Thrombocytopenia Induced by Combination Administration of Romidepsin and Bortezomib In Mice Is Rapidly Reversible After Drug Treatment

The proteasome inhibitor bortezomib and the histone deacetylase inhibitor (HDACi) romidepsin are both able to induce tumor cell death in single-agent clinical studies. In vitro studies using the combination of bortezomib and romidepsin have demonstrated synergistic activity against tumor cell lines, and clinical trials using both classes of agents have demonstrated efficacy. Bortezomib and romidepsin induce thrombocytopenia that is transient and reversible. It is thought that these agents temporarily disrupt the process of platelet budding from megakaryocytes, in contrast to long-term thrombocytopenia induced by classical cytotoxic chemotherapeutic agents that deplete the megakaryocyte population. However, thrombocytopenia has been a dose-limiting factor when proteasome inhibition is combined with HDACi. We have previously shown that platelet recovery after bortezomib exposure in mice results in transient high elevation of circulating platelets and that the megakaryocyte population is preserved, with increased numbers of immature forms suggesting a rebound effect (Lonial et al., Blood, 2005). Here, we conducted additional murine studies to determine whether combination bortezomib + romidepsin induces increased thrombocytopenia compared to the single-agent drugs, examine platelet recovery kinetics, and test effects on bone marrow megakaryocytes.

Six groups of 12 female BALB/c mice were used: Control, 1mg/kg bortezomib, 2mg/kg bortezomib, 1mg/kg romidepsin, 2mg/kg romidepsin, and the combination of 1mg/kg bortezomib + 1mg/kg romidepsin. Only one injection timepoint was used (d1, via tail vein) to narrow the focus of thrombocytopenic effects. Three mice per group were bled on subsequent days (to d11) for CBC including WBC and platelet counts. Plasma samples were frozen for batch analysis of thrombopoietin (TPO) levels by ELISA. Subgroups of 2–3 mice were sacrificed at d3, 5, and 8 for analysis of bone marrow megakaryocyte ploidy. Bone marrow cells were fixed in ethanol and analyzed by flow cytometry to determine the representation of CD41-positive megakaryocytes in the different 7-AAD ploidy categories based upon 7-AAD-stained DNA content. Bone marrow H&E histological analysis of marrow megakaryocyte content is ongoing.

WBC counts for controls and mice treated with bortezomib alone were at baseline values or greater during the course of the experiment. Mice treated with romidepsin, or the combination of bortezomib + romidepsin, showed an initial drop in d2 WBC counts to 50% of baseline. The platelet nadir for most groups was at d3, with very similar counts for all single-agent groups near 500 × 10³ per mcL (Figure 1, bars represent standard deviation). Counts for the groups treated with 2mg/kg bortezomib or romidepsin were only slightly lower than for groups treated with 1mg/kg. However, the average day 3 platelet count for the combination group was significantly lower at 250 × 10³ per mcL (Figure 1, * indicates p=0.0018). Platelet recovery was very rapid in the mice that received bortezomib alone, with d6 levels 150% of baseline. Platelets remained low in romidepsin-treated mice until d5 and then began increasing. Interestingly, platelet counts in the combination group also remained low until d5, similar to romidepsin alone, but then rose rapidly with kinetics reminiscent of the rebound seen in the bortezomib-treated groups (Figure 1). On d3, thrombocytopenia was associated with an increase in plasma TPO, especially in the bortezomib + romidepsin group, indicating that platelet reduction induced by the combination of both agents is not the result of TPO suppression. At d5 and d8, drug-treated groups had lower percentages of 8N/16N megakaryocytes and corresponding increases in the 2N/4N category, suggesting a megakaryocyte rebound effect in response to thrombocytopenia.

The combination of bortezomib + romidepsin induces more profound thrombocytopenia in mice compared to either drug alone, but platelets recover to baseline levels or greater by 7 days after treatment. The platelet recovery phase is characterized by a rapid increase in platelet counts exceeding baseline values, similar to the rebound effect induced by bortezomib alone. This suggests that the platelet-producing megakaryocyte population is maintained after bortezomib + romidepsin treatment.

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Nix: Gloucester, Celgene: Consultancy, Employment. Lonial: Gloucester, Celgene: Consultancy, Research Funding.