Diffuse Intrinsic Pontine Glioma Cells Research Articles

Synergistic imipridone‐based drug combinations for treatment of pediatric H3K27M mutant diffuse intrinsic pontine glioma (DIPG)

The H3K27M oncohistone mutation has been identified in approximately 80% of diffuse intrinsic pontine gliomas (DIPG), and now represents a potential target for urgently needed new therapies. ONC201, a first-in-class imipridone therapy, is known to selectively induce apoptosis of cancer cells independent of p53, and has shown efficacy against H3K27M mutant DIPG. We sought to identify synergy between ONC201 or second generation imipridones (ONC206 and ONC212), and other chemotherapeutics with known preclinical activity against DIPG. We hypothesized that second generation imipridones would demonstrate superior activity to ONC201, and that the combination of imipridones and other chemotherapeutics may be superior to single agent treatment in H3K27M mutant DIPG. Seven patient derived DIPG cell lines (SU-DIPG-IV, SU-DIPG-13, SU-DIPG-25, SUDIPG-27, SU-DIPG-29, SU-DIPG-36, SF8628) were grown in culture and exposed to imipridones as a monotherapy and in combination with other FDA approved chemotherapeutics (histone de-acetylase inhibitors [HDACi], marizomib, etoposide, and temozolomide). Cell viability was measured by CellTiter-Glo. Dose dependent response to imipridone therapy was noted in all cell lines with half maximal inhibitory concentration (IC50) of 1.46 µM, 0.11 µM, and 0.03 µM, in ONC201, ONC206, and ONC212 respectively. Synergy was identified via combination analyses, with combination indices of <1 indicating some degree of synergy, and <0.5 indicating very strong synergy. Strong synergy was identified between ONC201 and HDACi Panobinostat (CI 0.01), Romidepsin (CI 0.08) and protease inhibitor Marizomib (CI 0.19). Lesser synergy was demonstrated between ONC201 and Entinostat (CI 0.71) or Etoposide (CI 0.54). Importantly, temozolomide and ONC201 showed a lack of combinational efficacy against H3K27M mutant DIPG, consistent with the literature to date. Second generation imipridones showed similar synergistic effects with Panobinostat, Romidepsin, and Marizomib. Additionally, induction of apoptosis as measured by the induction of poly (ADP-ribose) polymerase (PARP) cleavage via immunoblotting, was demonstrated with a combination of imipridones and Panobinostat. These results suggest increased sensitivity of H3K27M mutant DIPG cell lines to second generation imipridone therapies, as compared to ONC201. Additionally, there is evidence of promising synergy between imipridones and Panobinostat, Romidepsin, or Marizomib, which should be taken into consideration for future clinical trials.

Evofosfamide Is Effective against Pediatric Aggressive Glioma Cell Lines in Hypoxic Conditions and Potentiates the Effect of Cytotoxic Chemotherapy and Ionizing Radiations.

Simple SummaryDespite many therapeutic approaches attempted over the last years, the prognosis of children with high-grade glioma or diffuse intrinsic pontine glioma remains dismal. Hypoxia-activated prodrugs (HAPs) were developed to target hypoxic areas within solid tumors as gliomas. Evofosfamide (Evo) is a 2nd generation HAP exhibiting significant preclinical and clinical activities against adult glioblastoma. We thus investigated the potential of Evo in six pediatric glioma cell lines. Interestingly, we showed that the growth of all cell lines was inhibited by Evo, mainly under hypoxia as expected. We also evidenced a significant synergism between Evo and three drugs widely used in pediatric oncology. Finally, Evo appeared able to potentiate the effect of ionizing radiations. Since these tumors are highly hypoxic and Evo appears effective in hypoxic glioma cells as a single drug and in combination with radio- and chemotherapy, hypoxia-activated prodrugs could represent a promising therapeutic option for children with brain tumors.Hypoxia is a hallmark of many solid tumors and is associated with resistance to anticancer treatments. Hypoxia-activated prodrugs (HAPs) were developed to target the hypoxic regions of these tumors. Among 2nd generation HAPs, Evofosfamide (Evo, also known as TH-302) exhibits preclinical and clinical activities against adult glioblastoma. In this study, we evaluated its potential in the field of pediatric neuro-oncology. We assessed the efficacy of Evo in vitro as a single drug, or in combination with SN38, doxorubicin, and etoposide, against three pediatric high-grade glioma (pHGG) and three diffuse intrinsic pontine glioma (DIPG) cell lines under hypoxic conditions. We also investigated radio-sensitizing effects using clonogenic assays. Evo inhibited the growth of all cell lines, mainly under hypoxia. We also highlighted a significant synergism between Evo and doxorubicin, SN38, or etoposide. Finally, Evo radio-sensitized the pHGG cell line tested, both with fractionated and single-dose irradiation schedules. Altogether, we report here the first preclinical proof of evidence about Evofosfamide efficiency against hypoxic pHGG and DIPG cells. Since such tumors are highly hypoxic, and Evo potentiates the effect of ionizing radiation and chemotherapy, it appears as a promising therapeutic strategy for children with brain tumors.

