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Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPG), are the most lethal of childhood cancers. Palliative radiotherapy is the only established treatment, with median patient survival of 9 to 11 months. ONC201 is a DRD2 antagonist and ClpP agonist that has shown preclinical and emerging clinical efficacy in DMG. However, further work is needed to identify the mechanisms of response of DIPGs to ONC201 treatment and to determine whether recurring genomic features influence response. Using a systems-biological approach, we showed that ONC201 elicits potent agonism of the mitochondrial protease ClpP to drive proteolysis of electron transport chain and tricarboxylic acid cycle proteins. DIPGs harboring PIK3CA mutations showed increased sensitivity to ONC201, whereas those harboring TP53 mutations were more resistant. Metabolic adaptation and reduced sensitivity to ONC201 was promoted by redox-activated PI3K/Akt signaling, which could be counteracted using the brain penetrant PI3K/Akt inhibitor, paxalisib. Together, these discoveries coupled with the powerful anti-DIPG/DMG pharmacokinetic and pharmacodynamic properties of ONC201 and paxalisib have provided the rationale for the ongoing DIPG/DMG phase II combination clinical trial NCT05009992. Significance: PI3K/Akt signaling promotes metabolic adaptation to ONC201-mediated disruption of mitochondrial energy homeostasis in diffuse intrinsic pontine glioma, highlighting the utility of a combination treatment strategy using ONC201 and the PI3K/Akt inhibitor paxalisib.

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Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPG), are the most lethal of childhood cancers. Palliative radiotherapy is the only established treatment, with median patient survival of 9 to 11 months. ONC201 is a DRD2 antagonist and ClpP agonist that has shown preclinical and emerging clinical efficacy in DMG. However, further work is needed to identify the mechanisms of response of DIPGs to ONC201 treatment and to determine whether recurring genomic features influence response. Using a systems-biological approach, we showed that ONC201 elicits potent agonism of the mitochondrial protease ClpP to drive proteolysis of electron transport chain and tricarboxylic acid cycle proteins. DIPGs harboring PIK3CA mutations showed increased sensitivity to ONC201, whereas those harboring TP53 mutations were more resistant. Metabolic adaptation and reduced sensitivity to ONC201 was promoted by redox-activated PI3K/Akt signaling, which could be counteracted using the brain penetrant PI3K/Akt inhibitor, paxalisib. Together, these discoveries coupled with the powerful anti-DIPG/DMG pharmacokinetic and pharmacodynamic properties of ONC201 and paxalisib have provided the rationale for the ongoing DIPG/DMG phase II combination clinical trial NCT05009992.Significance:PI3K/Akt signaling promotes metabolic adaptation to ONC201-mediated disruption of mitochondrial energy homeostasis in diffuse intrinsic pontine glioma, highlighting the utility of a combination treatment strategy using ONC201 and the PI3K/Akt inhibitor paxalisib.

Diffuse midline glioma (DMG), including tumors diagnosed in the brainstem (diffuse intrinsic pontine glioma – DIPG), are uniformly fatal brain tumors that lack effective pharmacological treatment. Analysis of pooled CRISPR-Cas9 loss-of-function gene deletion screen datasets, identifiedPIK3CAandMTORas targetable molecular dependencies across DIPG patient derived models, highlighting the therapeutic potential of the blood-brain barrier penetrant PI3K/Akt/mTOR inhibitor paxalisib. At the human equivalent maximum tolerated dose, mice treated with paxalisib experienced systemic feedback resulting in increased blood glucose and insulin levels, commensurate with DIPG patients in Phase 1b clinical trials who experienced hyperglycemia/hyperinsulinemia. To exploit genetic dependences, but maintain compliance and benefit, we optimized a paxalisib treatment regimen that employed reduced dosing more frequently, in combination with the anti-hyperglycemic drug, metformin. Combining optimized dosing with metformin restored glucose homeostasis and decreased phosphorylation of the insulin receptorin vivo, a common mechanism of PI3K-inhibitor resistance, extending the survival of DIPG xenograft models. RNA sequencing and phosphoproteomic profiling of DIPG models treated with paxalisib identified increased calcium-activated PKC signaling. Using the brain penetrant PKC inhibitor, enzastaurin in combination with paxalisib, we synergistically extended the survival of orthotopic xenograft models, benefits further promoted by metformin; thus, identifying a clinically relevant DIPG combinatorial approach.Brief SummaryDiffuse intrinsic pontine glioma is a lethal childhood brain tumor. Here we identifyPIK3CAas a genetic dependency targeted by the brain penetrant pan-PI3K-inhibitor paxalisib.

Figure S1. Pracinostat reactivates acetylation of non-histone protein tubulin at higher doses. Figure S2. Alteration in malignant behavior of cancer cell lines in vitro with Pracinostat treatment. Figure S3. ATF3 regulates the transcriptional activation of RB1. Figure S4. Pracinostat treatment inhibits in vivo tumor growth. Figure S5. Pracinostat treatment inhibits the production of proangiogenic factor VEGF- A in vitro. Figure S6. ATF3 reactivation is crucial in determining the sensitivity of the HDACi treatment in bladder cancer. Figure S7. The level of expression of HDAC enzymes may dictate the degree of sensitivity in different cell lines in vitro. Supplementary Methods: Generation of stable knockdown cells; Quantitative RT-PCR.

This file contains the following supplemental data: Supplemental Table 1. Specific information for MTT cell viability assays with single agents. Supplemental Table 2. Patient characteristics of synovial sarcoma cohort. Supplemental Table 3. Specifications of antibodies used for IHC and Western Blot. Supplemental Table 6. Summary of SS cell line sensitivity to specific tyrosine kinase inhibitors. Supplemental Figure 1. Integrated protein-protein interaction network for all sarcoma subgroups. Supplemental Figure 2. ALK expression and phosphorylation in ES and RMS cell lines. Supplemental Figure 3. Schematic representation of ALK exon 2-17 deletion in Aska-SS cells. Supplemental Figure 4. Deglycosylation of ALK in Aska-SS cells. Supplemental Figure 5. SS cell line sensitivity to foretinib, gefitinib and erlotinib. Supplemental Figure 6. Effects of ALK inhibition on cell cycle and apoptotic markers in Aska-SS cells. Supplemental Figure 7. Effects of ALK inhibition on Aska-SS in vivo tumors. Supplemental Figure 8. ALK, MET and PDGFRA mRNA expression in clinical SS versus other STS samples. Supplemental Figure 9. ALK IHC and patient characteristics of ALK-positive SS tumors. Supplemental Figure 10. Effect of PDGFR and MET inhibition on apoptosis in Yamato-SS cells. Brief sections of Supplementary methods and Supplementary references are included in this file as well.

