DIPG-22. DISSECTING THE ONCOGENIC ROLE OF FOXR2 IN DIFFUSE INTRINSIC PONTINE GLIOMA

Abstract BACKGROUND Diffuse intrinsic pontine gliomas (DIPGs) pose particular challenges for treatment. We recently completed a genomic analysis of close to 200 DIPGs and high-grade gliomas. We identified that nearly 10% of all DIPGs have increased expression of the fork head domain transcription factor FOXR2. We hypothesize that FOXR2 accelerates gliomagenesis in histone mutant DIPGs and represents a previously unexplored therapeutic target. METHODS To determine whether FOXR2 is sufficient to mediate gliomagenesis, we applied an integrative genomics approach using both in vitro and in vivo DIPG models: mouse neural stem cell models expressing FOXR2, in vivo mouse models using in utero brainstem electroporation, patient-derived DIPG cell lines, and RNA sequencing analysis of human and mouse tumors expressing FOXR2. RESULTS Our data shows that FOXR2 indeed is an oncogene that rapidly accelerates gliomagenesis using an in vivo brainstem in utero electroporation model of DIPG. In human tumors, increased FOXR2 expression is mutually exclusive with MYC amplification suggesting functional redundancy. In vivo, FOXR2 results in large brainstem gliomas and rapid neurologic decline of animals. Transcriptional profiling of these tumors demonstrates activation of MYC signaling pathways. In vitro, we have further identified patient-derived cell lines with increased expression of FOXR2. CONCLUSION FOXR2 is sufficient to enhance gliomagenesis and represents a previously understudied therapeutic target for patients with the devastating disease DIPG.

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Global Genomic Analysis of Newly Diagnosed t(4 ;14) Multiple Myeloma Reveals a Specific Mutational Spectrum and Identifies PKD2 As a Potential Therapeutic Target

Blood ◽

10.1182/blood.v128.22.4462.4462 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4462-4462

Author(s):

Xiu Ly Song ◽

Raphaël Szalat ◽

Alexis Talbot ◽

HaiVu Nguyen ◽

Mehmet K. Samur ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Therapeutic Target ◽

Plasma Cells ◽

Genomic Analysis ◽

Sequencing Analysis ◽

Newly Diagnosed ◽

Potential Therapeutic Target ◽

Mutational Landscape

Abstract In Multiple Myeloma (MM), the t(4;14) translocation is associated with a poor outcome. However, beside this translocation, the genetic events which determine the adverse evolution of the disease and the resistance to treatments remain elusive. In this study we performed whole exome or RNA sequencing analysis of samples from 65 newly diagnosed t(4;14) MM. We found that NRAS, KRAS, MAPK and FGFR3 are frequently mutated (12%, 9%, 13.8%, and 20% respectively). Overall, the FGFR3/RAS/BRAF/MAPK genes were mutated in 36 cases (54%). There was a negative correlation between mutations in FGFR3 and those occurring in NRAS, KRAS and BRAF as expected from the mutually exclusive occurrence of mutations in these genes. In addition to alterations in TP53 and DIS3, we found marked elevated frequency of mutations in PRKD2 (10.7%), ATM/ATR (10.7%) and MYCBP2 (7.6%), reduced frequency in FAM46C (1.5%) and no mutation in TRAF3 and CCND1. Mutations in ATM/ATR were strongly associated with the MB4-2 breakpoint (Bp) (p = 1.62 10-4) and significantly correlated with mutations affecting genes coding for members of the MAPK family. We observed a positive correlation between non-silent mutations in PRKD2 and the MB4-1 or MB4-3 Bp (p = 1.3 10-2). Of note, PRKD2 mutations are exclusively found in 3 t(4;14) MM cell lines and among the 84 MM sequenced by Bolli et al. (1), none of the non t(4;14) patient were mutated in PRKD2, indicating that this genetic lesion is associated with t(4;14) MM. In the NCI-H929 t(4;14) MM cell line, which is mutated for PRKD2, encoding the PKD2 serine/threonine kinase, we observed elevated levels of phosphorylated PKD2. Furthermore, inhibition of PKD, decreased PKD2 phosphorylation and triggered reduced proliferation and apoptosis of MM cell lines and fresh plasma cells from patients in vitro. These results define a specific mutational landscape for t(4;14) MM and identify PKD2 as a potential therapeutic target in MM patients. Altogether, these results define a specific mutational landscape for t(4;14) MM and identify PKD2 as a potential therapeutic target in MM patients. Reference 1. Bolli, N., Avet-Loiseau, H., Wedge, D.C., Van Loo, P., Alexandrov, L.B., Martincorena, I., Dawson, K.J., Iorio, F., Nik-Zainal, S., Bignell, G.R., et al. (2014). Heterogeneity of genomic evolution and mutational profiles in multiple myeloma. Nat Commun 5, 2997. Disclosures Munshi: Janssen: Consultancy; Takeda: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Merck: Consultancy; Pfizer: Consultancy; Oncopep: Patents & Royalties.

