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CHK1 Inhibitor Blocks Phosphorylation of FAM122A and Promotes Replication Stress

ABSTRACT: While effective anti-cancer drugs targeting the CHK1 kinase are advancing in the clinic, drug resistance is rapidly emerging. Here, we demonstrate that CRISPR-mediated knockout of the little-known gene FAM122A confers cellular resistance to CHK1 inhibitors and cross-resistance to ATR inhibitors. Knockout of FAM122A results in activation of PP2A-B55α, a phosphatase which dephosphorylates the WEE1 protein and rescues WEE1 from Ubiquitin-mediated degradation. The resulting increase in WEE1 protein expression reduces replication stress, activates the G2/M checkpoint, and confers cellular resistance to CHK1 inhibitors. Interestingly, in tumor cells with oncogene-driven replication stress, CHK1 can directly phosphorylate FAM122A, leading to activation of the PP2A-B55α phosphatase and increased WEE1 expression. A combination of a CHK1 inhibitor plus a WEE1 inhibitor can overcome CHK1 inhibitor resistance of these tumor cells, thereby enhancing anti-cancer activity. The FAM122A expression level in a tumor cell can serve as a useful biomarker for predicting CHK1 inhibitor sensitivity or resistance.

ORGANISM(S): Homo sapiens

PROVIDER: GSE158338 | GEO | 2021/01/01

REPOSITORIES: GEO

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Project description:The Ser/Thr protein phosphatase 2A (PP2A) is a highly conserved collection of heterotrimeric holoenzymes responsible for the dephosphorylation of many regulated phosphoproteins. Substrate recognition and the integration of regulatory cues are mediated by B regulatory subunits that are complexed to the catalytic subunit (C) by a scaffold protein (A). PP2A/B55 substrate recruitment was thought to be mediated by charge-charge interactions between the surface of B55a and its substrates. Challenging this view, we recently discovered a conserved SLiM [RK]-V-x-x-[VI]-R in a range of proteins, including substrates such as the retinoblastoma-related protein p107 and TAU (Fowle et al. eLife 2021;10:e63181). Here we report the identification of this SLiM in FAM122A, an inhibitor of B55a/PP2A. This conserved SLiM is necessary for FAM122A binding to B55a in vitro and in cells. Computational structure prediction with AlphaFold-Multimer predicts an interaction consistent with the mutational and biochemical data and supports a mechanism whereby FAM122A uses the 'SLiM' in the form of a short a-helix (helix 1) to dock to the B55a top groove, thereby blocking substrate binding to the same site. In this model, FAM122A also spatially constrains substrate access by occluding the catalytic subunit with a second a-helix immediately adjacent to helix 1. Consistently, FAM122A functions as a competitive inhibitor as it prevents the binding of substrates in in vitro competition assays and the dephosphorylation of CDK substrates by B55a/PP2A in cell lysates. The ablation of FAM122A in human cell lines reduces the rate of proliferation, progression through cell cycle transitions, and abrogates G1/S and intra-S phase cell cycle checkpoints. FAM122A-KO in HEK293 cells results in attenuation of CHK1 and CHK2 activation in response to replication stress. Overall, these data strongly suggest that FAM122A is a short helical motif (SHeM)-dependent, substrate-competitive inhibitor of B55a/PP2A that suppresses multiple functions of B55a in the DNA damage response and in timely progression through the cell cycle interphase.

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CHK1 Inhibitor Blocks Phosphorylation of FAM122A and Promotes Replication Stress

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