Guiding ATR and PARP inhibitor combinations with chemogenomic screens

Combinations of inhibitors of Ataxia Telangiectasia- and Rad3-related kinase (ATRi) and poly(ADP-ribose) polymerases (PARPi) synergistically kill tumor cells through modulation of complementary DNA repair pathways, but their tolerability is limited by hematological toxicities. To address this we performed a genome-wide CRISPR/Cas9 screen to identify genetic alterations that hypersensitize cells to a combination of the ATRi RP-3500 with PARPi, including deficiency in RNase H2, RAD51 paralog mutations or the Alternative Lengthening of Telomeres telomere maintenance mechanism. We show that RP-3500 and PARPi combinations kill cells carrying these genetic alterations at doses sub-therapeutic as single agents. We also demonstrate the mechanism of combination hypersensitivity in RNase H2-deficient cells, where we observe an irreversible replication catastrophe, allowing us to design a highly efficacious and tolerable in vivo dosing schedule. Altogether, we present a comprehensive dataset to inform development of ATRi and PARPi combinations and an experimental framework applicable to other drug combination strategies.

Telomerase and Alternative Lengthening of Telomeres coexist in the regenerating zebrafish caudal fins

Telomeres are essential for chromosome protection and genomic stability, and telomerase function is critical to organ homeostasis. Zebrafish has become a useful vertebrate model for understanding the cellular and molecular mechanisms of regeneration. The regeneration capacity of the caudal fin of wild-type zebrafish is not affected by repetitive amputation, but the behavior of telomeres during this process has not yet been studied. In this study, the regeneration process was characterized in a telomerase deficient zebrafish model. Moreover, the regenerative capacity after repetitive amputations and at different ages was studied. Regenerative efficiency decreases with aging in all genotypes and surprisingly, telomere length is maintained even in telomerase deficient genotypes. Our results suggest that telomere length can be maintained by the regenerating cells through the recombination-mediated Alternative Lengthening of Telomeres (ALT) pathway, which is likely to support high rates of cell proliferation during the tailfin regeneration process. As far as we know, this is the first animal model to study ALT mechanism in regeneration, which opens a wealth of possibilities to study new treatments of ALT dependent processes.

ATRX proximal protein associations boast roles beyond histone deposition

The ATRX ATP-dependent chromatin remodelling/helicase protein associates with the DAXX histone chaperone to deposit histone H3.3 over repetitive DNA regions. Because ATRX-protein interactions impart functions, such as histone deposition, we used proximity-dependent biotinylation (BioID) to identify proximal associations for ATRX. The proteomic screen captured known interactors, such as DAXX, NBS1, and PML, but also identified a range of new associating proteins. To gauge the scope of their roles, we examined three novel ATRX-associating proteins that likely differed in function, and for which little data were available. We found CCDC71 to associate with ATRX, but also HP1 and NAP1, suggesting a role in chromatin maintenance. Contrastingly, FAM207A associated with proteins involved in ribosome biosynthesis and localized to the nucleolus. ATRX proximal associations with the SLF2 DNA damage response factor help inhibit telomere exchanges. We further screened for the proteomic changes at telomeres when ATRX, SLF2, or both proteins were deleted. The loss caused important changes in the abundance of chromatin remodelling, DNA replication, and DNA repair factors at telomeres. Interestingly, several of these have previously been implicated in alternative lengthening of telomeres. Altogether, this study expands the repertoire of ATRX-associating proteins and functions.

CBIO-04. G-QUADRUPLEX STABILIZATION ENHANCES REPLICATION STRESS AND DNA DAMAGE IN ATRX-DEFICIENT HIGH-GRADE GLIOMA

Loss of function mutations in α-thalassaemia/mental retardation X-linked (ATRX) are a critical molecular hallmark for invariably fatal high-grade glioma (HGG). Mutational inactivation of histone chaperone ATRX leads to accumulations of abnormal DNA secondary structures known as G-quadruplexes (G4s), thereby inducing replication stress and DNA damage. As G4s arise at GC-rich regions (i.e., pericentromeric and telomeric regions), ATRX-deficiency alters genome-wide accessibility of chromatin, leads to transcriptional dysregulation, and induces alternative lengthening of telomeres (ALT). Our goal is to target ATRX deficiency through G4 stabilizers, which represent a class of novel small molecule compounds that selectively bind to and stabilize G4 structures. However, the genomic consequences and efficacy of this therapy for ATRX-deficient HGG are poorly understood. We therefore sought to evaluate the molecular mechanisms that drive selective lethality in patient-derived ATRX-deficient glioma stem cells (GSCs), following G4 stabilization. We found that ATRX-deficient GSCs demonstrate dose-dependent enhanced sensitivity to G4 stabilization, compared to ATRX-intact controls. Cell viability assays confirmed the specificity of our G4 stabilizer in comparison to other commonly used G4 stabilizers. Interestingly, G4 stabilization activated p53-independent apoptosis in ATRX-deficient GSCs. Furthermore, ATRX-deficient GSCs exhibit upregulated expression of both ATR and ATM pathways upon G4 stabilization, indicating enhanced replication stress and DNA damage via double-stranded breaks, respectively. Our preliminary findings suggest that ATR and ATM activation leads to the inhibition of transcription factor NF-κB, which in turn drives apoptosis. Lastly, our data indicate that G4 stabilization perturbs the ALT phenotype in ATRX-deficient GSCs, likely contributing to telomeric dysfunction. Taken together, these findings suggest that G4 stabilizers could synergize with ionizing radiation, the standard of care, as they are both DNA-damaging therapies. Our work defines mechanisms of action and efficacy of a novel therapeutic strategy for ATRX-deficient HGG, with strong implications for other ATRX-deficient cancers.

