Selinexor-Induced Thrombocytopenia Results from the Inhibition of Megakaryocyte Progenitor Cells in the Early Stage of Megakaryopoiesis

Background

Selinexor (KPT-330), a selective inhibitor of nuclear export (SINE) Exportin 1 (XPO1, CRM1) antagonist, has shown potent activity against solid and hematological malignancies in phase 1 clinical trials. Inactivation of XPO1 by selinexor results in accumulation of tumor suppressor proteins in the nucleus and activation of cell cycle checkpoints. This leads to transient cell cycle arrest in normal cells and irreversible arrest and cell death in cancer cells. Thrombocytopenia is a common side effect of selinexor and we aim to define the mechanism of selinexor-induced thrombocytopenia to potentially help in its clinical management. In this study, we have examined the effects of selinexor on platelet count in humans and mice. Additionally, we investigated the effects of selinexor on differentiation and maturation of megakaryocytes (MKs) in culture as a model system for understanding the effects on platelet levels in humans.

Methods

Platelet counts were measured in solid tumor patients in a Phase 1 trial of selinexor QoDx2 weekly. For in vivo studies, CD1 mice were treated with high-dose selinexor (20 mg/kg QoDx3) for 3 weeks and platelet number was determined in peripheral blood. In addition, femur bone marrow samples were analyzed histologically. In vitro, MK progenitors were isolated from fetal mouse livers, and MK maturation and platelet production was analyzed by microscopy, flow cytometry and immunofluorescence. In addition, whole protein lysates from selinexor-treated MKs were analyzed to detect changes in XPO1 and its cargo proteins.

Results

A majority of solid tumor patients treated with selinexor QoDx2 weekly in a Phase 1 trial for at least 1 cycle (4 wks) had >40% reduction in platelet count within the first cycle (72%, N=50), with an average maximal reduction of 50% after 20 days. These reductions were independent of pre-dose platelet count, did not typically decrease further after the first cycle, and recovered following dose reduction or cessation of treatment. Also, administration of the thrombopoietin (TPO) receptor agonists romiplostim and eltrombopag were found to reduce selinexor mediated thrombocytopenia. As in the clinical studies, selinexor reduced the platelets in mice by 40-50% within 2-3 weeks. In studies of MKs in cell culture, selinexor inhibited XPO1 mediated nuclear export as shown by nuclear localization of IkBa and FOXO3a proteins, but was not cytotoxic to MKs or platelets in vitro and did not affect platelet activation. However, MK progenitor cell development was significantly blocked in a dose dependent fashion, with 200 nM selinexor inducing a reduction of 81% in the number of progenitor cells that differentiated into MKs. The same dose of selinexor also affected endomitosis, a marker of MK maturation, shifting ploidy from predominantly 16N to 2N. Importantly, these effects were substantially reversible. When selinexor was washed out after 6 hours of treatment, the number of progenitor cells that differentiated into MKs was 65%, accompanied by reversion to 16N ploidy. Interestingly, MK inhibition was decreased when cells were treated later in the differentiation process and treatment of mature MKs did not decrease pro-platelet formation or release, suggesting selinexor affected an earlier stage in MKs development.

Conclusions

Selinexor induces reductions in platelets in humans and mice. Our results suggest that this adverse effect is likely due to inhibition of the early commitment and differentiation phase of MK maturation from progenitor cells, and not a cytotoxic effect on normal stem cells or MKs. The reversibility of the effect in cell culture is congruent with the observations that selinexor-induced thrombocytopenia is reversible in humans upon dose interruption or reduction and/or the use of TPO receptor agonists by relieving the progenitor cell to MK differentiation block. Based on these results, the recommended Phase 2 dosing regimen of selinexor is twice weekly (days 1 and 3) for 3 weeks, followed by a 10-day dosing holiday and treatment with a TPO agonist if platelet counts are very low.