Presentation Details
| DNA Damage Repair Inhibition: A Catalyst for Enhanced Megakaryopoiesis and Thrombopoiesis Roelof H Bekendam1, 2, Andrew Stone1, Clementine Payne1, Virginia Camacho1, Isabelle Becker1, Estelle Carminita1, Maria Barrachina1, Kellie Machlus1, Joseph Italiano1. 1Vascular Biology Program, Boston Children's Hospital, Boston, MA, USA.2Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Boston, MA, USA |
Abstract
Background: Megakaryocytes are large hematopoietic cells that can reach polyploid states greater than 64n. This is notable, as cancer cells with a polyploid DNA complement of even 4n are more susceptible to DNA damage accumulation. Poly-ADP-ribose-polymerase (PARP) inhibitors exhibit their anti-tumor effects in solid tumors through inducing DNA damage by interfering with DNA repair mechanisms. The bone marrow compartment is extremely susceptible to PARP inhibition. Clinically, higher dose PARP inhibitors have led to thrombocytopenia as a major dose-limiting toxicity. However, DNA damage accumulation can also induce stress hematopoiesis. To date, the role of low amounts of DNA damage accumulation on megakaryopoiesis has not been investigated.
Objectives: To investigate the role of DNA damage repair inhibition on megakaryocyte development and platelet production. We hypothesize that low dose PARP inhibition stimulates megakaryopoiesis through accumulation of DNA damage.
Methods: PARP inhibitors were administered to C57BL/6J or vWF-GFP reporter mice using intraperitoneal injection. Murine long bones and bone marrow were harvested for evaluation of hematopoietic stem and progenitor cell (HSPC) and megakaryocyte populations, megakaryocyte differentiation, polyploidization, and platelet formation. DNA damage was measured using antibodies against γH2AX - a marker of DNA strand breaks. Platelets were washed, followed by counting and analysis using flow cytometry.
Results: To assess DNA damage in megakaryocytes, immunofluorescence staining of γH2AX in femurs of C57BL/6J mice was performed in situ, showing a significant number of γH2AX foci in mature megakaryocytes. Flow cytometry revealed that γH2AX intensity increased with increasing ploidy in both murine and human megakaryocytes. To examine the effect of increasing DNA damage in megakaryocytes, C57BL/6J mice were treated with high and low doses of FDA-approved PARP inhibitors for both 3- and 11-day intervals. High dose PARP inhibition led to severe pancytopenia - consistent with the clinical phenotype. After 3 days of lower dose PARP inhibitor treatment, there was a significant expansion in CD41+-biased HSCs, the multipotent progenitor 2 cell population (MPP-2) and mature (CD41+ CD42d+) megakaryocytes. This correlated with increased γH2AX in long term HSCs and CD41+ HSCs, confirming accumulation of DNA damage. However, platelet counts were not changed. After 11 days, there was a significant decrease in CD41+ HSCs and MPP-2 cells in the PARP inhibitor treated group. Further, both megakaryocytes and (immature) platelets were elevated ~2-fold. Strikingly, there was a significant increase in >32n megakaryocytes in the PARP inhibitor treated group, suggesting enhanced DNA damage led to an increase in megakaryocyte maturation. To observe live platelet production by megakaryocytes in vivo, vWF-GFP reporter mice underwent 2-photon intravital microscopy in their calvaria after treatment with lower dose PARP inhibitors for 7 days. These mice showed increased megakaryocyte number, proplatelet production, and platelet counts. To test whether PARP inhibitors alter platelet structure/function we performed platelet activation assays, which confirmed normal functionality of platelets in the PARP inhibitor-treated group.
Conclusions: Our data suggest that DNA damage repair inhibition using PARP inhibitors increases murine thrombopoiesis via enhanced megakaryopoiesis. Further, the resulting megakaryocytes and platelets are functionally active. Targeting DNA damage repair pathways may represent a novel therapeutic strategy in thrombocytopenic states.
No part of this publication may be reproduced, distributed, or transmitted in any form or by any means, including photocopying, recording, or other electronic or mechanical methods, without the prior written permission of the author.
Objectives: To investigate the role of DNA damage repair inhibition on megakaryocyte development and platelet production. We hypothesize that low dose PARP inhibition stimulates megakaryopoiesis through accumulation of DNA damage.
Methods: PARP inhibitors were administered to C57BL/6J or vWF-GFP reporter mice using intraperitoneal injection. Murine long bones and bone marrow were harvested for evaluation of hematopoietic stem and progenitor cell (HSPC) and megakaryocyte populations, megakaryocyte differentiation, polyploidization, and platelet formation. DNA damage was measured using antibodies against γH2AX - a marker of DNA strand breaks. Platelets were washed, followed by counting and analysis using flow cytometry.
Results: To assess DNA damage in megakaryocytes, immunofluorescence staining of γH2AX in femurs of C57BL/6J mice was performed in situ, showing a significant number of γH2AX foci in mature megakaryocytes. Flow cytometry revealed that γH2AX intensity increased with increasing ploidy in both murine and human megakaryocytes. To examine the effect of increasing DNA damage in megakaryocytes, C57BL/6J mice were treated with high and low doses of FDA-approved PARP inhibitors for both 3- and 11-day intervals. High dose PARP inhibition led to severe pancytopenia - consistent with the clinical phenotype. After 3 days of lower dose PARP inhibitor treatment, there was a significant expansion in CD41+-biased HSCs, the multipotent progenitor 2 cell population (MPP-2) and mature (CD41+ CD42d+) megakaryocytes. This correlated with increased γH2AX in long term HSCs and CD41+ HSCs, confirming accumulation of DNA damage. However, platelet counts were not changed. After 11 days, there was a significant decrease in CD41+ HSCs and MPP-2 cells in the PARP inhibitor treated group. Further, both megakaryocytes and (immature) platelets were elevated ~2-fold. Strikingly, there was a significant increase in >32n megakaryocytes in the PARP inhibitor treated group, suggesting enhanced DNA damage led to an increase in megakaryocyte maturation. To observe live platelet production by megakaryocytes in vivo, vWF-GFP reporter mice underwent 2-photon intravital microscopy in their calvaria after treatment with lower dose PARP inhibitors for 7 days. These mice showed increased megakaryocyte number, proplatelet production, and platelet counts. To test whether PARP inhibitors alter platelet structure/function we performed platelet activation assays, which confirmed normal functionality of platelets in the PARP inhibitor-treated group.
Conclusions: Our data suggest that DNA damage repair inhibition using PARP inhibitors increases murine thrombopoiesis via enhanced megakaryopoiesis. Further, the resulting megakaryocytes and platelets are functionally active. Targeting DNA damage repair pathways may represent a novel therapeutic strategy in thrombocytopenic states.
No part of this publication may be reproduced, distributed, or transmitted in any form or by any means, including photocopying, recording, or other electronic or mechanical methods, without the prior written permission of the author.
Content Locked. Log into a registered attendee account to access this presentation.