Spi-1/PU.1 Accelerates Replication Fork Elongation through PP1a Phosphatase-Associated Dephosphorylation of CHK1 in Erythroleukemic Cells

The multistage process of leukemic formation is driven by the progressive acquisition of somatic mutations. Replication stress creates genomic instability in mammals. Oncogenes and tumor suppressors trigger a replicative stress, that will consequently participates to the progression of cancer partly through this increased genetic instability. Thus, determining how cells cope with replicative stress should help understanding leukemogenesis and lead to the identification of new targets for antitumor treatments. Using a multistep leukemia model driven by Spi-1/PU.1 overexpression, we investigated the relationship between DNA replication and cancer progression. We have previously identified that the constitutive overexpression of the oncogenic transcription factor Spi-1/PU.1 is associated with an increased speed of DNA chain elongation favoring genetic instability without inducing DNA strand breaks. The Spi-1-induced replicative stress is peculiar because, in contrast to most of stress triggered by oncogenes or tumor suppressor, it is associated with an increase fork progression speed instead of alteration in the program of origin firing.

Here, we bring evidence that the S phase checkpoint protein, CHK1, is maintained in the inactive dephosphorylated form in Spi-1/PU.1 overexpressing pre-leukemic cells inducing and/or maintaining the observed high speed of DNA chain elongation. CHK1 under-phosphorylation is not due to defects in ATR signaling, its main regulator. Moreover, pharmacological inhibition of the kinases, ATM and DNA-PK, did not decrease CHK1 phosphorylation in the preleukemic cells overexpressing Spi-1. These findings are not consistent with an involvement of DNA damage response kinases in Spi-1-mediated modulation of CHK1 phosphorylation.We found that PP1a expression is increased in Spi-1/PU.1 overexpressing pre-leukemic cells compared to cells in which Spi-1/PU.1 was down-regulated. Two functional assays bring arguments that PP1 activity mediates the Spi-1/PU.1 effect on CHK1 dephosphorylation. Indeed, inhibition of PP1activity in cells overexpressing Spi-1 promoted the phosphorylation of CHK1, while the overexpression of PP1a led to the loss of a correlation between CHK1 phosphorylation and Spi-1 expression. In addition, PP1a inhibition and overexpression, not only acted on the CHK1 phosphorylation status controlled by Spi-1 but also inversely modified the progression of replication. Altogether, these results support the existence of a pathway linking Spi-1/PU.1 expression to acceleration of DNA replication via a PP1-mediated control of CHK1 phosphorylation in the pre-leukemic cells. These results identified a new pathway by which an oncogene influences replicative stress and favors the leukemic progression by fostering the incidence of genomic instability.