Histone Mutations Research Articles

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

CNSC-26. NETWORK SIGNATURES OF SURVIVAL IN BRAIN TUMOUR GENETICS

Abstract Tumor heterogeneity is increasingly recognized as a major obstacle to therapeutic innovation across neuro-oncology. Gliomas typically exhibit a spectrum of possible genetic mutations, reflecting multiple, potentially complex interactions across distinct molecular pathways. Predicting disease evolution and prescribing individually optimal treatment plausibly requires statistical models complex enough to capture the intricate genetic structure underpinning oncogenesis. Here we formalize this task as the discovery of distinct patterns of connectivity within network representations of the interactions between genetic loci. Evaluating multi-institutional clinical, genetic, and outcome data from 7847 consecutive patients over a 14-year period, we employ Bayesian stochastic block modelling to reveal a hierarchical network structure of tumor genetic interactions spanning (molecularly confirmed) IDH-wildtype glioblastoma, IDH-mutant oligodendroglioma and astrocytoma. We show that this structure not only identifies the patients’ diagnosis, but predicts individual survival with greater fidelity (Cox’s proportional hazard cross-validated (CPH-CV) concordance = 0.78; Bayesian 12, 24 and 36-month survival models = R2 0.31, 0.40 and 0.46, respectively) than the latest WHO tumor classification (CPH-CV concordance = 0.77; Bayesian 12, 24 and 36-month survival models = R2 0.26, 0.36 and 0.39, respectively) or linear models of genetic features (CPH-CV concordance = 0.76; Bayesian 12, 24 and 36-month survival models = R2 0.26, 0.35 and 0.37, respectively). For example, a diagnosis of IDH-wildtype glioblastoma yielded median survivals varying from 289 days in patients with the signature of marked EGFR amplification, MGMT methylation, and histone mutations, to 425 days in those with the signature of TERT mutations, histone and EGFR wild-types, and variable MGMT methylation. These findings suggest network-based analysis can reveal distinct signatures of survival with better prognostic fidelity than current gold standard diagnostic categories.

TMET-09. LOSS OF MAT2A COMPROMISES METHIONINE METABOLISM AND REPRESENTS A VULNERABILITY IN H3K27M MUTANT GLIOMAS

Abstract H3K27-mutant diffuse midline gliomas (DMGs) are defined as grade IV tumors by the World Health Organization. DMGs are inoperable and resistant to chemo/radio therapies. Median survival ranges from 8-11 months, with 2% of patients surviving beyond 5 years. H3K27M mutations lead to global epigenetic and transcriptional reprogramming driven by global loss of negative transcriptional regulator H3K27 trimethylation (H3K27me3). Loss of H3K27me3 is an initiating event in gliomagenesis. This disease lacks appropriate models to predict disease biology and response to treatment. Therefore, we developed a novel syngeneic H3K27M mouse model. An unbiased integrated systems biology approach identified that H3K27M but not isogenic controls relied on the amino acid methionine and the enzyme Methionine Adenosyltransferase 2A (MAT2A). MAT2A is a central regulator of one-carbon metabolism by converting methionine to S-adenosylmethionine (SAM), the universal methyl-donor for protein and nucleotide methylation reactions. In complementary genetic approaches, we applied these findings to patient-derived cell lines with the H3K27M mutation. We hypothesize that MAT2A abrogation, genetic/pharmacological, would alter DMG viability by disrupting the methylome. The current MAT2A sensitivity paradigm is based on Methylthioadenosine Phosphorylase (MTAP) deletion through a synthetic lethal mechanism. We provide a novel mechanism whereby H3K27M cells are sensitive to MAT2A loss, independent of MTAP and through Adenosylmethionine Decarboxylase 1 (AMD1) overexpression disrupting MAT2A regulation. This results in H3K27M cells having lower MAT2A protein levels, conferring a sensitivity by inhibiting residual MAT2A. Genetic/pharmacological aberrations to MAT2A resulted in reduced proliferation. Parallel H3K36me3 ChIP and RNA-sequencing identified loss of oncogenic and developmental transcriptional programs associated with MAT2A loss. In vivo syngeneic and patient-derived xenograft models with both inducible MAT2A knockdown or methionine restricted diets showed extended survival. These results suggest novel interactions between methionine metabolism and the epigenome of H3K27M gliomas and provide evidence that MAT2A, presents exploitable therapeutic vulnerabilities in histone mutant gliomas.