Doxorubicin-Loaded Gold Nanoarchitectures as a Therapeutic Strategy against Diffuse Intrinsic Pontine Glioma.

Simple SummaryDiffuse intrinsic pontine gliomas (DIPGs) are the most aggressive high-grade gliomas known to affect children. Due to the infiltrative nature of the DIPG tumours in the brainstem, they are very difficult to treat. Unfortunately, children succumb to their disease within 1–2 years from their diagnosis. Novel therapeutic treatments are, thus, urgently needed. Using primary cultures and orthotopic models of DIPG, we evaluated the therapeutic efficacy of passionfruit like nanoarchitectures functionalized with human serum albumin and loaded with doxorubicin (NA-HSA-Dox). We found that NA-HSA-Dox were significantly effective at penetrating DIPG spheroids and subsequently at reducing the proliferation and colony formation in DIPG cells. Although an antitumour effect was not observed in orthotopic models of DIPG, NA-HSA-Dox was well tolerated. These study demonstrates the importance of employing brain tumour orthotopic models for the identification of novel therapies and the need to develop treatments that effectively cross the blood brain barrier.Diffuse Intrinsic Pontine Gliomas (DIPGs) are highly aggressive paediatric brain tumours. Currently, irradiation is the only standard treatment, but is palliative in nature and most patients die within 12 months of diagnosis. Novel therapeutic approaches are urgently needed for the treatment of this devastating disease. We have developed non-persistent gold nano-architectures (NAs) functionalised with human serum albumin (HSA) for the delivery of doxorubicin. Doxorubicin has been previously reported to be cytotoxic in DIPG cells. In this study, we have preclinically evaluated the cytotoxic efficacy of doxorubicin delivered through gold nanoarchitectures (NAs-HSA-Dox). We found that DIPG neurospheres were equally sensitive to doxorubicin and doxorubicin-loaded NAs. Colony formation assays demonstrated greater potency of NAs-HSA-Dox on colony formation compared to doxorubicin. Western blot analysis indicated increased apoptotic markers cleaved Parp, cleaved caspase 3 and phosphorylated H2AX in NAs-HSA-Dox treated DIPG neurospheres. Live cell content and confocal imaging demonstrated significantly higher uptake of NAs-HSA-Dox into DIPG neurospheres compared to doxorubicin alone. Despite the potency of the NAs in vitro, treatment of an orthotopic model of DIPG showed no antitumour effect. This disparate outcome may be due to the integrity of the blood-brain barrier and highlights the need to develop therapies to enhance penetration of drugs into DIPG.

Dual targeting of polyamine synthesis and uptake in diffuse intrinsic pontine gliomas

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.

OLIG2 maintenance is not essential for diffuse intrinsic pontine glioma cell line growth but regulates tumor phenotypes.

Diffuse intrinsic pontine glioma (DIPG) is a pediatric lethal high-grade brainstem glioma with no effective therapies. OLIG2 (oligodendrocyte transcription factor 2) was reported to be critical for the growth of a DIPG cell line CCHMC-DIPG-1. Surprisingly, we found that the CCHMC-DIPG-1 cells express little OLIG2 and exhibit a mesenchymal phenotype, which raised a question regarding the role of OLIG2 in the growth of DIPG cells. We evaluated the function of OLIG2 in different DIPG cell lines through molecular and genetic approaches and performed transcriptomic and genomic landscape profiling including whole-genome bisulfite sequencing, RNA-seq, ATAC-seq, and ChIP-seq. shRNA-mediated knockdown and CRISPR-Cas9-mediated knockout approaches were utilized to assess OLIG2 functions in DIPG cell growth. We found that DIPG cells are phenotypically heterogeneous and exhibit the characteristics of distinct malignant gliomas including proneural, classical, and mesenchymal subtypes. OLIG2 knockdown did not impact the growth of CCHMC-DIPG-1 cells, wherein OLIG2 is epigenetically silenced. Moreover, OLIG2 deletion did not substantially impair OLIG2-expressing proneural-like DIPG growth but led to an upregulation of HIPPO-YAP1 and epidermal growth factor receptor (EGFR) signaling and a tumor phenotype shift. Targeting HIPPO-YAP1 and EGFR signaling in OLIG2-deficient DIPG cells inhibited tumor cell growth. Our data indicate that OLIG2 is dispensable for DIPG growth but regulates the phenotypic switch of DIPG tumor cells. OLIG2 downregulation leads to deregulation of adaptive YAP1 and EGFR signaling. Targeting YAP1 and EGFR pathways inhibits the growth of OLIG2-deficient DIPG cells, pointing to a therapeutic potential by targeting adaptive signaling to treat DIPG tumors with nominal OLIG2 expression.

