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.

Graphical Abstract

Figure 1.

Tethering of orphan NRs to telomeres triggers ALT induction in human fibroblasts. (A) Representative images showing PML and telomere co-localization in BJ^T cells expressing the full-length orphan NRs COUP-TF1, COUP-TF2, TR2, TR4 or EAR-2. 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 yellow. White arrows indicate APBs. ‘APB + cells (%)’ values reflect that more than five telomere + PML co-localization events were observed in a given cell. Quantification of APBs (B) and telomere numbers (C) in individual BJ^T cells (n > 100) expressing the full-length orphan NRs COUP-TF1, COUP-TF2, TR2, TR4 or EAR2. (D) Representative images showing co-localization of PML and telomeres in BJ^T cells expressing the LBD of orphan NRs COUP-TF1, COUP-TF2, TR2, TR4 or EAR2. Quantification of APBs (E) and telomere numbers (F) in individual BJ^T cells (n > 100) expressing LBDs of orphan NRs COUP-TF1, COUP-TF2, TR2, TR4 or EAR2. (G) Representative images showing EdU at telomeres and PML in BJ^T-COUP-TF1^LBD-TRF1 and BJ^T-COUP-TF2^LBD-TRF1 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. Quantification of telomere and EdU co-localization (H) and telomere, PML and EdU co-localization (I) in BJ^T-COUP-TF1^LBD-TRF1 and BJ^T-COUP-TF2^LBD-TRF1 cells (n > 100). (J) C-circles detected in BJ^T-COUP-TF1^LBD-TRF1 and BJ^T-COUP-TF2^LBD-TRF1 cells. (K) Quantification of C-circles detected in (J). (L) Representative images showing T-SCEs in BJ^T-COUP-TF2^LBD-TRF1 cells. Co-localization of TelG (green) and TelC (red) appears yellow. White arrows indicate T-SCEs. (M) Zoom-in view of the boxed region in (L) and quantification of T-SCEs detected in (L) from two independent experiments (n > 500). Red lines represent median of two independent experiments. ns P> 0.05, *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001, as determined by Mann–Whitney U test.

Figure 2.

Orphan NRs mediate APB formation and ALT telomere DNA synthesis in ALT cells. (A) Representative images showing PML and telomere co-localization in U2OS cells overexpressing COUP-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^ΔAF2 and TR4^ΔAF2. (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< 0.0001, as determined by Mann–Whitney U test.

Figure 3.

The AF2 domain of orphan NRs is critical for ALT induction. (A) Representative images showing loss of APBs in BJ^T-COUP-TF1^LBD/ΔAF2-TRF1 and BJ^T-COUP-TF2^LBD/ΔAF2-TRF1 cells. 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 yellow. White arrows indicate APBs. Quantification of APBs (B) and telomere numbers (C) in individual BJ^T cells (n > 100) expressing COUP-TF1^LBD-TRF1, COUP-TF2^LBD-TRF1, COUP-TF1^LBD/ΔAF2-TRF1 or COUP-TF2^LBD/ΔAF2-TRF1. (D) Relative ZNF827 expression in BJ^T cells expressing COUP-TF1^LBD-TRF1 or COUP-TF2^LBD-TRF1, as measured by qPCR. (E) Representative images showing reduced APB formation in BJ^T-COUP-TF1^LBDTRF1 and BJ^T-COUP-TF2^LBDTRF1 cells upon treatment with siRNAs against ZNF827. Quantification of APBs (F), telomere numbers (G), and telomere and EdU co-localization (H) in individual BJ^T cells (n > 100) expressing COUP-TF1^LBD-TRF1 or COUP-TF2^LBD-TRF1 upon treatment with siRNAs against ZNF827. Red lines represent median of two independent experiments. ns P> 0.05, *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001, as determined by Mann–Whitney U test.

Figure 4.

ATRX/DAXX depletion combined with orphan NR recruitment to telomeres promotes features of ALT activity more dramatically. (A) Western blot showing the loss of protein expression in BJ^T-COUP-TF1^LBD-TRF1 and BJ^T-COUP-TF2^LBD-TRF1 cells upon treatment with siRNAs against ATRX, DAXX or H3.3. Quantification of APBs (B) and telomere numbers (C) in BJ^T-COUP-TF1^LBD-TRF1 and BJ^T-COUP-TF2^LBD-TRF1 cells (n > 100) upon treatment with siRNAs against ATRX, DAXX, or H3.3. (D) Representative images showing EdU at telomeres and PML in BJ^T-COUP-TF1^LBD-TRF1 and BJ^T-COUP-TF2^LBD-TRF1 cells upon treatment with siRNAs against ATRX, DAXX or H3.3. 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. Quantification of telomere and EdU co-localization (E) and of telomere, PML and EdU co-localization (F) in BJ^T-COUP-TF1^LBD-TRF1 and BJ^T-COUP-TF2^LBD-TRF1 cells (n > 100) upon treatment with siRNAs against ATRX, DAXX or H3.3. Cells were synchronized in G2 phase by means of 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< 0.0001, as determined by Mann–Whitney U test.

Figure 5.

PML is critical for orphan NR-induced telomeric DNA synthesis at APBs. (A) Representative images of PML(+) and PML(–) BJ^T-COUP-TF2^LBD-TRF1 cells. PML was detected by IF. (B) Western blot showing PML protein expression in PML(+) and PML(–) BJ^T cells. (C) Quantification of telomere number in PML(+) and PML(–) BJ^T cells (n > 100). (D) Representative images showing EdU at telomeres in PML(+) and PML(–) BJ^T cells. EdU were detected by IF, and telomeres were detected by FISH using the TelC PNA probe. Co-localization of EdU (green) and telomeres (red) appears yellow. White arrows indicate telomeric DNA synthesis. (E) Quantification of telomere and EdU co-localization in PML(+) and PML(–) BJ^T cells (n > 100). Cells were synchronized in G2 phase by means of 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< 0.0001, as determined by Mann–Whitney U test.

Figure 6.

Arsenic trioxide disrupts APBs and inhibits ALT telomere DNA synthesis in ALT cells. (A) Western blot showing loss of PML isoforms upon 1 μM or 2 μM As₂O₃ treatment of U2OS, WI38-VA13/2RA, and SaOS2 cells. (B) Representative images showing loss of PML and telomere + PML co-localization in U2OS, WI38-VA13/2RA and SaOS2 cells upon As₂O₃ treatment. 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 yellow. White arrows indicate APBs. Quantification of PML bodies (C) and APBs (D) in individual U2OS (n > 100), WI38-VA13/2RA (n > 100), and SaOS2 (n > 100) cells. (E) Representative images showing loss of EdU at telomeres in U2OS, WI38-VA13/2RA and SaOS2 cells upon As₂O₃ treatment. EdU were detected by IF and telomeres were detected by FISH using the TelC PNA probe. Co-localization of EdU (green) and telomeres (red) appears yellow. White arrows indicate telomeric DNA synthesis. (F) Quantification of telomere and EdU co-localization in individual U2OS (n > 100), WI38-VA13/2RA (n > 100) and SaOS2 (n > 100). Red lines indicate median of two independent experiments. ns P> 0.05, *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001, as determined by Mann–Whitney U test.

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 As₂O₃ (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 yellow. White arrows indicate APBs. Loss of PML bodies (B) and telomere + PML co-localization (C) in mouse xenografts treated with As₂O₃. (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 As₂O₃. Red lines indicate the mean. ns P> 0.05, *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001, as determined by unpaired t-test.

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.