Effects of CHD2 depletion on the growth of H3K27M DMG xenografts.

CHD2 regulates neuron-induced proliferation of H3.1K27M DMG cells.

FOSL1 colocalizes with CHD2 to regulate cell viability and gene expression.

Validation of hits from CRISPR/Cas9 screens.

CHD2 and FOSL1 regulate H3K27me3 and H3K27ac in H3.1K27M DMG cells.

Analysis for DEGs of CHD2-depleted H3.1K27M DMG cells.

CHD2 binds to the gene regulatory elements of axon-guidance and synaptic genes.

Calcium imaging in brain slices with SU-DIPG36 xenografts in hippocampal CA1.

Analysis for shared DEGs between CHD2 and FOSL1 depletion in H3.1K27M DMG cells.

Abstract Diffuse midline glioma (DMG) is one of the most devastating childhood cancers with a median survival of < 1year from diagnosis. There is a critical need for new therapeutics for improving treatment outcomes for DMG patients. We performed genome-wide CRISPR/Cas9 screening and identified RAD52 as a potential therapeutic target in DMG cells. RAD52 inhibition, using shRNA-mediated RAD52 depletion and treatment with RAD52 inhibitor, D-I03, suppressed DMG cell proliferation. Western blotting revealed that RAD52 inhibition increased DNA double-starand breaks (DSBs) marker γH2AX while it decreased repair markers BRCA1 and RAD51. Also, RAD52 inhibition causes a sustained level of phosphorylated RAD50 and γH2AX in irradiated DMG cells over 24 hours. Immunocytochemistry (ICC) of γH2AX and repair marker 53BP1 showed that RAD52 inhibition sustained DNA damage with high levels of γH2AX at 24 hours following radiation while the level of 53BPI was decreased, thereby inhibiting DNA DSB repair. DNA repair assay showed that RAD52 inhibition suppressed DNA DSB repair through a homologous recombination pathway. RNA sequencing showed that RAD52 inhibition downregulated genes associated with the DNA repair pathway. Importantly, RAD52 inhibition increased the radiosensitivity of DMG cells. To understand the mechanism of RAD52 inhibition on DNA damage, we performed dot plot assay and ICC of S9.6 which recognizes DNA-RNA hybrids (also known as R-loops) and found accumulation of R-loop formation in DMG cells treated with RAD52 inhibitor. In addition, RAD52 inhibition decreased the expression of RNA polymerase II. Finally, the combination therapy of RAD52 inhibitor and radiation inhibited tumor growth and increased survival of mice bearing DMG patient-derived xenografts, outperforming either monotherapy. Together, our results highlight that RAD52 inhibition induces DNA damage by accumulating R-loops, results in reducing DMG cell proliferation, while also increase DMG radiosensitivty, and providing a rationale for developing combination therapy with radiation for the treatment of DMG.

Abstract Introduction. Pediatric high-grade gliomas are the most common cause of cancer-related death in children, of which the most malignant and devastating tumors include diffuse midline glioma (DMG). Recent availability of tumor samples and advances in next-generation sequencing enable the profiling of thousands of molecular features in DMG. We employ a system-based approach that uses gene expression as a representation of the molecular state, providing an avenue for therapeutic discovery. Previously, we used this approach to identify repurposed candidates for DMG; however, the final candidate had poor brain penetration, prompting us to discover novel compounds with better brain penetration. To apply it to novel compound discovery, we need the expression profiles of all library compounds, which is impractical for millions of compounds. To overcome this, we have developed a platform, Gene expression profiles Predictor on chemical Structures (GPS), that incorporates machine learning algorithms which leverage existing drug-induced gene expression profiles to predict gene expression based on compound structure. methods. DMG RNA-Seq samples were acquired from St. Jude Children’s Research Hospital and Children’s Brain Tumor Network (CBTN). We developed deep learning autoencoder to search reference normal tissues from Genotype-Tissue Expression (GTEx). A DMG meta-signature was created and fed into GPS to predict drug candidates from the Enamine CNS library, seven of which were experimentally tested in vitro and the most promising one subsequently validated in vivo. Results. Cell proliferation analyzed by MTS assay showed three compounds with IC50< 50 µM in K27M-mutant DMG cell lines (SF8628, DIPG007, SU-DIPG36). Toxicity study showed no adverse effects in the mice treated with 100-200 mg/kg of the most promising compound (IC50 = 1.9 µM in DMG cells). Conclusion. Our novel computational framework leverages deep learning to fill the gap in drug discovery in DMG. Work is underway to determine antitumor activity, brain penetration (via HPLC) and survival benefit in the mice bearing DMG PDX treated with the lead compound.

