PB0378 - DNA Damage Accumulation in Polyploid Megakaryocytes: Investigating the Impact of Poly (ADP-ribose) polymerase (PARP) Inhibitors

Background: Megakaryocytes are large hematopoietic cells that can reach ploidy levels greater than 64n. The potential for DNA damage accumulation in polyploid megakaryocytes is of interest as cancer cells with a DNA complement of even 4N are more susceptible to DNA damage. Poly (ADP-ribose) polymerase (PARP) inhibitors exhibit anti-tumor effects in solid tumors through induction of single stranded DNA breaks, ultimately leading to double stranded breaks and cell death. Hematological side effects including thrombocytopenia are the main dose-limiting toxicity in the clinic. However, the molecular mechanisms behind this thrombocytopenia remain largely unknown.

Aims: Investigate the potential for DNA damage accumulation in megakaryocytes and understand the effect of PARP inhibitor-induced thrombocytopenia by assessing megakaryocytic function and platelet production.

Methods: Immunofluorescence staining of yH2AX, a marker of DNA damage, was performed on native and in-vitro matured murine and human megakaryocytes. The effect of three clinically relevant PARP inhibitors was assessed on platelet functional assays and in vitro megakaryopoiesis. Finally, PARP inhibitors were administered to mice to investigate how they affect megakaryocyte differentiation, polyploidization, and platelet formation.

Results: Analysis of wildtype murine femurs revealed that megakaryocytes were positive for yH2AX staining (Figure 1). Human megakaryocytes, differentiated from bone marrow derived CD34+ cells, similarly showed yH2AX foci. Increased megakaryocyte yH2AX was associated with higher ploidy. Addition of PARP inhibitors to murine lineage depleted bone marrow cells resulted in reduced megakaryopoiesis (Figure 2A) and decreased megakaryocyte PARylation. Platelet function, however, remained intact (Figure 2B-C). In vivo application of PARP inhibitors to healthy C57BL/6J mice led to a significant shift toward higher ploidy megakaryocytes (>32n).

Conclusion(s): Our data provide evidence that higher ploidy correlates with increased DNA damage and PARP inhibitors may exert their hematological toxicity through increasing DNA damage to megakaryocytes. Further study will clarify the effect of DNA damage accumulation and repair on platelet production in megakaryocytes.