Cis- and trans-regulation by histone H4 basic patch R17/R19 in metazoan development.

The histone H4 basic patch is critical for chromatin structure and regulation of the chromatin machinery. However, the biological roles of these positively charged residues and the mechanisms by which they regulate gene expression remain unclear. In this study, we used histone mutagenesis to investigate the physiological function and downstream regulatory genes of H4 residues R17 and R19 in Drosophila. We found all histone mutations including R17A/E/H and R19A/E/H (R17 and R19 of H4 are substituted by A, E and H respectively) result in a range of growth defects and abnormalities in chromosomal high-order structures, whereas R17E mutation is embryonic lethal. RNA-seq demonstrates that downregulated genes in both R17A and R19A show significant overlap and are enriched in development-related pathways. In addition, Western and cytological analyses showed that the R17A mutation resulted in a significant reduction in H4K16 acetylation and male offspring, implying that the R17 may be involved in male dosage compensation mechanisms. R19 mutation on the other hand strongly affect Gpp (Dot1 homologue in flies)-mediated H3K79 methylation, possibly through histone crosstalk. Together these results provide insights into the differential impacts of positive charges of H4 basic patch R17/R19 on regulation of gene transcription during developmental processes.

Redefining a Rare CNS Tumour Through Targeted Genetic Sequencing

Abstract AIMS Isolated cerebellar vermis lesions in adults is a rare location and entity with few literature case reports detailing gangliogliomas and gangliocytomas. Using targeted genetic sequencing, traditional histological diagnosis can be refined further, with a clear impact on prognosis as well as possible genetic counselling implications. METHOD A single case of cerebellar vermis lesion, identified incidentally through imaging of a neck nodule, with subtle features of ataxia and mild impairment of initiation of speech. Sub-total resection of lesion underwent standard histopathological work-up, followed by targeted genetic sequencing to obtain an integrated diagnosis. RESULTS Histological assessment identified atypical ganglion cells labelled with Synaptophysin on immunohistochemistry. Features closely resembled dysplastic cerebellar gangliocytoma (Lhermitte-Duclos Disease). Further targeted sequencing showed no evidence of IDH1, IDH2, TERT promoter, or histone mutation, however, BRAF mutation was present, supporting an alternate diagnosis of ganglioglioma. CONCLUSION Precision medicine is facilitated with advanced diagnostic techniques. This redefines categorisation of some CNS tumours, particularly rare entities. Techniques are especially valuable to individual patient management as they can have a direct impact on aspects of clinical work-up, prognosis, follow-up, and in some cases, genetic counselling.

Comparative Analysis of the Histone H3 Mutant Protein Interactome Landscape in Paediatric High-Grade Gliomas

Abstract AIMS There have been no significant improvements in the treatments for childhood and adolescent High-Grade Glioma (pHGG) and Diffuse Intrinsic Pontine Glioblastoma (DIPG), which have a very poor prognosis. These cancers harbour mutations affecting histone 3 (H3) proteins, 80% of DIPGs harbour histone H3.1 and H3.3 K27M somatic mutations whilst 30% of pHGGs exhibit H3.3 G34R or G34V mutations. Several studies have highlighted the epigenetic changes associated with these mutations, however their precise role in tumourigenesis is still unknown. We hypothesize that H3 mutations promote an aberrant interaction landscape and analysis of these interactome will highlight important pathophysiological consequences in these tumours. METHOD Two different affinity chromatographic proteomic analyses were performed using different recombinant H3 wild type (WT) and H3 mutant histone proteins (K27M, G34R and G34V) to purify their binding partners, from cell extracts of control and pHGG/DIPG patients cell lines. The purified samples were then subjected to Liquid Chromatography-Mass Spectrometry (LC-MS) to identify their co-bound partners. RESULTS The results have isolated unique and common differential interaction partners of the H3 mutants and with cellular proteins in comparison to H3 WT protein, in the pHGG tumour cells. CONCLUSION The role these altered protein interactions are being investigating in tumour initiation, progression and/or maintenance of H3 mutated pHGG/DIPG, with the aim to manipulate their function as alternate therapeutic intervention. Results of these analyses/experiments will be presented.