Abstract High-grade gliomas (HGG) are deadly diseases for both adult and pediatric patients. Recently, it has been shown that neuronal activity promotes the progression of multiple subgroups of HGG. However, epigenetic mechanisms that govern this process remain elusive. Here we report that the chromatin remodeler chromodomain helicase DNA-binding protein 2 (CHD2) regulates neuron–glioma interactions in diffuse midline glioma (DMG) characterized by onco-histone H3.1K27M. Depletion of CHD2 in H3.1K27M DMG cells compromises cell viability and neuron-to-glioma synaptic connections in vitro, neuron-induced proliferation of H3.1K27M DMG cells in vitro and in vivo, activity-dependent calcium transients in vivo, and extends the survival of H3.1K27M DMG-bearing mice. Mechanistically, CHD2 coordinates with the transcription factor FOSL1 to control the expression of axon-guidance and synaptic genes in H3.1K27M DMG cells. Together, our study reveals a mechanism whereby CHD2 controls the intrinsic gene program of the H3.1K27M DMG subtype, which in turn regulates the tumor growth-promoting interactions of glioma cells with neurons. Significance: Neurons drive the proliferation and invasion of glioma cells. Here we show that chromatin remodeler chromodomain helicase DNA-binding protein 2 controls the epigenome and expression of axon-guidance and synaptic genes, thereby promoting neuron-induced proliferation of H3.1K27M diffuse midline glioma and the pathogenesis of this deadly disease.

Purpose Liquid biopsy of cyst fluid in brain tumors has not been extensively studied to date. The present study was performed to see whether diagnostic genetic alterations found in brain tumor tissue DNA could also be detected in cell-free DNA (cfDNA) of cyst fluid in cystic brain tumors. Methods Cyst fluid was obtained from 22 patients undergoing surgery for a cystic brain tumor with confirmed genetic alterations in tumor DNA. Pathological diagnoses based on WHO 2021 classification and diagnostic alterations in the tumor DNA, such as IDH1 R132H and TERT promoter mutation for oligodendrogliomas, were detected by Sanger sequencing. The same alterations were analyzed by both droplet digital PCR (ddPCR) and Sanger sequencing in cyst fluid cfDNA. Additionally, multiplex ligation-dependent probe amplification (MLPA) assays were performed to assess 1p/19q status, presence of CDKN2A loss, PTEN loss and EGFR amplification, to assess whether differentiating between astrocytomas and oligodendrogliomas and grading is possible from cyst fluid cfDNA. Results Twenty-five genetic alterations were found in 22 tumor samples. All (100%) alterations were detected in cyst fluid cfDNA by ddPCR. Twenty of the 25 (80%) alterations were also detected by Sanger sequencing of cyst fluid cfDNA. Variant allele frequency (VAF) in cyst fluid cfDNA was comparable to that of tumor DNA (R = 0.62, Pearson’s correlation). MLPA was feasible in 11 out of 17 (65%) diffuse gliomas, with close correlation of results between tumor DNA and cyst fluid cfDNA. Conclusion Cell-free DNA obtained from cyst fluid in cystic brain tumors is a reliable alternative to tumor DNA when diagnosing brain tumors.

