ALT-associated PML Bodies Research Articles

TRIM24 directs replicative stress responses to maintain ALT telomeres via chromatin signaling.

An inability to replicate the genome can cause replication stress and genome instability. Here, we develop BLOCK-ID, a proteomic method to identify and visualize proteins at stressed replication forks. This approach successfully identified novel mediators of the replication stress response, including the chromatin acetylation reader protein TRIM24. In validating TRIM24 function, we uncovered its crucial role in coordinating Alternative Lengthening of Telomeres (ALT), a cancer-specific telomere extension pathway involving replication stress. Our data reveal that TRIM24 is directed to telomeres via a p300/CBP-dependent acetylation chromatin signaling cascade, where it organizes ALT-associated PML bodies (APBs) to promote telomere DNA synthesis. Strikingly, we demonstrate that when artificially tethered at telomeres, TRIM24 can stimulate new telomere DNA synthesis in a SUMO-dependent manner, independently of p300/CBP or PML-dependent APBs. Thus, this study identifies a TRIM24 chromatin signaling pathway required for ALT telomere maintenance.

Polyubiquitinated PCNA triggers SLX4-mediated break-induced replication in alternative lengthening of telomeres (ALT)cancer cells.

Replication stresses are the major source of break-induced replication (BIR). Here, we show that in alternative lengthening of telomeres (ALT) cells, replication stress-induced polyubiquitinated proliferating cell nuclear antigen (PCNA) (polyUb-PCNA) triggers BIR at telomeres and the common fragile site (CFS). Consistently, depleting RAD18, a PCNA ubiquitinating enzyme, reduces the occurrence of ALT-associated promyelocytic leukemia (PML) bodies (APBs) and mitotic DNA synthesis at telomeres and CFS, both of which are mediated by BIR. In contrast, inhibiting ubiquitin-specific protease 1 (USP1), an Ub-PCNA deubiquitinating enzyme, results in an increase in the above phenotypes in a RAD18- and UBE2N (the PCNA polyubiquitinating enzyme)-dependent manner. Furthermore, deficiency of ATAD5, which facilitates USP1 activity and unloads PCNAs, augments recombination-associated phenotypes. Mechanistically, telomeric polyUb-PCNA accumulates SLX4, a nuclease scaffold, at telomeres through its ubiquitin-binding domain and increases telomere damage. Consistently, APB increase induced by Ub-PCNA depends on SLX4 and structure-specific endonucleases. Taken together, our results identified the polyUb-PCNA-SLX4 axis as a trigger for directing BIR.

ALTering Cancer by Triggering Telomere Replication Stress through the Stabilization of Promoter G-Quadruplex in SMARCAL1.

Most of the human cancers are dependent on telomerase to extend the telomeres. But ∼10% of all cancers use a telomerase-independent, homologous recombination mediated pathway called alternative lengthening of telomeres (ALT). Due to the poor prognosis, ALT status is not being considered yet in the diagnosis of cancer. No such specific treatment is available to date for ALT positive cancers. ALT positive cancers are dependent on replication stress to deploy DNA repair pathways to the telomeres to execute homologous recombination mediated telomere extension. SMARCAL1 (SWI/SNF related, matrix-associated, actin-dependent regulator of chromatin, subfamily A-like 1) is associated with the ALT telomeres to resolve replication stress thus providing telomere stability. Thus, the dependency on replication stress regulatory factors like SMARCAL1 made it a suitable therapeutic target for the treatment of ALT positive cancers. In this study, we found a significant downregulation of SMARCAL1 expression by stabilizing the G-quadruplex (G4) motif found in the promoter of SMARCAL1 by potent G4 stabilizers, like TMPyP4 and BRACO-19. SMARCAL1 downregulation led toward the increased localization of PML (promyelocytic leukemia) bodies in ALT telomeres and triggered the formation of APBs (ALT-associated promyelocytic leukemia bodies) in ALT positive cell lines, increasing telomere replication stress and DNA damage at a genomic level. Induction of replication stress and hyper-recombinogenic phenotype in ALT positive cells mediated by G4 stabilizing molecules already highlighted their possible application as a new therapeutic window to target ALT positive tumors. In accordance with this, our study will also provide a valuable insight toward the development of G4-based ALT therapeutics targeting SMARCAL1.

Orphan nuclear receptors-induced ALT-associated PML bodies are targets for ALT inhibition.