Despite intensive multimodal treatment of sarcomas, a heterogeneous group of malignant tumors arising from connective tissue, survival remains poor. Candidate-based targeted treatments have demonstrated limited clinical success, urging an unbiased and comprehensive analysis of oncogenic signaling networks to reveal therapeutic targets and personalized treatment strategies. Here we applied mass spectrometry–based phosphoproteomic profiling to the largest and most heterogeneous set of sarcoma cell lines characterized to date and identified novel tyrosine phosphorylation patterns, enhanced tyrosine kinases in specific subtypes, and potential driver kinases. ALK was identified as a novel driver in the Aska-SS synovial sarcoma (SS) cell line via expression of an ALK variant with a large extracellular domain deletion (ALKΔ2–17). Functional ALK dependency was confirmed in vitro and in vivo with selective inhibitors. Importantly, ALK immunopositivity was detected in 6 of 43 (14%) of SS patient specimens, one of which exhibited an ALK rearrangement. High PDGFRα phosphorylation also characterized SS cell lines, which was accompanied by enhanced MET activation in Yamato-SS cells. Although Yamato-SS cells were sensitive to crizotinib (ALK/MET-inhibitor) but not pazopanib (VEGFR/PDGFR-inhibitor) monotherapy in vitro, synergistic effects were observed upon drug combination. In vivo, both drugs were individually effective, with pazopanib efficacy likely attributable to reduced angiogenesis. MET or PDGFRα expression was detected in 58% and 84% of SS patients, respectively, with coexpression in 56%. Consequently, our integrated approach has led to the identification of ALK and MET as promising therapeutic targets in SS. Cancer Res; 77(16); 4279–92. ©2017 AACR.

Protein-protein network analysis

Small cell lung cancer (SCLC) is an aggressive neuroendocrine cancer characterized by loss of function TP53 and RB1 mutations in addition to mutations in other oncogenes including MYC. Overexpression of MYC together with Trp53 and Rb1 loss in pulmonary neuroendocrine cells of the mouse lung drives an aggressive neuroendocrine low variant subtype of SCLC. However, the transforming potential of MYC amplification alone on airway epithelium is unclear. Therefore, we selectively and conditionally overexpressed MYC stochastically throughout the airway or specifically in neuroendocrine, club, or alveolar type II cells in the adult mouse lung. We observed that MYC overexpression induced carcinoma in situ which did not progress to invasive disease. The formation of adenoma or SCLC carcinoma in situ was dependent on the cell of origin. In contrast, MYC overexpression combined with conditional deletion of both Trp53 and Rb1 exclusively gave rise to SCLC, irrespective of the cell lineage of origin. However, cell of origin influenced disease latency, metastatic potential, and the transcriptional profile of the SCLC phenotype. Together this reveals that MYC overexpression alone provides a proliferative advantage but when combined with deletion of Trp53 and Rb1 it facilitates the formation of aggressive SCLC from multiple cell lineages.

Purpose: Eryaspase is composed of l-asparaginase encapsulated in erythrocytes and has demonstrated significant efficacy in a randomized phase II trial. We assessed the prognostic and predictive value of circulating tumor DNA (ctDNA) in patients, plasma included in this trial. Experimental Design: Samples prospectively collected pretreatment were centrally analyzed by next-generation sequencing. Prognostic values of baseline ctDNA and ctDNA early changes between day 0 and 28 were assessed in both arms combined on objective response rate (ORR), progression-free survival (PFS), and overall survival (OS); three groups were defined: negative ctDNA (Neg), ctDNA responders (Resp), and ctDNA nonresponders (NResp). Predictive value of ctDNA for eryaspase efficacy was investigated. Results: ctDNA was positive at baseline in 77 patients of the 113 tested patients (68%). Detectable ctDNA was an independent negative prognostic factor for OS (4.6 vs. 8.8 months; P = 0.0025) and PFS (1.6 vs. 3.3 months; P = 0.00043). Early change in ctDNA levels was correlated with ORR (20%, 26%, 0%; P < 0.04), PFS (3.7, 3.4, 1.6 months; P < 0.0001), and OS (11.7, 6.5, 4.3 months; P < 0.0001) according to the three defined groups (Neg, Res, NResp, respectively). In patients with ctDNA detectable at baseline, eryaspase was associated with better PFS [HR = 0.53; 95% confidence interval (CI): 0.3–0.94)] and OS (HR = 0.52; 95% CI: 0.29–0.91). Conclusions: We confirm from a prospective randomized trial that: (i) the presence of ctDNA at baseline is a major prognostic factor, (ii) the early change of ctDNA correlates with treatment outcome, and (iii) the ctDNA could be a predictive biomarker of eryaspase efficacy.

Diffuse midline glioma (DMG) is a deadly pediatric and adolescent central nervous system (CNS) tumor localized along the midline structures of the brain atop the spinal cord. With a median overall survival (OS) of just 9–11-months, DMG is characterized by global hypomethylation of histone H3 at lysine 27 (H3K27me3), driven by recurring somatic mutations in H3 genes including, HIST1H3B/C (H3.1K27M) or H3F3A (H3.3K27M), or through overexpression of EZHIP in patients harboring wildtype H3. The recent World Health Organization’s 5th Classification of CNS Tumors now designates DMG as, ‘H3 K27-altered’, suggesting that global H3K27me3 hypomethylation is a ubiquitous feature of DMG and drives devastating transcriptional programs for which there are no treatments. H3-alterations co-segregate with various other somatic driver mutations, highlighting the high-level of intertumoral heterogeneity of DMG. Furthermore, DMG is also characterized by very high-level intratumoral diversity with tumors harboring multiple subclones within each primary tumor. Each subclone contains their own combinations of driver and passenger lesions that continually evolve, making precision-based medicine challenging to successful execute. Whilst the intertumoral heterogeneity of DMG has been extensively investigated, this is yet to translate to an increase in patient survival. Conversely, our understanding of the non-genomic factors that drive the rapid growth and fatal nature of DMG, including endogenous and exogenous microenvironmental influences, neurological cues, and the posttranscriptional and posttranslational architecture of DMG remains enigmatic or at best, immature. However, these factors are likely to play a significant role in the complex biological sequelae that drives the disease. Here we summarize the heterogeneity of DMG and emphasize how analysis of the posttranslational architecture may improve treatment paradigms. We describe factors that contribute to treatment response and disease progression, as well as highlight the potential for pharmaco-proteogenomics (i.e., the integration of genomics, proteomics and pharmacology) in the management of this uniformly fatal cancer.