Abstract Pediatric high-grade glioma (pHGG) is the most common cause of cancer-related death in children, of which the most malignant and devastating tumors include diffuse midline glioma (DMG). Its anatomical location in the brainstem, infiltrative nature, and blood-brain barrier (BBB) limit surgical resection and the distribution of systemically administered drugs. Intranasal delivery (IND) is a noninvasive method that bypasses the BBB, by leveraging the unique anatomic connections of the olfactory and trigeminal nerve pathways with the nasal cavity. We previously demonstrated the efficacy of IND of nanoliposomal (LS) formulation of the active metabolite of the DNA topoisomerase I inhibitor, irinotecan, LS-SN-38, in orthotopic human brainstem xenograft models. The specificity of liposomes can be enhanced by using immunoliposome (iLS) formulation, which are coated with antibodies specific to the tumor cells leading to targeted drug delivery and reduced toxicity to normal cells. Platelet-derived growth factor receptor alpha (PDGFRA) plays a role in DMG oncogenesis and is expressed in as much as 70% of DMG by immunohistochemistry. In this study, we evaluated the effect of anti-PDGFRA monoclonal antibody-conjugated immunoliposome encapsulated SN-38 (PDGFRA-iLS-SN-38) in pHGG cell lines. PDGFRA signals were assessed in pHGG cells using western blotting. Cells were treated with rhodamine (Rho)-labeled PDGFRA-iLS and cellular uptake was confirmed with a fluorescent microscope. PDGFRA-iLS-SN38 had the additive effect on the LS-SN-38 in DIPG007 cells (PDGFRA positive), but not in SF8628 cells (PDGFRA negative) in vitro. We treated mice bearing human DMG xenografts with IND of PDGFRA-iLS-SN38. Tumor growth and response to therapy are quantitatively measured by bioluminescence imaging, and efficacy is assessed by survival analysis. Results of survival analysis will be reported at the meeting. PDGFRA-targeted nanotherapeutics represented a specific effect on glioma cells expressing PDGFRA, providing a promising therapeutic approach for targeting pHGG.

Abstract BACKGROUND Diffuse midline glioma (DMG) is one of the most devastating childhood cancers that limited response to radiation therapy (RT). There is a critical need for new therapeutics that enhance the radiation effect. We, therefore, tested the hypothesis that targeting RAD52 activity sensitizes the radiation response of DMG. METHODS Genome-wide CRISPR/Cas9 screening was performed to identify the potential therapeutic target. RAD52 inhibition was used by shRNA-mediated RAD52 depletion and treatment with RAD52 inhibitor, D-I03. The protein expression, cell proliferation, DNA damage marker expression, DNA repair pathway, and transcriptional alteration were analyzed by western blotting, MTS assay, colony formation assay, immunocytochemistry, DNA repair pathway assay, and RNA sequencing. Mice with DMG patient-derived xenograft (PDX) models were treated with RAD52 inhibitor alone or combination with RT. RESULTS Genome-wide CRISPR/Cas9 screening identified RAD52 as a potential therapeutic target in DMG cells. RAD52 inhibition suppressed DMG cell proliferation. Importantly, RAD52 inhibition in combination with RT further increased the radiosensitivity of DMG cells. Immunocytochemistry of DNA double-strand breaks (DSB) marker γH2AX and repair marker 53BP1 showed that RAD52 inhibition sustained DNA damage with high levels of γH2AX at 24 hours following radiation while the level of 53BPI was decreased, thereby inhibiting DNA DSB repair. Western blotting also revealed that RAD52 inhibition causes a sustained level of phosphorylated Rad50 and γH2AX in irradiated DIPG cells over 24 hours. DNA repair assay showed that RAD52 inhibition suppressed homologous recombination DNA repair pathway. RNA sequencing showed that RAD52 inhibition downregulated genes associated with the DNA repair pathway. Finally, the combination therapy of RAD52 inhibitor and RT further suppressed tumor growth and increased survival of mice bearing DMG PDXs, outperforming either monotherapy. CONCLUSIONS these results highlight RAD52 inhibition as a potential radiosensitization and provide a rationale for developing combination therapy with radiation in the treatment of DMG.

