Priming of Megakaryopoiesis and Platelet Production through DNA Damage Repair Inhibition

Megakaryocytes (MKs) are large, hematopoietic cells that primarily reside in the bone marrow and release platelets into the circulation. MKs can reach extreme states of polyploidy (with a DNA content of up to 128n) and contain their nuclear lobes within a single nuclear membrane. While DNA replication in MKs is well studied, the impact of DNA damage and repair inhibition on megakaryopoiesis. To determine if modulating DNA damage within the hematopoietic stem cell and progenitor compartment affects megakaryocyte development, we employed a pharmacological approach using poly-ADP ribose polymerase inhibitors (PARPi). PARP, a sensor within the DNA damage repair pathway, binds to sites of DNA damage and recruits DNA repair effectors. PARPi, such as niraparib and olaparib, can trap PARP at site of lesions, leading to stalling of DNA replication forks and accumulation of DNA damage. Clinically, PARP inhibition is known to cause thrombocytopenia in patients, postulated to be due to effects on megakaryocytes and progenitors as platelets themselves do not have DNA. Higher dosing of PARPi in C57BL/6J mice mimicked this clinical phenotype. Surprisingly, treatment of C57BL/6J mice with lower doses of the FDA-approved PARP inhibitors niraparib and olaparib led to a doubling of the platelet count after 11 days. Flow cytometric analysis of niraparib-treated platelets revealed a larger and younger phenotype, as indicated by increased thiazole orange (TO) incorporation. Functional evaluation showed an increased sensitivity to GPVI stimulation as measured by CD62P expression and GPIIbIIIA activation. Cell surface expression of GPIIb, GPIIIA and GPVI were unaltered. Splenic weights of the mice were unaltered, decreasing the likelihood that the observed increase in platelets is due to splenic platelet pool mobilization. Together, these findings suggests that lower dose niraparib exposure in mice leads to increased functional platelets, by enhancing platelet production. To determine the root cause of this increased production, we evaluated the hematopoietic stem cell and progenitor compartment. Initially, we observed increased MKs after niraparib treatment via 2-photon intravital microscopy (2PIVM) in the calvaria of MK/platelet (vWF-GFP) reporter mice. These findings were validated by immunofluorescent visualization of MKs in femoral cryosections, revealing an increase in both the number, and size of MKs in niraparib-treated mice after 11 days of treatment. In vivo niraparib treatment also resulted in an increase in MK ploidy from day 3 to day 11, after which we observed a stark shift towards high ploidy MKs, at the expense of 2n MKs. While MK ploidy is not directly causative of platelet production, higher ploidy MKs are associated with increased platelet production and more terminal MK maturation. To directly analyze DNA damage in our cellular populations, we isolated native bone marrow MKs from niraparib- and vehicle-treated mice and performed comet assays. This indicated increased DNA damage in MKs isolated from niraparib-treated mice, as these cells showed significantly longer tail lengths when compared to control. In line with our comet assays, we then measured accumulation of phosphorylated histone H2 family member X (γ-H2AX), a marker of DNA breaks, by immunofluorescence. In situ, we observed a higher proportion of γ-H2AX positive MKs as well as more foci per MK after 11 days of treatment with niraparib. Evaluation of the bone marrow compartment revealed that after three days of niraparib treatment, and preceding the increase in platelet counts, there was a pronounced increase in the number of γ-H2AX-positive LT-HSCs, CD41+ HSCs, and MK progenitors (MkP), indicating increased DNA damage accumulation. In addition, after 11 days of treatment, there was an absolute increase in all multipotent progenitor populations (MPP2-4), LT-HSCs, MkPs and MKs. Together, these data suggest that low dose niraparib treatment causes DNA damage in HSPCs and MkPs and enhances canonical megakaryopoiesis, leading to increased platelet production.