ALC1 links chromatin accessibility to PARP inhibitor response in homologous recombination deficient cells
Posted on: 31 December 2020
Preprint posted on 16 December 2020
Article now published in Nature Cell Biology at http://dx.doi.org/10.1038/s41556-020-00624-3
The chromatin remodeler ALC1 contributes to PARPi toxicity in BRCA-deficient cancer cells.
Selected by Giuseppina D'Alessandro
Categories: cancer biology, cell biology
Background
Everyday thousands of lesions challenge the stability of our DNA. To preserve genome integrity, our cells activate a number of dedicated DNA repair pathways. The poly(ADP-ribose) polymerase (PARP) enzymes, in particular PARP1 and PARP2, recognize single-strand DNA breaks (SSB) and other DNA lesions and deposit poly(ADP-ribose) (PAR) chains that modulate the DNA damage response and chromatin remodelling (Ray Chaudhuri and Nussenzweig, 2017). PARP inhibitors (PARPi) not only impair the enzymes catalytic activity but can also “trap” them on chromatin, thus leading to the generation of DNA lesions that require the homologous recombination (HR) proteins BRCA1 and BRCA2 for repair (Murai, 2012). This makes HR-deficient cancer cells specifically sensitive to PARPi.
Despite numerous studies investigated the impact of PARylation on chromatin accessibility, the contribution of chromatin remodelling to the toxicity of PARPi in HR deficient cells is less clear. In this preprint, the authors demonstrate that loss of the PAR-dependent chromatin remodeler ALC1 contributes to PARPi toxicity in BRCA-deficient cancer cells.
Figure 1: PARP-mediated PARylation induces chromatin de-condensation and mediates ALC1 recruitment, which, in turn, further increases chromatin accessibility. In the absence of ALC1 and PARP activities, recruitment of base damage repair factors to the chromatin is reduced. During S-phase, unrepaired lesions generate replication coupled single-strand gaps and DSBs that rely on BRCA-dependent HR repair (From this preprint under the CC-BY-NC-ND 4.0 International license).
Key findings:
- ALC1 loss is synthetically lethal with BRCA loss, hypersensitizes BRCA-deficient cells to PARPi, and overcomes known mechanisms of resistance to PARPi
By performing a CRISPR-screen, the authors identified the chromatin remodeler ALC1/CHD1L as a factor that further sensitized BRCA-mutant cancer cells to the PARPi, olaparib. PARPi hyper-sensitivity of ALC1 knockout cells was not due to defective SSB or DNA double-strand breaks (DSB) response, as demonstrated by additivity between loss of ALC1 and SSBR/HR proteins. The authors also demonstrated that ALC1 loss overcame known mechanisms of resistance to PARPi, such as 53BP1 loss, which removes the barrier to DNA end resection, thus partially restoring HR in BRCA1-deficient cells. ALC1 depletion was also able to overcome olaparib resistance due to PARP1 loss, thus suggesting a role for PARP2 in mediating PARPi toxicity in this context, likely caused by the increased PARP2 trapping observed in ALC1 deficient cells.
- ALC1 loss increases genomic instability and reliance on BRCA-dependent HR.
ALC1 loss in BRCA-mutant cells increased single-strand DNA (as monitored by non-denaturing CIdU immunofluorescence), DSBs in S-phase (as monitored by γH2AX immunofluorescence), and radial chromosomes. ALC1 deficiency hyper-sensitized cells to SSBs induced by the alkylating agent methyl methanesulfonate (MMS), but not by the topoisomerase I poison camptothecin. Based on these data, the authors argue that ALC1 remodels chromatin around complex lesions, as those generated by alkylating agents, rather than being generally required for SSB repair. ALC1-deficient cells accumulated replication-coupled single-strand gaps, as shown by the higher S1 nuclease sensitivity of nascent replication tracts, that require SSB and HR repair mechanisms.
- ALC1 localizes to chromatin via PARP1 and PARP2-dependent PARylation.
Immunofluorescence of undamaged cells revealed ALC1 localization on S-phase chromatin in the presence of PARG inhibitor, which enhances PARylation. Differently, ALC1 chromatinization was detected in both S- and non-S- phase cells upon treatment with MMS and was abolished in PARP1 and PARP2 knockout cells.
- ALC1 chromatin remodeling and PAR binding activities are essential to protect BRCA-mutant cells from PARPi and alkylation damage
As a member of the SNF2 superfamily, ALC1 uses its ATPase activity to remodel nucleosome in vitro and it also contains a PAR-binding macrodomain that controls its recruitment to chromatin and its allosteric activation (Ahel, 2009; Gottschalk, 2009). To study how each of these domains affected PARPi hypersensitivity of BRCA-mutant cells, the authors complemented ALC1 knockout cells with different ALC1 mutants. ALC1 mutants lacking nucleosome sliding activity (by either mutation within the ATPase domain or mutation of the residues responsible for the interaction with the basic histone H4) or mutants unable to bind PAR (by mutations within the macrodomain) did not rescue PARPi sensitivity of BRCA mutant cells, thus suggesting that both the nucleosome remodeling and PAR-binding activity are required to protect BRCA-mutant cells from PARPi.
- ALC1 and PARP activity cooperate to promote chromatin accessibility
ALC1 loss reduced overall chromatin accessibility, as monitored by ATAC-seq (Assay for Transpose-Accessible Chromatin), while not causing locus-specific changes or transcriptional changes. As a consequence, the chromatin association of base damage repair factors, including the apurinic/apyrimidinic endonuclease APE1 and XRCC1, was reduced. Reduced chromatin accessibility and recruitment of base damage repair protein were exacerbated by PARPi, thus suggesting that ALC1 and PARP cooperate to promote chromatin accessibility.
Future perspectives and questions for the authors
In summary, the authors demonstrated that ALC1 loss reduces the viability of BRCA-mutant cells and further sensitizes them to PARPi, thus revealing a new vulnerability in HR deficient cancers. Since loss of ALC1 ATPase activity phenocopies ALC1-loss, ALC1 inhibitors could represent a future opportunity to overcome acquired PARPi resistance in BRCA-deficient cells. The authors propose that ALC1 chromatin remodeling activity favors the processing of complex lesions and, together with PARP1 and PARP2 activity, enhances chromatin recruitment of base repair factors. PARP inhibition in ALC1 deficient cells causes accumulation of lesions that rely on BRCA-mediated HR during S-phase.
- ALC1 loss hyper-sensitizes BRCA-deficient cells to all the tested PARPi: veliparib, olaparib and talazoparib. Interestingly, a smaller sensitivity window is observed with the strongest PARP trapper talazoparib: could this suggest that PARP trapping is less crucial in the context of ALC1 loss?
- It is interesting that ALC1 loss increases PARP2 trapping, but does not seem to affect PARP1, as observed in chromatin fractionation experiment. It would be interesting to hear the author’s opinion on this, also in the light of the recent publication reporting a role for ALC1 in releasing PARP2 but not PARP1 from chromatin (Blessing, 2020).
- ALC1 loss is synthetically lethal with BRCA deficiency even in the absence of PARPi: could this suggest that ALC1 inhibitors would be effective in BRCA deficient tumors even as an alternative to PARPi? What could be the underlying mechanism responsible for this synthetic lethality?
doi: https://doi.org/10.1242/prelights.26696
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