Transcriptomic Analysis of Diffuse Intrinsic Pontine Glioma (DIPG) Identifies a Targetable ALDH-Positive Subset of Highly Tumorigenic Cancer Stem-like Cells.

Understanding the cancer stem cell (CSC) landscape in diffuse intrinsic pontine glioma (DIPG) is desperately needed to address treatment resistance and identify novel therapeutic approaches. Patient-derived DIPG cells demonstrated heterogeneous expression of aldehyde dehydrogenase (ALDH) and CD133 by flow cytometry. Transcriptome-level characterization identified elevated mRNA levels of MYC, E2F, DNA damage repair (DDR) genes, glycolytic metabolism, and mTOR signaling in ALDH+ compared with ALDH-, supporting a stem-like phenotype and indicating a druggable target. ALDH+ cells demonstrated increased proliferation, neurosphere formation, and initiated tumors that resulted in decreased survival when orthotopically implanted. Pharmacologic MAPK/PI3K/mTOR targeting downregulated MYC, E2F, and DDR mRNAs and reduced glycolytic metabolism. In vivo PI3K/mTOR targeting inhibited tumor growth in both flank and an ALDH+ orthotopic tumor model likely by reducing cancer stemness. In summary, we describe existence of ALDH+ DIPGs with proliferative properties due to increased metabolism, which may be regulated by the microenvironment and likely contributing to drug resistance and tumor recurrence. IMPLICATIONS: Characterization of ALDH+ DIPGs coupled with targeting MAPK/PI3K/mTOR signaling provides an impetus for molecularly targeted therapy aimed at addressing the CSC phenotype in DIPG.

Therapeutic targeting of transcriptional elongation in diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine glioma (DIPG) is associated with transcriptional dysregulation driven by H3K27 mutation. The super elongation complex (SEC) is required for transcriptional elongation through release of RNA polymerase II (Pol II). Inhibition of transcription elongation by SEC disruption can be an effective therapeutic strategy of H3K27M-mutant DIPG. Here, we tested the effect of pharmacological disruption of the SEC in H3K27M-mutant DIPG to advance understanding of the molecular mechanism and as a new therapeutic strategy for DIPG. Short hairpin RNAs (shRNAs) were used to suppress the expression of AF4/FMR2 4 (AFF4), a central SEC component, in H3K27M-mutant DIPG cells. A peptidomimetic lead compound KL-1 was used to disrupt a functional component of SEC. Cell viability assay, colony formation assay, and apoptosis assay were utilized to analyze the effects of KL-1 treatment. RNA- and ChIP-sequencing were used to determine the effects of KL-1 on gene expression and chromatin occupancy. We treated mice bearing H3K27M-mutant DIPG patient-derived xenografts (PDXs) with KL-1. Intracranial tumor growth was monitored by bioluminescence image and therapeutic response was evaluated by animal survival. Depletion of AFF4 significantly reduced the cell growth of H3K27M-mutant DIPG. KL-1 increased genome-wide Pol II occupancy and suppressed transcription involving multiple cellular processes that promote cell proliferation and differentiation of DIPG. KL-1 treatment suppressed DIPG cell growth, increased apoptosis, and prolonged animal survival with H3K27M-mutant DIPG PDXs. SEC disruption by KL-1 increased therapeutic benefit in vitro and in vivo, supporting a potential therapeutic activity of KL-1 in H3K27M-mutant DIPG.