Orphan nuclear receptors (NRs), such as COUP-TF1, COUP-TF2, EAR2, TR2and TR4, are implicated in telomerase-negative cancers that maintain their telomeres through the alternative lengthening of telomeres (ALT) mechanism. However, how telomere association of orphan NRs is involved in ALT activation remains unclear. Here, we demonstrate that telomeric tethering of orphan NRs in human fibroblasts initiates formation of ALT-associated PML bodies (APBs) and features of ALT activity, including ALT telomere DNA synthesis, telomere sister chromatid exchange, and telomeric C-circle generation, suggesting de novo ALT induction. Overexpression of orphan NRs exacerbates ALT phenotypes in ALT cells, while their depletion limits ALT. Orphan NRs initiate ALT via the zinc finger protein 827, suggesting the involvement of chromatin structure alterations for ALT activation. Furthermore, we found that orphan NRs and deficiency of the ALT suppressor ATRX-DAXX complex operate in concert to promote ALT activation. Moreover, PML depletion by gene knockout or arsenic trioxide treatment inhibited ALT induction in fibroblasts and ALT cancer cells, suggesting that APB formation underlies the orphan NR-induced ALT activation. Importantly, arsenic trioxide administration abolished APB formation and features of ALT activity in ALT cancer cell line-derived mouse xenografts, suggesting its potential for further therapeutic development to treat ALT cancers.

Effects of p53 and ATRX inhibition on telomeric recombination in aging fibroblasts

In order to avoid replicative senescence, tumor cells must acquire a telomere maintenance mechanism. Beside telomerase activation, a minority of tumors employs a recombinational mechanism called Alternative Lengthening of Telomeres (ALT). Several studies have investigated the potential ALT stimulation by inactivation of ATRX in tumor cells, obtaining contrasting results. Differently, since ALT can be viewed as a mechanism to overcome telomere shortening-mediated replicative senescence, we have investigated the effects of the inhibition of ATRX and p53 in aging primary fibroblasts. We observed that senescence leads to a phenotype that seems permissive for ALT activity, i.e. high levels of ALT-associated PML bodies (APB), telomeric damage and telomeric cohesion. On the other hand, RAD51 is highly repressed and thus telomeric recombination, upon which the ALT machinery relies, is almost absent. Silencing of ATRX greatly increases telomeric recombination in young cells, but is not able to overcome senescence-induced repression of homologous recombination. Conversely, inhibition of both p53 and ATRX leads to a phenotype reminiscent of some aspects of ALT activity, with a further increase of APB, a decrease of telomere shortening (and increased proliferation) and, above all, an increase of telomeric recombination.

DNAR-07. MOLECULAR BIOMARKERS ASSOCIATED WITH TARGETED DISRUPTION OF ALTERNATIVE LENGTHENING OF TELOMERES IN ATRX-DEFICIENT GLIOMA MODELS

Abstract Alternative lengthening of telomeres (ALT) is a telomerase-independent mechanism of telomere maintenance that occurs in approximately 10-15 percent of all cancers. ALT enables the replicative immortality of cancer cells and is characterized by elevated levels of DNA replication stress and telomere-localized DNA double-strand breaks (DSBs). In gliomas, ALT frequently co-occurs with inactivating mutations in ATRX, which is thought to promote ALT induction by disrupting ATRX-mediated histone H3.3 deposition at telomeres, thus leading to telomere synthesis by homology-directed repair mechanisms. To identify potential therapeutic vulnerabilities specific to ALT-positive tumors, we identified candidate ALT-positive cancer cell lines in the Cancer Dependency Map (DepMap) dataset and validated the presence or absence of ALT phenotypic markers, including ALT-associated PML bodies (APBs) and nuclear foci of single-stranded telomeric DNA (ssTeloC). Comparing ALT-positive cells to all other cancer cell lines in the DepMap dataset, we identified the replication fork remodeling enzymes SMARCAL1 and FANCM as gene dependencies with the highest degree of ALT-specificity. To validate SMARCAL1 as an ALT-specific dependency, we used inducible RNAi to deplete SMARCAL1 in ALT-positive glioma cells. SMARCAL1 depletion caused a marked increase in the abundance and intensity of ssTeloC nuclear foci, as well as increased γH2AX abundance in ALT+ glioma cells but not telomerase-positive cells. Inducible SMARCAL1 depletion did not coincide with detectable increases in apoptotic markers (e.g. caspase-3 cleavage). However, SMARCAL1 depletion caused a significant increase in the abundance of nuclear abnormalities indicative of mitotic catastrophe (e.g. micronuclei, nucleoplasmic bridges, and fragmented/multilobular nuclei). In an orthotopic model of ALT-positive glioma, doxycycline-inducible RNAi targeting SMARCAL1 extended the median survival of tumor-bearing animals to 155 days compared to 111 days in the non-targeting control condition. These data validate the therapeutic targetability of SMARCAL1 in ALT-positive gliomas and support the development of small molecule SMARCAL1 inhibitors for treatment of ALT-positive tumors.