Molecular basis of epigenetic regulation in cancer diagnosis and treatment.

The global cancer cases and mortality rates are increasing and demand efficient biomarkers for accurate screening, detection, diagnosis, and prognosis. Recent studies have demonstrated that variations in epigenetic mechanisms like aberrant promoter methylation, altered histone modification and mutations in ATP-dependent chromatin remodelling complexes play an important role in the development of carcinogenic events. However, the influence of other epigenetic alterations in various cancers was confirmed with evolving research and the emergence of high throughput technologies. Therefore, alterations in epigenetic marks may have clinical utility as potential biomarkers for early cancer detection and diagnosis. In this review, an outline of the key epigenetic mechanism(s), and their deregulation in cancer etiology have been discussed to decipher the future prospects in cancer therapeutics including precision medicine. Also, this review attempts to highlight the gaps in epigenetic drug development with emphasis on integrative analysis of epigenetic biomarkers to establish minimally non-invasive biomarkers with clinical applications.

Genomic and microenvironmental landscape of stage I follicular lymphoma, compared with stage III/IV

AbstractAlthough the genomic and immune microenvironmental landscape of follicular lymphoma (FL) has been extensively investigated, little is known about the potential biological differences between stage I and stage III/IV disease. Using next-generation sequencing and immunohistochemistry, 82 FL nodal stage I cases were analyzed and compared with 139 FL stage III/IV nodal cases. Many similarities in mutations, chromosomal copy number aberrations, and microenvironmental cell populations were detected. However, there were also significant differences in microenvironmental and genomic features. CD8+ T cells (P = .02) and STAT6 mutations (false discovery rate [FDR] <0.001) were more frequent in stage I FL. In contrast, programmed cell death protein 1–positive T cells, CD68+/CD163+ macrophages (P < .001), BCL2 translocation (BCL2trl+) (P < .0001), and KMT2D (FDR = 0.003) and CREBBP (FDR = 0.04) mutations were found more frequently in stage III/IV FL. Using clustering, we identified 3 clusters within stage I, and 2 clusters within stage III/IV. The BLC2trl+ stage I cluster was comparable to the BCL2trl+ cluster in stage III/IV. The two BCL2trl– stage I clusters were unique for stage I. One was enriched for CREBBP (95%) and STAT6 (64%) mutations, without BLC6 translocation (BCL6trl), whereas the BCL2trl– stage III/IV cluster contained BCL6trl (64%) with fewer CREBBP (45%) and STAT6 (9%) mutations. The other BCL2trl– stage I cluster was relatively heterogeneous with more copy number aberrations and linker histone mutations. This exploratory study shows that stage I FL is genetically heterogeneous with different underlying oncogenic pathways. Stage I FL BCL2trl– is likely STAT6 driven, whereas BCL2trl– stage III/IV appears to be more BCL6trl driven.

Structural variants shape driver combinations and outcomes in pediatric high-grade glioma.