DIPG-71. SELECTIVE HDAC INHIBITOR RG2833 INDUCES DIPG CELL DEATH VIA DOWNREGULATION OF THE NFĸB PATHWAY

Histone deacetylase (HDAC) inhibitor panobinostat demonstrated activity against diffuse intrinsic pontine glioma (DIPG) in vitro, but its efficacy in vivo was limited by toxicity and poor blood brain barrier penetration. RG2833 (RGFP109) is a selective HDAC1/3 inhibitor that has established brain penetration. In clinical trials, the Cmax (plasma) of RG2833 was 32uM. RG2833 demonstrated cytotoxicity against temozolomide-resistant glioblastoma and downregulated the NFĸB pathway. Because this pathway is overexpressed in DIPG and may play a role in DIPG cell growth and survival, we hypothesized that RG2833 would kill DIPG cells. Treatment of DIPG cell lines with RG2833 as a single agent suppresses cell proliferation in the 5–10μM range (MTS assay for HSJD007 p=0.0004 10μM vs DMSO, JHH-DIPG1 p=0.001 10μM vs DMSO, SF-7761 p=0.04 10μM vs DMSO, SU-DIPG13 p=0.01 10μM vs DMSO by t-test). RG2833 induces apoptosis by 48 hours as measured by Western blot for cPARP and cleaved caspase 3 immunofluorescence (HSJD007 p<0.003 8μM vs DMSO, JHH-DIPG1 p=0.0026 10μM vs DMSO by t-test). RG2833 also slows cell proliferation as measured by Western blot for pRb and immunofluorescence for BrdU (HSJD007 p=0.008 8μM vs DMSO, JHH-DIPG1 p=0.0002 10μM vs DMSO by t-test). Western blot confirmed a dose-dependent increase in histone 3 acetylation with RG2833 treatment at 5 hours. We detected increased acetylated p65 and decreased expression of the NFĸB regulated pro-survival genes BCL2, BCL-xL, and XIAP with RG2833 treatment. Together, this data shows that HDAC inhibitor RG2833 may be a promising therapeutic candidate for DIPG via downregulation of the NFĸB pathway.

DIPG-29. PHOSPHATIDYLINOSITOL-4,5-BISPHOSPHATE 3-KINASE (PI3K) INHIBITION DRIVES PROTEIN KINASE C ACTIVATION (PKC) IN DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG)

Recurring somatic mutations and gene amplifications to members of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) signaling axis are overarching contributors to the aggressive growth and survival of diffuse intrinsic pontine gliomas (DIPG). However, targeting PI3K has thus far failed to improve outcomes for patients in the clinic. To identify the mechanisms underpinning PI3K/AKT/mTOR treatment failure in DIPG, we have employed high-resolution quantitative phosphoproteomic profiling in patient-derived DIPG cell lines harboring H3K27M and PI3K mutations, +/- the blood-brain barrier permeable PI3K inhibitor, paxalisib (previously “GDC-0084”, currently in Phase I trials - NCT03696355) and rapamycin. Paxalisib was significantly more potent than rapamycin at inducing PI3K/AKT/mTOR inhibition, however, both simultaneously activated protein kinase C signaling (pT500PKCβ +8.2 and +4.5 fold, respectively). PKC lies directly upstream of myristoylated alanine-rich C-kinase substrate (MARCKs), which was phosphorylated at Ser170 by +9.4 and +4.7 fold, respectively; promoting actin cytoskeletal remodeling and cellular migration. Indeed, activation of PKC signaling using phorbol 12-myristate 13-acetate (PMA), increased DIPG cell growth and migration by >3 fold. Targeting PKC using midostaurin (FDA-approved for acute myeloid leukemia), and enzastaurin (blood-brain barrier penetrant inhibitor of PKCβ), in combination with paxalisib was highly synergistic (CI=<0.9), reducing proliferation and driving apoptosis. Mechanistically, compensatory activation of PKC signaling following PI3K inhibition was regulated by the accumulation of Ca+2 ions, as chelation using BAPTA-AM significantly reduced PKC activity following PI3K inhibition. These data highlight the power of phosphoproteomic profiling for the rational design of drug combination strategies, which need to be tested in vivo prior to clinical trials for DIPG.

MODL-29. EVALUATING TUMOR-IMMUNE INTERACTIONS IN MOUSE MODELS OF DIFFUSE INTRINSIC PONTINE GLIOMA

BACKGROUNDWhile adult gliomas show some level of immune cell infiltration, diffuse intrinsic pontine glioma (DIPG) is characterized as having an “immune cold” state. We have developed new immunocompetent mouse models of DIPG. These models faithfully recapitulate the pathological hallmarks of DIPG and provides a unique platform to investigate immune modulatory therapies and potential therapeutic benefits of check point inhibitor combination therapies.METHODSTo evaluate the effects of CDK4/6 inhibition (CDK4/6i) on cell proliferation and immune interactions we performed a series of in vitro and in vivo studies using DIPG mouse models. In vitro assays included dose response curves, transcriptional profiling, and MHC1 expression. In vivo preclinical studies treated mouse models with CDK4/6i with or without immune check-point inhibitors (ICI). We also examined other candidate immune modulatory therapies in vitro.RESULTSCDK4/6i (Abemeciclib) reduced proliferation of DIPG cells derived from mouse models, and displayed a modest increase in immune activation by MHC1 expression and transcriptome. Pilot in vivo preclinical studies did not show any significant changes in DIPG proliferation or immune changes with CDK4/6i treatment, ICI treatment, or the combination of CDK4/6i + ICI. In vitro testing of other immune-modulatory drugs identified additional candidates that can be tested in vivo.CONCLUSIONCDK4/6i displayed in vitro action, but lacked efficacy in DIPG mouse models in vivo. Further use of spontaneous DIPG mouse models will provide a rapid preclinical platform to evaluate in vivo tumor-immune interactions, drug efficacy, and mechanisms of resistance.