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CBMT-31. HDAC INHIBITION DIMINISHES THE GROWTH OF ENDOGENOUS IDH MUTANT GLIOMAS

Neuro-Oncology ◽

10.1093/neuonc/noz175.153 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi39-vi40

Author(s):

Lubayna Elahi ◽

Matthew Garrett ◽

Lea Guo ◽

Michael Condro ◽

Riki Kawaguchi ◽

...

Keyword(s):

Cell Lines ◽

Transcriptional Repression ◽

Glioma Cell ◽

Histone Deacetylase Inhibitors ◽

Sequencing Analysis ◽

Hdac Inhibition ◽

In Vivo Experiments ◽

Glioma Cell Lines

Abstract Histone deacetylase inhibitors (HDACi’s) have emerged as a promising class of drugs for treatment of malignancies such as glioblastoma (GBM). Several studies have demonstrated the anti-tumor property of HDACi’s against GBM in both in vitro and in vivo experiments. Nonetheless, in clinical trials, HDACi only marginally increased overall survival of patients with GBM. The mixed results of trials with HDACi’s in glioma have prompted us to hypothesize that improved selection of patients by tumor characteristics could enhance the efficacy of therapy. We specifically tested the effects of valproic acid (VPA), a HDACi and an antiepileptic drug against IDH mutant gliomas. We have previously demonstrated that our IDH mutant glioma cell lines have gene expression and methylation patterns highly similar to IDH mutant tumors in situ. Mutant IDH1 alters the epigenetic landscape of gliomas leading to the hypermethylation phenotype and transcriptional repression of genes. This repression of genes may contribute to tumorigenesis and progression of IDH mutant gliomas. We found that VPA inhibits the growth of patient-derived IDH1 mutant glioma lines. In addition, RNA sequencing analysis of vehicle and VPA-treated IDH1 mutant glioma cells showed de-repression of several genes previously shown to be downregulated in IDH1 mutant glioma cell lines. We also treated cells with another HDACi LBH589 and found that both VPA and LBH589 upregulates similar gene sets suggesting that HDAC inhibition promotes de-repression of previously repressed genes. Ongoing studies are aimed at determining the molecular mechanism by which VPA regulates the growth of IDH1 mutant tumors.

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Overexpression of the Ubiquitin Specific Proteases USP43, USP41, USP27x and USP6 in Osteosarcoma Cell Lines: Inhibition of Osteosarcoma Tumor Growth and Lung Metastasis Development by the USP Antagonist PR619

Cells ◽

10.3390/cells10092268 ◽

2021 ◽

Vol 10 (9) ◽

pp. 2268

Author(s):

Mélanie Lavaud ◽

Mathilde Mullard ◽

Robel Tesfaye ◽

Jérôme Amiaud ◽

Mélanie Legrand ◽

...

