which is a key driver of polyamine synthesis. We investigated the efficacy of polyamine pathway inhibitors as a therapeutic strategy against DIPG. We found high expression levels of synthetic enzymes in the polyamine pathway in primary patient samples and cultures. Using cytotoxicity and clonogenic assays, we found that DFMO inhibited the proliferation of DIPG neurospheres. However, DIPG cells compensated for DFMO inhibition by increasing expression of the polyamine transporter SLC3A2. Gene expression analysis showed that the polyamine transporter, SLC3A2, was significantly overexpressed in DIPG compared with all other high-risk childhood cancers. Addition of polyamine transporter inhibitor AMXT 1501 to DFMO led to synergistic inhibition of DIPG proliferation. Consistent with the in vitro results, the combination treatment significantly prolonged the survival of mice bearing 3 different DIPG orthografts with 2/3 of the animals surviving up to 160 days. Addition of irradiation further improved the survival of mice treated with DFMO and AMXT 1501. Our results suggest that DIPG tumours are exquisitely sensitive to polyamine inhibitors and that dual blockade of polyamine synthesis and transport is a promising novel therapeutic strategy. AMXT 1501 is currently in clinical development for adult cancers (NCT03536728). A clinical trial for DIPG patients is planned through the CONNECT consortium.

In adult cancer, immune signatures such as the T cell-inflamed gene expression profile (GEP) have been developed to predict which patients are likely to respond to immune checkpoint inhibitors (ICIs) beyond high tumor mutation burden (TMB) and PD-L1 expression. The GEP infers T cell infiltration and activation in the tumor microenvironment (TME) from transcriptomic data. However, it is not known whether tools such as GEP are applicable in pediatric cancer, as the TME in childhood cancers is largely unexplored and response to ICIs are rare. We have undertaken an integrated analysis of the pediatric TME using RNA-sequencing (RNA-seq) and immunohistochemistry (IHC). Our goal is to identify patients with T cell-inflamed or “hot” tumors who may benefit from ICIs. Through Australia's ZERO childhood cancer precision medicine program we performed RNA-seq on 347 high-risk pediatric cancers (estimated Citation Format: Chelsea Mayoh, Rachael L. Terry, Marie Wong, Loretta M. Lau, Dong Anh Khuong-Quang, Marion K. Mateos, Vanessa Tyrrell, Michelle Haber, David S. Ziegler, Mark J. Cowley, Joseph A. Trapani, Paul J. Neeson, Paul G. Ekert. The unexplored immune landscape of high-risk pediatric cancers [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 3044.

Background: Minimal Residual Disease (MRD) detection is vital for therapy monitoring and relapse prediction in cancers. While RNA-based MRD assays are shown to be effective at diagnostic time-points, serial response monitoring does not provide additional predictive value.DNA-based assays may be more sensitive and allow longitudinal monitoring of the disease trajectory. This study aimed to develop MRD assays based on whole genome sequencing (WGS) data for high-risk neuroblastoma (HRNB) and Ewing sarcoma (ES) patients, to establish their sensitivity in quantitative PCR (qPCR) anddroplet digital PCR (ddPCR) formats, and to directly compare against RNA-based MRD assays. Methodology: We analyzed WGS data to identify patient-specific chromosomal breakpoints in HRNB (N=6) and ES (N=6) patients, then established qPCR and ddPCR assays. Assay performance was validated using patient-derived cells spiked into bone marrow samples and in serially collected clinical samples as available. The DNA-MRD markers were compared against a panel of RNA-MRD markers for HRNB (TH/DCX/PHOX2B) and ES (EWS-FLI1/ERG/ETV1). Results: DNA-MRD markers showed high sensitivity and quantitation ranging from 10-4(0.01%) to 10-5(0.001%) in qPCR assays and ddPCR assays. In clinical samples of HRNB and ES, DNA-MRD markers were detected in diagnosis specimens and predicted disease trajectory. Among RNA-MRD markers, TH & EWS-FLI1 transcripts showed the maximum detection sensitivity (0.01%), however was less sensitive than the patient specific DNA-MRD markers. Conclusion: This study identifies DNA-MRD analysis as a reliable complimentary approach to RNA-MRD assays and highlights its enhanced sensitivity and clinical utility for monitoring treatment response and disease progression in solid pediatric tumors. Citation Format: Vinod Vijay Subhash, Alvin Kamili, Marie-Wong Erasmus, Dan Chen, Libby Huang, Vanessa Tyrell, Caroline Atkinson, Nicola Venn, Mark Cowley, Glenn Marshall, Paul Ekert, Michelle Henderson, Rosemary Sutton, Murray Norris, Michelle Haber, Jamie Fletcher, Toby Trahair. Analysis of DNA minimal residual disease markers in pediatric solid cancers using quantitative real time PCR and droplet digital PCR [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 639.