Rare Brain Tumor Registry., [Background] Embryonal tumours with multi-layered rosettes (ETMRs) are a newly recognised, rare paediatric brain tumour with alterations of the C19MC microRNA locus. Due to varied diagnostic practices and scarce clinical data, disease features and determinants of outcomes for these tumours are poorly defined. We did an integrated clinicopathological and molecular analysis of primary ETMRs to define clinical phenotypes, and to identify prognostic factors of survival and key treatment modalities for this orphan disease., [Methods] Paediatric patients with primary ETMRs and tissue available for analyses were identified from the Rare Brain Tumor Consortium global registry. The institutional histopathological diagnoses were centrally re-reviewed as per the current WHO CNS tumour guidelines, using histopathological and molecular assays. Only patients with complete clinical, treatment, and survival data on Nov 30, 2019, were included in clinicopathological analyses. Among patients who received primary multi-modal curative regimens, event-free survival and overall survival were determined using Cox proportional hazard and log-rank analyses. Univariate and multivariable Cox proportional hazard regression was used to estimate hazard ratios (HRs) with 95% CIs for clinical, molecular, or treatment-related prognostic factors., [Findings] 159 patients had a confirmed molecular diagnosis of primary ETMRs (median age at diagnosis 26 months, IQR 18–36) and were included in our clinicopathological analysis. ETMRs were predominantly non-metastatic (94 [73%] of 128 patients), arising from multiple sites; 84 (55%) of 154 were cerebral tumours and 70 (45%) of 154 arose at sites characteristic of other brain tumours. Hallmark C19MC alterations were seen in 144 (91%) of 159 patients; 15 (9%) were ETMR not otherwise specified. In patients treated with curative intent, event-free survival was 57% (95% CI 47–68) at 6 months and 31% (21–42) at 2 years; overall survival was 29% (20–38) at 2 years and 27% (18–37) at 4 years. Overall survival was associated with non-metastatic disease (HR 0·48, 95% CI 0·28–0·80; p=0·0057) and non-brainstem location (0·42 [0·22–0·81]; p=0·013) on univariate analysis, as well as with gross total resection (0·30, 0·16–0·58; p=0·0014), high-dose chemotherapy (0·35, 0·19–0·67; p=0·0020), and radiotherapy (0·21, 0·10–0·41; p, [Interpretation] Prompt molecular diagnosis and post-surgical treatment with intensive multi-modal therapy tailored to patient-specific risk features could improve ETMR survival., Canadian Institute of Health Research, Canada Research Chair Awards, Australian Lions Childhood Cancer Research Foundation, Spanish Society of Pediatrics, Consejería de Salud y Familias de la Junta de Andalucía, Miracle Marnie, Phoebe Rose Rocks, Tali's Funds, Garron Cancer Centre, Grace's Walk, Meagan's Hug, Brainchild, Nelina's Hope, and Jean Martel Foundation.

Objectives Primary cilia are sensory organelles which co-ordinate several developmental/repair pathways including hedgehog signalling. Studies of human renal allografts suffering acute tubular necrosis have shown that length of primary cilia borne by epithelial cells doubles throughout the nephron and collecting duct, and then normalises as renal function returns. Conversely the loss of primary cilia has been reported in chronic allograft rejection and linked to defective hedgehog signalling. We investigated the fate of primary cilia in renal allografts suffering acute rejection. Results Here we observed that in renal allografts undergoing acute rejection, primary cilia were retained, with their length increasing 1 week after transplantation and remaining elevated. We used a mouse model of acute renal injury to demonstrate that elongated renal primary cilia in the injured renal tubule show evidence of smoothened accumulation, a biomarker for activation of hedgehog signalling. We conclude that primary cilium-mediated activation of hedgehog signalling is still possible during the acute phase of renal allograft rejection.

Background Different cell types possess different copies of mtDNA to support their specific requirements for cellular metabolism. Cell-specific mtDNA copy numbers are established through cell-specific mtDNA replication during cell differentiation. However, cancer cells are trapped in a “pseudo-differentiated” state as they fail to expand mtDNA copy number. Global DNA methylation can regulate this process, as induced DNA demethylation promotes differentiation of cancer cells and expansion of mtDNA copy number. Results To determine the role that mtDNA methylation plays in regulating mtDNA replication during tumorigenesis, we have characterized the patterns of mtDNA methylation using glioblastoma and osteosarcoma tumor models that have different combinations of mtDNA genotypes and copy number against common nuclear genome backgrounds at different stages of tumor progression. To ensure the reliability of the findings, we have applied a robust experimental pipeline including three approaches, namely whole-mtDNA bisulfite-sequencing with mtDNA-genotype-specific analysis, pyrosequencing, and methylated immunoprecipitation against 5mC and 5hmC. We have determined genotype-specific methylation profiles, which were modulated through tumor progression. Moreover, a strong influence from the nuclear genome was also observed on mtDNA methylation patterns using the same mtDNA genotype under different nuclear genomes. Furthermore, the numbers of mtDNA copy in tumor-initiating cells affected mtDNA methylation levels in late-stage tumors. Conclusions Our findings highlight the influences that the nuclear and mitochondrial genomes have in setting mtDNA methylation patterns to regulate mtDNA copy number in tumorigenesis. They have important implications for assessing global DNA methylation patterns in tumorigenesis and the availability of mtDNA template for mtDNA replication. Electronic supplementary material The online version of this article (10.1186/s13148-018-0590-0) contains supplementary material, which is available to authorized users.

Background Diffuse intrinsic pontine glioma (DIPG) is a fatal childhood brainstem tumor for which radiation is the only treatment. Case studies report a clinical response to ONC201 for patients with H3K27M-mutant gliomas. Oncoceutics (ONC201) is only available in the United States and Japan; however, in Germany, DIPG patients can be prescribed and dispensed a locally produced compound—ONC201 German-sourced ONC201 (GsONC201). Pediatric oncologists face the dilemma of supporting the administration of GsONC201 as conjecture surrounds its authenticity. Therefore, we compared GsONC201 to original ONC201 manufactured by Oncoceutics Inc. Methods Authenticity of GsONC201 was determined by high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy. Biological activity was shown via assessment of on-target effects, in vitro growth, proliferation, and apoptosis analysis. Patient-derived xenograft mouse models were used to assess plasma and brain tissue pharmacokinetics, pharmacodynamics, and overall survival (OS). The clinical experience of 28 H3K27M+ mutant DIPG patients who received GsONC201 (2017–2020) was analyzed. Results GsONC201 harbored the authentic structure, however, was formulated as a free base rather than the dihydrochloride salt used in clinical trials. GsONC201 in vitro and in vivo efficacy and drug bioavailability studies showed no difference compared to Oncoceutics ONC201. Patients treated with GsONC201 (n = 28) showed a median OS of 18 months (P = .0007). GsONC201 patients who underwent reirradiation showed a median OS of 22 months compared to 12 months for GsONC201 patients who did not (P = .012). Conclusions This study confirms the biological activity of GsONC201 and documents the OS of patients who received the drug; however, GsONC201 was never used as a monotherapy.

We previously used a pulse-based in vitro assay to unveil targetable signalling pathways associated with innate cisplatin resistance in lung adenocarcinoma (Hastings et al., 2020). Here, we advanced this model system and identified a non-genetic mechanism of resistance that drives recovery and regrowth in a subset of cells. Using RNAseq and a suite of biosensors to track single-cell fates both in vitro and in vivo, we identified that early S phase cells have a greater ability to maintain proliferative capacity, which correlated with reduced DNA damage over multiple generations. In contrast, cells in G1, late S or those treated with PARP/RAD51 inhibitors, maintained higher levels of DNA damage and underwent prolonged S/G2 phase arrest and senescence. Combined with our previous work, these data indicate that there is a non-genetic mechanism of resistance in human lung adenocarcinoma that is dependent on the cell cycle stage at the time of cisplatin exposure.