We analyzed the contributions of structural variants (SVs) to gliomagenesis across 179 pediatric high-grade gliomas (pHGGs). The most recurrent SVs targeted MYC isoforms and receptor tyrosine kinases (RTKs), including an SV amplifying a MYC enhancer in 12% of diffuse midline gliomas (DMG), indicating an underappreciated role for MYC in pHGG. SV signature analysis revealed that tumors with simple signatures were TP53 wild type (TP53WT) but showed alterations in TP53 pathway members PPM1D and MDM4. Complex signatures were associated with direct aberrations in TP53, CDKN2A and RB1 early in tumor evolution and with later-occurring extrachromosomal amplicons. All pHGGs exhibited at least one simple-SV signature, but complex-SV signatures were primarily restricted to subsets of H3.3K27M DMGs and hemispheric pHGGs. Importantly, DMGs with complex-SV signatures were associated with shorter overall survival independent of histone mutation and TP53 status. These data provide insight into the impact of SVs on gliomagenesis and the mechanisms that shape them.

Giant cell tumor of bone: An update, including spectrum of pathological features, pathogenesis, molecular profile and the differential diagnoses.

Giant cell tumor of bone (GCTB) is an enigmatic tumor. Despite its benign histological appearance and clinical behavior in most cases, it is associated with recurrences, uncommonly metastasis, and rarely with a malignant transformation. During the last few years, there has been a significant evolution in the diagnosis and management of GCTB, including discoveries related to the underlying pathogenesis (RANK/RANK/OPG pathway), with treatment-related implications in the form of denosumab (approved inhibitor for targeting RANKL), leading to improved surgical resections, especially in cases of recurrent, large and borderline resectable tumors. Lately, a specific Histone mutation, namely H3.3G34W underlying almost all GCTBs has been discovered, further leading to the identification of a highly sensitive and specific immunohistochemical antibody marker, H3.3G34W, which is very useful for an exact diagnosis of a GCTB, including its differentiation from its various mimics, which has significant implications. This review describes clinicopathological features of a GCTB, including its variable features, recent concepts, underlying pathogenesis, post-denosumab related changes and various entities that constitute its differential diagnosis, including their molecular signatures, with treatment-related implications.

Single-cell epigenetic analysis reveals principles of chromatin states in H3.3-K27M gliomas

Cancer cells are highly heterogeneous at the transcriptional level and epigenetic state. Methods to study epigenetic heterogeneity are limited in throughput and information obtained per cell. Here, we adapted cytometry by time-of-flight (CyTOF) to analyze a wide panel of histone modifications in primary tumor-derived lines of diffused intrinsic pontine glioma (DIPG). DIPG is a lethal glioma, driven by a histone H3 lysine 27 mutation (H3-K27M). We identified two epigenetically distinct subpopulations in DIPG, reflecting inherent heterogeneity in expression of the mutant histone. These two subpopulations are robust across tumor lines derived from different patients and show differential proliferation capacity and expression of stem cell and differentiation markers. Moreover, we demonstrate the use of these high-dimensional data to elucidate potential interactions between histone modifications and epigenetic alterations during the cell cycle. Our work establishes new concepts for the analysis of epigenetic heterogeneity in cancer that could be applied to diverse biological systems.

The histone H2B Arg95 residue links the pheromone response pathway to rapamycin-induced G1 arrest in yeast

Rapamycin is an immunosuppressant used for treating many types of diseases such as kidney carcinomas. In yeast, rapamycin inhibits the TORC1 kinase signaling pathway causing rapid alteration in gene expression and ultimately cell cycle arrest in G1 through mechanisms that are not fully understood. Herein, we screened a histone mutant collection and report that one of the mutants, H2B R95A, is strikingly resistant to rapamycin due to a defective cell cycle arrest. We show that the H2B R95A causes defects in the expression of a subset of genes of the pheromone pathway required for α factor-induced G1 arrest. The expression of the STE5 gene and its encoded scaffold protein Ste5, required for the sequential activation of the MAPKs of the pheromone pathway, is greatly reduced in the H2B R95A mutant. Similar to the H2B R95A mutant, cells devoid of Ste5 are also resistant to rapamycin. Rapamycin-induced G1 arrest does not involve detectable phosphorylation of the MAPKs, Kss1, and Fus3, as reported for α factor-induced G1 arrest. However, we observed a sharp induction of the G1 cyclin Cln2 (~ 3- to 4-fold) in the ste5Δ mutant within 30 min of exposure to rapamycin. Our data provide a new insight whereby rapamycin signaling via the Torc1 kinase may exploit the pheromone pathway to arrest cells in the G1 phase.