EPCO-08. ATRX DEFICIENCY INDUCES DYSFUNCTIONAL HETEROCHROMATIN ARCHITECTURE IN GLIOMAS AND ESTABLISHES DISEASE-DEFINING TRANSCRIPTIONAL NETWORKS

Loss of ATRX (Alpha Thalassemia/Mental Retardation Syndrome X, a member of SWI/SNF family chromatin regulator is altered in diffuse gliomas and defines molecular subtypes with aggressive behavior. Mechanistically, ATRX regulates incorporation of histone H3.3 into chromatin sites across the genome, maintains alternative lengthening of telomeres and establishes genomic distribution of polycomb responsive genes. We have recently reported Atrx deficiency induces glioma oncogenic features via widespread alterations in chromatin accessibility using mouse Neural Progenitor Cells (mNPCs- Tp53 -/-,Atrx -/-). Surprisingly, Atrx was found to be associated with transcription start site and enhancer regions, suggesting their strong association with epigenome architecture. In this background, we have recently performed ChIP-seq for histone marks that define active transcription, enhancers, repressors and gene bodies and Cohesion molecules on Atrx intact and deficient mNPCs. Our integrated analysis reports depletion of H3K9me3 loci’s with enrichment of H3K27me3 marks that coincidently enriched with Atrx binding sites and Lamina-Associated Domains (LADs). GSEA confirmed that the genes corresponding to “newly formed LADs” in mNPC-to-astrocyte differentiation are significantly enriched for genes down-regulated in Atrx deficient mNPCs and belongs to Gene Ontology categories such as cell cycle, chromosome organization and DNA damage. Alternatively, genes corresponding to decreased LAD association are enriched for up-regulated genes in Atrx deficient mNPCs and for regulation of differentiation, adhesion and cell death. Additionally, whole-genome bisulphite sequencing further demonstrated loss of methylation marks at H3K9me3 sites in Atrx deficient mNPCs, suggesting perturbations of heterochromatin regions leading to activation of canonical signals that define glioma phenotype and disease-state. To summarize, our data establishes tangible links between Atrx deficiency and dysregulated chromatin and heterochromatin architecture in gliomas and suggests the role of Atrx in establishing global chromatin features and transcriptional networks. Further, our data unravel novel therapeutic molecules/pathways for engineering potential.

CBIO-08. ENDOGENOUS DNA DOUBLE STRAND BREAKS ACTIVATE HETEROGENOUS DNA DAMAGE SIGNALING IN IDH1/2 MUTANT GLIOMAS

Isocitrate dehydrogenase 1/2 (IDH1/2) mutations are common in astrocytic glioma and are frequently coupled with TP53 and ATRX mutations. Collectively, these alterations cause genomic instability leading to high basal DNA double strand breaks (DSBs). Understanding how IDH/TP53/ATRX mutant cells process endogenous DSBs may help exploit inhibitors of DNA damage response (DDR) for the treatment of patients with IDH mutant gliomas. Through systematic effort to uncover the mechanisms involved in repair of endogenous DSBs in IDH1/2 mutant GBMs, we have discovered that high basal phosphorylated DNA-PK (p-DNA-PK) was characteristic of an IDH1/TP53/ATRX mutant GBM164 patient derived xenograft (PDX) but not in another IDH1 mutant GBM196 PDX. Immunofluorescence (IF) studies in patient specimen from which GBM164 was derived showed that p-DNA-PK co-localized with g-H2AX, 53BP1 or H4K20me2 (but not p-RPA) the known surrogates of DSBs. In contrast, p-DNA-PK was absent in the patient specimen from which GBM196 was derived, which otherwise had equally intense g-H2AX immunostaining colocalized with p-RPA. An independent IF study involving 11 IDH1 wild-type (WT) and 11 IDH1 mutant GBM patient samples, the p-DNA-PK was observed in 3 (27%) of 11 IDH1 mutant samples while IDH1 WT tumors were negative for p-DNA-PK. A telomere specific fluorescence in situ hybridization (Tel-FISH) confirmed elevated alternative lengthening of telomere (ALT) activity in GBM196 (but not in GBM164) indicative of HR proficiency. Consistently, HR related genes, including BRCA1 and MRE11A, were found upregulated in ALT-positive GBM196 as compared to those in GBM164. Interestingly, ALT+ GBM196 cells were highly vulnerable to inhibitors of ATM and ATR pathways. In conclusion, IDH1/TP53/ATRX mutant gliomas can be subdivided into HR-mediated ALT-positive group, which repairs the endogenous DSBs by HR (e.g. GBM196) and an ALT-negative/p-DNA-PK group, which repairs DSBs by c-NHEJ (e.g. GBM164) and this subdivision can be developed as a prescient biomarker of sensitivity to DDR inhibitors.