Diffuse midline gliomas (DMGs) remain incurable cancers and new treatments are urgently needed. One promising new therapeutic avenue for these cancers is targeting of metabolic vulnerabilities including a heightened dependence on mitochondrial metabolism. We and others have shown that the oral, brain-penetrant imipridone drugs ONC201 and ONC206 target mitochondrial metabolism in cancer cells. In particular, ONC201 and ONC206 hyper-activate the mitochondrial protease ClpP, impair mitochondrial oxidative phosphorylation (OXPHOS), activate the integrated stress response (ISR) signaling pathway, and induce apoptosis in DMG preclinical models. We validated ClpP as a key target of ONC206 by showing that CRISPR/Cas9-mediated CLPP knockout significantly decreased ONC206 sensitivity in DMG cells. We further showed that imipridone-mediated ClpP activation resulted in significant degradation of the chaperone protein ClpX. Moreover, ONC201 and ONC206 treatment inhibited mitochondrial respiration, decreased mitochondrial membrane potential and triggered extensive mitochondrial structural damage, including disintegration of mitochondrial cristae. Time-course RNA sequencing of five DMG cell lines treated with ONC201 and ONC206, alone or in combination, revealed robust ATF4 and CHOP upregulation, indicating potent activation of ISR signaling. Notably, ATF4/CHOP upregulation was strongest in ONC201/6 combination-treated cells, indicating synergy between the two drugs. We further explored drug combinations by testing ONC201 together with ONC206, Panobinostat, JQ1, and Osimertinib to identify synergistic combination treatments. The strongest synergistic effect was found over a broad IC50 range for ONC201 and ONC206. Finally, we showed that ONC201 and ONC206 significantly prolonged survival of mice bearing brainstem DIPG xenografts. Ongoing studies include assessment of the in vivo efficacy of ONC201 and ONC206 across different CNS tumor models, as well as investigation and validation of clinically relevant biomarkers of response to treatment. In summary, our preclinical data strongly support the utility of the mitochondrial targeting agents ONC201 and ONC206 for the treatment of DMG and other malignant brain tumors.

Small cell lung cancer (SCLC) is an aggressive neuroendocrine cancer characterized by loss of function TP53 and RB1 mutations in addition to mutations in other oncogenes including MYC. Overexpression of MYC together with Trp53 and Rb1 loss in pulmonary neuroendocrine cells of the mouse lung drives an aggressive neuroendocrine low variant subtype of SCLC. However, the transforming potential of MYC amplification alone on airway epithelium is unclear. Therefore, we selectively and conditionally overexpressed MYC stochastically throughout the airway or specifically in neuroendocrine, club, or alveolar type II cells in the adult mouse lung. We observed that MYC overexpression induced carcinoma in situ which did not progress to invasive disease. The formation of adenoma or SCLC carcinoma in situ was dependent on the cell of origin. In contrast, MYC overexpression combined with conditional deletion of both Trp53 and Rb1 exclusively gave rise to SCLC, irrespective of the cell lineage of origin. However, cell of origin influenced disease latency, metastatic potential, and the transcriptional profile of the SCLC phenotype. Together this reveals that MYC overexpression alone provides a proliferative advantage but when combined with deletion of Trp53 and Rb1 it facilitates the formation of aggressive SCLC from multiple cell lineages.

Ephrin receptor A2 (EphA2) is a member of the Ephrin/Eph receptor cell-to-cell signaling family of molecules, and it plays a key role in cell proliferation, differentiation, and migration. EphA2 is overexpressed in a broad range of cancers, and its expression is in many cases associated with poor prognosis. We recently developed a novel EphA2-targeting antibody-directed nanotherapeutic encapsulating a labile prodrug of docetaxel (EphA2-ILs-DTXp) for the treatment of EphA2-expressing malignancies. Here, we characterized the expression of EphA2 in bladder cancer using immunohistochemistry in 177 human bladder cancer samples and determined the preclinical efficacy of EphA2-ILs-DTXp in four EphA2-positive patient-derived xenograft (PDX) models of the disease, either as a monotherapy, or in combination with gemcitabine. EphA2 expression was detected in 80&ndash, 100% of bladder cancer samples and correlated with shorter patient survival. EphA2 was found to be expressed in tumor cells and/or tumor-associated blood vessels in both primary and metastatic lesions with a concordance rate of approximately 90%. The EphA2-targeted antibody-directed nanotherapeutic EphA2-ILs-DTXp controlled tumor growth, mediated greater regression, and was more active than free docetaxel at equitoxic dosing in all four EphA2-positive bladder cancer PDX models. Combination of EphA2-ILs-DTXp and gemcitabine in one PDX model led to improved tumor growth control compared to monotherapies or the combination of free docetaxel and gemcitabine. These data demonstrating the prevalence of EphA2 in bladder cancers and efficacy of EphA2-ILs-DTXp in PDX models support the clinical exploration of EphA2 targeting in bladder cancer.

BackgroundETMRs are a newly recognized rare paediatric brain tumor with alterations of the C19MC microRNA locus. Due to varied diagnostic practices and limited clinical data, disease features and determinants of outcome are poorly defined. We performed an integrated clinico-pathologic and molecular analyses of 159 primary ETMRs to define clinical phenotypes, identify predictors of survival and critical treatment modalities for this orphan disease.MethodsPrimary ETMR patients were identified from the Rare Brain Tumor Consortium (rarebraintumorconsortium.ca) global registry using histopathologic and molecular assays. Event-Free (EFS) and Overall Survival (OS) for 108 patients treated with curative multi-modal regimens were determined using Cox proportional hazard and log rank analyses.FindingsETMRs were predominantly non-metastatic (73%) tumors arising from multiple sites; 55% were cerebral tumors, 45% arose at sites characteristic of other brain tumors. Hallmark C19MC alterations were seen in 91%; 9% were ETMR-NOS. Survival and hazard analyses showed a 6 month median EFS and 2-4yr OS of 27-29% with metastatic disease (HR=0.44, 95% CI 0.26-0.74; p=0.002) and brainstem location (HR=0.40, 95% CI 0.021-0.75; p=0.005) correlating with adverse OS. Gross total resection (GTR: HR=0.38, 95% CI 0.21-0.68; p=0.001), high dose chemotherapy (HDC: HR=0.55, 95% CI 0.31-0.97; p=0.04) and radiation (RT: HR=0.32, 95% CI 0.16-0.60; p=InterpretationPrompt molecular diagnosis and post-surgical treatment with multi-modal therapy tailored to patient-specific risk features improves ETMR survival.FundingThis work was supported by the Canadian Institute of Health Research Grant No. 137011, Canada Research Chair Awards to AH. Funds from Miracle Marnie, Phoebe Rose Rocks, Tali’s Funds, Garron Cancer Centre, Grace’s Walk, Meagan’s Walk, Nelina’s Hope and Jean Martel Foundation are gratefully acknowledged. SK and PS were respectively supported by the Australian Lions Children’s Cancer Foundation and the Spanish Society of Pediatrics, Consejería de Salud y Familias de la Junta de Andalucía Project EF-0451-2017.