DIPG-51. ACVR1 MUTATIONS PROMOTE TUMOR GROWTH IN MODELS OF DIFFUSE INTRINSIC PONTINE GLIOMA

Mutations in the gene encoding activin A receptor type 1 (ACVR1) are found in approximately 25% of diffuse intrinsic pontine gliomas (DIPGs), a pediatric glioma with 2-year survival rate of less than 10%. ACVR1mutations frequently coincide with activating PIK3CA or PIK3R1 mutations, indicating a potential cooperative effect of BMP and PI3K signaling in gliomagenesis. We used genetically engineered mice with inducible knock-in of Acvr1R206H or Pik3caE545K alleles, such that cre-mediated recombination resulted in expression of the gain of function mutated genes from their endogenous promoters at physiological levels. Cre-mediated deletion in GFAP-CreER;Pik3caE545K/+;p53cKO mice (Pik3ca;p53) mediated Trp53 deletion and expression of Pik3caE545K in glial progenitors, and spontaneously induced high-grade glioma (HGG) in mice with complete penetrance. Heterozygous knock-in of the Acvr1R206H allele accelerated tumorigenesis and impaired survival in Pik3ca;p53 mice (Acvr1;Pik3ca;p53). Transcriptomic analysis of Acvr1;Pik3ca;p53 tumors compared to Pik3ca;p53 littermate controls, as in patient-derived tumors, revealed broad molecular signatures associated with cell fate commitment and chromosome maintenance. Pharmacologic inhibition of ACVR1 was sufficient to impair growth in human patient-derived DIPG cell lines. Together, our studies show that ACVR1 activation promotes tumor growth in spontaneous mouse HGG and patient-derived DIPG cells, suggesting that ACVR1 inhibition may produce a clinically significant therapeutic effect in DIPG.

Mesenchymal Stem Cells Successfully Deliver Oncolytic Virotherapy to Diffuse Intrinsic Pontine Glioma

Diffuse intrinsic pontine glioma (DIPG) is among the deadliest of pediatric brain tumors. Radiotherapy is the standard-of-care treatment for DIPG, but offers only transient relief of symptoms for patients with DIPG without providing significant survival benefit. Oncolytic virotherapy is an anticancer treatment that has been investigated for treating various types of brain tumors. Here, we have explored the use of mesenchymal stem cells (MSC) for oncolytic virus (OV) delivery and evaluated treatment efficacy using preclinical models of DIPG. The survivin promoter drives the conditional replication of OV used in our studies. The efficiency of OV entry into the cells is mediated by fiber modification with seven lysine residues (CRAd.S.pK7). Patients' samples and cell lines were analyzed for the expression of viral entry proteins and survivin. The ability of MSCs to deliver OV to DIPG was studied in the context of a low dose of irradiation. Our results show that DIPG cells and tumors exhibit robust expression of cell surface proteins and survivin that enable efficient OV entry and replication in DIPG cells. MSCs loaded with OV disseminate within a tumor and release OV throughout the DIPG brainstem xenografts in mice. Administration of OV-loaded MSCs with radiotherapy to mice bearing brainstem DIPG xenografts results in more prolonged survival relative to that conferred by either therapy alone (P < 0.01). Our study supports OV, CRAd.S.pK7, encapsulated within MSCs as a therapeutic strategy that merits further investigation and potential translation for DIPG treatment.

EXTH-67. PHARMACOLOGIC INHIBITION OF LYSINE SPECIFIC DEMETHYLASE-1 (LSD1) AS AN ADJUVANT IMMUNE-SENSITIZATION STRATEGY IN DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG)

