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

Abstract Orphan nuclear receptors (NRs), such as COUP-TF1, COUP-TF2, EAR2, TR2 and 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.


Introduction
Telomeres are protective nucleoprotein structures at chromosome termini that prevent activation of the DNA damage response and chromosomal fusion.In humans, telomeres consist of 5 TTAGGG 3 DNA sequence repeats and telomerebinding proteins of the shelterin complex ( 1 ,2 ).Due to incom-plete replication of the lagging DNA strand, telomeres shorten after each round of cell division, termed the 'end-replication problem'.When telomeres become too short to maintain chromosome integrity and genome stability, cells enter replicative senescence and death ( 3 ,4 ).Cancer cells overcome this problem by activating a telomere maintenance mechanism for their unlimited proliferation.In ∼85% of cancers, telomere length is maintained by reactivating telomerase.However, ∼15% of cancers, particularly in tumors of mesenchymal or neuroepithelial origin, utilize the alternative lengthening of telomeres (ALT) mechanism for telomere maintenance, independent of telomerase.The ALT pathway is a homologous recombination-based telomere maintenance mechanism relying on break-induced replication (BIR)-mediated ALT telomere DNA synthesis (5)(6)(7).However, the initiating events that lead to activation of the ALT pathway remain unclear.
ALT cancer cells exhibit associations between the promyelocytic leukemia nuclear bodies (PML-NBs) and telomeres ( 8 ), forming the ALT-associated PML-NBs (APBs).PML-NBs are composed of the structural proteins PML and SP100, as well as associated proteins such as DAXX and SUMO.PML-NBs assemble via multivalent SUMO-SUMO-interacting motif (SIM) interactions, which may depend on liquid-liquid phase separation (LLPS) ( 9 ,10 ).APB formation can be promoted by replication stress, with DNA damage agents and loss of several replication stress response proteins increasing APBs in ALT cells (11)(12)(13)(14).Formation of APBs is regulated by SUMO modifications on the shelterin components TRF1 and TRF2, with mutations of the sumoylation sites preventing APB formation ( 15 ).Aside from their PML-NB components, APBs also contain recombination and repair proteins, supporting that APBs are sites for homologous recombination where a self-perpetuating loop of ALT activity may occur (16)(17)(18).Knockdown or knockout of PML in the U2OS ALT cell line reduced recombination-mediated telomere synthesis, suggesting that APBs are critical for ALT activity ( 7 ,19 ).However, APB induction is insufficient to induce ALT telomere DNA synthesis in non-ALT cells ( 18 ), indicating that additional factors are important for ALT activation.In addition to APB formation, spontaneous telomere DNA damage response and accumulation of extrachromosomal telomere repeat DNA are ALT characteristics that may arise from and positively regulate ALT activation ( 11 ,20-22 ).
Genetic alterations may underlie ALT development.The most common mutations reported in ALT cancers and cell lines are mutations in genes encoding for αthalassemia / mental retardation syndrome X-linked (ATRX) and death-domain-associated protein (DAXX) (23)(24)(25)(26).Together, ATRX and DAXX form a histone chaperone complex that deposits histone variant H3.3 on telomeres and other heterochromatic regions (27)(28)(29)(30).ATRX and DAXX are known to be ALT suppressors, as their re-expression suppresses ALT phenotypes ( 24 , 31 , 32 ).An in vitro cell immortalization study revealed that spontaneous loss of ATRX is an early event during ALT activation ( 31 ).ATRX deficiency results in the accumulation of RNA:DNA hybrids and G-quadruplexes at telomeres, which induces replication stress and ALT phenotypes ( 32 ,33 ).Prolonged loss of ATRX or DAXX also leads to progressive telomere decompaction, which gradually induces telomere dysfunction ( 34 ) .However, loss of ATRX or DAXX is insufficient to cause ALT telomere elongation, implying that additional genetic or epigenetic events may cooperate with ATRX and DAXX deficiency to promote ALT development ( 24 , 31 , 33 , 35 ).
Orphan nuclear receptors (NRs), including COUP-TF1, COUP-TF2, TR2, TR4 and EAR2, have been shown to associate with telomeres in ALT cells ( 36 ) and are implicated in ALT development.These orphan NRs belong to the nuclear hormone receptor superfamily and NR2C / F classes and bind to telomeres via variant TCAGGG telomeric repeats, which are abundant in ALT telomeres but not in normal or telomerase-positive cells ( 37 ).Several lines of evidence support a critical role of orphan NRs in ALT regulation.COUP-TF2 and TR4 promote the telomeric localization of the zincfinger protein 827 (ZNF827), which recruits the nucleosome remodeling and histone deacetylation (NuRD) complex to remodel telomeric chromatin structures and render telomeres more permissive for recombination ( 38 ).In addition, COUP-TF2 and TR4 can interact directly with FANCD2, a protein involved in the Fanconi anemia DNA repair pathway, to induce a DNA damage response that contributes to the ALT activity in ALT cells ( 39 ).Moreover, through bridging the interactions between ALT telomeres and their transcriptionally regulated sites, orphan NRs promote insertions of telomeric sequence throughout the genome, causing genomic instability ( 40 ).
Nevertheless, studies on the role of orphan NRs in the ALT pathway have primarily been performed on ALT cell lines, which engender confounding factors such as genetic mutations, epigenetic alterations, and telomere structural instabilities that may impair efforts to dissect the direct function of orphan NRs in ALT development.Here, we present a unique model system for ALT activation in human fibroblasts by tethering orphan NRs to telomeres.Using this system, we characterized the direct role of orphan NRs in inducing ALT phenotypes and features of ALT activity and revealed coordination between ATRX-DAXX deficiency and orphan NRs for AL T activation.Lastly , we elucidated the function of APBs in orphan NR-mediated ALT activation and the therapeutic potential of targeting APBs for ALT inhibition.

RNA interference
siRNA transfections were done by reverse transfection with Lipofectamine RNAiMax (Invitrogen).All siRNAs were transfected at 25 μM as per the manufacturer's recommendations.For synchronization using thymidine and CDK1 inhibitor (CDK1i, RO-3306), cells were treated with thymidine 48 h post transfection.