Abstract Diffuse midline gliomas (DMG) are lethal pediatric brain tumours, the majority of which have truncal histone (H3K27M) mutations. To investigate cellular vulnerabilities of histone mutant cells we generated oligodendrocyte precursor cells (OPC) isogenic cell lines and performed a high-throughput synthetic lethality drug screen (2400 small molecules). This identified HSP90 inhibitors as a potential therapeutic option for H3K27M DMG. We hypothesize that epichaperome HSP90 inhibitors present a new therapeutic approach targeting multiple aspects of DMG oncogenicity. HSP90 inhibitors, PU-H71 and PU-HZ151, effectively reduced viability of DMG lines in vitro in the low nanomolar range and result in caspase mediated cell death (p<0.0001 1 uM, p<0.05 500 nM, n=6). PU-HZ151 treated mice had a survival benefit vs control mice injected with orthotopic DMG xenografts (p=0.0012). In diseased cells, activated HSP90 forms a scaffold defined as the epichaperome. To unravel the molecular mechanisms of epichaperome inhibition in DMG cells, we performed epichaperomics – a pull down of epichaperome HSP90 with co-chaperones and clients followed by LC-MS/MS analysis. We identified an interactome enriched with proteins involved in cell cycling, RAS/MAPK signaling, immune response, and metabolic reprogramming. Proteomic and phosphoproteomic profiling of DMG cells post-PU-HZ151 treatment provided insights into the direct downstream effects critical for maintaining DMG viability, identified functional vulnerabilities primed by epichaperome inhibition including downregulation of cell cycling, and upregulation of HSF1 mediated cellular response to stress. In silico screening and kinase enrichment analysis highlighted CSNK2A1 as a top active kinase in DMG cells following PU-HZ151 treatment. To enhance the therapeutic efficacy of epichaperome inhibition, we employed the CSNK2A1 kinase inhibitor, CX-4945, in combination with PU-HZ151, resulting in the effective ablation of DMG patient cells in vitro. These data highlight the potential of epichaperome inhibitors for DMG treatment, as they target multiple DMG oncogenic pathways.

Abstract INTRODUCTION pHGGs are the most common cause of cancer-related death in children, of which the most malignant and devastating tumors include diffuse midline glioma (DMG). Its anatomical location in the braistem, infiltrative nature, and blood-brain barrier (BBB) limit surgical resection and the distribution of systemically administered drugs. Intranasal delivery (IND) is a noninvasive method that bypasses the BBB, by leveraging the unique anatomic connections of the olfactory and trigeminal nerve pathways with the nasal cavity. We previously demonstrated the efficacy of IND of liposomal (LS) formulation of the active metabolite of the DNA topoisomerase I inhibitor, irinotecan, LS-SN-38, in orthotopic human brainstem xenograft models. The specificity of liposomes can be enhanced by using immunoliposome (iLS) formulation, which are coated with antibodies specific to the tumor cells leading to targeted drug delivery and reduced toxicity to normal cells. Platelet-derived growth factor receptor alpha (PDGFRA) plays a role in DMG oncogenesis and is expressed in as much as 70% of DMG by immunohistochemistry. In this study, we evaluated the specificity of anti-PDGFRA monoclonal antibody conjugated immunoliposome (PDGFRA-iLS) in pHGG cell lines. METHODS PDGFRA signals were assessed in pHGG cells using western blotting. Cells with high and low expression of PDGFRA were treated with rhodamine (Rho) labeled PDGFRA-iLS and imaged with fluorescent microscope. RESULTS DIPG007, SF7761 and KNS42 expressed high levels of PDGFRA, while SF8628 and DIPG36 cells showed low expression. Fluorescence microscopy revealed high PDGFRA-rhodamine (Rho)-iLS expression in DIPG007 cells (PDGFRA high), relative to SF8628 and DIPG36 cells (PDGFRA low). CONCLUSION PDGFRA-Rho-iLS shows increase cellular uptake in DIPG cells. Work is underway to determine the biodistribution, host toxicity, and anti-tumor activity of IND of PDGFRA-iLS-SN- 38 alone and in combination with radiation therapy in pHGG xenograft models.