Abstract Diffuse midline gliomas (DMG), including brainstem DIPG (diffuse intrinsic pontine glioma), are incurable pediatric brain tumors. Mutations in the H3 histone tail (H3.1/3.3-K27M) are a feature of DIPG, highlighting epigenetic aberrations in these tumors. Lysine specific demethylase-1 (LSD1), is an enzyme that acts upon mono and di-methylated histone H3K4/K9, and pharmacological inhibitors of its enzymatic and scaffolding domains are clinically viable. Patient-derived DIPG cell lines, orthotopic mouse models, and pediatric high-grade glioma (pHGG) datasets were used to evaluate effects of LSD1 inhibitors on cytotoxicity and immune gene expression. Because an unique immune signature was sen with enzymatic LSD1 inhibitors in DIPG cells in vitro, immune cell cytotoxicity was assessed in DIPG cells pre-treated with LSD1 inhibitors and informatics platforms were used to determine immune infiltration of pHGG. Selective cytotoxicity and an immunogenic gene expression signature was established in DIPG cell lines and in brainstem tumor-bearing mice using clinically-relevant LSD1 inhibitors. Pediatric HGG patient sequencing data demonstrated survival benefit using this LSD1-dependent gene expression signature. On-target binding of catalytic LSD1 inhibitors was confirmed in DIPG and pre-treatment of DIPG with these inhibitors increased lysis by natural killer (NK) cells. CIBERSORT analysis of patient data confirmed NK infiltration is beneficial to patient survival while CD8 T-cells are negatively prognostic. Catalytic LSD1 inhibitors are non-perturbing to NK cells while scaffolding LSD1 inhibitors are toxic to NK cells and do not induce the gene expression signature in DIPG cells. Treatment of orthotopic xenograft bearing DIPG mouse models with catalytic LSD1 inhibitors and NK cells showed a significant reduction in tumor burden. In summary, we find that LSD1 inhibition using catalytic inhibitors is both selectively cytotoxic and promotes a functional immune gene expression signature that is associated with NK cell killing, representing a therapeutic opportunity for pHGG.

EXTH-46. ARTIFICIAL INTELLIGENCE-BASED IDENTIFICATION OF COMBINED VANDETANIB AND EVEROLIMUS IN THE TREATMENT OF ACVR1-MUTANT DIFFUSE INTRINSIC PONTINE GLIOMA

Abstract Somatic mutations in ACVR1, encoding the serine/threonine kinase ALK2 receptor, are found in a quarter of children with the currently incurable brain tumour diffuse intrinsic pontine glioma (DIPG). Treatment of ACVR1-mutant DIPG patient-derived models with multiple inhibitor chemotypes leads to a reduction in cell viability in vitro and extended survival in orthotopic xenografts in vivo, though there are currently no specific ACVR1 inhibitors licensed for DIPG. Using an Artificial Intelligence-based platform to search for approved compounds which could be used to treat ACVR1-mutant DIPG, the combination of vandetanib and everolimus was identified as a possible therapeutic approach. Vandetanib, an approved inhibitor of VEGFR/RET/EGFR, was found to target ACVR1 (Kd=150nM) and reduce DIPG cell viability in vitro, but has been trialed in DIPG patients with limited success, in part due to an inability to cross the blood-brain-barrier. In addition to mTOR, everolimus inhibits both ABCG2 (BCRP) and ABCB1 (P-gp) transporter, and was synergistic in DIPG cells when combined with vandetanib in vitro. This combination is well-tolerated in vivo, and significantly extended survival and reduced tumour burden in an orthotopic ACVR1-mutant patient-derived DIPG xenograft model. Based on these preclinical data, three patients with ACVR1-mutant DIPG were treated with vandetanib and everolimus. These cases may inform on the dosing and the toxicity profile of this combination for future clinical studies. This bench-to-bedside approach represents a rapidly translatable therapeutic strategy in children with ACVR1 mutant DIPG.

Diffuse Intrinsic Pontine Glioma Cells Are Vulnerable to Mitotic Abnormalities Associated with BMI-1 Modulation.

Diffuse intrinsic pontine glioma (DIPG) is a poor-prognosis pediatric brain tumor with a median survival of less than 1 year. No effective therapy is currently available, and no therapeutic advances have been made in several decades. We have previously identified BMI-1 as a potential therapeutic target in DIPG and have shown that BMI-1 is highly expressed in DIPG tumors regardless of histone 3 subtype. In the present study, we show that the modulation of BMI-1 leads to DNA damage, M phase cell-cycle arrest, chromosome scattering, and cell death. Interestingly, EZH2 inhibition did not alter these effects. Furthermore, modulation of BMI-1 sensitizes DIPG patient-derived stem-like cells to ionizing radiation (IR). Treatment of DIPG stem-like cells with PTC596, a BMI-1 modulator, and IR impairs the kinetics of DNA damage response (DDR). Both DDR foci formation and resolution were delayed, resulting in further reduction in cell viability compared with either treatment alone. In vivo, treatment of mice bearing DIPG xenografts with PTC596 leads to decreased tumor volume and growth kinetics, increased intratumoral apoptosis, and sustained animal survival benefit. Gene expression analysis indicates that BMI-1 expression correlates positively with DIPG stemness and BMI-1 signature. At the single-cell level, the analysis reveals that BMI-1 pathway is upregulated in undifferentiated cells and positively correlates with stemness in DIPG tumors. IMPLICATIONS: Together, our findings indicate that BMI-1 modulation is associated with mitotic abnormalities, impaired DDR, and cell death, supporting the combination of BMI-1 modulation and radiation as a promising novel therapy for children with DIPG.