Keyword(s):

Tumor Growth ◽

Cell Lines ◽

Lung Metastases ◽

Poor Response ◽

Response To Treatment ◽

Osteosarcoma Cell ◽

Sequencing Analysis ◽

Protein Ubiquitination

Osteosarcoma (OS) is the most common malignant bone tumor in children and teenagers. In many cases, such as poor response to treatment or the presence of metastases at diagnosis, the survival rate of patients remains very low. Although in the literature, more and more studies are emerging on the role of Ubiquitin-Specific Proteases (USPs) in the development of many cancers, few data exist regarding OS. In this context, RNA-sequencing analysis of OS cells and mesenchymal stem cells differentiated or not differentiated into osteoblasts reveals increased expression of four USPs in OS tumor cells: USP6, USP27x, USP41 and USP43. Tissue microarray analysis of patient biopsies demonstrates the nucleic and/or cytoplasmic expression of these four USPs at the protein level. Interestingly, Kaplan–Meyer analysis shows that the expression of two USPs, USP6 and USP41, is correlated with patient survival. In vivo experiments using a preclinical OS model, finally demonstrate that PR619, a USP inhibitor able to enhance protein ubiquitination in OS cell lines, reduces primary OS tumor growth and the development of lung metastases. In this context, in vitro experiments show that PR619 decreases the viability of OS cells, mainly by inducing a caspase3/7-dependent cell apoptosis. Overall, these results demonstrate the relevance of targeting USPs in OS.

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TAMI-79. THERAPEUTIC REVERSAL OF PRENATAL PONTINE ID1 SIGNALING IN DIPG

Neuro-Oncology ◽

10.1093/neuonc/noab196.861 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi215-vi215

Author(s):

Viveka Nand Yadav ◽

Micah K Harris ◽

Dana Messinger ◽

Chase Thomas ◽

Jessica R Cummings ◽

...

Keyword(s):

Tumor Model ◽

Pediatric Brain Tumor ◽

Diffuse Intrinsic Pontine Glioma ◽

Normal Brain ◽

Sequencing Analysis ◽

In Vivo Models ◽

Chip Sequencing ◽

Tumor Expression

Abstract Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brain tumor with rare survival beyond two years. This poor prognosis is largely due to the tumor's highly infiltrative and invasive nature. Nearly 80% of DMGs harbor K27M mutation in the genes encoding histone H3.1 (H3F3A) or H3.3 (HISTIH3B), often with concurrent ACVR1 mutation. Inhibitor of DNA-binding (ID) proteins are key transcriptional regulators of genes involved in lineage commitment and are associated with invasiveness and poor clinical outcomes in multiple human cancers. Introduction of H3K27M and ACVR1 mutations increase ID1 expression in cultured astrocytes, but this has not been confirmed in human tumors or targeted therapeutically. We developed an in-utero electroporation (IUE) murine H3K27M-driven tumor model, which demonstrates increased ID1 expression in H3K27M- and ACVR1-mutated tumor cells. Exome and transcriptome sequencing analysis of multi-focal DMG tumors (n=52) and normal brain tissue revealed that increased ID1 expression is associated with H3K27M/ACVR1-mutation and brainstem location, and correlates with poor survival in patients. ChIP-sequencing for H3K27ac and H3K27me3 in multiple DMG tumors (n=5) revealed that the ID1 gene is epigenetically active, which matches the epigenetic state of murine prenatal hindbrain cells. Higher ID1-expressing astrocyte-like DIPG cells share a similar transcriptional program with ID1+/SPARCL1+ positive oligo/astrocyte-precursor (OAPC) cells from the developing human brain and demonstrate upregulation of gene sets involved in regulation of cell migration. Both genetic and pharmacologic [cannabidiol (CBD)] suppression of ID1 result in decreased DIPG cell invasion/migration in vitro and invasion/tumor growth in multiple in vivo models. Mechanistically, CBD reduces proliferation through production of reactive oxygen species. Further, DIPG patients treated off-trial with CBD (n=15) displayed reduced ID1 tumor expression and improved overall survival. In summary, ID1 is upregulated in DIPG through K27M-mediated epigenetic reactivation of a developmental OAPC-like transcriptional state, and ID1-driven invasiveness of DIPG is therapeutically targetable with CBD.

