Perturbation of PALB2 function by the T413S mutation found in small cell lung cancer

Abstract

Background: Germline mutations in the PALB2 gene are associated with the genetic disorder Fanconi anaemia and increased predisposition to cancer. Disease-associated variants are mainly protein-truncating mutations, whereas a few missense substitutions are reported to perturb its interaction with breast cancer susceptibility proteins BRCA1 and BRCA2, which play essential roles in homology-directed repair (HDR). More recently, PALB2 was shown to associate with active genes independently of BRCA1, and through this mechanism, safeguards these regions from DNA replicative stresses. However, it is unknown whether PALB2 tumour suppressor function requires its chromatin association. Methods: Mining the public database of cancer mutations, we identified four potentially deleterious cancer-associated missense mutations within the PALB2 chromatin association motif (ChAM). To assess the impact of these mutations on PALB2 function, we generated cell lines expressing PALB2 variants harbouring corresponding ChAM mutations, and evaluated PALB2 chromatin association properties and the cellular resistance to camptothecin (CPT). Additionally, we examined the accumulation of γH2A.X and the RAD51 recombinase as readouts of DNA damage signalling and HDR, respectively. Results: We demonstrate that a small-cell lung cancer (SCLC)-associated T413S mutation in PALB2 impairs its chromatin association and confers reduced resistance to CPT, the only FDA-approved drug for relapsed SCLC. Unexpectedly, we found a less efficient γH2A.X nuclear foci formation in PALB2 T413S expressing cells, whereas a near-normal level of RAD51 nuclear foci was visible. Conclusions: These findings support the importance of PALB2 chromatin association in the suppression of tumours, including SCLC, an unusually aggressive type of cancer with poor prognosis. PALB2 T413S has little impact on RAD51 recruitment, likely due to its intact interaction with BRCA1 and BRCA2. However, this mutant shows inefficient DNA stress signalling. This finding sheds new light on the function of PALB2, playing a role in efficient DNA stress signalling through constitutive chromatin association.

PALB2 chromatin recruitment restores homologous recombination in BRCA1-deficient cells depleted of 53BP1

“…NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14563-y ARTICLE expected 26,29 , in BRCA1/53BP1-proficient (WT) RPE1 cells, deletion of either the ChAM or MRG15-binding domain had a modest effect on PALB2 IRIF formation, whereas lack of both regions had a more pronounced effect (Fig. 3b, c and Supplementary Fig.…”

“…4g). Interestingly, cancer-associated mutations in this region have been previously reported to exhibit reduced chromatin association in chromatin fractionation studies 29 . In line with this, we observed that reported missense mutations 35 T413S, R414Q and S417Y, but not V425M, displayed reduced interaction with NCPs in vitro ( Supplementary Fig.…”

KAT2-mediated acetylation switches the mode of PALB2 chromatin association to safeguard genome integrity

“…While PALB2 7R appeared to maintain global chromatin association equivalent to PALB2 WT ( Fig. 4B and C), PALB2 function in gene protection requires its enriched association with a subset of actively transcribed genes (Bleuyard et al, 2017a). Markedly, our ChIP-qPCR revealed reduced enrichment of both PALB2 7Q and 7R at known PALB2-bound loci, namely the ACTB, TCOF1 and WEE1 genes ( Fig.…”

“…In light of our previous finding that steady-state PALB2 chromatin association is important for the protection of actively transcribed genes during DNA replication (Bleuyard et al, 2017a), we assessed cell cycle progression of U2OS-shPALB2 cells complemented with either PALB2 WT, 7Q and 7R. To this end, cells were arrested at G1/S boundary by double thymidine block, in the presence of doxycycline throughout (43 h), and were synchronously released into S phase.…”

“…PALB2 ChAM 7K-patch is required to maintain genome stability.ChIP-qPCR analysis defecting FLAG-PALB2 WT, 7Q and 7R enrichment at known PALB2-bound loci(Bleuyard et al, 2017a), namely the coding regions of ACTB (A), TCOF1 (B) and WEE1 (C) genes.…”