Dual Targeting Of Mutant p53 Protein And Jumonji Family Histone Demethylase Sensitizes H3K27M Diffuse Intrinsic Pontine Glioma Cells To Radiation

Dual Targeting Of Mutant p53 Protein And Jumonji Family Histone Demethylase Sensitizes H3K27M Diffuse Intrinsic Pontine Glioma Cells To Radiation

Focused Ultrasound-Mediated Blood Brain Barrier Opening is Safe and Feasible in Diffuse Intrinsic Pontine Glioma after Radiation Treatment

Diffuse intrinsic pontine glioma (DIPG), also known as diffuse midline glioma, is a fatal disease in children. Unlike glioblastoma for adults, there are no effective systemic therapies and radiotherapy is the only modality of treatment. With radiation, overall survival improves as compared to supportive care; however, all patients succumb to disease recurrence and ultimately die. The challenge is that the disease is heterogeneous with extensive infiltrative microscopic disease that extends to regions in which the blood brain barrier (BBB) remains relatively intact. Focused ultrasound (FUS) is a novel technology that allows for non-invasive opening of the BBB that is reversible. The question remains whether this can be performed safely in the brainstem after standard radiation. The purpose of this study is to examine the safety and feasibility of BBB-opening with FUS following radiation in a mouse model. A mouse syngeneic DIPG cell line harboring H3.3K27M, PDGF-B (+), and p53 (-/-) was used to factor the potential inflammatory effects of radiation and ultrasound that may contribute to harm. 10,000 cells in 1 μl was injected into the brainstem. At 3 weeks after implantation, magnetic resonance imaging (MRI) T1-post contrast and T2 sequences were obtained to confirm tumor growth. Using the small animal radiation research platform (SARRP), 9 Gy in 3 fractions was delivered to the brainstem. FUS-mediated BBB-opening with microbubbles was performed within 5 day of completion of radiation. Vitals were continuously monitored during FUS. MRI was obtained to confirm BBB-opening. An additional MRI was obtained at 48 hours to show BBB-closure. Mice were observed daily for neurological deficits, including motor dysfunction, seizure, irregular breathing and death. Daily weights were recorded. Kondziela’s inverted screen test was performed before and after FUS to assess for motor strength. Kaplan Meier methods were used to assess survival. Using MRI guidance, radiation to the brainstem was tolerated by mice harboring DIPG. FUS-mediated BBB-opening in the brainstem was feasible and confirmed on MRI. BBB closure was seen by 48-hours after BBB-opening. Compared to radiation alone, FUS did not alter vitals, motor function, weight change, neurological deficit and survival. This is the first study to demonstrate that FUS-guided BBB-opening in the brainstem to target DIPG is safe and feasible after radiation. These results are currently being translated into the clinic to assess the safety and feasibility of FUS in children with DIPG.

Senescence Induced by BMI1 Inhibition Is a Therapeutic Vulnerability in H3K27M-Mutant DIPG

Diffuse intrinsic pontine glioma (DIPG) is an incurable brain tumor of childhood characterized by histone mutations at lysine 27, which results in epigenomic dysregulation. There has been a failure to develop effective treatment for this tumor. Using a combined RNAi and chemical screen targeting epigenomic regulators, we identify the polycomb repressive complex 1 (PRC1) component BMI1 as a critical factor for DIPG tumor maintenance in vivo. BMI1 chromatin occupancy is enriched at genes associated with differentiation and tumor suppressors in DIPG cells. Inhibition of BMI1 decreases cell self-renewal and attenuates tumor growth due to induction of senescence. Prolonged BMI1 inhibition induces a senescence-associated secretory phenotype, which promotes tumor recurrence. Clearance of senescent cells using BH3 protein mimetics co-operates with BMI1 inhibition to enhance tumor cell killing in vivo.