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Preclinical evaluation of KZR-261, a novel small molecule inhibitor of Sec61.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.15_suppl.3582 ◽

2020 ◽

Vol 38 (15_suppl) ◽

pp. 3582-3582

Author(s):

Eric Lowe ◽

R. Andrea Fan ◽

Jing Jiang ◽

Henry W. B. Johnson ◽

Christopher J. Kirk ◽

...

Keyword(s):

Gene Expression ◽

Gene Expression Profiling ◽

Cell Lines ◽

Therapeutic Target ◽

Expression Profiling ◽

Single Agent ◽

Tumor Types ◽

Anti Tumor Activity

3582 Background: Secreted and transmembrane proteins play key roles in malignant transformation and growth, including in autocrine growth factor expression, receptor oncogene signaling, and immune system evasion. Biogenesis of these proteins involves translocation of the nascent polypeptides into the endoplasmic reticulum (ER) through the Sec61 channel, providing an untapped therapeutic target for a broad spectrum of malignancies. Here we describe preclinical activity of KZR-261 and related inhibitors of Sec61-dependent protein secretion. Methods: Sec61 inhibition with KZR-261 and related analog KZR-834 were evaluated using cell lines overexpressing proteins of interest tagged with luciferase. In vitro anti-tumor activity was assessed against a panel of 346 cell lines across 25 tumor types. Quantitative proteomic profiling by mass spec and gene expression profiling by RNAseq were conducted following treatment in multiple solid and heme tumor cell lines. Anti-tumor efficacy was evaluated in athymic nude mice implanted with the cancer cell lines H82 (SCLC), HT29 (CRC), BxPC3 (Pancreatic), 22RV1 (Prostate), H929 (Myeloma) and RL (NHL). Activity was also evaluated in a MC38 syngeneic colon tumor model. Results: KZR-261 and KZR-834 exhibited nanomolar potency against many therapeutic targets, including immune checkpoints, VEGF-A, VEGFR and EGFR. Broad in vitro anti-cancer activity was observed with KZR-834, which potently decreased cell viability across both solid and heme tumor types including CRC, Pancreatic, HNSCC, HCC, Lymphoma and Myeloma. Global proteomic analysis observed more than 1.5 fold downregulation of < 10% of detected Sec61 client proteins following treatment, while gene expression profiling revealed upregulation of ER stress response genes in sensitive versus resistant cell lines. Analysis of the TCGA database also found these genes upregulated in a number of different tumor types. In vivo, weekly IV administration was well tolerated and induced a dose dependent anti-tumor response at doses below the MTD in solid and heme xenograft models. In the syngeneic MC38 model, administration of KZR-834 in combination with anti-PD1 antibody resulted in greater anti-tumor activity than either single agent. Conclusions: Novel Sec61 inhibitors potently block expression of secreted and membrane proteins, translating into anti-tumor activity against many tumor types in vitro and in vivo, suggesting broad therapeutic potential. Clinical trials are being planned with KZR-261 to understand safety and early efficacy of this novel compound and therapeutic target.

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Integrative genomic analysis of early neurogenesis reveals a temporal genetic program for differentiation and specification of preplate and Cajal-Retzius neurons

10.1101/331900 ◽

2018 ◽

Author(s):

Jia Li ◽

Lei Sun ◽

Xue-Liang Peng ◽

Xiao-Ming Yu ◽

Shao-Jun Qi ◽

...