Abstract 1340: The role of chemokines CCL8 and CCL12 in diffuse intrinsic pontine gliomas

Abstract Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brainstem glioma, which carries the deadliest prognosis of all childhood neoplasms. Despite recent advances in molecular profiling and characterization of pediatric high-grade gliomas, patient outcomes have not improved. One critical reason why current therapeutic approaches fail to provide a durable response is the adaptive nature of the highly understudied tumor microenvironment (TME). The most abundant non-neoplastic infiltrates in the DIPG TME are the tumor-associated macrophages (TAMs). There is limited data available on the role of TAMs in DIPG tumor growth and response to therapy, and the chemokines responsible for driving the infiltration of both innate and adaptive immune cells into these tumors remain unidentified.To accurately model DIPGs, we utilized immunocompetent genetically engineered mouse models (GEMMs) based on the RCAS/tv-a gene transfer system. At postnatal days 0-2, mice were injected with retrovirus-producing DF-1 cells directly in the brainstem. Tumorigenesis was driven by PDGFB overexpression and Tp53 silencing, together with human DIPG-specific mutations including: H3 histones with K27M substitution (H3.1K27M/H3.3K27M) and Acvr1R206H. Our data using these GEMMs show that mouse tumors are strikingly similar to their human counterparts as demonstrated by hallmark pathological observations including T2-contrast enhancement, highly infiltrating margins, and pseudopalisading necrosis. Loss of H3K27 trimethylation in histone mutant tumors is also observed in these tumors. Utilizing these GEMMs, we have identified CCL8 and CCL12 as important chemokines in H3.1K27M-DIPGs. Specifically, we observed longer survival in Ccl8/Ccl12 double knockout (DKO) tumor-bearing mice compared to wild-type (WT) mice. Interestingly, spectral flow cytometry analysis revealed no differences in myeloid cell infiltration but an increased number of NK cells, CD4+, and CD8+ T cells in tumors generated in Ccl8/Ccl12 DKO mice compared to WT. We also tested the efficacy of inhibiting two CCL8 receptors using a syngeneic mouse model of DIPG. Pharmacological inhibition of CCR1 and CCR5 rendered a reduction in macrophage presence in the tumors, and resulted in an extension of survival in tumor-bearing mice comparable to the effect of standard of care, radiation therapy. Our ongoing experiments aim to elucidate the mechanisms by which CCL8 and CCL12 modulate immune cell infiltration, and determine whether the effect of CCL8/CCL12 loss on survival seen in H3.1K27M-DIPG holds true for H3.3K27M-DIPG-bearing mice. Citation Format: Gonzalo M. Pinero, Montserrat Puigdelloses Vallcorba, James L. Ross, Zhihong Chen, Tanvi Joshi, Dolores Hambardzumyan. The role of chemokines CCL8 and CCL12 in diffuse intrinsic pontine gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1340.

Patterns of care in adult histone mutant gliomas: Results of an international survey.

Histone mutant gliomas (HMG) with histone H3 K27 and G34 mutations are recognized as biologically discrete entities with distinct anatomical locations, younger age at presentation (in comparison to the most common high-grade gliomas, IDH wildtype glioblastoma), and poor prognosis. There is a paucity of data regarding the management of adult HMG patients and no consensus on management. This study aims to identify current patterns of Australian and US neuro-oncology clinical practice for this entity. Following institutional approvals, patterns of care questionnaire designed to capture relevant clinical variables was circulated through the Cooperative Trials Group for Neuro-Oncology (COGNO) in Australia and the Caris Precision Oncology Alliance in the United States (US). Between 4/2021 and 10/2021, 43 responses were collected. 33% (n = 14) of responders tested all patients for HMGs routinely; 40.92% (n = 18) tested in select patients 26% (n = 11) did not test for HMGs. The common indications for testing selected patients were midline anatomic location (n = 18) and age (n = 11) (<50 years). 23 used molecular sequencing, 22 used IHC at their centers. Nine participants stated knowledge of histone H3 mutations did not affect their management of these gliomas, 11 said it affected their management at the time of recurrence, 23 stated it affected the management of midline K27M patients, 11 participants stated it affected the management of K27M mutant gliomas in other locations, and 3 felt it affected the management of G34R/V mutant gliomas. Here we present a description of how the discovery of a new molecular subtype of primary glial tumors, histone mutated gliomas in adults, is being introduced into clinical practice.