Myc transcriptional activity is frequently deregulated in human cancers, but a Myc-driven gene signature with prognostic ability across multiple tumor types remains lacking. Here, we selected 18 Myc-regulated genes from published studies of Myc family targets in epithelial ovarian cancer (EOC) and neuroblastoma. A Myc family activity score derived from the 18 genes was correlated to MYC/MYCN/MYCL1 expression in a panel of 35 cancer cell lines. The prognostic ability of this signature was evaluated in neuroblastoma, medulloblastoma, diffuse large B-cell lymphoma (DLBCL), and EOC microarray gene expression datasets using Kaplan–Meier and multivariate Cox regression analyses and was further validated in 42 primary neuroblastomas using qPCR. Cell lines with high MYC, MYCN, and/or MYCL1 gene expression exhibited elevated expression of the signature genes. Survival analysis showed that the signature was associated with poor outcome independently of well-defined prognostic factors in neuroblastoma, breast cancer, DLBCL, and medulloblastoma. In EOC, the 18-gene Myc activity signature was capable of identifying a group of patients with poor prognosis in a "high-MYCN" molecular subtype but not in the overall cohort. The predictive ability of this signature was reproduced using qPCR analysis of an independent cohort of neuroblastomas, including a subset of tumors without MYCN amplification. These data reveal an 18-gene Myc activity signature that is highly predictive of poor prognosis in diverse Myc-associated malignancies and suggest its potential clinical application in the identification of Myc-driven tumors that might respond to Myc-targeted therapies. Cancer Res; 77(4); 971–81. ©2016 AACR.

BACKGROUND: The current study was conducted to expedite international standardized reporting of bone marrow disease in children with neuroblastoma and to improve equivalence of care. METHODS: A multidisciplinary International Neuroblastoma Response Criteria Bone Marrow Working Group was convened by the US National Cancer Institute in January 2012 with representation from Europe, North America, and Australia. Practical transferable recommendations to standardize the reporting of bone marrow disease were developed. RESULTS: To the authors' knowledge, the current study is the first to comprehensively present consensus criteria for the collection, analysis, and reporting of the percentage area of bone marrow parenchyma occupied by tumor cells in trephine-biopsies. The quantitative analysis of neuroblastoma content in bone marrow aspirates by immunocytology and reverse transcriptase-quantitative polymerase chain reaction are revised. The inclusion of paired-like homeobox 2b (PHOX2B) for immunohistochemistry and reverse transcriptase-quantitative polymerase chain reaction is recommended. Recommendations for recording bone marrow response are provided. The authors endorse the quantitative assessment of neuroblastoma cell content in bilateral core needle biopsies-trephines and aspirates in all children with neuroblastoma, with the exception of infants, in whom the evaluation of aspirates alone is advised. It is interesting to note that 5% disease is accepted as an internationally achievable level for disease assessment. CONCLUSIONS: The quantitative assessment of neuroblastoma cells is recommended to provide data from which evidence-based numerical criteria for the reporting of bone marrow response can be realized. This is particularly important in the minimal disease setting and when neuroblastoma detection in bone marrow is intermittent, where clinical impact has yet to be validated. The wide adoption of these harmonized criteria will enhance the ability to compare outcomes from different trials and facilitate collaborative trial design.

Philadelphia (Ph)-like acute lymphoblastic leukemia (ALL) is characterized by aberrant activation of signaling pathways and high risk of relapse. Approximately 50% of Ph-like ALL cases overexpress cytokine receptor-like factor 2 (CRLF2) associated with gene rearrangement. Activated by its ligand thymic stromal lymphopoietin (TSLP), CRLF2 signaling is critical for the development, proliferation, and survival of normal lymphocytes. To examine activation of tyrosine kinases regulated by TSLP/CRLF2, phosphotyrosine (P-Tyr) profiling coupled with stable isotope labeling of amino acids in cell culture (SILAC) was conducted using two CRLF2-rearranged (CRLF2r) Ph-like ALL cell lines stimulated with TSLP. As a result, increased P-Tyr was detected in previously reported TSLP-activated tyrosine kinases and substrates, including JAK1, JAK2, STAT5, and ERK1/2. Interestingly, TSLP also increased P-Tyr of insulin growth factor 1 receptor (IGF1R) and fibroblast growth factor receptor 1 (FGFR1), both of which can be targeted with small-molecule inhibitors. Fixed-ratio combination cytotoxicity assays using the tyrosine kinase inhibitors BMS-754807 and ponatinib that target IGF1R and FGFR1, respectively, revealed strong synergy against both cell line and patient-derived xenograft (PDX) models of CRLF2r Ph-like ALL. Further analyses also indicated off-target effects of ponatinib in the synergy, and novel association of the Ras-associated protein-1 (Rap1) signaling pathway with TSLP signaling in CRLF2r Ph-like ALL. When tested