Generation of CRISPR PML-knockout cell lines
BJ T cells stably expressing doxycycline-inducible Cas9 were first generated by lentiviral transduction.The stable cells were transfected with PML guide RNAs using Lipofectamine RNAiMax, according to the manufacturer's instructions (Invitrogen, USA), in parallel with doxycycline treatment (50 ng / ml) for 3 days.For U2OS and WI38-VA13 / 2RA PMLknockout cells, U2OS and WI38-VA13 / 2RA doxycyclineinducible Cas9 cells were transduced with PML guide RNAs, and then treated with doxycycline (50 ng / ml) for 3 days.Single clones were isolated using limiting dilution.Knockout efficiency was checked by immunofluorescence and western blot.Retroviral transduction was then used to stably express COUP-TF1 LBD -TRF1, or COUP-TF2 LBD -TRF1 in PML(+) and PML(-) BJ T cells.

Detection of telomeric DNA synthesis
To synchronize cells at G2 phase, cells were treated with 2 mM thymidine for 21 h, released into fresh medium for 4 h, and then treated with 15 mM CDK1i for 12 h.To visualize DNA synthesis, cells were incubated with 20 mM EdU for 3 h.EdU was labeled with the fluorescent dye picolyl azide via Click-it reaction (Invitrogen).

Immunofluorescence (IF) and fluorescence in situ hybridization (FISH)
Cells were seeded onto coverslips inserted into 12-well plates and then fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.5% Triton-X-100 (diluted in phosphate buffered saline, PBS) for 5 min.The cells were then blocked with 2% FBS (diluted in PBS) for 30 min, and incubated for 1 h each with anti-flag or anti-PML primary antibodies (1:200) and Alexa 488-conjugated secondary antibodies (1:800) diluted in 2% FBS.The cells were then fixed again with 4% paraformaldehyde for 10 min and subjected to serial dehydration in 70% EtOH, 95% EtOH and 100% EtOH for 5 min at each concentration before air-drying the coverslips.The cells were then subjected to denaturation at 80 • C on a heat-plate for 3 min with hybridization mix (10 mM Tris-HCl pH 7.5, 70% formamide, 10% blocking reagent (Roche), 0.25 μM TMR-conjugated (CCCTAA) 3 telomere PNA probe) on the slide, followed by overnight hybridization at room temperature.They were then washed twice with wash buffer A (70% formamide, 10 mM Tris-HCl pH 7.5), and then three times with wash buffer B (10 mM Tris-HCl pH 7.5, 4 M NaCl, 20% Tween-20).DAPI (Life Technology) was used to stain nuclei.Serial alcohol dehydration was performed before again air-drying the coverslips and then mounting on a slide with Prolong Gold Antifade Mountant (Life Technology).

Chromosome orientation fluorescence in situ hybridization (CO-FISH)
Cells were labeled with 10 μM 3:1 BrdU: BrdC for 16 hr and then treated with 0.2 μg / ml colcemid for 4 h prior to harvesting.Cells were trypsinized and resuspended with 75 mM KCl prior to overnight fixation in 3:1 methanol / acetic acid.Cells were then dropped onto glass slides and air-dried overnight.To degrade the newly synthesized stands, slides were first rehydrated with PBS for 5 min, treated with 0.5 mg / ml RNase A for 10 min at 30 • C, and labeled with 0.5 μg / ml Hoechst in 2X SSC for 15 min at RT.The slides were then exposed to long wave (365 nm) UV light for 1 h to generate DNA nicks and these were digested with 10 U / μl of Exonuclease III (Promega) for 30 min at 37 • C. Slides were washed with PBS and subjected to serial dehydration in 70% EtOH, 95% EtOH and 100% EtOH for 5 min at each concentration before air-drying.For hybridization, slides were first hybridized with FAM-00-[TTAGGG] 3 PNA probe in hybridization mix (10 mM Tris-HCl pH 7.5, 70% formamide, 10% blocking reagent (Roche) for 2 h at RT, washed with wash buffer A (70% formamide, 10 mM Tris-HCl pH 7.5), then hybridized again with TMR-00-[CCCTAA] 3 in hybridization mix for 2 h at RT. Slides were then washed and mounted following the protocol described for FISH.

C-circle assay
Cellular DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega) and was digested using RsaI and HinfI restriction enzymes (New England BioLabs).C-circle amplifications were then performed by incubating a 40μl reaction mixture containing 100 ng digested cellular DNA, 15U phi29 DNA polymerase (Thermo), 1 × phi29 reaction buffer, and 500 μM dNTPs at 30 • C for 10 h.Following amplification, products were detected by dot blotting using 32 P-labeled telomeric probes.

Image acquisition and quantification
Fluorescence 3D images were acquired using a GE Healthcare DeltaVision Deconvolution microscope.Images were taken with 0.2 μm spacing for a total of 5 μm.The images were analyzed using softWoRx 5.5.1 for deconvolution and ImageJ / FIJI for quantification.First, a maximum intensity projection was applied to visualize the acquired Z-stack as a 2D-image.Then, the 'tophat' filter was used for background subtraction.The 'find maxima' command was used to identify the local maxima of signal intensity in each image, wherein the results are the segmented objects identified for each telomere or each PML-NB.Finally, overlapping signals for objects in the telomere and PML-NB images were recognized as APBs.To automatically quantify telomere and APB numbers, we utilized the ImageJ Macro language (IJM), which is a scripting language built into ImageJ.

Xenograft model
Nude mice were obtained from the National Laboratory Animal Center, Taiwan.Thirteen 6-week-old male mice were subcutaneously injected with 1 × 10 7 SaOS2 cells suspended in serum free DMEM on either flank.The mice were randomly allocated to treatment with either As 2 O 3 (2 mg / kg) or equivalent volume of PBS.As 2 O 3 and PBS treatment was started 1 week after inoculation when the mice had developed palpable tumors.Both As 2 O 3 and PBS were administered intraperitoneally for 3-5 consecutive days.This study was carried out in strict adherence with the recommendations from the IACUC (21-12-1754) of Academia Sinica, Taiwan, and efforts were made to minimize the number of animals used for the study.

Statistical analyses
GraphPad Prism 6 and Microsoft Excel were used to generate tables and graphs.For cell-based imaging experiments, each experiment was conducted two to three times, with a minimum of 100 cells analyzed per experiment, resulting in approximately 200-300 cells analyzed per sample.In the scatter plots, each dot represents the quantified number per cell and the red lines indicate either the median or mean to facilitate comparison between samples.Statistical analyses were performed using either two-tailed Mann-Whitney U tests or unpaired t-tests as described in the figure legends.For mouse-based imaging experiments, a total of seven tumors from six PBS-treated mice were compared to eight tumors from seven As 2 O 3 -treated mice.A minimum of 200 cells were analyzed for each tumor.In the graphs, each dot indicates the average number quantified per cell within one tumor.Statistical analyses were performed using unpaired t -tests.