Keyword(s):

Neuronal Differentiation ◽

Molecular Mechanisms ◽

Genomic Analysis ◽

Molecular Heterogeneity ◽

Sequencing Analysis ◽

Cortical Function ◽

Early Neurogenesis ◽

And Function

AbstractNeurogenesis in the developing neocortex begins with the generation of the preplate, which consists of early born neurons including Cajal-Retzius (CR) cells and subplate neurons. Here, utilizing the Ebf2-EGFP transgenic mouse in which EGFP initially labels the preplate neurons then persists in CR cells, we reveal the dynamic transcriptome profiles of early neurogenesis and CR cell differentiation. At E15.5 when Ebf2-EGFP+ cells are mostly CR neurons, single-cell sequencing analysis of purified Ebf2-EGFP+ cells uncovers molecular heterogeneity in CR neurons, but without apparent clustering of cells with distinct regional origins. Along a pseudotemporal trajectory these cells are classified into three different developing states, revealing genetic cascades from early generic neuronal differentiation to late fate specification during the establishment of CR neuron identity and function. Further genome-wide RNA-seq and ChIP-seq analyses at multiple early neurogenic stages have revealed the temporal gene expression dynamics of early neurogenesis and distinct histone modification patterns in early differentiating neurons. We have also identified a new set of coding genes and lncRNAs involved in early neuronal differentiation and validated with functional assays In Vitro and In Vivo. Our findings shed light on the molecular mechanisms governing the early differentiation steps during cortical development, especially CR neuron biology, and help understand the developmental basis for cortical function and diseases.

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A Deptor Inhibitor Induces Its Degradation with Resulting Anti-Myeloma Cytotoxicity in Vitro and In Vivo

Blood ◽

10.1182/blood-2019-121480 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1821-1821

Author(s):

Mario I Vega ◽

Yijiang Shi ◽

Patrick Frost ◽

Sara Huerta-Yepez ◽

Alan Lichtenstein

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Therapeutic Target ◽

Cytotoxic Effect ◽

Plasma Cells ◽

Conflicts Of Interest ◽

Inhibition Of Proliferation ◽

Induction Of Apoptosis

Multiple myeloma (MM) is a hematological disorder characterized by a proliferation of malignant monoclonal plasma cells in the bone marrow (BM) and / or in extramedullary sites. Despite recent progress in OS rates, MM remains an incurable disease and most patients will relapse and require treatment. Deptor is a component of mTOR complexes and a constitutive inhibitor of their activities. It is known that the inhibition of Deptor results in the inhibition of the proliferation and induction of apoptosis in MM cells. In addition, high levels of Deptor are predictive of a poor response to conventional therapies, indicating that Deptor expression are important as a prognostic marker for patients with myeloma and is a possible therapeutic target. Our group previously identified a drug which prevents mTOR-Deptor binding (NSC126405) and induces cellular cytotoxicity in MM (Shi Y, et al 2016). In this study, we developed a new related chemical inhibitor (43 M) capable of inducing the inhibition of the mTOR / Deptor interaction and results in the negative regulation of Deptor that leads to the inhibition of proliferation and induces apoptosis in several MM cell lines. The cytotoxic effect of 43 M is not dependent of caspase activation and induces the activation of p70 and AKT (T308). This leads to the induction of apoptosis in MM cell lines and tumor cells derived from MM patients. The degradation of Deptor induced by 43 M is dependent on the proteasome complex since it was prevented in the presence of MG132. In vivo, 43 M prevents the expression of Deptor in a xenograft tumor, and delayed tumor growth and interestingly, induces the eradication of tumors in 40% of mice in a murine model of MM, without significant toxic implications. Recent studies show that Deptor expression protects MM cells against Bortezomib treatment, suggesting that anti-Deptor drugs can synergize with proteasome inhibitors (PIs). However, the combination of 43 M + Bortezomib was not synergistic, and was antagonistic in vitro. These results are probably due to the prevention of the proteasomal degradation of Deptor, suggesting a possible use of the 43 M inhibitor in MM in the absence of the current PIs. This study describes for the first time the possible role of Deptor as a therapeutic target using a chemical inhibitor capable of degrading and inducing a cytotoxic effect in MM cell lines. In addition, Deptor is reported as an important therapeutic target in an in vivo MM model. Shi Y, Daniels-Wells TR, Frost P, Lee J, Finn RS, Bardeleben C, Penichet ML, Jung ME, Gera J, Lichtenstein A. Cytotoxic Properties of a DEPTOR-mTOR Inhibitor in Multiple Myeloma Cells. Cancer Res. 2016 Oct 1;76(19):5822-5831 Disclosures No relevant conflicts of interest to declare.