Ligand-dependent activation of Hedgehog (Hh) signaling in cancer occurs without mutations in canonical pathway genes. Consequently, the genetic basis of Hh pathway activation in adult solid tumors, such as small-cell lung cancer (SCLC), is unknown. Here we show that combined inactivation of Trp53 and Rb1, a defining genetic feature of SCLC, leads to hypersensitivity to Hh ligand in vitro, and during neural tube development in vivo. This response is associated with the aberrant formation of primary cilia, an organelle essential for canonical Hh signaling through smoothened, a transmembrane protein targeted by small-molecule Hh inhibitors. We further show that loss of both Trp53 and Rb1 disables transcription of genes in the autophagic machinery necessary for the degradation of primary cilia. In turn, we also demonstrate a requirement for Kif3a, a gene essential for the formation of primary cilia, in a mouse model of SCLC induced by conditional deletion of both Trp53 and Rb1 in the adult airway. Our results provide a mechanistic framework for therapeutic targeting of ligand-dependent Hh signaling in human cancers with somatic mutations in both TP53 and RB1.

Maintenance of male fertility requires spermatogonial stem cells (SSCs) that self-renew and generate differentiating germ cells for production of spermatozoa. Germline cells are sensitive to genotoxic drugs and patients receiving chemotherapy can become infertile. SSCs surviving treatment mediate germline recovery but pathways driving SSC regenerative responses remain poorly understood. Using models of chemotherapy-induced germline damage and recovery, here we identify unique molecular features of regenerative SSCs and characterise changes in composition of the undifferentiated spermatogonial pool during germline recovery by single-cell analysis. Increased mitotic activity of SSCs mediating regeneration is accompanied by alterations in growth factor signalling including PI3K/AKT and mTORC1 pathways. While sustained mTORC1 signalling is detrimental for SSC maintenance, transient mTORC1 activation is critical for the regenerative response. Concerted inhibition of growth factor signalling disrupts core features of the regenerative state and limits germline recovery. We also demonstrate that the FOXM1 transcription factor is a target of growth factor signalling in undifferentiated spermatogonia and provide evidence for a role in regeneration. Our data confirm dynamic changes in SSC functional properties following damage and support an essential role for microenvironmental growth factors in promoting a regenerative state.

SUMMARY Over the past decade, wingless-activated (WNT) medulloblastoma has been identified as a candidate for therapy de-escalation based on excellent survival; however, a paucity of relapses has precluded additional analyses of markers of relapse. To address this gap in knowledge, an international cohort of 93 molecularly confirmed WNT MB was assembled, where 5-year progression-free survival is 0.84 (95%, 0.763–0.925) with 15 relapsed individuals identified. Maintenance chemotherapy is identified as a strong predictor of relapse, with individuals receiving high doses of cyclophosphamide or ifosphamide having only one very late molecularly confirmed relapse (p = 0.032). The anatomical location of recurrence is metastatic in 12 of 15 relapses, with 8 of 12 metastatic relapses in the lateral ventricles. Maintenance chemotherapy, specifically cumulative cyclophosphamide doses, is a significant predictor of relapse across WNT MB. Future efforts to de-escalate therapy need to carefully consider not only the radiation dose but also the chemotherapy regimen and the propensity for metastatic relapses., Graphical Abstract, In Brief Nobre et al. analyze a clinically annotated cohort of 93 WNT-activated medulloblastoma using an integrated genomic approach. A maintenance chemotherapy regimen is the strongest predictor of relapse, and cumulative cyclophosphamide doses confer a reduced risk of relapse. Unlike other medulloblastoma subgroups, WNT-activated medulloblastoma recurs most frequently in the lateral ventricles.

Our understanding of genomic heterogeneity in lung cancer is largely based on the analysis of early-stage surgical specimens. Here we used endoscopic sampling of paired primary and intrathoracic metastatic tumors from 11 lung cancer patients to map genomic heterogeneity inoperable lung cancer with deep whole-genome sequencing. Intra-patient heterogeneity in driver or targetable mutations was predominantly in the form of copy number gain. Private mutation signatures, including patterns consistent with defects in homologous recombination, were highly variable both within and between patients. Irrespective of histotype, we observed a smaller than expected number of private mutations, suggesting that ancestral clones accumulated large mutation burdens immediately prior to metastasis. Single-region whole-genome sequencing of from 20 patients showed that tumors in ever-smokers with the strongest tobacco signatures were associated with germline variants in genes implicated in the repair of cigarette-induced DNA damage. Our results suggest that lung cancer precursors in ever-smokers accumulate large numbers of mutations prior to the formation of frank malignancy followed by rapid metastatic spread. In advanced lung cancer, germline variants in DNA repair genes may interact with the airway environment to influence the pattern of founder mutations, whereas similar interactions with the tumor microenvironment may play a role in the acquisition of mutations following metastasis.

Simple Summary Atypical teratoid rhabdoid tumour (ATRT) is an aggressive undifferentiated malignancy of the central nervous system in children. A defining feature of ATRT is the loss of the SMARCB1 gene that is essential for regulating gene expression required for normal developmental processes. We show that treatment of human ATRT cell models with the histone deacetylate inhibitor, panobinostat, inhibits tumour growth, reactivates the expression of developmental genes, and drives neuronal differentiation. These results demonstrate the therapeutic potential of panobinostat for the treatment of ATRT. Abstract Atypical teratoid rhabdoid tumour (ATRT) is a rare but highly aggressive undifferentiated solid tumour arising in the central nervous system and predominantly affecting infants and young children. ATRT is exclusively characterized by the inactivation of SMARCB1, a member of the SWI/SNF chromatin remodelling complex that is essential for the regulation of large sets of genes required for normal development and differentiation. Histone deacetylase inhibitors (HDACi) are a promising anticancer therapy and are able to mimic the normal acetylation functions of SMARCB1 in SMARCB1-deficient cells and drive multilineage differentiation in extracranial rhabdoid tumours. However, the potential efficacy of HDACi in ATRT is unknown. Here, we show that human ATRT cells are highly responsive to the HDACi panobinostat and that sustained treatment leads to growth arrest, increased cell senescence, decreased clonogenicity and induction of a neurogenesis gene-expression profile. Furthermore, in an orthotopic ATRT xenograft model, continuous panobinostat treatment inhibits tumour growth, increases survival and drives neuronal differentiation as shown by the expression of the neuronal marker, TUJ1. Collectively, this preclinical study supports the therapeutic potential of panobinostat-mediated differentiation therapy for ATRT.

Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive and infiltrative pediatric brainstem tumor with universal lethality. Current treatment is limited to palliative radiotherapy. Molecular characterization of DIPG has revealed recurrent heterozygous mutation (p.K27M) in the Histone H3 variant genes; H3.1 (HIST1H3B, HIS1H3C) or H3.3 (H3F3A) in >80% of cases. While H3K27M function is still under heavy investigation, accumulating molecular evidence now suggests its subsequent role in tumorigenesis is driven through epigenetic dysregulation of the chromatin via the loss of tri-methylation markers. Co-occurring mutations in known oncogenes and amplification in cancer prone proliferative pathways further contribute to the DIPG's diverse molecular landscape and may represent therapeutic vulnerabilities. The purpose of this present study is to utilize a functional genomics pipeline to investigate the intrinsic molecular mechanisms of H3K27M DIPG. Identification of novel molecular dependencies and pathways that contribute to overall disease progression serve as potential targets for therapeutic exploitation and development for clinical utility. Sixteen established and validated patient derived cell lines (10 H3.3K27M, 4 H3.1K27M and 2 Wt), obtained from external collaborators were used in this study. High throughput screening for DIPG drug sensitivity was carried out using a panel of 2048 molecular compounds. Cell viability data following 72 hours treatment was z- score transformed and potential compounds identified based on a z-score ≤-1.5 and no effect in a neural stem cell (NSC) control line. Highly prevalent targets identified from this primary screen include HDAC, Proteasome, Topoisomerase and Microtubule Associated inhibitors. In parallel, a 300 gene pooled CRISPR/Cas9 loss of function screen was used to determine key DIPG survival dependencies. Cas9 expressing DIPG cell lines (3 H3.1K27M and 3 H3.3K27M) were transduced with 1200 gRNAs and cultured for 21-28 day period. Next Generation Sequencing (NGS) of samples was used to determine gRNA depletion and a set z score threshold of ≤-1.5 to distinguish potential “hits”. Hierarchical clustering revealed genotype specific patterns suggesting differing tumorigenic mechanisms between H3.1 and H3.3 K27M DIPG. Comparison between CRISPR and drug screens revealed 24 molecular targets with therapeutic sensitivity and DIPG cell survival dependency. We are currently carrying out validation studies of these targets for therapeutic development, effectively demonstrating a functional genomics pipeline is able to identify therapeutic dependencies in DIPG. Citation Format: Sarah Parackal, Gabrielle Bradshaw, Claire Sun, Wai Chin Chong, Paul Daniel, Samantha Jayasekara, Duncan Crombie, Ron Firestein, Jason Cain, Department of Molecular Translational Science, Monash University. Functional genomics pipeline reveals therapeutic dependencies in diffuse intrinsic pontine glioma [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 3029.

There is a growing role for mutations affecting histone linker and histone core-encoding genes across several adult and pediatric cancers. However, the extent to which somatic histone mutations may bridge across different cancers as common tumorigenic events – particularly in the context of pediatric CNS tumors – remains unclear. To address this knowledge gap, we set out to define a comprehensive pan-cancer landscape of somatic histone mutations. We first queried the ICGC PCAWG and TCGA Pan-Cancer Atlas representing >12,500 adult and pediatric cancer patients. We found lymphomas to be most enriched for histone mutations (50–75%) and, in particular, for mutations in linker histones (HIST1H1B-E), yet also in specific core histone genes (eg, HIST2H2BE). Moreover, we observed a significant enrichment of histone mutations in adult high-grade vs low-grade gliomas (10% vs 6%, P

Intrinsic ureteropelvic junction obstruction is the most common cause of congenital hydronephrosis, yet the underlying pathogenesis is undefined. Hedgehog proteins control morphogenesis by promoting GLI-dependent transcriptional activation and inhibiting the formation of the GLI3 transcriptional repressor. Hedgehog regulates differentiation and proliferation of ureteric smooth muscle progenitor cells during murine kidney-ureter development. Histopathologic findings of smooth muscle cell hypertrophy and stroma-like cells, consistently observed in obstructing tissue at the time of surgical correction, suggest that Hedgehog signaling is abnormally regulated during the genesis of congenital intrinsic ureteropelvic junction obstruction. Here, we demonstrate that constitutively active Hedgehog signaling in murine intermediate mesoderm–derived renal progenitors results in hydronephrosis and failure to develop a patent pelvic-ureteric junction. Tissue obstructing the ureteropelvic junction was marked as early as E13.5 by an ectopic population of cells expressing Ptch2, a Hedgehog signaling target. Constitutive expression of GLI3 repressor in Ptch1-deficient mice rescued ectopic Ptch2 expression and obstructive hydronephrosis. Whole transcriptome analysis of isolated Ptch2+ cells revealed coexpression of genes characteristic of stromal progenitor cells. Genetic lineage tracing indicated that stromal cells blocking the ureteropelvic junction were derived from intermediate mesoderm–derived renal progenitors and were distinct from the smooth muscle or epithelial lineages. Analysis of obstructive ureteric tissue resected from children with congenital intrinsic ureteropelvic junction obstruction revealed a molecular signature similar to that observed in Ptch1-deficient mice. Together, these results demonstrate a Hedgehog-dependent mechanism underlying mammalian intrinsic ureteropelvic junction obstruction.

Purpose: To determine whether deposition characteristics of ferumoxytol (FMX) iron nanoparticles in tumors, identified by quantitative MRI, may predict tumor lesion response to nanoliposomal irinotecan (nal-IRI).Experimental Design: Eligible patients with previously treated solid tumors had FMX-MRI scans before and following (1, 24, and 72 hours) FMX injection. After MRI acquisition, R2* signal was used to calculate FMX levels in plasma, reference tissue, and tumor lesions by comparison with a phantom-based standard curve. Patients then received nal-IRI (70 mg/m2 free base strength) biweekly until progression. Two percutaneous core biopsies were collected from selected tumor lesions 72 hours after FMX or nal-IRI.Results: Iron particle levels were quantified by FMX-MRI in plasma, reference tissues, and tumor lesions in 13 of 15 eligible patients. On the basis of a mechanistic pharmacokinetic model, tissue permeability to FMX correlated with early FMX-MRI signals at 1 and 24 hours, while FMX tissue binding contributed at 72 hours. Higher FMX levels (ranked relative to median value of multiple evaluable lesions from 9 patients) were significantly associated with reduction in lesion size by RECIST v1.1 at early time points (P < 0.001 at 1 hour and P < 0.003 at 24 hours FMX-MRI, one-way ANOVA). No association was observed with post-FMX levels at 72 hours. Irinotecan drug levels in lesions correlated with patient's time on treatment (Spearman ρ = 0.7824; P = 0.0016).Conclusions: Correlation between FMX levels in tumor lesions and nal-IRI activity suggests that lesion permeability to FMX and subsequent tumor uptake may be a useful noninvasive and predictive biomarker for nal-IRI response in patients with solid tumors. Clin Cancer Res; 23(14); 3638–48. ©2017 AACR.