Tethering orphan NRs to telomeres triggers ALT induction in human fibroblasts
We established an experimental model to recapitulate telomeric recruitment of orphan NRs in primary fibroblast cells immortalized with telomerase (BJ T ) for the investigation of how orphan NRs contribute to ALT activation.The orphan NRs COUP-TF1, COUP-TF2, TR2, TR4 and EAR2 were tethered to telomeres in BJ T cells through ectopic stable expression as a fusion protein with the telomere binding protein TRF1.We visualized the cellular localization of the orphan NR-TRF1 fusion proteins by immunofluorescence (IF) staining.Our IF data revealed orphan NR nuclear foci co-localized with telomeric signals from fluorescence in situ hybridization (FISH), indicating the telomeric localization of orphan NR-TRF1 fusion proteins ( Supplementary Figure S1 A).Next, we investigated the ability of orphan NRs to induce ALT phenotypes upon their targeting to telomeres in BJ T cells.Using IF with antibodies against PML in combination with telomere FISH, we observed robust co-localization between PML-NBs and telomeres in BJ T cells expressing COUP-TF1-TRF1, COUP-TF2-TRF1, TR2-TRF1, TR4-TRF1 or EAR2-TRF1 relative to vector control cells or cells overexpressing TRF1 (Figure 1 A,B), suggesting that targeting orphan NRs to telomeres induces APB formation.Using an established protocol for quantifying telomere FISH signal ( 42 ), we assessed telomere clustering upon telomeric targeting of orphan NRs.Our results show that expression of orphan NR-TRF1 fusion proteins in BJ T cells caused a decrease in the number of telomere foci in comparison to cells overexpressing TRF1, indicating that telomere targeting of orphan NRs induces telomere clustering (Figure 1 C).
The C-terminal ligand binding domain (LBD) of orphan NRs has been shown to be critical for their transcriptional activity ( 43 ).Therefore, we coupled the LBDs of COUP-TF1, COUP-TF2, TR2, TR4 and EAR2 with TRF1 and generated BJ T cells expressing orphan NR LBD-TRF1 fusion proteins ( Supplementary Figure S1 B).We also found that upon targeting to telomeres, the LBDs of orphan NRs is sufficient to induce APB formation (Figure 1 D,E) and telomere clustering, evidenced by a decrease in telomere number (Figure 1 F) and an increase in telomere volume ( Supplementary Figure S2 A).This ALT induction by LBDs of orphan NRs is often to a higher degree than that detected for the full-length proteins.This is presumably due to the specific association of LBD-TRF1 proteins with telomeres, whereas full-length orphan NR-TRF1 proteins may engage in both telomere and non-telomere bindings.
We have shown above that telomere tethering of orphan NRs in human fibroblast cells induces ALT phenotypes, including APB formation and telomere clustering, so we were interested in investigating if this then induces AL T activity .To examine that possibility, we investigated the induction of other ALT phenotypes, such as ALT telomere DNA synthesis, telomeric C-circle generation, and telomere sister chromatid exchange (T-SCEs), which are features of ALT activity associated with telomere recombination and elongation ( 44 ).Specifically, we determined levels of recombination-based ALT telomere DNA synthesis during the G2 phase of the cell cycle in BJ T cells upon orphan NR recruitment.The cells were synchronized in G2 phase by means of thymidine and CDK1 inhibitor treatments, followed by addition of EdU (5-ethynyl-2 -deoxyuridine) to detect DNA synthesis ( 7 ).Upon conducting Click chemistry for EdU visualization and telomeric FISH, co-localizations between telomeres and EdU foci were identified in BJ T cells expressing COUP-TF1 LBD -TRF1 and COUP-TF2 LBD -TRF1, indicative of non-S phase DNA synthesis at telomeres (Figure 1 G,H).Moreover, EdU-associated telomeres also co-localized with PML foci, indicating telomere synthesis at APBs (Figure 1 G,I).Thus, our data indicate that targeting orphan NRs to telomeres induces ALT telomere DNA synthesis at APBs in human fibroblasts.Moreover, accumulation of extra chromosomal telomeric C-circles as recombination byproducts has been shown to be associated with ALT telomere recombination ( 45 ).Accordingly, we extracted genomic DNA from control BJ T cells, as well as TRF1, COUP-TF1 LBD -TRF1 and COUP-TF2 LBD -TRF1-expressing BJ T cells, and used it for C-circle assay.Our results show that telomeric targeting of the LBDs of COUP-TF1 or COUP-TF2 induced Ccircle accumulation in BJ T cells (Figure 1 J,K).Additionally, T-SCEs are direct evidence of ALT recombination ( 46 ).We conducted chromosome orientation FISH (CO-FISH) and found that tethering COUP-TF2 LBD to telomeres of BJ T cells induces T-SCEs (Figure 1 L,M).Thus, together, our data indicate that telomeric recruitment of orphan NRs induces features of ALT activity , suggesting AL T induction.
To demonstrate the reproducibility of our ALT-inducing model, we also targeted the COUP-TF2 LBD to the telomeres of three additional primary fibroblast cells-BJ, IMR90 and WI38 ( Supplementary Figure S1 C).We observed ALT induction evidenced by APB formation ( Supplementary Figure S2 B-D), telomere clustering ( Supplementary Figure S2 E), and telomere DNA synthesis ( Supplementary Figure S2 F-G) upon expression of COUP-TF2 LBD -TRF1 in BJ, IMR90 and WI38 cells.ALT induction analysis in the additional fibroblasts revealed consistent results with BJ T cells, validating that telomeric targeting of orphan NRs induces ALT in human fibroblasts.By performing IF against shelterin proteins TRF2 and RAP1 with telomere FISH, we found that telomeric targeting of COUP-TF2 LBD by TRF1 does not prevent shelterin localization to telomeres ( Supplementary Figure S1 D).Upon investigating for DNA damage marked by colocalization of phosphorylated histone H2AX ( γH2AX) and telomeres or telomere dysfunction-induced foci (TIFs), which has been previously shown to be elevated upon ALT induction ( 11-14 ,37 ), we did not observe an increase of DNA damage response (DDR) upon tethering of orphan NRs to telomeres of fibroblasts ( Supplementary Figure S3 A-D).This discovery suggests our innovative model as a unique mechanism independent of telomere DDR for ALT induction.