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Downregulation of PRAME Suppresses Proliferation and Promotes Apoptosis in Hepatocellular Carcinoma Through the Activation of P53 Mediated Pathway

Cellular Physiology and Biochemistry ◽

10.1159/000487353 ◽

2018 ◽

Vol 45 (3) ◽

pp. 1121-1135 ◽

Cited By ~ 9

Author(s):

Hanzhang Zhu ◽

Jingrui Wang ◽

Junjie Yin ◽

Bei Lu ◽

Qijun Yang ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Lines ◽

Therapeutic Target ◽

Cell Apoptosis ◽

Poor Prognosis ◽

P53 Pathway ◽

Tissue Samples ◽

Potential Biomarker

Background/Aims: The expression of PRAME and its role in hepatocellular carcinoma (HCC) remain unknown. The aim of this study was to examine the functional role of PRAME in HCC development and exploring the molecular mechanism. Methods: We first detected PRAME expression in 96 human HCC tissue samples and correlated with clinicopathological characteristics and prognosis of the patients. We then established stable HCC cell lines with PRAME overexpression and knockdown followed by functional analysis in vitro. Further, we examined the relationship between PRAME and p53 pathway in vitro by using Western blotting. Finally, PRAME expression was detected to evaluate its correlation with p-p53 and p53 pathway related apoptotic proteins in xenograft tumor mouse model using immunohistochemistry. Results: PRAME expression was significantly higher in HCC tissues than in adjacent non-tumor tissues and their expression was positively correlated with alpha fetoprotein levels and tumor size. In addition, PRAME expression was associated with AJCC stage and is a potential biomarker of poor prognosis regarding 5-year overall survival in HCC. In vitro studies, we found that PRAME expression was higher in HCC cell lines than in normal hepatic cell line. Inhibited cell proliferation and increased cell apoptosis was observed in PRAME knockdown HCC cells. Futher, increased cell apoptosis was correlated with the proportion of cells in G0/G1 stage, activated p53 mediated apoptosis, and increased cyclin p21 expression. Xenograft analysis in nude mice also found that PRAME knockdown inhibited tumorigenesis while PRAME overexpression had opposite effect. Conclusions: In HCC, PRAME serves as a potential biomarker for poor prognosis and novel therapeutic target in treating this cancer. PRAME is a potential biomarker of poor prognosis in HCC. PRAME surpresses HCC cell death in vitro and in vivo by regulating p53 apoptotic signaling and may serve as a potential therapeutic target in HCC.

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RARE-22. THERAPEUTIC TARGETING OF PURINE METABOLISM IN DIPG

Neuro-Oncology ◽

10.1093/neuonc/noab090.183 ◽

2021 ◽

Vol 23 (Supplement_1) ◽

pp. i45-i45

Author(s):

Ian Mersich ◽

Biplab Dasgupta

Keyword(s):