Colorectal cancer patient-derived organoids and cell lines harboring ATRX and/or DAXX mutations lack Alternative Lengthening of Telomeres (ALT)

Telomere maintenance is necessary to maintain cancer cell unlimited viability. However, the mechanisms maintaining telomere length in colorectal cancer (CRC) have not been extensively investigated. Telomere maintenance mechanisms (TMM) include the re-expression of telomerase or alternative lengthening of telomeres (ALT). ALT is genetically associated with somatic alterations in alpha-thalassemia/mental retardation X-linked (ATRX) and death domain-associated protein (DAXX) genes. Cells displaying ALT present distinctive features including C-circles made of telomeric DNA, long and heterogenous telomeric tracts, and telomeric DNA co-localized with promyelocytic leukemia (PML) bodies forming so-called ALT-associated PML bodies (APBs). Here, we identified mutations in ATRX and/or DAXX genes in an extensive collection of CRC samples including 119 patient-derived organoids (PDOs) and 232 established CRC cell lines. C-circles measured in CRC PDOs and cell lines showed low levels overall. We also observed that CRC PDOs and cell lines did not display a significant accumulation of APBs or long telomeres with no appreciable differences between wild-type and mutated ATRX/DAXX samples. Overall, our extensive analyses indicate that CRC is not prone to engage ALT, even when carrying genetic lesions in ATRX and/or DAXX, and support the notion that ATRX/DAXX genomic footprints are not reliable predictors of ALT.

Potential clinical treatment prospects behind the molecular mechanism of alternative lengthening of telomeres (ALT).

Normal somatic cells inevitably experience replicative stress and senescence during proliferation. Somatic cell carcinogenesis can be prevented in part by limiting the reproduction of damaged or old cells and removing them from the cell cycle [1, 2]. However, Cancer cells must overcome the issues of replication pressure and senescence as well as preserve telomere length in order to achieve immortality, in contrast to normal somatic cells [1, 2]. Although telomerase accounts for the bulk of telomere lengthening methods in human cancer cells, there is a non-negligible portion of telomere lengthening pathways that depend on alternative lengthening of telomeres (ALT) [3]. For the selection of novel possible therapeutic targets for ALT-related disorders, a thorough understanding of the molecular biology of these diseases is crucial [4]. The roles of ALT, typical ALT tumor cell traits, the pathophysiology and molecular mechanisms of ALT tumor disorders, such as adrenocortical carcinoma (ACC), are all summarized in this work. Additionally, this research compiles as many of its hypothetically viable but unproven treatment targets as it can (ALT-associated PML bodies (APB), etc.). This review is intended to contribute as much as possible to the development of research, while also trying to provide a partial information for prospective investigations on ALT pathways and associated diseases.