Orphan NRs mediate APB formation and ALT telomere DNA synthesis in ALT cells
We have shown that targeting orphan NRs to telomeres in human fibroblasts induces APB formation, telomere clustering, C-circle generation, and telomeric DNA synthesis at APBs.
To determine if all these roles of orphan NRs are consistent in ALT cells, we overexpressed COUP-TF2 and TR4 in U2OS and WI38-VA13 / 2RA ALT cell lines, which exhibit pre-existing ALT phenotypes and activity.Telomeric localization of ectopically expressed COUP-TF2 and TR4 proteins was observed ( Supplementary Figure S1 E), which is consistent with the presence of abundant TCAGGG repeat sequences in ALT telomeres ( 37 ).Furthermore, co-expression of COUP-TF2 and TR4 significantly enhanced APB formation (Figure 2 A, B) and telomere DNA synthesis (Figure 2 D,E), while only minimally increased telomere clustering (Figure 2 C), in U2OS cells.In WI38-VA13 / 2RA cells, simultaneous overexpression of COUP-TF2 and TR4 resulted in a marginal increase of APB formation ( Supplementary Figure S4 A), but did not promote further telomere clustering ( Supplementary Figure S4 B) and telomere DNA synthesis ( Supplementary Figure S4 C), presumably due to pre-existing high-level of ALT phenotypes and telomeric association of endogenous orphan NRs.Our overexpression experiments indicate that orphan NRs may promote APB formation and telomere DNA synthesis when they are localized to telomeres in ALT cells.
To delineate the contribution of specific orphan NRs, particularly COUP-TF2 and TR4, in promoting ALT, we depleted COUP-TF2 and / or TR4 from ALT cell lines by siRNAs ( Supplementary Figure S4 D) and then subjected them to ALT induction analyses.In U2OS cells, we observed that depletion of COUP-TF2 reduced APB formation ( Supplementary Figure S4 E) and telomeric DNA synthesis (Figure 2 F, G), but not telomere clustering ( Supplementary Figure S4 F).However, depletion of TR4 did not lead to a decrease in APB formation ( Supplementary Figure S4 E) and telomere clustering ( Supplementary Figure S4 F) but it slightly reduced telomere DNA synthesis (Figure 2 F, G).Upon co-depletion of COUP-TF2 and TR4, a further reduction in telomere DNA synthesis was observed (Figure 2 F, G).These findings suggest that COUP-TF2 contributes to ALT activation in U2OS cells more significantly than TR4.In WI38-VA13 / 2RA cells, similar to what was identified in U2OS cells, COUP-TF2 depletion resulted in significant reductions of APB formation ( Supplementary Figure S4 G) and telomere DNA synthesis ( Supplementary Figure S4 H) but not telomere clustering ( Supplementary Figure S4 I).These knockdown experiments highlight the role of orphan NRs, particularly COUP-TF2, in promoting APB formation and telomere DNA synthesis in U2OS and WI38-VA13 / 2RA cells.However, our findings suggest that orphan NRs may not play a major role in telomere clustering in these ALT cells, which differs from our observation in fibroblasts (Figure 1 C, F).Our findings also reveal that specific orphan NRs may have different and / or compensatory roles in promoting ALT activation.

The AF2 domain of orphan NRs and ZNF827 are critical for ALT induction
Next, we investigated how orphan NRs promote ALT activation.The LBD of orphan NRs contains the transcriptional function AF2 domain that is responsible for recruiting transcriptional co-regulators ( 43 ).We reasoned that the AF2 domain might mediate the ALT induction triggered by telomeric targeting of orphan NRs.To examine this possibility, the AF2 domain at the C-terminus of the LBDs of COUP-TF1 and COUP-TF2 were deleted to generate BJ T cells expressing COUP-TF1 LBD / AF2 -TRF1 and COUP-TF2 LBD / AF2 -TRF1 ( Supplementary Figure S1 F).We found that removing the AF2 domain from COUP-TF1 LBD and COUP-TF1 LBD prevented APB formation (Figure 3 A,B), telomere clustering (Figure 3 C), C-circle formation (Figure 1 J, K), and telomere DNA synthesis (Figure 1 H, I) upon their telomeric targeting through TRF1 fusion in BJ T cells.Similarly, ectopic expression of COUP-TF2 AF2 and TR4 AF2 in U2OS and WI38-VA13 / 2RA cells failed to induce features of ALT activity (Figure 2 A-E, Supplementary Figure S4 A-C).These data support that the AF2 domain of orphan NRs is critical for their ability to induce ALT upon telomere recruitment.
Moreover, previous studies have shown that APB formation and ALT activity are regulated by cellular processes such as chromatin remodeling and sumoylation ( 15 ,38 ).Therefore, we reasoned that depleting proteins involved in these processes from BJ T cells expressing COUP-TF1 LBD -TRF1 and COUP-TF2 LBD -TRF1 might perturb ALT induction.It has been shown previously that the zinc finger protein ZNF827 is localized at the telomeres of ALT cells, where it promotes APB formation and recruits the nucleosome remodeling and histone deacetylation (NuRD) complex to facilitate homologous recombination ( 38 ).We used siRNAs to deplete ZNF827 from BJ T -COUP-TF1 LBD -TRF1 and BJ T -COUP-TF2 LBD -TRF1 cells (Figure 3 D) and then assessed ALT induction.We found that ZNF827 depletion significantly reduced APB formation (Figure 3 E,F), increased telomere number (Figure 3 G), and decreased telomere DNA synthesis (Figure 3 H).In addition, overexpression of ZNF827 in BJ T -COUP-TF2 LBD -TRF1 cells enhanced APB formation but its overexpression in BJ T -COUP-TF2 LBD / AF2 -TRF1 could not rescue the ALT phenotype ( Supplementary Figure S5 A).These findings indicate that the ability of orphan NRs to induce ALT activation in BJ T cells is dependent on ZNF827.Moreover, it has been shown previously that the Fanconi anemia protein FANCD2 interacts with COUP-TF2 and TR4 to promote ALT activity in ALT cells ( 39 ).Using siRNAs to deplete FANCD2 from BJ T -COUP-TF2 LBD -TRF1 cells, we observed that loss of FANCD2 expression did not affect APB formation ( Supplementary Figure S5 B), telomere clustering ( Supplementary Figure S5 C), or telomere DNA synthesis ( Supplementary Figure S5 D).In addition, the MMS21-SMC5-SMC6 SUMO E3 ligase complex has been shown to promote APB formation in ALT cells by sumoylating TRF1 and TRF2 ( 15 ).Using IF staining, we found that the recombination proteins SMC5, SMC6 and NBS1 localized at PML-NBs in parental BJ T fibroblasts ( Supplementary Figure S5 E), and were further identified at telomeres upon expressing COUP-TF1 LBD -TRF1 or COUP-TF2 LBD -TRF1 ( Supplementary Figure S5 F-H), suggesting that APB formation results in their telomeric localization.However, depletion of NBS1, SMC5 or SMC6 by siRNA transfection ( Supplementary Figure S5 I) in BJ T -COUP-TF1 LBD -TRF1 and BJ T -COUP-TF2 LBD -TRF1 cells did not prevent either APB formation ( Supplementary Figure S5 J) or telomere clustering ( Supplementary Figure S5 K), indicating that these recombination and repair proteins may not be involved in orphan NR-mediated ALT activation.Thus, these findings suggest that orphan NRs induce ALT activation in primary fibroblasts via their AF2 domains and this activity is dependent on ZNF827.