DIPG-38. Significant tumor regression of H3K27M-mutated diffuse midline glioma of the brainstem with panobinostat: a case report.

INTRODUCTION: Diffuse midline glioma is a fatal CNS tumor that chiefly occurs in children. To date, standard of care has been limited to local field radiation. A histone mutation in H3K27M is seen in 80% of diffuse midline gliomas of the brainstem, also called diffuse intrinsic pontine glioma (DIPG). Panobinostat, a potent inhibitor of histone deacetylases, has shown moderate efficacy in DIPG/DMGs in preclinical models, and a dose-finding clinical trial (PBTC-047) is ongoing. We report on a case of a child with biopsy-proven H3K27M-mutated DMG of the brainstem treated off-trial with panobinostat monotherapy after radiation. METHODS: This is a case report of a 12-year-old female with H3K27M-mutated DMG of the medulla and cerebellum that exhibited gadolinium-enhancement on MRI. The patient was treated with 54 Gy focal photon radiation to the tumor field over six weeks after biopsy demonstrating the H3.3K27M mutation (H3F3A), as well as mutations in TP53, PIK3R1 and AXSL1 genes. She was not eligible for PBTC-047 due to uncontrolled hypertension. She received panobinostat as per the ongoing PBTC protocol at a dose of 28 mg/m2 given on Monday, Wednesday, and Friday on alternating weeks. She received three 28-day cycles over three months. RESULTS: The patient tolerated the therapy well, with minimal adverse reactions of low-grade abdominal pain and constipation. MRI imaging after three cycles revealed an 80% reduction in tumor volume. Together with this radiographic response, she exhibited improvement in left-sided motor strength and improved left facial sensation. Due to an unrelated need for oral surgery, panobinostat was held. MRI imaging six weeks after cessation of panobinostat revealed extensive relapse with supratentorial and spinal disease. CONCLUSION: Systemic panobinostat may demonstrate anti-tumor efficacy in some cases of H3K27M-mutated DMG. Future work is required to determine factors predictive of favorable response.

HGG-27. Understanding the role of PLAG family transcription factors in cortex development and tumorigenesis

Gliomas are the most common type of central nervous system (CNS) tumors in children. Therapy and outcome often reflect the grade of the tumor, with high grade glioma (HGG) leading to a significantly worse survival prognosis than low grade glioma. However, HGG are highly diverse regarding their molecular entities and clinical associations. In some pediatric-type histone 3 K27M mutated HGG, the zinc-finger transcription factor PLAG1 was previously found to be overexpressed, which is also confirmed by patient data from the INFORM program. Further, in a novel type of embryonic CNS tumors without histone mutations, the PLAG1-related genes PLAGL1/PLAGL2 are amplified. However, the consequences of aberrant PLAG gene expression on CNS tumor formation during development are unknown. Especially the understanding of downstream signaling pathways that are commonly altered by this transcription factor family could provide necessary starting points for more specific therapy. We use transgenic mouse models with targeted PLAG1 overexpression in neural stem and progenitor cells to investigate the function of PLAG1 in cerebral cortex development and tumorigenesis. We found that PLAG1 overexpression together with heterozygous loss of p53 activity causes neurological defects and impaired cortex development. Next, to understand how PLAG gene overexpression affects neural specification, we will overexpress the PLAG genes in embryonic neural stem and progenitor cells using in utero electroporation. After FACS isolation of electroporated cells and single nucleus RNA sequencing, we will assess how the overexpression changes the transcriptional trajectories of neural development in mouse cortex stem and progenitor cells. We hypothesize that an early event leads to the constitutive upregulation of PLAG genes, which are usually tightly regulated during development, in patients with pediatric brain tumors harboring aberrant PLAG gene expression. Deciphering the transcriptional downstream pathways of this aberrant expression in neural stem and progenitor cells seems promising to find new potential therapeutic targets.

