From in silico to in Vitro to In Vivo: Increasing Megakaryocytes to Treat Thrombocytopenia

Hematopoietic stem cells undergo a multi-step differentiation program through several precursor stages to give rise to megakaryocytes (Mk), which ultimately release platelets. Thrombocytopenia can manifest in various clinical settings and can adversely affect a patient's quality of life through easy bruising and risk of bleeding. An example is chemotherapy-induced thrombocytopenia (CIT) which occurs when myelosuppressive chemotherapy is given, especially with certain agents such as platinum analogs and cyclophosphamide. We used a single-cell RNA-sequencing approach to study the differentiation of hematopoietic stem cells to Mk progenitors (MkP) and delineated the trajectory that defines the transition of cells to the megakaryocytic lineage. Using our proprietary platform which pairs new approaches in computation and machine learning in drug discovery, we predicted small molecule interventions that could modulate the hematopoietic lineage direction to increase the differentiation towards MkP and subsequent increases in platelet counts. Using an in vitro 5-day CD34-based Mk differentiation assay we demonstrated that some of our predicted interventions increased generation of MkP from stem cells. Analysis of early MkP (CD34-CD235a-CD71+CD41b+CD42b-) and late MkP (CD34-CD235a-CD71-CD41b+CD42b+) by flow cytometry elucidated differential mechanisms of action such as induction of early or late MKP or both. We performed an 8-day study in healthy mice, which revealed that a selected set of compounds increased MkPs in vivo, consistent with our predicted cell behavior and in vitro findings.

To evaluate a possible application for this MkP and platelet increases we tested the efficacy of one of our hit compounds CP3 in a CIT mouse model using Carboplatin. In this model, we recorded a >70 % reduction in platelet counts on day 9 post carboplatin administration, with platelet numbers recovering on day 13 and return to basal levels by day 21. To assess the efficacy of CP3, mice dosed first with this compound were challenged with carboplatin. The primary endpoint of this study was the ability of the compound to rescue mice from nadir of thrombocytopenia and accelerate recovery to baseline. Prior to carboplatin dosing, 7 days of CP3 dosing showed a dose dependent increase in platelet counts confirming our initial studies. However, CP3 dosing did not rescue platelet nadir or accelerate recovery to baseline in the CIT model. This study demonstrated the capacity of CP3 to increase platelets in a healthy BM environment but not under conditions of myelosuppression as seen in the CIT mouse model.

Taken together, these findings highlight the benefits of our platform to predict compounds that modulate lineage differentiation towards MkP, and a resultant increase in platelet numbers in an animal model.