ATRX / DAXX depletion synergizes with telomeric recruitment of orphan NRs for ALT activation
The ALT suppressors ATRX and DAXX form a histone chaperone complex that deposits histone variant H3.3 on telomeres ( 23 , 24 , 27 , 28 , 30 ).It has been shown previously that loss of ATRX or DAXX may promote ALT phenotypes and ALT activation ( 34 ).As described above, we found that tethering orphan NRs to telomeres in BJ T fibroblast cells induced ALT induction.Therefore, we wondered if these two ALT events, i.e. orphan NR recruitment to telomeres and loss of ATRX or DAXX, can coordinate to promote ALT activation in BJ T cells.To examine this possibility, we depleted ATRX, DAXX, as well as histone H3.3 expression by siRNA transfection in BJ T cells and then conducted ALT induction analyses (Figure 4 A).We observed that depletion of ATRX or DAXX from BJ T cells elicited low-level APB formation (Figure 4

APB is critical for orphan NR-induced telomeric DNA synthesis
We have shown that tethering orphan NRs to telomeres induces features of ALT activity in BJ T cells.To investigate the significance of APB formation for ALT induction, we generated PML knockout BJ T cells by means of CRISPR / Cas9 technology using guide RNAs targeting exons 1-3 of the PML gene for expressing COUP-TF1 LBD -TRF1 and COUP-TF2 LBD -TRF1.Our IF staining (Figure 5 A) and Western blotting (Figure 5 B) analyses showed that the knockout cells lacked expression of PML isoforms, indicating loss of APBs.Upon expression of COUP-TF1 LBD -TRF1 or COUP-TF2 LBD -TRF1, telomere clustering partially decreased in the BJ T PML knockout cells (Figure 5 C), in agreement with previous reports that APB formation promotes telomere clustering ( 18 ,47 ).Interestingly, substantial telomere clustering still occurred in the BJ T PML knockout cells expressing COUP-TF1 LBD -TRF1 or COUP-TF2 LBD -TRF1 (Figure 5 C), suggesting that there may be an APB-independent mechanism for telomere clustering.Furthermore, we investigated if the telomere DNA synthesis induced by orphan NRs requires PML.Intriguingly, telomere DNA synthesis was not observed in PML knockout BJ T cells expressing COUP-TF1 LBD -TRF1 or COUP-TF2 LBD -TRF1 (Figure 5 D,E), suggesting that APB is critical for ALT activation induced by recruitment of orphan NRs to telomeres.To investigate if PML is required for ALT telomere DNA synthesis in ALT cells, we also generated PML knockout U2OS and WI38-VA13 / 2RA cells ( Supplementary Figure S6 A) and again observed that telomere clustering was unaffected ( Supplementary Figure S6 B,C) and telomeric DNA synthesis was abolished ( Supplementary Figure S6 D,E).These results are consistent with our experiments on BJ T cells and support findings by others ( 7 ,19 ) that PML is critical for telomeric DNA synthesis in ALT cells.

Arsenic trioxide disrupts APBs and inhibits ALT telomere DNA synthesis in ALT cells
As described above, APBs are critical to orphan NR-mediated ALT induction, so we hypothesized that drugs targeting PML for degradation could also inhibit AL T activity .Arsenic trioxide (As 2 O 3 ) is a therapeutic drug used for the treatment of acute promyelocytic leukemia (APL) by promoting degradation of the PML-retinoic acid receptorα (PML-RAR) oncogene ( 48 ).It can also bind to PML and promote its proteolysis ( 49 ,50 ).Aside from its use in APL, As 2 O 3 has been demonstrated to kill various types of cancer cells, such as liver cancer , lung cancer , breast cancer , and glioma, among others, and thus also has been subjected to clinical trials for these multiple cancer types ( 51 ).As 2 O 3 has also been reported to inhibit cell proliferation and induce cell death in osteosarcoma cell lines, including U2OS and SaOS2 ALT cells ( 52), but whether As 2 O 3 has the ability to inhibit ALT in ALT cells has not been explored.Thus, we sought to investigate the possibility of targeting ALT using As 2 O 3. As 2 O 3 is a poison with profound cell cytotoxicity through inducing oxidative stress and cell apoptosis ( 50 ,53 ).We reasoned that severe cell death could prevent accurate measurements of ALT phenotypes.Therefore, we treated cells with various concentrations of As 2 O 3 over a 48-h period and observed cell growth, in order to identify optimal conditions for the experiments.Consistent with its general hypertoxicity, we observed that As 2 O 3 at 10 μM elicited a robust cell death of U2OS, WI38-VA13 / 2RA and SaOS2 ALT cells within 24 h ( Supplementary Figure S7 A).Although a shorter treatment duration of 4 h at 10 μM allowed for improved cell survival ( Supplementary Figure S7 A), this was not sufficient to degrade PML isoforms (Figure 6 A).At lower concentrations, we observed that significant cell death  still occurred upon treatments of 1 μM As 2 O 3 over 48 hours or 2 μM over 24 h, whereas cells could tolerate 1 μM of As 2 O 3 for 24 h ( Supplementary Figure S7 A).Moreover, Western blotting and IF-telomere FISH revealed that treatment with 1 μM of As 2 O 3 for 24 h was sufficient to degrade PML proteins (Figure 6 A) and to disrupt PML bodies in U2OS, WI38-VA13 / 2RA and SaOS2 ALT cells (Figure 6 B, C), resulting in loss of APBs in these ALT cells (Figure 6 B, D).Following this loss of APBs, we also observed that As 2 O 3 treatment prevented telomere DNA synthesis (Figure 6 E, F).Thus, our in vitro analysis indicates that As 2 O 3 may inhibit ALT activity by disrupting APB formation in ALT cells.