Cell Lines ◽

De Novo ◽

Treatment Strategies ◽

Diffuse Intrinsic Pontine Glioma ◽

Purine Metabolism ◽

In Vivo Studies ◽

Molecule Inhibitor ◽

Clinical Benefits

Abstract Diffuse intrinsic pontine glioma is a universally lethal disease primarily impacting pediatric patients. There are currently no targeted therapies increasing overall for patients with these tumors; therefore, our lab set out to elucidate metabolic dependencies in DIPG patient-derived cell lines with the ultimate goal of identifying novel therapeutic targets. Through untargeted metabolomics and gene expression analyses, we have identified the purine metabolism gene ATIC to be important for DIPG tumor cell survival and proliferation. Anti-folate drugs such as methotrexate target de novo purine biosynthesis and are used to treat other pediatric cancers; however, we have identified a small molecule inhibitor of ATIC that may offer clinical benefits over other inhibitors of this pathway. In vitro cell viability experiments have demonstrated DIPG cell lines are much more sensitive to the ATIC inhibitor relative to normal neural stem cells and glial cell lines. Furthermore, we have started in vivo studies on pre-clinical mouse models of DIPG with promising results. Treatment with the ATIC inhibitor has significantly increased overall survival relative to control and vehicle treated mice. The dosage we started at was well tolerated in these mice so we are following up on this in vivo work through dose-escalation studies as well as combination treatment strategies. Mechanistically, the ATIC inhibitor works differently than anti-folate compounds such as methotrexate; therefore we are also elucidating why cancer cells are much more sensitive to this compound.

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HGG-30. BRAIN PENETRANT HDAC INHIBITOR RG2833 SYNERGIZES WITH LOMUSTINE AND RADIATION TO INDUCE DIPG CELL DEATH

Neuro-Oncology ◽

10.1093/neuonc/noab090.094 ◽

2021 ◽

Vol 23 (Supplement_1) ◽

pp. i23-i23

Author(s):

Katherine Barnett ◽

Orlandi Novak ◽

Charles Eberhart ◽

Eric Raabe

Keyword(s):

Cell Growth ◽

Western Blot ◽

Cell Lines ◽

Hdac Inhibitor ◽

Diffuse Intrinsic Pontine Glioma ◽

T Test ◽

Tumor Mouse Model ◽

Apoptotic Effects

Abstract Diffuse intrinsic pontine glioma (DIPG) is driven by epigenetic dysregulation. The pan-HDAC inhibitor panobinostat showed pre-clinical efficacy against DIPG, but was limited by toxicity in clinical trials. RG2833 (RGFP109) is a selective HDAC1/3 inhibitor with established brain penetration. RG2833 reverses temozolomide-resistance in glioblastoma through downregulation of the NFĸB pathway. As this pathway is upregulated in DIPG and may contribute to tumorigenesis, we hypothesized that RG2833 would kill DIPG cells. We found that RG2833 treatment inhibits cell growth in the 4 to 9μM range in both autopsy and biopsy-derived DIPG cell lines (MTS assay for HSJD007 p=0.0004, JHH-DIPG1 p=0.001, SF-7761 p=0.04, SU-DIPG13 p=0.01 by t-test). Western blot confirmed on target activity by increased histone 3 acetylation at IC50 doses. RG2833 induces apoptosis (cPARP Western blot, cleaved caspase 3 immunofluorescence HSJD007 p<0.003, JHH-DIPG1 p=0.0026 by t-test) and slows cell proliferation (phospho-Rb Western blot, BrdU immunofluorescence HSJD007 p=0.008, JHH-DIPG1 p=0.0002 by t-test) in multiple DIPG cell lines. RG2833 disrupts the NFĸB pathway through acetylation of p65, resulting in decreased expression of NFĸB regulated pro-survival genes (Western blot for BCL2, BCL-xL, and XIAP). In a DIPG flank tumor mouse model, treatment with RG2833 alone for 1 week suppresses flank tumor growth (p< 0.005 by t-test) and induces apoptosis (Western blot for cPARP). We next assessed whether RG2833 combines synergistically with conventional therapies. We found that RG2833 has strong synergism with both lomustine and radiation to slow DIPG cell growth in vitro (ZIP synergy scores by SynergyFinder for RG2833+lomustine in JHH-DIPG1 17.8 and HSJD007 17.7, RG2833+radiation in JHH-DIPG1 9.7 and JHH-DIPG16A 10.9). Evaluation of combination treatment for apoptotic effects in vitro and in vivo are ongoing. This data identifies selective HDAC inhibitor RG2833 as a promising therapy for DIPG that combines synergistically with conventional therapies.

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