DNAR-08. THE ROLE OF SMARCAL1 AS A SYNTHETIC LETHAL VULNERABILITY IN ATRX-DEFICIENT GLIOMAS

Abstract Mechanisms to maintain telomere length over successive cell divisions are a requirement for cancer cell immortalization. Although many cancers maintain telomere length through the activation of telomerase, ~10-15% of human cancers use telomerase-independent mechanisms of telomere maintenance, termed Alternative Lengthening of Telomeres (ALT). In gliomas, the ALT phenotype is associated with loss-of-function mutations in the ATRX gene in IDH-mutant astrocytomas, adult and pediatric glioblastomas (GBM), high-grade astrocytomas with piloid features, and pleomorphic xanthoastrocytomas. In adult GBM, we previously identified a relatively rare subset of ALT-positive ATRX wildtype tumors that harbored loss-of-function mutations in the SMARCAL1 gene, and we found that loss of SMARCAL1 activity plays a causative role in the onset of ALT in these tumors. SMARCAL1 is an annealing helicase that localizes to sites of DNA damage and replication stress and catalyzes the reversal of stalled replication forks. To better understand the relationship between ATRX mutations, SMARCAL1 activity, and the ALT phenotype, we investigated the localization and function of SMARCAL1 in ALT-positive glioma cells and xenografts with native ATRX deficiency. We found that SMARCAL1 localizes to ALT-associated PML bodies in astrocytoma cell lines with concurrent ATRX and IDH1 mutations (derived from grade 3 and 4 tumors). Our data show that inducible suppression of SMARCAL1 via doxycycline-induced RNAi leads to increased DNA double-strand breaks, increased abundance of extrachromosomal telomeric repeats (c-circles), and increased sensitivity to irinotecan, a topoisomerase I inhibitor. In an orthotopic mouse model, SMARCAL1 depletion exhibited a chemosensitizing effect in combination with irinotecan against a patient-derived xenograft (astrocytoma, IDH-mutant Grade 4). Based on these data, we hypothesize that SMARCAL1 activity is critical for resolving ALT-associated replication stress in ATRX-deficient malignant gliomas. We therefore propose that SMARCAL1 functions as a synthetic lethal vulnerability in ATRX-deficient ALT-positive gliomas and that SMARCAL1 inhibition is a viable therapeutic strategy in these tumors.

XPF activates break-induced telomere synthesis

Alternative Lengthening of Telomeres (ALT) utilizes a recombination mechanism and break-induced DNA synthesis to maintain telomere length without telomerase, but it is unclear how cells initiate ALT. TERRA, telomeric repeat-containing RNA, forms RNA:DNA hybrids (R-loops) at ALT telomeres. We show that depleting TERRA using an RNA-targeting Cas9 system reduces ALT-associated PML bodies, telomere clustering, and telomere lengthening. TERRA interactome reveals that TERRA interacts with an extensive subset of DNA repair proteins in ALT cells. One of TERRA interacting proteins, the endonuclease XPF, is highly enriched at ALT telomeres and recruited by telomeric R-loops to induce DNA damage response (DDR) independent of CSB and SLX4, and thus triggers break-induced telomere synthesis and lengthening. The attraction of BRCA1 and RAD51 at telomeres requires XPF in FANCM-deficient cells that accumulate telomeric R-loops. Our results suggest that telomeric R-loops activate DDR via XPF to promote homologous recombination and telomere replication to drive ALT.

Consecutive Inhibition of Telomerase and Alternative Lengthening Pathway Promotes Hodgkin's Lymphoma Cell Death.

Telomere maintenance is key during cancer development. Malignant cells can either use telomerase or an alternative lengthening of telomere (ALT) pathway to maintain their telomere length. In Hodgkin’s Lymphoma (HL), the presence of telomerase activation is established. The activation of ALT has been reported recently. Our data confirm this notion describing co-localization of the phosphorylated form of telomeric repeat-binding factor 1 (pT371-TRF1) with ALT-associated promyelocytic leukemia bodies. Surprisingly, to our knowledge, there are no published studies targeting both telomere maintenance pathways in HL. Consequently, we investigated, for the first time, the effects of both telomerase and ALT inhibition on HL cell viability: We inhibited telomerase and/or ALT, given either individually, simultaneously, or consecutively. We report that the inhibition of telomerase using BIBR1532 followed by ALT inhibition, using trabectedin, caused a decrease of greater than 90% in cell viability in three patient-derived HL cell lines. Our results suggest that HL cells are most vulnerable to the consecutive inhibition of telomerase followed by ALT inhibition.

TIN2 deficiency leads to ALT-associated phenotypes and differentiation defects in embryonic stem cells.

SummaryTelomere integrity is critical for embryonic development, and core telomere-binding proteins, such as TIN2, are key to maintaining telomere stability. Here, we report that homozygous Tin2S341X resulted in embryonic lethality in mice and reduced expression of Tin2 in the derived mouse embryonic stem cells (mESCs). Homozygous mutant mESCs were able to self-renew and remain undifferentiated but displayed many phenotypes associated with alternative lengthening of telomeres (ALT), including excessively long and heterogeneous telomeres, increased ALT-associated promyelocytic leukemia (PML) bodies, and unstable chromosomal ends. These cells also showed upregulation of Zscan4 expression and elevated targeting of DAXX/ATRX and H3K9me3 marks on telomeres. Furthermore, the mutant mESCs were impeded in their differentiation capacity. Upon differentiation, DAXX/ATRX and PML bodies disassociated from telomeres in these cells, where elevated DNA damage was also apparent. Our results reveal differential responses to telomere dysfunction in mESCs versus differentiated cells and highlight the critical role of TIN2 in embryonic development.