Arsenic trioxide suppresses APB formation and features of ALT activity in SaOS2 xenografts in mice
To investigate the therapeutic potential of As 2 O 3 on ALT inhibition, we examined the ability of As 2 O 3 to target PML bodies and APB formation in tumor xenografts generated by subcutaneously injecting SaOS2 ALT cancer cells into nude mice.The mice carrying tumors were treated with PBS as a control or As 2 O 3 for 3-5 consecutive days.We collected the tumors and performed PML IF staining and telomere FISH to investigate APB formation.PML bodies could be visualized in control SaOS2 tumors, and they co-localized with telomere signals, indicative of APB formation in SaOS2 mouse xenografts (Figure 7 A-C).However, following As 2 O 3 treatment, we detected a significant reduction in PML-NBs (Figure 7 A, B) and a subsequent reduction in APBs in SaOS2 tumors (Figure 7 A, C), indicating that As 2 O 3 treatment disrupts APB formation in mouse xenografts in vivo .In addition, we examined whether ALT activity is abolished upon As 2 O 3 administration.We utilized the native FISH assay to detect single-stranded Crich telomeric DNA (ssTeloC), such as C-circles, which are features of ALT activity ( 19 , 54 , 55 ).Our findings demonstrate that As 2 O 3 treatment led to a reduction of ssTeloC in SaOS2 xenografts (Figure 7 D, E), suggesting a loss of AL T activity .Together, these data suggest that As 2 O 3 may represent a potential ALT inhibitor via targeting APB formation.This offers preliminary support for its future preclinical / clinical development aimed at exploring its efficacy in ALT cancer treatment.