HGG-47. Comparative analysis of the Histone H3 mutant protein interactome landscape in paediatric high-grade gliomas

Abstract There have been no significant improvements in the treatments for childhood and adolescent High-Grade Glioma (pHGG) and Diffuse Intrinsic Pontine Glioblastoma (DIPG), which have a very poor prognosis. These cancers harbour mutations affecting histone 3 (H3) proteins, 80% of DIPGs harbour histone H3.1 and H3.3 K27M somatic mutations whilst 30% of pHGGs exhibit H3.3 G34R or G34V mutations. Several studies have highlighted the epigenetic changes associated with these mutations, however their precise role in tumourigenesis is still unknown. We hypothesize that H3 mutations promote an aberrant interaction landscape and analysis of these interactome will highlight important pathophysiological consequences in these tumours. Two different affinity chromatographic proteomic analyses were performed using different recombinant H3 wild type (WT) and H3 mutant histone proteins (K27M, G34R and G34V) to purify their binding partners, from cell extracts of control and pHGG/DIPG patients cell lines. The purified samples were then subjected to Liquid Chromatography-Mass Spectrometry (LC-MS) to identify their co-bound partners. The results have isolated unique and common differential interaction partners of the H3 mutants and with cellular proteins in comparison to H3 WT protein, in the pHGG tumour cells. The role these altered protein interactions are being investigating in tumour initiation, progression and/or maintenance of H3 mutated pHGG/DIPG, with the aim to manipulate their function as alternate therapeutic intervention. Results of these analyses/experiments will be presented.

DIPG-32. Paediatric histone-mutant diffuse midline glioma: real world outcomes from a low-middle income country (LMIC) setting

Abstract BACKGROUND: Histone-mutant diffuse midline glioma (H3K27M) is a distinct high-grade central nervous system (CNS) tumor arising in the midline structures of the brain and/or spinal cord in children and young adults with dismal clinical outcomes. Real world data on this entity, particularly from the low-middle income countries (LMICs) is lacking. We report outcomes of pediatric histone-mutant diffuse midline gliomas treated between 2014-2021 at our comprehensive cancer centre in India. METHODS: Children (&amp;lt;16 years) with biopsy-proven diffuse midline glioma demonstrating histone mutation (H3K27M) on gene expression profiling were identified through electronic search of neuro-pathology database. Clinico-demographic characteristics, histo-molecular features, treatment details, and survival outcomes were retrieved retrospectively from institutional records. All children had undergone biopsy and occasionally debulking surgery prior to definitive radiotherapy (RT). Upfront temozolomide (TMZ) either concurrent and/or adjuvant was at the discretion of treating oncologist. Progression-free survival (PFS) and overall survival (OS) were calculated using Kaplan-Meier methods and expressed as point estimates with 95% confidence interval (CI). RESULTS: Median age of the study cohort (N=51) was 11 years (inter-quartile range 6-14 years) with no gender predilection. Metastatic disease (leptomeningeal dissemination, sub-ependymal spread, or distant brain involvement) was seen at presentation in 7 (13.7%) patients and at first progression after definitive RT in 10 (19.6%) patients. At a median follow up of 10.3 months, 1-year Kaplan-Meier estimates of PFS and OS were 21.5% (95%CI: 12.2-37.9%) and 45.6% (33.1-62%) yielding median PFS and OS of 7.8 months (95%CI: 5.6-11 months) and 11.3 months (95%CI: 9.4-14.9 months) respectively. CONCLUSIONS: We report aggressive biological course and dismal outcomes in pediatric histone-mutant diffuse midline gliomas (H3K27M) treated in a LMIC setting. High incidence of metastatic CNS failure in our cohort suggests a potential role for testing neuraxial irradiation and aggressive systemic therapy to improve survival.

