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

Abstract

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.

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

“…( D , E ) Volcano plots demonstrating a significant number of CCT244747 effects in Eμ-Myc T505A lymphomas on both the total ( D ) and phospho ( E ) proteome, with both down- (red dots) and up-regulation (blue dots) being observed. Please note that figures ( B , C ), showing data from WT Eμ-Myc lymphomas are replicated in another manuscript [ 24 ], where they are used for comparison with data from Eμ-Myc /cRel −/− lymphomas.…”

“…Strikingly, comparatively few significant changes relative to WT cells were seen on the total and phosphoproteomes following acute CCT244747 treatment of RelA T505A Eμ-Myc lymphomas, with only 157 proteins and 315 phosphopeptides being differentially regulated ( P ≤ 0.05) ( Figure 5D,E , Supplementary Data File S1 ). Please note that the data from WT Eμ-Myc mice shown here is also used in our study on cRel −/− Eμ-Myc lymphomas [ 24 ]. These experiments were performed in parallel as part of the same larger study.…”

“…Western blot analysis confirmed reduced levels of Claspin protein in reimplanted Eμ-Myc/ Rela T505A lymphomas, while in Eμ-Myc/ cRel −/− extracts it was almost totally absent ( Supplementary Figure S9C ). In our parallel study, we have also observed reduced Claspin mRNA levels in isolates of the osteosarcoma cell line, U2OS that we have adapted over time to develop resistance to the CCT244747 [ 24 ].…”

“…This manuscript is one of four reports that together reveal both the complexity of NF-κB regulation of DNA replication stress and CHK1 signalling, as well as how defects in these pathways can lead to CHK1 inhibitor (CHK1i) resistance. Related to this manuscript we show elsewhere, also using the Eμ-Myc model of B-cell lymphoma, that deletion of the c-Rel NF-κB subunit results in loss of CHK1 protein, at least in part due to down-regulation of the deubiquitinase USP1 [ 24 ]. There, we propose that an important component of CHK1i resistance is loss of CHK1 itself.…”

“…The focus of this current manuscript and our report investigating resistance to CHK1 inhibition in the c-Rel null mouse model [ 24 ], are the mechanisms that lead to defects in CHK1 activity. This removal or alteration of the target of the CHK1i is an important component in the development of resistance but is not the only change these cells need to undergo.…”