Abstract 347: Neogenin as an anti-invasive target for diffuse intrinsic pontine gliomas

Abstract Introduction. Diffuse Intrinsic Pontine Glioma (DIPG) is a rare high-grade tumor of the brainstem that occurs almost exclusively in children. The significant mortality rates of DIPG stem largely from its capacity to invade adjacent normal brain. We have previously published studies identifying the axon guidance factor netrin-1 and its receptor neogenin as key drivers regulating brain tumor invasion in medulloblastoma and glioblastoma. Here we expand this work to investigate the role of netrin-1 and neogenin in DIPG and identify them as putative biomarkers and governors of tumor invasion. Methods. Urine of 49 DIPG patients and 42 age- and sex-matched controls was collected. Netrin-1 expression was quantified by ELISA. Results were subjected to univariate and multivariate statistical analyses and compared between clinical cohorts. In vitro experiments with established DIPG cell lines utilized RTqPCR, Western blot and flow cytometry to quantify expression of netrin-1 and neogenin. The effect of neogenin blockade on viability and invasion was assessed by flow cytometry (n=3) and transwell chamber assays (n=3) respectively. Results. Patient samples revealed a significantly low level of urinary netrin-1 compared to matched controls (p&amp;lt;0.005). This result was corroborated with cell lines in vitro. Interestingly, its receptor neogenin was upregulated in these cells when compared to control glial cells when quantified by mRNA, protein and FACS analysis. Blockade of neogenin in DIPG cell lines resulted in an increase of cell death and a significant decrease of cell invasion. Conclusion. Expanding on previous work, this data suggests that urinary levels of netrin-1 may have utility as a non-invasive biomarker for DIPG. Our data demonstrates the vital role of neogenin as a regulator of tumor invasion and survival in DIPG. The blockade of neogenin in DIPG cells also demonstrates the potential utility of neogenin as a cell-surface druggable target, capable of reducing tumor invasion and increasing tumor cell death. Citation Format: Julie Sesen, Jessica Driscoll, Edward Smith. Neogenin as an anti-invasive target for diffuse intrinsic pontine gliomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 347.

Abstract 5416: Positioning the NQO1-bioactivatable drug isobutyl-deoxynyboquinone in diffuse intrinsic pontine glioma (DIPG): an exceptional therapeutic opportunity in pediatric brain tumor

Abstract Purpose: Diffuse Intrinsic Pontine Glioma (DIPG), is a very aggressive pediatric cancer with poor overall survival and no effective treatment. Despite numerous clinical trials, the overall survival remains at 9 months after diagnosis and only radiotherapy has shown a relative efficacy. Therefore, it is primordial to increase our understanding of the biology of DIPG tumors, as well as finding new healthcare strategies to tackle this pediatric disease. Recently, we discovered that NQO1, a stress-related protein, was overexpressed in 3 out of 6 DIPG patient-derived cell lines, as well as in half of the primary tissues. Interestingly, NQO1 overexpression can be targeted by substrates that induce an excessive oxidative stress into the cells that finally push them to apoptosis, while normal surrounding tissues with basal expression of NQO1 would be keep safe. Experimental procedure: This discovery prompted us to test in in vitro and in vivo assays a promising drug named isobutyl-deoxynyboquinone (IB-DNQ): a NQO1 bioactivatable substrate. We first tested the drug response of the 6 cell lines before testing it on orthotopic xenograft mouse models. We also investigated by which mechanism NQO1 is regulated in DIPG. Finally, we performed experiments in order to decipher by which mechanism NQO1 is regulated in DIPG. Results: We saw that NQO1 overexpressing cells are very sensitive to the drug, compared to the cell lines with normal expression. Moreover, we started to validate the use of IB-DNQ in vivo using a DIPG cell line treated or not (mock) with IB-DNQ and also a NQO1-knockdown model in orthotopic xenograft mice models, confirming that IB-DNQ crosses the blood brain barrier and increases the overall survival. NQO1 increased expression in DIPG is surprisingly not due to a NRF2-mediated transcriptional regulation as we did not observe a correlation between NQO1 transcript abundance and protein expression in DIPG cell lines, indicating that NRF2 pathway could not be implicated, but translational or post-translational regulation could be operating. We performed a polysome profiling of 4 patient-derived cell lines, a protein stability assay and a large scale proteome for the 6 cell lines (pending results). Conclusion: Our preliminary results, including drug efficacy, are very encouraging for the development of this new therapeutic in DIPG. This discovery represents a promising opportunity to tackle this devastating disease and a new hope for the patients and their families. Citation Format: Maxime Henri Janin, Vanessa Ortiz Barahona, Pere Llinas Arias, Carolina De La Torre, Angel Montero Carcaboso, Andres Morales La Madrid, Paul Hergenrother, Manel Esteller. Positioning the NQO1-bioactivatable drug isobutyl-deoxynyboquinone in diffuse intrinsic pontine glioma (DIPG): an exceptional therapeutic opportunity in pediatric brain tumor [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5416.