HGG-36. Elucidating the role of long non-coding RNAs in pediatric high grade gliomas

Abstract BACKGROUND: Genomic and transcriptomic studies have elucidated new insights into the landscape of diffuse intrinsic pontine glioma (DIPG). However, the role of long non-coding RNAs (lncRNAs) has not been explored at depth in these tumors, and there have not been studies focused on how lncRNAs interact with the K27M histone mutation. In a recent analysis of nearly 200 DIPGs and pediatric high-grade gliomas (pHGG), we previously detected a novel, recurring structural variant in the lncRNA CCDC26. This rearrangement occurs in nearly 10% of all DIPGs, and we have furthermore identified alterations in more than 100 lncRNAs in DIPG. METHODS: To identify lncRNAs required for proliferation of patient-derived DIPG cancer cells, we designed two custom genome-scale lncRNA libraries. We generated a genome-scale lncRNA CRISPR-Cas9 knockout pooled library, consisting of 45,766 single guide RNAs (sgRNAs). Additionally, we generated a genome-wide CRISPR interference pooled library consisting of 45,608 sgRNAs, targeting lncRNA transcription start sites (TSS). RESULTS: We utilized in vitro histone-mutant pHGG models as well as edited clones of these models with the K27M mutant corrected in order to compare lncRNA dependencies in these two contexts. We have successfully performed genome-scale CRISPR-Cas9 knockout and CRISPR interference screens targeting lncRNAs in these cell lines, revealing lncRNA dependencies. Candidate dependencies in our CRISPR-Cas9 knockout screen include LOC100507412, LOC105379524, and LINC02193. CONCLUSION: Genome-wide lncRNA CRISPR knock-out and CRISPR interference screens are a novel approach for the unbiased identification of lncRNAs that are required for pediatric high-grade glioma proliferation. Further validation of specific lncRNAs is required, and these lncRNA dependencies represent potential novel therapeutic targets.

HGG-33. Prognostic factors of H3K27M histone-mutant diffuse midline gliomas in patients ≤18yrs

OBJECTIVE：Diffuse midline gliomas associated with high malignancy and poor prognosis. The primary treatment modalities include surgery, radiotherapy and chemotherapy. The purpose of this study was to investigate the prognostic factors of diffuse intracranial midline glioma. METHODS: A retrospective analysis was performed on 44 cases younger than 18 yrs of H3K27M histone-mutant diffuse midline gliomas diagnosed in Guangdong Sanjiu Brain Hospital from November 2017 to November 2021. The median age was 9 years (range:3-18), including 24 males and 20 females, lesions located in thalamus were 9, while in brain stem were 35. Treatment methods: 35 cases received radiotherapy, 9 cases did not. Among the patients who received RT, 26 cases with concurrent chemoradiotherapy + adjuvant chemotherapy, 8 cases with concurrent chemoradiotherapy only, and 1 case only with radiotherapy. Kaplan-meier method was used to calculate overall survival (OS), and log-rank test was used to test. P < 0.05 was considered statistically significant. RESULTS: By January 27, 2022, 13 cases survived, 25 cases died, and 6 cases were lost to follow-up. The median survival time of 44 patients was 6.95 months (range：1-23.5 months). The median survival was 8.4 months in the radiotherapy group vs 3.7 months in the non-radiotherapy group (P＜0.001); The median survival time of radiotherapy without adjuvant chemotherapy vs radiotherapy with adjuvant chemotherapy was 6.2 months vs 9.35 months (P=0.479). CONCLUSION: Radiotherapy can prolong the survival time of diffuse midline glioma in children with H3K27M histone mutant but no survival benefit was observed in patients with concurrent chemotherapy.