Discussion
Telomeric association of orphan NRs has been suggested to regulate the ALT pathway (36)(37)(38)(39)(40).In the present study, by tethering orphan NRs to telomeres, we observed ALT induction in human fibroblasts.Then we explored the role of orphan NRs in ALT induction using this in vitro system.Our results reveal that the telomeric localization of orphan NRs induces the formation of APBs and telomere clustering, which subsequently promotes ALT telomere recombination as evidenced by C-circle formation and ALT telomere DNA synthesis.PML knockout from human fibroblasts abolished ALT telomere DNA synthesis, supporting the critical role of APBs in orphan NR-induced ALT activation.These roles of orphan NRs are also consistent in ALT cell lines.Depletion of orphan NR COUP-TF2 from U2OS and WI38-VA13 / 2RA cells diminished APB formation, as well as ALT telomere DNA synthesis, supporting a dependency on orphan NRs for the ALT activation in AL T cells.Similarly , the AL T telomere DNA synthesis in ALT cells was inhibited upon silencing their PML expression.These findings are in agreement with previous studies ( 7 ,19 ) and further highlight a critical role for PML / APBs in mediating ALT activation in cancers via the telomeric association of orphan NRs.
Our system in which orphan NRs are tethered to telomeres for ALT induction in human fibroblasts has enabled us to dissect the mechanism directly contributing to orphan NRmediated ALT activation.It has been shown previously that orphan NRs bind to the variant TCAGGG repeats of telomeres in ALT cells and they recruit the NuRD-ZNF827 complex, which may alter chromatin structures by impeding shelterin binding and histone deacetylase-mediated hypoacetylation to promote telomere recombination ( 37 ,38 ).Similarly, we found that ZNF827 is critical for ALT induction by orphan NRs in human fibroblasts, consistent with the notion that chromatin alteration may underlie orphan NR-induced AL T activation.Interestingly , we showed that orphan NRmediated APB formation in human fibroblasts is also regulated by ZNF827, but it remains to be determined if this is also a consequence of changes in chromatin structure.It has also been shown previously that the orphan NRs COUP-TF2 and TR4 directly interact with FANCD2 to induce a DNA damage response and to contribute to the ALT pathway in ALT cells ( 39 ).However, our results show that depletion of FANCD2 does not affect APB formation, telomere clustering, and telomere DNA synthesis in fibroblasts.It is worth noting that FANCD2 has also been shown to restrain ALT activity by resolving replication stress at ALT telomeres, with data supporting that depletion of FANCD2 leads to more ALT activity ( 56 ).This discrepancy in the role of FANCD2 from three studies highlights the complexity of the function of FANCD2 in ALT activation, suggesting the necessity for additional investigation.Particularly, factors such as DDR activation at telomeres and the levels of FANCD2 expression may influence the role of FANCD2 in ALT induction.Our differing results from previous studies may arise due to the following factors: the use of different cell models-specifically, human fibroblasts versus U2OS and WI38-VA13 / 2RA ALT cells; the absence of detectable telomeric DDR in primary fibroblasts upon orphan NR-induced ALT ( Supplementary Figure S3 ), contrasting with findings in ALT cells; varying levels of FANCD2 expression between non-transformed fibroblasts and ALT cell lines; and differences in methodology for FANCD2 depletion (RNAi vs. CRISPR).Moreover, the MMS21-SMC5-SMC6 SUMO E3 ligase complex has been shown to promote APB formation in ALT cells via sumoylation of TRF1 and TRF2 ( 15 ).Similar to FANCD2, MMS21-SMC5-SMC6 complex is not required APB formation and telomere clustering our system.Thus, these proteins do not seem to play direct roles in orphan NR-mediated ALT activation.The discrepancy observed from different studies may again be due to cell line-dependent roles of these proteins or experimental methodologies.Nevertheless, we do not rule out the possibility that these proteins may promote maintenance and stabilization of APBs and telomere clusters in ALT cells.
It has been shown previously that ALT cells exhibit spontaneous telomere DDRs, likely arising from replication stress or telomere trimming ( 11 ).Thus, DDR-related proteins may be preferentially recruited to and activated on ALT telomeres to modulate ALT activities.In this study, we present ALT induction by orphan NRs in non-transformed BJ T cells, as well as in three additional primary cell lines BJ, IMR90, and WI38, which have intact p16 and p53.Remarkably, these primary cells expressing COUP-TF2 LBD -TRF1 exhibited sustained normal cell growth in culture, suggesting that intact p16 and p53 do not prevent ALT induction in primary fibroblasts ( Supplementary Figure S2 ).Our results demonstrate that DNA damage was not detected upon tethering orphan NRs to human primary fibroblasts ( Supplementary Figure S3 ), implying that ALT activation by orphan NRs in primary cells might occur even in the absence of telomere DNA damage responses.Transient DDR has been reported to occur at functional telomeres in the G2 phase of the cell cycle after DNA replication, enabling the formation of the t-loop structure ( 57 ).Accordingly, we anticipate that clustered telomeres with altered chromatin structures may engage with the intrinsic telomere DDR in APBs to initiate ALT recombination for subsequent ALT induction.This scenario may explain how the telomeric localization of orphan NRs promotes ALT induction in human fibroblasts in the absence of profound DDR.
Recently, several studies employing divergent strategies have reported induction of ALT phenotypes and activity in cultured cells ( 6 , 18 , 35 , 37 , 47 , 58 , 59 ).By incorporating the variant telomeric sequence TCAGGG or TGAGGG into telomeres, various ALT characteristics-including telomere dysfunction-induced foci (TIFs), C-circles, and heterogenous telomeres-have been induced in HT1080 sarcoma cells, albeit doing so was insufficient to trigger ALT telomere recombination ( 37 ).Directly inducing APB formation ( 18 ,58 ) or telomere double strand breaks ( 6 ) promotes ALT activity in ALT cell lines, but not in cells that utilize telomerase for telomere maintenance.Depletion of the ASF1 histone chaperone to perturb DNA replication elicited ALT phenotypes and telomere recombination in HeLa and immortalized (by hTERT and HPV E6-E7) human fibroblasts ( 35 ).These systems have been used for the elucidation of the ALT mechanism, but they have some caveats.For instance, these models often induce severe genomic and / or telomeric DNA damage, so they have to be established in cancer cell lines (telomerase or ALT) or transformed primary cells that are defective in cell cycle checkpoints.Selectively activating particular ALT fea-tures, e.g.APBs, in ALT cells promotes higher ALT activity, but they cannot trigger ALT induction in non-ALT cells ( 18 ,58 ).In contrast, our system for ALT induction in human fibroblasts circumvents these limitations.First, tethering orphan NRs to telomeres did not trigger a significant telomere DDR in human fibroblasts, so the cells could proliferate normally.Targeting orphan NRs to telomeres triggered ALT phenotypes and ALT telomere DNA synthesis in ALT cells, as well as in primary human fibroblasts, indicating that orphan NRs coordinate multiple pathways for AL T induction.Consistently , the features of ALT activity we observed was dependent on both ZNF827 and PML, so ALT induction may result from the cooperative effects of the structural changes in chromatin and APB formation following telomeric targeting of orphan NRs.Thus, our system represents a unique in vitro strategy for ALT activation and can be deployed to investigate the ALT mechanism and ALT cancer development.
Our findings suggest that orphan NRs can promote telomere clustering upon their localization to telomeres in human fibroblasts.In the absence of PML, orphan NR-mediated telomere clustering was partly reduced in human fibroblasts, an outcome consistent with a previous finding showing that APB formation promotes telomere clustering ( 18 ,47 ).Intriguingly, we still detected substantial telomere clustering in PML knockout human fibroblasts, implying that the telomere clustering induced by orphan NRs can occur in an APB-independent manner .However , unlike for APB formation and ALT telomere DNA synthesis, telomere clustering in ALT cells was not affected by overexpression or knockdown of orphan NRs (Figure 2 , Supplementary Figure S4 ).Moreover, telomere clustering in ALT cells was not affected upon depletion of PML, although it did abolish ALT telomere DNA synthesis.These results indicate that telomere clustering in ALT cells may occur independently of orphan NR functions and APB formation.It would be intriguing to further elucidate the underlying mechanism of telomere clustering in ALT cells, which is critical for understanding its role in ALT activation.Moreover, future work is also required to investigate the cell functions of orphan NR-mediated telomere clustering.
ATRX / DAXX mutations are the most frequently reported genetic alterations associated with ALT .However , it has been shown previously that ATRX depletion from mortal or telomerase-positive immortal cells is insufficient to activate ALT ( 31 ).Instead, ATRX loss progressively changes telomere chromatin structures and induces telomere dysfunction and cell growth arrest ( 34 ).We have demonstrated herein that combinatorial orphan NR targeting to telomeres and depletion of ATRX DAXX induced ALT DNA synthesis in human fibroblasts (Figure 4 ), supporting that orphan NR re-cruitment to telomeres and ATRX / DAXX dysfunction represent two triggering events that may cooperatively promote AL T activation (Figure 8 ).Consistently , ATRX / DAXX depletion and telomeric targeting of orphan NRs elicited additive effects of APB formation and telomere clustering.However, ATRX / DAXX loss can also lead to telomere decompaction ( 34 ).Additionally, we observed that orphan NRs stimulate ALT phenotypes through ZNF827 (Figure 3 ), which has been shown to enhance chromatin compaction through the NuRD complex ( 38 ).However, it remains to be determined how chromatin compaction is altered under our experimental settings.Nevertheless, our findings suggest that the effects of telomere compaction upon ATRX / DAXX dysfunction and telomere recruitment of orphan NRs do not counteract ALT activation.In addition to telomere compaction, ATRX / DAXX loss has been implicated in promoting replication stress to stimulate ALT by increasing the expression of telomeric long noncoding RNAs (TERRA, telomeric repeat-containing RNA), R-loops ( 33 ,60 ,61 ) and Gquadruplex structures ( 32 ,62 ), as well as promoting loss of telomere cohesion ( 63 ,64 ), and dynamic exchange of histone macroH2A1.2( 65 ).Further comprehensive study is required to understand how ATRX dysfunction-associated changes interplay with orphan NRs to regulate ALT.It is worth noting that although ATRX / DAXX is a chaperone complex for telomeric deposition of histone H3.3, we did not observe changes in ALT phenotypes upon knockdown of histone H3.3 by siRNAs in control human fibroblasts or those expressing TRF1-orphan NRs, suggesting that transient depletion of histone H3.3 does not alter telomere chromatin structures.This outcome supports that dysregulation of the nonchaperone function of ATRX / DAXX can mediate ALT activation.However, we cannot rule out the possibility that prolonged ATRX / DAXX dysfunction can result in decreased histone H3.3 deposition at telomeres to promote ALT activation.Furthermore, while we observed induction of multiple features of ALT activity upon tethering of orphan NRs to telomeres in fibroblasts, this was not sufficient to counteract the effects of replication-associated telomere shortening and directly result in cell immortalization (unpublished data).Establishing whether the telomeric localization of orphan NRs promotes telomere lengthening upon ATRX / DAXX loss warrants further investigation.
Ultimately, we have demonstrated that APBs are critical for orphan NR-mediated ALT induction of human fibroblast cells and for enhancing features of ALT activity in ALT cells.APBs are sites of homologous recombination and are critical for ALT activity ( 7 , 8 , 19 , 66 ).We and others ( 7 ,19 ) have shown that depletion of PML from ALT cell lines abolishes ALT telomeric DNA synthesis.Significantly, PML is required for the ALT induction in our primary fibroblast cells based ALT model, and depletion of orphan NRs from ALT cells limits APB formation and ALT telomere DNA synthesis.It has been postulated previously that PML is important in ALT because it localizes the BLM-TOP3A-RMI (BTR) complex to ALT telomeres ( 19 ).Identifying that PML is critical for orphan NR-mediated ALT induction led us to explore if targeting APBs or PML is a potential anti-AL T strategy .Arsenic trioxide, which promotes PML degradation, has long been used as an APL treatment and, recently, it has been repurposed for other cancer types such as osteosarcoma and glioblastoma ( 51 ,52 ).Here, we demonstrate that arsenic trioxide can act as a potential ALT inhibitor given that it degrades PML to prevent APB formation and ALT telomere DNA synthesis in both primary fibroblasts and ALT cell lines.Aside from our in vitro experiments, we extended our investigation to demonstrate that arsenic trioxide also disrupts APBs and ssTeloC in mouse xenografts.Although multiple compounds have been investigated for ALT targeting, these studies have predominantly been conducted in cultured cells, serving primarily as proof of concept ( 44 ,67 ).Nevertheless, our study demonstrates for the first time that a clinically approved drug can inhibit features of ALT activity in vivo .Consequently, we propose a novel anti-ALT cancer strategy involving repurposing arsenic trioxide, which could undergo further clinical development for ALT cancer patients.Overall, our significant findings illuminate the role of orphan NRs in ALT cancer development and provide valuable information that may be utilized for ALT cancer treatment.