NIMG-50. DEUTERIUM METABOLIC IMAGING OF THE ALTERNATIVE LENGTHENING OF TELOMERES PATHWAY REPORTS ON TUMOR BURDEN AND PSEUDOPROGRESSION IN LOW-GRADE GLIOMAS

Glioma patient management relies heavily on magnetic resonance imaging (MRI). However, MRI is often inadequate for assessment of tumor burden and pseudoprogression. Non-invasive methods that report on molecular pathways such as telomere maintenance that drive tumor proliferation are needed. Among brain tumors, low-grade astrocytomas (LGAs) use the alternative lengthening of telomeres (ALT) pathway for telomere maintenance. The goal of this study was to identify ALT-linked metabolic alterations that can be exploited for non-invasive magnetic resonance spectroscopy (MRS)-based imaging of LGAs. We examined the patient-derived BT257 model and compared neurospheres that are ALT-dependent (BT257 ALT+) with those in which the ALT pathway has been silenced (BT257 ALT-). Our studies suggest that expression and activity of the rate-limiting glycolytic enzyme phosphofructokinase-1 are significantly higher in BT257 ALT+ neurospheres relative to ALT-, an effect that is associated with elevated glucose flux to lactate. Studies indicate that poly(ADP-ribose) polymerase inhibitors such as niraparib selectively induce telomeric fusion and cell death in ALT-dependent cells. We find that the telomeric fusion-mediated cytotoxicity of niraparib is associated with significantly reduced glycolytic flux in BT257 ALT+ neurospheres. We then examined whether 2H-MRS using [6,6’-2H]-glucose, which is a clinically translatable method of imaging glycolytic flux, can be used to monitor the ALT pathway in vivo. [6,6’-2H]-glucose flux to lactate is elevated in tumor relative to normal brain in mice bearing orthotopic BT257 tumors. Importantly, following treatment of BT257 tumor-bearing mice with niraparib, lactate production from [6,6’-2H]-glucose is significantly reduced at early timepoints when alterations in tumor volume cannot be observed by MRI, pointing to the ability of [6,6’-2H]-glucose to report on pseudoprogression in vivo. Collectively, our studies mechanistically link the ALT pathway with elevated glycolytic flux via phosphofructokinase-1 and identify deuterium metabolic imaging as a novel, non-invasive method of imaging tumor burden and treatment response in LGAs in vivo.

Alternative Lengthening of Telomeres: Lessons to Be Learned from Telomeric DNA Double-Strand Break Repair

The study of the molecular pathways underlying cancer has given us important insights into how breaks in our DNA are repaired and the dire consequences that can occur when these processes are perturbed. Extensive research over the past 20 years has shown that the key molecular event underpinning a subset of cancers involves the deregulated repair of DNA double-strand breaks (DSBs) at telomeres, which in turn leads to telomere lengthening and the potential for replicative immortality. Here we discuss, in-depth, recent major breakthroughs in our understanding of the mechanisms underpinning this pathway known as the alternative lengthening of telomeres (ALT). We explore how this gives us important insights into how DSB repair at telomeres is regulated, with relevance to the cell-cycle-dependent regulation of repair, repair of stalled replication forks and the spatial regulation of DSB repair.

DAXX-ATRX-H3.3 Regulation of p53 Chromatin Binding and DNA Damage Response

DAXX and ATRX are tumor suppressor proteins that form a histone H3.3 chaperone complex and are frequently mutated in cancers with the alternative lengthening of telomeres (ALT), such as pediatric glioblastoma. Rapid loss of function of either DAXX or ATRX are not by themselves sufficient to induce the ALT phenotype. However, cells lacking DAXX or ATRX can be readily selected for ALT-like features. Here, we show that DAXX and ATRX null glioblastoma cells with ALT-like features have defects in p53 chromatin binding and DNA damage response regulation. RNA-seq analysis of DAXX or ATRX null U87 glioblastoma cells with ALT-like features revealed that p53 pathway is among perturbed. ALT-selected DAXX and ATRX-null cells had aberrant response to DNA damaging agent etoposide. Both DAXX and ATRX-null ALT cells showed a loss of p53 binding at a subset of response elements. Complementation of DAXX null cells with a wild-type DAXX transgene rescued p53 binding and transcription, while the tumor associated mutation L130R that disrupts ATRX binding was incapable of rescuing p53 chromatin binding. We show that histone H3.3 binding is reduced in DAXX-null cells especially at subtelomeric p53 binding sites and telomere repeats. These findings indicate that DAXX and ATRX function to enable p53 chromatin binding through modulation of histone H3.3 binding, especially at sub-telomeric sites.