Mutation of the RelA(p65) Thr505 phosphosite disrupts the DNA replication stress response leading to CHK1 inhibitor resistance

Hunter, Jill E.; Campbell, Amy E.; Butterworth, Jacqueline; Sellier, Hélène; Hannaway, Nicola; Luli, Saimir; Floudas, Achilleas; Kenneth, Niall S.; Moore, Adam J.; Brownridge, Philip; Thomas, Huw D.; Coxhead, Jonathan; Taylor, Leigh; Leary, Peter; Hasoon, Megan; Knight, Andrew M.; Garrett, Michelle D.; Collins, Ian; Eyers, Patrick A.; Perkins, Neil D.

doi:10.1042/bcj20220089

Cited by 9 publications

(60 citation statements)

References 65 publications

(115 reference statements)

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“…We also observed loss of CLSPN in these extracts [19], in agreement with our proteomics data which revealed ∼1.6-fold lower levels (P-value = 5.36 × 10 −4 ). Levels of the checkpoint kinase CHK2, which functions downstream of ATM in response to double strand DNA breaks, appeared broadly comparable, albeit variable, in the untreated Em-Myc WT and Em-Myc/cRel −/− lymphoma cells.…”

Section: Analysis Of Rna Seq Data From Em-myc Lymphoma Cellssupporting

confidence: 92%

“…Our analysis of this data demonstrated a significant number of CCT244747 effects in WT Em-Myc lymphomas, with 622 proteins and 625 phosphopeptides exhibiting a significant up-or down-regulation (P-value ≤0.05) (Supplementary Figure S2B,C, also shown in [19]). Strikingly, in comparison, relatively few significant changes were seen on the total and phospho proteomes following acute CCT244747 treatment of Em-Myc/ cRel −/− lymphomas, with only 162 proteins and 89 phosphopeptides being significantly differentially regulated (P-value ≤0.05) (Figure 2A,B).…”

Section: Em-myc Lymphomas Lacking C-rel Exhibit Altered Cell Signalli...mentioning

confidence: 81%

“…We and others have shown that there is significant cross-talk, between the ATR-CHK1 and NF-κB pathways. For example, phosphorylation of the putative CHK1 Thr 505 (T505) phosphosite in the RelA transactivation domain in vitro results in inhibition of tumour promoting activities of NF-κB, including resistance to apoptosis, autophagy, cell proliferation and cell migration [9][10][11][17][18][19]. Direct phosphorylation of the p50 NF-κB subunit on Ser 329 by CHK1 was demonstrated following DNA damage [20,21].…”

Section: Introductionmentioning

confidence: 99%

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Regulation of CHK1 inhibitor resistance by a c-Rel and USP1 dependent pathway

Hunter

Campbell

Hannaway

et al. 2022

Biochemical Journal

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Previously, we discovered that deletion of c-Rel in the Eµ-Myc mouse model of lymphoma results in earlier onset of disease, a finding that contrasted with the expected function of this NF-κB subunit in B-cell malignancies. Here we report that Eµ-Myc/cRel−/− cells have an unexpected and major defect in the CHK1 pathway. Total and phospho proteomic analysis revealed that Eµ-Myc/cRel−/− lymphomas highly resemble wild-type (WT) Eµ-Myc lymphomas treated with an acute dose of the CHK1 inhibitor (CHK1i) CCT244747. Further analysis demonstrated that this is a consequence of Eµ-Myc/cRel−/− lymphomas having lost expression of CHK1 protein itself, an effect that also results in resistance to CCT244747 treatment in vivo. Similar down-regulation of CHK1 protein levels was also seen in CHK1i resistant U2OS osteosarcoma and Huh7 hepatocellular carcinoma cells. Further investigation revealed that the deubiquitinase USP1 regulates CHK1 proteolytic degradation and that its down-regulation in our model systems is responsible, at least in part, for these effects. We demonstrate that treating WT Eµ-Myc lymphoma cells with the USP1 inhibitor ML323 was highly effective at reducing tumour burden in vivo. Targeting USP1 activity may thus be an alternative therapeutic strategy in MYC-driven tumours.

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Section: Analysis Of Rna Seq Data From Em-myc Lymphoma Cellssupporting

confidence: 92%

Section: Em-myc Lymphomas Lacking C-rel Exhibit Altered Cell Signalli...mentioning

confidence: 81%

Section: Introductionmentioning

confidence: 99%

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Regulation of CHK1 inhibitor resistance by a c-Rel and USP1 dependent pathway

Hunter

Campbell

Hannaway

et al. 2022

Biochemical Journal

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“…Parallel studies using the Eμ-Myc mouse model of B-cell lymphoma demonstrated that low levels of Clspn mRNA were associated with significantly reduced overall survival times. Mice with low levels of Clspn transcript had a median survival of 82 days compared with 138 days for animals expressing higher levels of Clspn in this model ( Figure 1A and [ 12 ]). This suggests that high levels of Claspin are protective and delay onset of disease in this particular mouse model.…”

Section: Resultsmentioning

confidence: 99%

“…Overall survival is significantly shorter in Eμ-Myc mice with low levels of Claspin transcript (** P = 0.005Mantel–Cox test and hazard ratio (HR) analysis indicate that these mice are at 3 times greater risk of dying earlier due to lymphoma compared with Eμ-Myc mice with high levels of Claspin transcript. Please note that this plot is also used in another study [ 12 ], Figure 8C. ( B ) Table summarising Kaplan–Meier plot analysis to determine the correlation of CLSPN expression levels with human cancer patient survival.…”

Section: Resultsmentioning

confidence: 99%

Claspin haploinsufficiency leads to defects in fertility, hyperplasia and an increased oncogenic potential

Madgwick

Luli

Sellier

et al. 2022

Biochemical Journal

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Claspin is an adaptor protein required for ATR-dependent phosphorylation of CHK1 during S-phase following DNA replication stress. Claspin expression is highly variable in cancer, with low levels frequently correlating with poor patient survival. To learn more about the biological consequences of reduced Claspin expression and its effects on tumorigenesis, we investigated mice with a heterozygous knockout of the Clspn gene. Claspin haploinsufficiency resulted in reduced female fertility and a maternally inherited defect in oocyte meiosis I cell cycle progression. Furthermore, aged Clspn+/− mice developed spontaneous lymphoid hyperplasia and increased susceptibility to non-alcoholic fatty liver disease. Importantly, we demonstrate a tumour suppressor role for Claspin. Reduced Claspin levels result in increased liver damage and tumourigenesis in the DEN model of hepatocellular carcinoma. These data reveal that Clspn haploinsufficiency has widespread unanticipated biological effects and establishes the importance of Claspin as a regulatory node controlling tumorigenesis and multiple disease aetiologies.

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The pathophysiology of the cell cycle in cancer and treatment strategies using various cell cycle checkpoint inhibitors

Almalki

2023

Pathology - Research and Practice

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Mutation of the RelA(p65) Thr505 phosphosite disrupts the DNA replication stress response leading to CHK1 inhibitor resistance

Cited by 9 publications

References 65 publications

Regulation of CHK1 inhibitor resistance by a c-Rel and USP1 dependent pathway

Regulation of CHK1 inhibitor resistance by a c-Rel and USP1 dependent pathway

Claspin haploinsufficiency leads to defects in fertility, hyperplasia and an increased oncogenic potential

The pathophysiology of the cell cycle in cancer and treatment strategies using various cell cycle checkpoint inhibitors

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