Hedgehog (Hh) signaling regulates cell fate and self-renewal in development and cancer. Canonical Hh signaling is mediated by Hh ligand binding to the receptor Patched (Ptch), which in turn activates Gli-mediated transcription through Smoothened (Smo), the molecular target of the Hh pathway inhibitors used as cancer therapeutics. Small cell lung cancer (SCLC) is a common, aggressive malignancy with universally poor prognosis. Although preclinical studies have shown that Hh inhibitors block the self-renewal capacity of SCLC cells, the lack of activating pathway mutations have cast doubt over the significance of these observations. In particular, the existence of autocrine, ligand-dependent Hh signaling in SCLC has been disputed. In a conditional Tp53;Rb1 mutant mouse model of SCLC, we now demonstrate a requirement for the Hh ligand Sonic Hedgehog (Shh) for the progression of SCLC. Conversely, we show that conditional Shh overexpression activates canonical Hh signaling in SCLC cells, and markedly accelerates tumor progression. When compared to mouse SCLC tumors expressing an activating, ligand-independent Smo mutant, tumors overexpressing Shh exhibited marked chromosomal instability and Smoothened-independent upregulation of Cyclin B1, a putative non-canonical arm of the Hh pathway. In turn, we show that overexpression of Cyclin B1 induces chromosomal instability in mouse embryonic fibroblasts lacking both Tp53 and Rb1. These results provide strong support for an autocrine, ligand-dependent model of Hh signaling in SCLC pathogenesis, and reveal a novel role for non-canonical Hh signaling through the induction of chromosomal instability.

BACKGROUND: Diffuse Intrinsic Pontine Glioma (DIPG) is a devastating, incurable childhood brain cancer. 80% of patients harbour a genetic lesion to Histone-H3 with a substitution of lysine 27 for a methionine (K27M) (H3F3A/H3.3 and HIST1H3B/H3.1) which results in the loss of methylation, interfering with chromatin function, activating oncogenic transcription. Unfortunately, H3 mutations are not directly targetable. RTK-RAS-PI3Kpathway alterations occur in more than half of these patients (69%), and may represent a more promising therapeutic target. However, PI3K inhibitors have been notoriously ineffective due to their inability to cross the blood-brain-barrier (BBB). Targeting downstream components of the PI3K-AKT-mTOR signalling axis is a promising paradigm for PI3K mutant patients. This project aims to overcome the limitations of targeting downstream effectors of the PI3K pathway by utilising a novel PIK3CA specific, BBB permeable inhibitor, GDC-0084, currently in clinical trials for Glioblastoma (GBM) and DIPG. METHODS: Utilising DIPG patient derived (PDX) cells harboringH3K27M mutations (n=6), and +/- PI3Kmutations, we examined the in-vitroefficacy of GDC-0084 on cell proliferation compared to Rapamycin. Phosphoproteomic profiling is currently underway investigating the role GDC-0084 has on inhibition of downstream oncogenic signalling pathways. Assessment of efficacy in a PDX in-vivo model is currently in progress. RESULTS: GDC-0084 significantly reduced the growth of all DIPG cell lines regardless of whether they harboredPI3K mutations (p=

Despite the rarity of renal tumors in children, many different types of malignant and nonmalignant renal neoplasms have been described. Therefore, the correct diagnosis and clinical management of these patients can represent a challenge. Here we provide a comprehensive review of the commonly diagnosed pediatric renal malignancies, including nephroblastoma (commonly known as Wilms tumor), clear cell sarcoma of the kidney, rhabdoid tumor of the kidney, several subtypes of renal cell tumors (often collectively termed renal cell carcinoma), and congenital mesoblastic nephroma. The epidemiology, pathology, treatments, underlying genetic and molecular mechanisms, and proposed developmental origins are discussed in detail, highlighting differential features and potential improved therapeutic strategies for affected individuals.

Oncogenic KRAS mutations are major drivers of lung adenocarcinoma (LAC), yet the direct therapeutic targeting of KRAS has been problematic. Here, we reveal an obligate requirement by oncogenic KRAS for the ADAM17 protease in LAC. In genetically engineered and xenograft (human cell line and patient‐derived) Kras G12D ‐driven LAC models, the specific blockade of ADAM17, including with a non‐toxic prodomain inhibitor, suppressed tumor burden by reducing cellular proliferation. The pro‐tumorigenic activity of ADAM17 was dependent upon its threonine phosphorylation by p38 MAPK, along with the preferential shedding of the ADAM17 substrate, IL‐6R, to release soluble IL‐6R that drives IL‐6 trans‐signaling via the ERK1/2 MAPK pathway. The requirement for ADAM17 in Kras G12D ‐driven LAC was independent of bone marrow‐derived immune cells. Furthermore, in KRAS mutant human LAC, there was a significant positive correlation between augmented phospho‐ADAM17 levels, observed primarily in epithelial rather than immune cells, and activation of ERK and p38 MAPK pathways. Collectively, these findings identify ADAM17 as a druggable target for oncogenic KRAS ‐driven LAC and provide the rationale to employ ADAM17‐based therapeutic strategies for targeting KRAS mutant cancers.

Atypical teratoid rhabdoid tumours (ATRTs) are high-grade malignant paediatric brain tumours with a less than one-year survival rate after diagnosis. Current treatment for ATRT, which includes high-intensive radiotherapy and chemotherapy, results in long-term side effects on ATRT patients. Hence, there is an urgent need to discover targeted therapies that could be used to treat patients with ATRT. As part of the Hudson Monash Paediatric Precision Medicine Program, we have collected 23 ATRT cell lines which we used to performed high-throughput small molecule and genetic (CRISPR) screening to identify new therapies and therapeutic targets. In parallel, we characterised the ATRT cell lines based on transcriptomic (RNA-seq) and epigenetic (methylation) signatures. An integrative multi-omic approach was then used to uncover discrete vulnerabilities in specific subsets of ATRT patients. Strikingly, these include a number of druggable dependencies, such as MEK, CDK, HDAC, and Topoisomerase, that offer a promise of rapid clinical translation. In our study, we focus on MEK dependency in a subtype of ATRT lines to further define the underlying mechanisms and biomarkers. While future studies validating the MEK/ERK signalling dependency in a wider cohort of patient models and in in vivo models are required, these data provide a framework for applying an integrative multi-omic approach in paediatric cancer precision medicine.

