Up-regulation of the PI3K/AKT and RHO/RAC/PAK signalling pathways in CHK1 inhibitor resistant Eµ-Myc lymphoma cells

Abstract

The development of resistance and the activation of bypass pathway signalling represents a major problem for the clinical application of protein kinase inhibitors. While investigating the effect of either a c-Rel deletion or RelAT505A phosphosite knockin on the Eµ-Myc mouse model of B-cell lymphoma, we discovered that both NF-κB subunit mutations resulted in CHK1 inhibitor resistance, arising from either loss or alteration of CHK1 activity, respectively. However, since Eµ-Myc lymphomas depend on CHK1 activity to cope with high levels of DNA replication stress and consequent genomic instability, it was not clear how these mutant NF-κB subunit lymphomas were able to survive. To understand these survival mechanisms and to identify potential compensatory bypass signalling pathways in these lymphomas, we applied a multi-omics strategy. With c-Rel−/− Eµ-Myc lymphomas we observed high levels of Phosphatidyl-inositol 3-kinase (PI3K) and AKT pathway activation. Moreover, treatment with the PI3K inhibitor Pictilisib (GDC-0941) selectively inhibited the growth of reimplanted c-Rel−/− and RelAT505A, but not wild type (WT) Eµ-Myc lymphomas. We also observed up-regulation of a RHO/RAC pathway gene expression signature in both Eµ-Myc NF-κB subunit mutation models. Further investigation demonstrated activation of the RHO/RAC effector p21-activated kinase (PAK) 2. Here, the PAK inhibitor, PF-3758309 successfully overcame resistance of RelAT505A but not WT lymphomas. These findings demonstrate that up-regulation of multiple bypass pathways occurs in CHK1 inhibitor resistant Eµ-Myc lymphomas. Consequently, drugs targeting these pathways could potentially be used as either second line or combinatorial therapies to aid the successful clinical application of CHK1 inhibitors.

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

“…While knockdown of CLSPN itself reduced the number of surviving U2OS clones by ∼50%, these cells were now CCT244747 resistant ( Figure 8E ). Although other factors are likely to be involved both in the earlier onset of lymphoma and CCT244747 resistance we see in RelA T505A Eμ-Myc lymphomas (see below and [ 24 , 26 ]), these data suggest that a reduction in Claspin expression makes a significant contribution towards these phenotypes ( Figure 9 ).…”

“…EnrichGO analysis of putative RelA regulated genes and down-regulated mRNAs in RelA T505A cells did not reveal any functional groups with a q -value < 0.05. In our parallel report examining up-regulated compensatory bypass pathways in CCT244747 resistant Eμ-Myc lymphomas we examine in more detail the up-regulated mRNAs in RelA T505A cells [ 26 ].…”

“…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. In the final paper in this series, we bring the Eμ-Myc RelA T505A and c-Rel −/− models together to consider how these lymphomas cope with these defects in CHK1 signalling, which in wild-type Eμ-Myc lymphomas is required for the survival [ 26 ]. We demonstrate that both models have up-regulated compensatory signalling pathways.…”

“…We demonstrate that both models have up-regulated compensatory signalling pathways. Moreover, we show that Eμ-Myc RelA T505A and c-Rel −/− lymphomas, while resistant to CHK1 inhibition are now sensitive to targeting these bypass pathways [ 26 ]. These results have implications for how CHK1i resistance might arise in human patients and, importantly, suggest potential combination or second line therapies to overcome this.…”

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

“…In this paper, genetic deletion of the c-Rel NF-κB subunit leads to loss of USP1 and consequently CHK1 expression, resulting in inactivation of this pathway. However, loss of this pathway can be overcome by activation of compensatory bypass pathways (not shown), which is described in Hunter et al [38]. Figure partially created using Biorender.…”