Figure 2 .Figure 3 .
Figure 2. Orphan NRs mediate APB formation and ALT telomere DNA synthesis in ALT cells.( A ) R epresentativ e images showing PML and telomere co-localization in U2OS cells o v ere xpressing CO UP-TF2 and TR4.Quantification of APBs ( B ) and telomere numbers ( C ) in individual U2OS cells ( n > 100) expressing orphan NRs COUP-TF2 and TR4 or COUP-TF2 AF2 and TR4 AF2 .( D ) Representative images showing EdU at telomeres and PML in U2OS-COUP-TF2 + TR4 cells.EdU and PML were detected by IF, and telomeres were detected by FISH using the TelC PNA probe.Co-localization of EdU (green), PML (magenta), and telomeres (red) appears white.White arrows indicate telomeric DNA synthesis at APBs. ( E ) Quantification of telomere and EdU co-localization in U2OS-COUP-TF2 + TR4 cells.( F ) Representative images showing reduced EdU signal at telomeres and PML levels in U2OS cells upon treatment with siRNAs against COUP-TF2 or TR4. ( G ) Quantification of telomere and EdU co-localization in U2OS cells upon treatment with siRNAs against COUP-TF2 or TR4.Cells were synchronized in G2 phase by thymidine and CDK1i treatments for 21 h and 12 h, respectively.Red lines represent median of two independent experiments.ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0 0 01, as determined by Mann-Whitney U test.
B).Moreover, in BJ T -COUP-TF1 LBD -TRF1 and BJ T -COUP-TF2 LBD -TRF1 cells, we detected a further increase in APBs and telomere clustering upon ATRX or DAXX depletion (Figure4 B, C).Furthermore, depletion of ATRX or DAXX triggered telomeric DNA synthesis at APBs (Figure4 D-F), which was further enhanced when combined with expression of COUP-TF1 LBD -TRF1 or COUP-TF2 LBD -TRF1 (Figure4 D-F).It is worth noting that depletion of histone H3.3 did not promote ALT induction in BJ T fibroblasts or in BJ T -COUP-TF1 LBD -TRF1 or BJ T -COUP-TF2 LBD -TRF1 cells.These combinatorial analyses indicate that orphan NRs may cooperate with ATRX and DAXX deficiencies to promote ALT activation, independently of histone H3.3 deposition.

Figure 7 .
Figure 7. Arsenic trioxide suppresses APB formation and features of ALT activity in SaOS2 xenografts in mice .( A ) Representative images APBs in mouse SaOS2 xenografts.ALT osteosarcoma cell lines were injected into nude mice for tumor formation and then 6 mice were treated with PBS as control (7 tumors) and 7 mice were treated with As 2 O 3 (8 tumors).PML was detected by IF, and telomeres were detected by FISH using the TelC PNA probe.Co-localization of PML (green) and telomeres (red) appears y ello w. White arrows indicate APBs.Loss of PML bodies ( B ) and telomere + PML co-localization ( C ) in mouse xenografts treated with As 2 O 3 .( D ) Representative images of single-stranded C-rich telomeric DNA (ssTeloC) detected in mouse SaOS2 xenografts.ssTeloC signals were detected by native FISH using the TelG PNA probe ( E ) Loss of ssTeloC or ALT activity detected by the native FISH assay in mouse xenografts treated with As 2 O 3 .Red lines indicate the mean.ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0 0 01, as determined by unpaired t -test.

Figure 8 .
Figure 8. Model of orphan NR-induced ALT activation.Orphan NRs bind to ALT telomeres via variant repeats.The orphan NRs at telomeres recruit ZNF827 and allow for APB formation, telomere clustering, C-circle formation, telomere sister chromatid exchange, and telomere DNA synthesis.The telomere localization of orphan NRs acts in concert with ATRX / DAXX loss to promote APB formation and telomere DNA synthesis.APBs are critical for orphan NR-mediated ALT induction.Arsenic trioxide is an ALT inhibitor that can target APBs and features of ALT activity.Solid arrows indicate that the relationship between ALT phenotypes is established by this study.Broken arrows indicate an undetermined causal relationship between ALT phenotypes.
• C (5 μg Vector, 10X CutSmart buffer 5 μl, BamHI 5 μl, H 2O 35 μl).A QIAquick PCR purification kit was used to purify the linearized vector.Target fragments were amplified by polymerase chain reaction (PCR), and PCR products were treated with DpnI before purification.The 5X InFusion HD Enzyme Premix, linearized vector, purified PCR fragment, and dH 2 O were mixed and incubated at 50 • C for 15 min.Transformation was conducted using stbl3 (ECOS™-competent cells) with ampicillin selection and overnight incubation at 37 • C. Single clones were selected and amplified by liquid culture.Plasmids were collected using a QIAprep Spin Miniprep Kit and were checked by sequencing and restriction digestion.Retroviral transduction was performed to stably express the plasmids in cells.TFORF0672 (ZNF827) was a gift from Feng Zhang (Addgene plasmid #141658;