SLX4IP N-terminus dictates telomeric localization in ALT-like castration-resistant prostate cancer cell lines.

To ensure replicative immortality in cancer, telomeres must be maintained through activation of telomere maintenance mechanisms (TMMs) that are dependent on telomerase or the alternative lengthening of telomeres (ALT) pathway. Although TMM pathways have traditionally been considered to be mutually exclusive, ALT hallmarks have been identified in cancers defined as being telomerase-positive, supporting TMM coexistence. In castration-resistant prostate cancer (CRPC), in vitro models were thought to be universally dependent on telomerase as the primary TMM; however, CRPC models with androgen receptor (AR) loss demonstrate ALT hallmarks with limited telomerase activity and require ALT-associated PML bodies (APBs) for sustained telomere maintenance. The TMM coexistence in AR-negative CRPC is reliant on the ALT regulator protein, SLX4IP. To identify the regions of SLX4IP responsible for the induction of APBs and telomere preservation in CRPC models, five 3xFLAG-tagged SLX4IP constructs were designed and stably introduced into parental C4-2B, DU145, and PC-3 cells. Once generated, these cell lines were interrogated for APB abundance and SLX4IP construct localization via immunofluorescence-fluorescence in situ hybridization (IF-FISH) and coimmunoprecipitation experiments for telomeric localization. Similarly, PC-3 cells with endogenous SLX4IP knockdown and SLX4IP construct introduction were interrogated for APB abundance, telomere length preservation, and senescent rescue. Here, we define the N-terminus of SLX4IP as being responsible for the promotion of the ALT-like phenotype of AR-negative CRPC models. Specifically, the N-terminus of SLX4IP was sufficient for promoting APB formation to a similar degree as full-length SLX4IP across CRPC cell lines. Additionally, APB promotion by the N-terminus of SLX4IP rescued telomere shortening and senescent induction triggered by SLX4IP knockdown in AR-negative CRPC cells. Moreover, APB formation and telomere maintenance were dependent on the ability of the N-terminus to direct SLX4IP localization at telomeres and APBs. These findings identify the role of the uncharacterized ALT regulator SLX4IP in the promotion of TMM coexistence to perpetuate replicative immortality in CRPC in vitro.

TRIM28 inhibits alternative lengthening of telomere phenotypes by protecting SETDB1 from degradation

BackgroundAbout 10–15% of tumor cells extend telomeres through the alternative lengthening of telomeres (ALT) mechanism, which is a recombination-dependent replication pathway. It is generally believed that ALT cells are related to the chromatin modification of telomeres. However, the mechanism of ALT needs to be further explored.ResultsHere we found that TRIM28/KAP1 is preferentially located on the telomeres of ALT cells and interacts with telomeric shelterin/telosome complex. Knocking down TRIM28 in ALT cells delayed cell growth, decreased the level of C-circle which is one kind of extrachromosomal circular telomeric DNA, increased the frequency of ALT-associated promyelocytic leukemia bodies (APBs), led to telomere prolongation and increased the telomere sister chromatid exchange in ALT cells. Mechanistically, TRIM28 protects telomere histone methyltransferase SETDB1 from degradation, thus maintaining the H3K9me3 heterochromatin state of telomere DNA.ConclusionsOur work provides a model that TRIM28 inhibits alternative lengthening of telomere phenotypes by protecting SETDB1 from degradation. In general, our results reveal the mechanism of telomere heterochromatin maintenance and its effect on ALT, and TRIM28 may serve as a target for the treatment of ALT tumor cells.

The Role of Non-Specific Interactions in Canonical and ALT-Associated PML-Bodies Formation and Dynamics.