We report a comprehensive proteogenomics analysis, including whole-genome sequencing, RNA sequencing, and proteomics and phosphoproteomics profiling, of 218 tumors across 7 histological types of childhood brain cancer: low-grade glioma (n = 93), ependymoma (32), high-grade glioma (25), medulloblastoma (22), ganglioglioma (18), craniopharyngioma (16), and atypical teratoid rhabdoid tumor (12). Proteomics data identify common biological themes that span histological boundaries, suggesting that treatments used for one histological type may be applied effectively to other tumors sharing similar proteomics features. Immune landscape characterization reveals diverse tumor microenvironments across and within diagnoses. Proteomics data further reveal functional effects of somatic mutations and copy number variations (CNVs) not evident in transcriptomics data. Kinase-substrate association and co-expression network analysis identify important biological mechanisms of tumorigenesis. This is the first large-scale proteogenomics analysis across traditional histological boundaries to uncover foundational pediatric brain tumor biology and inform rational treatment selection.

INTRODUCTION DMG-ACT (DMG- multi-arm Adaptive and Combinatorial Trial) aims to implement a highly innovative clinical trial design of combinatorial arms for patients with diffuse midline gliomas (DMGs) at all disease stages that is adaptive to pre-clinical data generated in eight collaborating institutions. The goals of the team are to: i) rapidly identify and validate promising drugs for clinical use, and ii) predict biomarkers for promising drugs. METHODS In vitro (n=15) and in vivo (n=8) models of DMGs across seven institutions were used to assess single and combination treatments with ONC201, ONC206, marizomib, panobinostat, Val-083, and TAK228. In vivo pharmacokinetic assays using clinically relevant dosing of ONC201, ONC206, and panobinostat were performed. Predictive biomarkers for ONC201 and ONC206 were identified using extensive molecular assays including CRISPR, RNAseq, ELISA, FACS, and IHC. RESULTS Inhibitory concentrations (IC50) were established and validated across participating sites. In vivo validation of single and combination drug assays confirmed drug efficacy as increased survival for: ONC201 (p=0.01), ONC206 (p=0.01), ONC201+ONC206 (p=0.02), and ONC201+panobinostat (p=0.01). Marizomib showed toxicity in murine/zebrafish PDXs models. Murine pharmacokinetic analysis showed peak brain levels of ONC201 and ONC206 above pre-clinical IC50. Molecular testing and analyses of existing drug screen across 537 cancer cell lines validated mitochondrial stress and ATF4 as the main targets induced by ONC201/6. CONCLUSION Thorough preclinical testing in a multi-site laboratory setting is feasible and identified ONC201 in combination with ONC206 as promising therapeutics for DMGs. Preclinical and correlative-clinical studies are ongoing.

The precise decoding of human genomes facilitated by the advancements in next-generation sequencing has led to a better understanding of genetic underpinnings of pediatric brain cancers. Indeed, it is now evident that tumours of the same type harbour distinct driving mutations and molecular aberrations that can result in different prognosis and treatment outcomes. The profounder insight into the the identity, amount and types of molecular aberrations has paved the way for the advent of targeted therapies in precision medicine. Nevertheless, less than 10% of pediatric cancer patients harbour actionable mutations. Strictly limited therapeutic options that are firstly available for brain cancers and secondly acceptable for children’s development further impede the breakthrough in the survival rate in pediatric brain cancers. This underscores a desperate need to delve beyond genomic sequencing to identify biomarker coupled therapies that not only featured with treatment efficacy in the central nervous system but also acceptable side effects for children. The Hudson-Monash Paediatric Precision Medicine (HMPPM) Program focuses on utilising genetic profiles of patients’ tumour models to identify new therapeutic targets and repurpose existing ones using high-throughput functional genomics screens (2220 drugs and CRISPR screen of 300 oncogenic genes). Using a large compendium of over sixty patient derived paediatric brain cancer models, we provide proof-of-concept data that shows an integrative pipeline for functional genomics with multi-omics datasets to perform genotype-phenotype correlations and, therefore, identify genetic dependencies. Herein, using several examples in ATRT, DIPG and HGG, we show how functional interrogations can better define molecular subclassification of tumours and identify unique vulnerabilities.

Atypical teratoid/rhabdoid tumors (ATRT), are the most common brain tumor in children under the age of 1 year with an overall survival of ~17%. Like extracranial rhabdoid tumors, ATRT is exclusively characterized by bi-allelic loss of SMARCB1, a critical subunit of the SWI/SNF chromatin remodeling complex, implicating epigenetic deregulation in the pathogenesis of disease. We have previously shown the ability of the histone deacetylase inhibitor, panobinostat, to mimic SMARCB1-mediated SWI/SNF functions in extracranial rhabdoid tumors to inhibit tumor growth by driving multi-lineage differentiation in vitro and in vivo. Whether this also applies to ATRT is unknown. Using a panel of human-derived ATRT cell lines, representing defined molecular subgroups, we have shown that prolonged treatment with panobinostat at nanomolar concentrations results in markedly reduced clonogenicity, and increased senescence, preceded by increased H3K27 acetylation, decreased H3K27 trimethylation and EZH2 expression. To determine potentially synergistic therapies, we performed high-throughput drug screening of 622 compounds already in advanced clinical trials or FDA-approved for other indications, across our panel of ATRT models and identified 30 common compounds, which decrease cell viability by >50%, with no effect on neural stem cell controls and 12 compounds which demonstrated subgroup specificity, highlighting the necessity to consider therapies in the molecular context. In addition to HDACi, consistent with our panobinostat in vitro findings, inhibitors of CDK, survivin and PI3K were the top hits. In vitro and in vivo validation of these compounds alone, and in combination with panobinostat is ongoing.

Diffuse Intrinsic Pontine Glioma (DIPG) is a highly aggressive paediatric brainstem tumour with a dismal prognosis. Recurrent heterozygous mutations (p.K27M) in Histone H3 variant genes have been identified in the majority of DIPG cases. While the exact mechanism of H3K27M’s function is poorly understood, evidence suggests a role for epigenetic dysregulation in disease pathogenesis. This study aims to use functional genomics to identify novel therapeutic dependencies in H3K27M DIPG. DIPG drug sensitivity screening was carried out in twelve established and validated patient derived cell lines (10 H3.3K27M and 2 Wt) using an FDA approved drug library containing 1480 compounds. Highly prevalent targets identified from this screen include HDAC, microtubule, proteasome and CDK inhibitors. Additionally, a custom pooled CRISPR knockout library of druggable targets (300 genes, 1200 guide RNAs) was used to identify key DIPG cell survival pathways. To date five DIPG cell lines (1 Wt; 1 H3.1; 3 H3.3) have undergone screening. Knockdown of known DIPG driver genes (TP53; PDGFRA; PIK3CA and PIK3CR1) resulted in reduced cell viability, consistent with their proposed function and validating knockout screen utility. Preliminary data demonstrates Wt and H3K27M DIPGs cluster independently based on genes required for survival, suggesting differing tumorigenesis mechanisms and the potential for therapeutically targeting genotype specific pathways. Correlation of parallel drug screen and RNA-seq data will potentially reveal H3-dependent pathways for therapeutic exploitation. Collectively, we show a functional genomics approach is able to identify genotype-specific pathway dependencies in DIPG, paving the way for molecularly informed personalized therapies for patients.