Abstract
Postnatal hematopoietic stem (and progenitor) cells (HS(P)Cs) are especially vulnerable to oxidative stress, leading to early hematopoietic senescence and/or malignant transformation. Elevated intracellular reactive oxygen species (ROS) can, among others, oxidize nucleotides, and thus can result in genotoxicity and mutagenesis if left unrepaired. Oxidized bases, as well as other spontaneous single base modifications, are recognized and repaired by the base excision repair (BER) pathway. Hence, the BER pathway is crucial to maintain genome integrity. In contrast to other DNA repair pathways however, the role of BER in maintaining HSPC functionality remains enigmatic, chiefly because knockout (KO) of BER genes is in many cases embryonic lethal.
BER is a complex multi-step repair process. After initial removal and excision of the damaged base, the apurinic/apyrimidinic (AP) site is processed by the AP endonuclease (APEX1) enzyme. At this point, the BER pathway branches into 2 sub-pathways, namely the short-patch (SP-BER; wherein DNA polymerase beta (Polβ), Ligase III (Lig3) together with X-ray repair cross-complementing protein 1 (Xrcc1) are active) and the long-patch BER (LP-BER; wherein Lig1, Flap Structure-Specific Endonuclease 1 (Fen1), and sometimes Polβ are active) for the repair synthesis and the gap filling steps.
In this study we wished to address the role of BER in adult hematopoiesis. Therefore, we used CRISPR-Cas9 to KO different BER genes in adult bone marrow (BM) HS(P)Cs, including two genes common to the BER (sub-)pathway(s) (Apex1 and Polβ) as well as one gene in the SP-BER (Xrcc1) and one gene in the LP-BER (Lig1) pathway. The effect thereof was evaluated on HS(P)C repopulation in vivo as well as on HS(P)C expansion during long-term in vitro culture (using the culture medium described by Wilkinson et al., Nature 2019). All CRISPR-Cas9 experiments were validated using a second sgRNA targeting the selected BER genes.
Lig1-KO caused in vivo HSPC dysfunction: at 20 weeks post-transplantation, significantly less Lig1 KO cells were observed in the committed progenitor (HPC) and lineage committed (Lin +) BM compartments. By contrast, KO of Xrcc1 had only minor effects on HS(P)C repopulation, but we observed increased HSC expansion and myeloid biased differentiation in some recipient mice, which might correspond to clonal hematopoiesis and is consistent with the finding of XRCC1 loss-of-function mutation in myelodysplastic patients (Joshi et al, Ann Hematol 2016). Knockout of Polβ did not affect hematopoiesis in vivo or in vitro. The most severe phenotype was observed when we knocked out Apex1, as Apex1-KO HS(P)Cs failed to repopulate irradiated recipient mice. Already after 2 weeks, significantly less Apex1 deficient cells were detected in the different blood lineages and nearly no CRISPR-Cas9 KO cells could be detected from 4 weeks onwards. This was confirmed in vitro, where reduced expansion of Apex1 KO BM cells was observed.
APEX1 has two major functional activities, namely its nuclease activity, involved in BER, and its redox activity (also called Ref-1 function) important in reducing oxidized transcription factors and therefore implicated in transcriptional regulation. However, little is known regarding the nuclease and Ref-1 function(s) in primary adult hematopoietic cells. We therefore cultured BM HS(P)Cs for 1 week in the continuous presence of 2 distinct chemicals blocking the APEX1 nucleases, or 2 different chemicals inhibiting specifically the Ref-1 function. We demonstrated that both APEX1 functions are essential for hematopoiesis, even if the 2 functions appear to support the survival, expansion and maintenance of HS(P)Cs through different mechanisms. While the Ref-1 function was essential for proliferation (as both Ref-1 inhibitors cause cell cycle arrest) of all the lineages (including the Lin + cells), both inhibitors of the nuclease function affected more the expansion/survival of the less committed HS(P)Cs without leading to any cell cycle arrest.
In conclusion, this study demonstrates for the first time the important role of BER genes in adult hematopoiesis, often deregulated in cancer, including hematopoietic malignancies. We observed a particularly severe phenotype upon loss of Apex1 in adult HSPCs, and ongoing studies (such as RNA sequencing analysis) should provide novel insights in underlying mechanisms of APEX1 deficiencies in HS(P)Cs.
Figure 1 Figure 1.
Disclosures
No relevant conflicts of interest to declare.
Nucleolar accumulation of APE1 depends on charged lysine residues that undergo acetylation upon genotoxic stress and modulate its BER activity in cells