In this work, we put forward a hypothesis about the decisive role of multivalent nonspecific interactions in the early stages of PML body formation. Our analysis of the PML isoform sequences showed that some of the PML isoforms, primarily PML-II, are prone to phase separation due to their polyampholytic properties and the disordered structure of their C-terminal domains. The similarity of the charge properties of the C-terminal domains of PML-II and PML-VI isoforms made it possible for the first time to detect migration of PML-VI from PML bodies to the periphery of the cell nucleus, similar to the migration of PML-II isoforms. We found a population of “small” (area less than 1 µm2) spherical PML bodies with high dynamics of PML isoforms exchange with nucleoplasm and a low fraction of immobilized proteins, which indicates their liquid state properties. Such structures can act as “seeds” of functionally active PML bodies, providing the necessary concentration of PML isoforms for the formation of intermolecular disulfide bonds between PML monomers. FRAP analysis of larger bodies of toroidal topology showed the existence of an insoluble scaffold in their structure. The hypothesis about the role of nonspecific multiple weak interactions in the formation of PML bodies is further supported by the change in the composition of the scaffold proteins of PML bodies, but not their solidification, under conditions of induction of dimerization of PML isoforms under oxidative stress. Using the colocalization of ALT-associated PML bodies (APBs) with TRF1, we identified APBs and showed the difference in the dynamic properties of APBs and canonical PML bodies.

Nuclear envelope tethering inhibits the formation of ALT-associated PML bodies in ALT cells.

Telomere length homeostasis is essential for maintaining genomic stability and cancer proliferation. Telomerase-negative cancer cells undergo recombination-mediated alternative lengthening of telomeres. Telomeres associate with the nuclear envelope through the shelterin RAP1 and nuclear envelope SUN1 proteins. However, how the associations between telomeres and the nuclear envelope affect the progression of telomere recombination is not understood. Here, we show that telomere anchorage might inhibit telomere-telomere recombination. SUN1 depletion stimulates the formation of alternative lengthening of telomeres-associated promyelocytic leukemia bodies in ALT cells. In contrast, overexpression of a telomere-nuclear envelope-tethering chimera protein, RAP1-SUN1, suppresses APB formation. Moreover, inhibition of this nuclear envelope attachment alleviates the requirement of TOP3α for resolving the supercoiling pressure during telomere recombination. A coimmunoprecipitation assay revealed that the SUN1 N-terminal nucleoplasmic domain interacts with the RAP1 middle coil domain, and phosphorylation-mimetic mutations in RAP1 inhibit this interaction. However, abolishing the RAP1-SUN1 interaction does not hinder APB formation, which hints at the existence of another SUN1-dependent telomere anchorage pathway. In summary, our results reveal an inhibitory role of telomere-nuclear envelope association in telomere-telomere recombination and imply the presence of redundant pathways for the telomere-nuclear envelope association in ALT cells.

Alternative lengthening of telomeres is a self-perpetuating process in ALT-associated PML bodies

Alternative lengthening of telomeres (ALT) is mediated by break-induced replication (BIR), but how BIR is regulated at telomeres is poorly understood. Here, we show that telomeric BIR is a self-perpetuating process. By tethering PML-IV to telomeres, we induced telomere clustering in ALT-associated PML bodies (APBs) and a POLD3-dependent ATR response at telomeres, showing that BIR generates replication stress. Ablation of BLM helicase activity in APBs abolishes telomere synthesis but causes multiple chromosome bridges between telomeres, revealing a function of BLM in processing inter-telomere BIR intermediates. Interestingly, the accumulation of BLM in APBs requires its own helicase activity and POLD3, suggesting that BIR triggers a feedforward loop to further recruit BLM. Enhancing BIR induces PIAS4-mediated TRF2 SUMOylation, and PIAS4 loss deprives APBs of repair proteins and compromises ALT telomere synthesis. Thus, a BLM-driven and PIAS4-mediated feedforward loop operates in APBs to perpetuate BIR, providing a critical mechanism to extend ALT telomeres.

Abstract 5821: The role of SMARCAL1 loss-of-function mutations in glioblastoma telomere maintenance

