Telomere is a nucleoprotein structure found at the end of the linear chromosome, which is composed of TTAGGG repetitive DNA and shelterin complex. Human shelterin is a six-subunit complex (TRF2, TRF1, RAP1, TPP1, TIN2, and POT1) that binds telomeres and shields the ends of chromosomes from degradation and end-to-end fusions 1. Due to the terminal replication problem, the length of the telomere gradually shortened with continuous cellular proliferation ,and the cells will eventually undergo cell cycle arrest then cellular senescence 2. Activation of telomere maintenance mechanism to prevent excessive telomere shortening is required for the sustained proliferation in cancer cells 3. Most of cancers use telomerase to extend their telomeres 4 but 10–15% of cancers elongate their telomere through telomerase independent pathway, which is named alternative lengthening of telomeres (ALT) 5,6.
ALT was firstly found in the telomerase mutational budding yeast 7 and then it was observed in human tumors. In the past decades, researchers have done a lot of work to uncover the molecular mechanism of ALT. Even though the precise molecular mechanism of ALT is still unclear. It is widely accepted that ALT is a DNA repair process dependent homologous recombination process 8, which is represented by the appearance of a variety of hallmarks, including extra chromosome telomere repeat (CTR, such as C-circle), ALT associated PML bodies (APBs), heterogeneity of telomere length, and telomere sister chromatid exchange (T-SCE) 5. In addition, accumulating evidence indicates that the long noncoding RNA (lncRNA) telomeric repeat-containing RNA (TERRA) are elevated in ALT cells9,10.
The current studies revealed that the presence of recombinase RAD51 and RAD52 are essential for ALT telomere maintenance11. Inhibition of RAD52 decreases the natural ALT telomere synthesis at APBs in G2 cells12, and the depletion of RAD51 leads to an increase of fragile telomeres and telomere dysfunction induced foci in ALT cells but not affects the mitotic DNA synthesis (MiDAS) at telomeres13. Instead of directly taking part in ALT DNA homologous recombination, RAD51 may mainly functions in lessening telomere fragility by suppressing the stalled replication forks. Moreover, RAD52-mediated telomeric MiDAS is observed in both ALT positive and telomerase positive cells when cells encounter with exogenous DNA damage or replication inhibitor, indicating that RAD52 is essential for break induce replication (BIR) at collapsed forks and fragile sites. Interestingly, a most recent study demonstrates that the c-circles levels are not altered in RAD52 knockdown cells, suggesting that there is a RAD52-independent ALT pathway, which is responsible for C-circles generation during telomere damage14. In addition, POLD3/POLD4 are reported to be required for the conservative DNA replication during BIR, which could be enhanced by BLM and reduced by SLX415. In ALT positive human cells, telomeres undergo conservative synthesis also rely on POLD3/4, thereby linking ALT to BIR.
53BP1 nuclear bodies are formed when DNA damage occurs in S phase, however it is excluded from DNA breaks during mitosis. In contrast, the ssDNA-binding protein RPA can bind to exposed ssDNA in M-phase16. Recent research reported that RPA plays critical roles in protecting resected ssDNA upon chromosome breakage and promoting the successful progress of telomeric MiDAS17. In addition, the elevated level of TERRA suggests it plays a crucial role in ALT telomeres recombinogenic by forming R-loop or RNA–DNA hybrids with the telomeric C-rich DNA strand, which contributes to activation of the DNA damage response (DDR) and promotes recruitment of the recombinase. 18,19. The aberrant accumulation of phosphorylated RPA at ALT telomeres when the RNaseH1 was deleted, which regulates the levels of RNA-DNA hybrids between telomeric, indicates that RPA itself could promote the forming of R-loop. Supporting this, it has been reported that the C-rich telomeric ssDNA accumulate in ALT cells when the RPA was depleted 20. Moreover, in yeast, the telomeres are bound by the Rap1 protein, Rap1 interacts with Sir3 and Sir4 that, together with the histone deacetylase Sir2, which initiate heterochromatin formation in subtelomeric regions21. The Sir2/Sir3/Sir4 complex also restricts TERRA expression via transcriptional repression22. So the roles of RPA and RAP1 in TERRA R-loop regulation in human ALT cells deserve further explore.
Previous studies have shown that Lamin A/C is involved in DNA damage repair and telomere maintenance 23–26. As a lamina associated protein, lamina associated polypeptide 2(LAP2) belongs to the LEM domain protein family. There are six splicing isoform encoded by the TMPO, including LAP2 α, β, γ, δ, ε,and ζ 27. All isoforms have LEM domain and LEM-like domain, by which to interact with BAF protein and bind to DNA 28,29. Unlike the other isoforms, LAP2α and ζ lacks transmembrane domain, thus they could only localize at nucleoplasm with Lamin A/C 30,31. In addition, it is reported that LAP2α interacted with WRN helicase and Ku86 25, which are essential for classical non-homologous end joining repair and telomere integrity during telomeric DNA damage repair 32–34. Moreover, it has been recently reported that LAP2α could promote the deposition of RPA on damaged DNA and facilitate the homologous recombination (HR) 35. It has been demonstrated that, the distribution of LAP2α in cells changes with cell cycles 36, and it binds to telomere or sub-telomere at the anaphase and telophase of mitosis 37. Additionally, it has demonstrated that LAP2α could located at the end of chromosome and colocalized with telomere, during anaphase and telophase of mitosis. Thus, we speculate that LAP2α might play a critical role in break induced replication mediated alternative telomere lengthening.
In this study, we demonstrated the interaction of LAP2α with the shelterin complex. The knockdown of LAP2α resulted in a significant increase in ALT-associated hallmarks, including APBs, C-circles, and T-SCE, emphasizing LAP2α's crucial role in ALT maintenance. Subsequent investigations unveiled that LAP2α depletion led to the recruitment of recombination factors, such as RPA and RAD52, to telomeres. Moreover, LAP2α was found to interact with HDAC1, potentially contributing to the de-repression of the heterochromatin state of telomeric DNA, promoting telomere recombination and ALT occurrence. This observation aligns with our findings that LAP2α expression is low in ALT-positive osteosarcoma patients. Furthermore, methotrexate (MTX), a widely used drug for treating osteosarcoma, has been reported to enhance DNA heterochromatinization. Our discovery revealed that MTX treatment increased the aggregation of H3K9 trimethylation and HP1 in the telomeric region. Notably, in our patient-derived xenograft (PDX) model, MTX exhibited specific inhibition of the proliferation of LAP2α-low-expressing ALT-positive osteosarcoma tumors. Mechanistically, our data provide initial evidence that the lamina-associated protein LAP2α intricately participates in ALT telomere synthesis by regulating telomeric heterochromatin status. This breakthrough opens new avenues for unraveling the precise molecular mechanisms underlying ALT, offering promising insights for future investigations. Importantly, our findings enhance the targeted use of MTX, providing a critical context for its improved clinical performance.