FANCM suppresses DNA replication stress at ALT telomeres by disrupting TERRA R-loops

Abstract

Cancer cells maintain their telomeres by either re-activating telomerase or adopting the homologous recombination (HR)-based Alternative Lengthening of Telomere (ALT) pathway. Among the many prominent features of ALT cells, C-circles (CC) formation is considered to be the most specific and quantifiable biomarker of ALT. However, the molecular mechanism behind the initiation and maintenance of CC formation in ALT cells is still largely unknown. We reported previously that depletion of the FANCM complex (FANCM-FAAP24-MHF1&2) in ALT cells induced pronounced replication stress, which primarily takes place at their telomeres. Here, we characterized the changes in ALT associated phenotypes in cells deficient of the FANCM complex. We found that depletion of FAAP24 or FANCM, but not MHF1&2, induces a dramatic increase of CC formation. Most importantly, we identified multiple DNA damage response (DDR) and DNA repair pathways that stimulate the dramatic increase of CC formation in FANCM deficient cells, including the dissolvase complex (BLM-TOP3A-RMI1/2, or BTR), DNA damage checkpoint kinases (ATR and Chk1), HR proteins (BRCA2, PALB2, and Rad51), as well as proteins involved in Break-Induced Replication (BIR) (POLD1 and POLD3). In addition, FANCD2, another Fanconi Anemia (FA) protein, is also required for CC formation, likely through promoting the recruitment of BLM to the replication stressed ALT telomeres. Finally, we demonstrated that TERRA R-loops accumulate at telomeres in FANCM deficient ALT cells and downregulation of which attenuates the ALT-associated PML bodies (APBs), replication stress and CC formation. Taken together, our data suggest that FANCM prevents replisomes from stalling/collapsing at ALT telomeres by disrupting TERRA R-loops.

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

“…Like its counterpart in yeast, the mammalian ortholog of Sgs1, the Bloom syndrome helicase (BLM), has been implicated in the mammalian ALT pathway. Depletion by siRNA in ALT-positive cells results in the reduction of ALT-associated phenotypes such as the accumulation of extrachromosomal telomeric repeats in the form of partially single-stranded Crich circles, termed C-circles, and G2/M telomere synthesis (O'Sullivan et al 2014;Pan et al 2019;Zhang et al). Notably, BLM also plays an important role at telomeres in cells that do not utilize ALT to maintain their telomeres, acting to facilitate telomere replication and suppressing rapid telomere deletions (Stavropoulos et al 2002;Sfeir et al 2009;Barefield and Karlseder 2012;Zimmermann et al 2014 ;Drosopoulos et al 2015;Pan et al 2017).…”

“…BLM is part of the BTR complex which also includes the topoisomerase TOP3a, and the OB-fold containing structural components RMI1 and RMI2 (Johnson et al 2000;Wu et al 2000;Yin et al 2005;Xu et al 2008). Interestingly, overexpression of BLM or dysregulation of the BTR complex induced by the loss of FANCM in ALT-positive cells has been shown to induce upregulation of ALT-associated phenotypes, suggesting that this factor is limiting for the ALTpathway and led to the proposal that the BTR complex acts in ALT to dissolve recombination intermediates into non-crossover products which results in telomere lengthening (Sobinoff et al 2017;Lu et al 2019;Min et al 2019;Pan et al 2019;Silva et al 2019).…”

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

“…Like its counterpart in yeast, the mammalian ortholog of Sgs1, the Bloom syndrome helicase (BLM), has been implicated in the mammalian ALT pathway. Depletion by siRNA in ALTpositive cells results in the reduction of ALT-associated phenotypes such as the accumulation of extrachromosomal telomeric repeats in the form of partially singlestranded C-rich circles, termed C-circles, and G2/M telomere synthesis (O'Sullivan et al 2014;Sobinoff et al 2017;Pan et al 2019;Zhang et al 2019). Notably, BLM also plays an important role at telomeres in cells that do not use ALT to maintain their telomeres, acting to facilitate telomere replication and suppressing rapid telomere deletions (Stavropoulos et al 2002;Sfeir et al 2009; Barefield and Karlseder 2012;Zimmermann et al 2014 ;Drosopoulos et al 2015;Pan et al 2017).…”

“…BLM is part of the BTR complex that also includes the topoisomerase TOP3α, and the OB-fold containing structural components RMI1 and RMI2 (Johnson et al 2000;Wu et al 2000;Yin et al 2005;Xu et al 2008). Interestingly, overexpression of BLM or dysregulation of the BTR complex induced by the loss of FANCM in ALT-positive cells has been shown to induce up-regulation of ALT-associated phenotypes, suggesting that this factor is limiting for the ALT pathway and led to the proposal that the BTR complex acts in ALT to dissolve recombination intermediates into noncrossover products, which results in telomere lengthening (Sobinoff et al 2017;Lu et al 2019;Min et al 2019;Pan et al 2019;Silva et al 2019).…”

Current understanding of extrachromosomal circular DNA in cancer pathogenesis and therapeutic resistance

“…The underlying molecular mechanisms that lead to the biogenesis of extrachromosomal circular DNA have yet to be fully clarified. Both DNA damage and the corresponding DNA repair strategies, the important events of genomic homeostasis observed particularly in tumorigenesis [23][24][25][26], are processes that have been proven to produce extrachromosomal circular DNA species [27][28][29] (Fig. 3).…”