Abstract Malignant tumor cells overcome the restriction of progressive telomere shortening by employing telomere maintenance mechanisms (TMMs) to achieve replicative immortality. Over 80% of human cancers maintain telomere length using the enzyme telomerase, which uses an RNA template subunit. However, other cancers maintain their telomeres in a telomerase-independent manner, also known as Alternative Lengthening of Telomeres (ALT). Recently, we identified loss-of-function mutations in the SMARCAL1 gene as novel genetic alterations associated with the ALT phenotype in a subset of GBM that is wildtype for IDH1 and TERT promoter mutations. SMARCAL1 encodes an annealing helicase that localizes to sites of DNA damage and/or replication stress and resolves stalled replication fork structures to facilitate fork progression within difficult-to-replicate DNA sequences, such as telomeres. However, limited models are available to study the underlying mechanisms associated with SMARCAL1 deficiency and ALT induction, and tumorigenic patient-derived xenograft (PDX) models of SMARCAL1 deficient GBM have yet to be established. In this study, we sought to generate an ALT-positive, SMARCAL1-deficient PDX by subcutaneously transplanting the D06MG cell line, which harbors a native SMARCAL1 loss-of-function mutation, into immune-compromised mice. Transplanted cells formed palpable subcutaneous tumors in nude mice within approximately 6 months. Tissue from these tumors was harvested, disaggregated, and intracranially transplanted into nude mice. Immune compromised mice bearing intracranial xenografts showed neurological symptoms due to tumor growth within three months. Cell lines were derived from PDX tissue for further characterization of telomere-related endpoints. PDX-derived D06MG cell lines maintained ALT status, as evidence by a lack of telomerase activity, presence of APBs (ALT-associated promyelocytic leukemia (PML) bodies), and extrachromosomal C-circles. PDX-derived D06MG cell lines were characterized relative to the parental cell line using SKY (spectral karyotyping) and RNA-seq analyses. SKY analysis revealed that, during the in vivo selection, a unique homogeneous subpopulation emerged, which remained genetically stable even after intracranial injection. The majority of PDX-derived cells are diploid and share similar chromosome aberrations. RNA-sequencing and pathway enrichment analyses revealed that several signal transduction pathways were upregulated in the D06MG PDX lines, while cytosolic DNA sensing pathways were partially suppressed. In summary, we have generated and characterized a PDX model of SMARCAL1-deficient GBM that maintains telomeres via the ALT pathway. Citation Format: Heng Liu, Cheng Xu, Lee H. Chen, Bill H. Diplas, Jinjie Ling, Paula K. Greer, Martin Roskoski, Stephen T. Keir, David M. Ashley, Hai Yan, Matthew S. Waitkus. The role of SMARCAL1 loss-of-function mutations in glioblastoma telomere maintenance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5821.

ORCA/LRWD1 Regulates Homologous Recombination at ALT-Telomeres by Modulating Heterochromatin Organization.

SummaryTelomeres are maintained by telomerase or in a subset of cancer cells by a homologous recombination (HR)-based mechanism, Alternative Lengthening of Telomeres (ALT). The mechanisms regulating telomere-homeostasis in ALT cells remain unclear. We report that a replication initiator protein, Origin Recognition Complex-Associated (ORCA/LRWD1), by localizing at the ALT-telomeres, modulates HR activity. ORCA's localization to the ALT-telomeres is facilitated by its interaction to SUMOylated shelterin components. The loss of ORCA in ALT-positive cells elevates the levels of two mediators of HR, RPA and RAD51, and consistent with this, we observe increased ALT-associated promyelocytic leukemia body formation and telomere sister chromatid exchange. ORCA binds to RPA and modulates the association of RPA to telomeres. Finally, the loss of ORCA causes global chromatin decondensation, including at the telomeres. Our results demonstrate that ORCA acts as an inhibitor of HR by modulating RPA binding to ssDNA and inducing chromatin compaction.

Telomere length heterogeneity in ALT cells is maintained by PML-dependent localization of the BTR complex to telomeres.

Telomeres consist of TTAGGG repeats bound by protein complexes that serve to protect the natural end of linear chromosomes. Most cells maintain telomere repeat lengths by using the enzyme telomerase, although there are some cancer cells that use a telomerase-independent mechanism of telomere extension, termed alternative lengthening of telomeres (ALT). Cells that use ALT are characterized, in part, by the presence of specialized PML nuclear bodies called ALT-associated PML bodies (APBs). APBs localize to and cluster telomeric ends together with telomeric and DNA damage factors, which led to the proposal that these bodies act as a platform on which ALT can occur. However, the necessity of APBs and their function in the ALT pathway has remained unclear. Here, we used CRISPR/Cas9 to delete PML and APB components from ALT-positive cells to cleanly define the function of APBs in ALT. We found that PML is required for the ALT mechanism, and that this necessity stems from APBs' role in localizing the BLM-TOP3A-RMI (BTR) complex to ALT telomere ends. Strikingly, recruitment of the BTR complex to telomeres in a PML-independent manner bypasses the need for PML in the ALT pathway, suggesting that BTR localization to telomeres is sufficient to sustain ALT activity.