Selinexor-induced thrombocytopenia results from inhibition of thrombopoietin signaling in early megakaryopoiesis

Abstract

Selinexor is the first oral selective inhibitor of nuclear export compound tested for cancer treatment. Selinexor has demonstrated a safety therapy profile with broad antitumor activity against solid and hematological malignancies in phases 2 and 3 clinical trials (#NCT03071276, #NCT02343042, #NCT02227251, #NCT03110562, and #NCT02606461). Although selinexor shows promising efficacy, its primary adverse effect is high-grade thrombocytopenia. Therefore, we aimed to identify the mechanism of selinexor-induced thrombocytopenia to relieve it and improve its clinical management. We determined that selinexor causes thrombocytopenia by blocking thrombopoietin (TPO) signaling and therefore differentiation of stem cells into megakaryocytes. We then used both in vitro and in vivo models and patient samples to show that selinexor-induced thrombocytopenia is indeed reversible when TPO agonists are administered in the absence of selinexor (drug holiday). In sum, these data reveal (1) the mechanism of selinexor-induced thrombocytopenia, (2) an effective way to reverse the dose-limiting thrombocytopenia, and (3) a novel role for XPO1 in megakaryopoiesis. The improved selinexor dosing regimen described herein is crucial to help reduce thrombocytopenia in selinexor patients, allowing them to continue their course of chemotherapy and have the best chance of survival. This trial was registered at www.clinicaltrials.gov as #NCT01607905.

Conflict of interest statement

Conflict-of-interest disclosure: E.S., T.J.U., T.K., B.K., M.C., and Y.L. are Karyopharm Therapeutics employees. J.E.I. has financial interest in and is a founder of Platelet BioGenesis, a company that aims to produce donor-independent human platelets from human-induced pluripotent stem cells at scale. J.E.I. is an inventor on this patent. The interests of J.E.I. were reviewed and are managed by the Brigham and Women’s Hospital and Partners HealthCare in accordance with their conflict-of-interest policies. The remaining authors declare no competing financial interests.

© 2017 by The American Society of Hematology.

Figures

Figure 1.

Selinexor affects the platelet counts of patients with solid tumors in a dose-dependent manner. (A) Absolute platelet count as a function of platelet count at baseline for patients treated with 7 to 8 high doses of selinexor (>100 mg) in the first 28-day cycle (N = 28). (B) Absolute platelet count as a function of platelet count at baseline for patients treated with 7 to 8 low doses of selinexor (50-70 mg) in the first 28-day cycle (N = 36). Each point represents the platelet count of an individual patient. The regression lines were derived from the average platelet count loss from baseline (slope values 0.24 and 0.53, R2 = 0.31 and R2 = 0.71, respectively). (C) Average (±SD) change in platelet count as function of time for patients treated with 7 to 8 doses of selinexor (30-145 mg) per 28-day cycle. The number of patients for each time point is listed on the graph.

Figure 2.

Selinexor does not affect MK apoptosis, proplatelet formation, or platelet activation. (A-B) Mature, round mouse MKs derived from murine fetal livers were treated with increasing doses of selinexor. Six hours later, viability of the MKs and the number of proplatelet-producing MKs were quantified. Bars represent 20 μm; n = 3. Mature donor human platelets were treated with increased concentrations of selinexor in the absence (C) or the presence (D) of thrombin, and platelet surface P-selectin was analyzed by fluorescence-activated cell sorter (FACS). Representative images of resting (C) and activated (D) platelets stained with actin (phalloidin, red) and anti-β1-tubulin antibody (green). Bars represent 2 μm; n = 3. DMSO, dimethyl sulfoxide.

Figure 3.

Selinexor treatment of murine fetal liver cells inhibits mature MK formation. Murine fetal liver cells were treated with selinexor at indicated doses on D1 of maturation, and the MK population was quantified with FACS (A) and visualized under the microscope on D4 (B). ****P < .0001 vs DMSO control. Bars represent 50 μm; n = 4. (C-E) Murine fetal liver cells were dosed with selinexor at D1, D2, or D3 of maturation either continuously until D4 (red bars) or selinexor was washed out after 6 hours (blue bars). On D4, the MK population was quantified with FACS. Values were normalized to DMSO control for each biological replicate, and then replicates averaged. *P < .05; **P < .01; ***P < .001; ****P < .0001 vs DMSO control; n = 3. (F) Human CD34+ peripheral blood cells were continuously treated with selinexor beginning on D3 of maturation at the indicated doses, and the number of mature MKs was quantified by FACS based on CD41/61 positivity on D6, D10, and D14. *P < .05; n = 4.

Figure 4.

MKs that overcome selinexor treatment have normal ploidy and morphology. Murine fetal liver cells were dosed with selinexor at D1 of maturation either continuously until D4 (A) or selinexor was washed out after 6 hours (B). On D4, the ploidy of the resulting MK population was quantified with FACS. *P < .05 vs vehicle control (DMSO); n = 3. (C) Representative transmission electron microscopy images of MKs showing polyploid nucleus (N), demarcation membrane (DMS), and granules (*). Bars represent 2 μM.

Figure 5.

Selinexor reduced blood cell counts in mice and the number of MKs in the bone marrow. (A) CD1 mice (N = 4 per cohort) were treated with saline vehicle (gray bars) or selinexor (red bars, 20 mg/kg by mouth, every other day, three times per week) for 3 weeks. Platelet counts were done at the end of each week of treatment. (B) Quantification of MKs in femur bone marrow sections. Images spanning 1 complete longitudinal cross section of the entire femur from 2 femurs per mouse were used for quantification. Each mouse (2 femurs) was considered 1 biological replicate. N = 4 per cohort; ***P < .001. (C) Representative hematoxylin and eosin images (×20) and high (×20) and low (×10) magnification immunofluorescence staining for von Willebrand factor showing MKs in bone marrow. N = 4 mice per cohort; bars represent 10 μm.

Figure 6.

Selinexor treatment inhibits TPO signaling in vitro. D1 cells derived from murine fetal livers were incubated with selinexor at indicated doses. (A) Apoptosis was measured by annexin V positivity in all cells by FACS on D4; n = 4. (B) TPO was added at 1× (70 ng/mL) or 10× on D1. In addition, selinexor was added at indicated doses and was either washed out after 6 hours or kept in culture continuously. MK number on D4 was quantified by FACS. Values were normalized to 1× TPO control for each biological replicate, and then replicates averaged; n = 3. (C) Immunofluorescence of pSTAT3 was visualized in MKs treated with 500 nM selinexor or vehicle control. pSTAT3 is shown in green, and nuclear staining (Hoechst) is shown in blue. Bars represent 10 μm. To quantify nuclear fluorescence, the nuclear area was determined by thresholding in ImageJ (white outline), and the nuclear area and average, minimum, and maximum fluorescent intensities of pSTAT3 staining in the nucleus (as gated by Hoechst) were quantified in ImageJ (D). n = 12 cells per group; 3 biological replicates; ***P < .001. (E) MKs were serum starved in TPO-free media and treated with 500 nM selinexor or vehicle control for 6 hours. TPO was then added for 15 minutes to probe TPO signaling, and then MKs were separated into nuclear (N) and cytoplasmic (C) fractions. Fractions were probed for STAT3 and pSTAT3. The loading controls were as follows: actin (total protein), histone H3 (nuclear protein), and GAPDH (cytoplasmic protein). (F) Positive selection of CD41+ cells was performed on D1 of maturation to isolate a population of predominantly MK progenitors. After selection, the cells were treated with TPO or TPO and selinexor. On D4, quantitative polymerase chain reaction was performed as described to measure the levels of Klf4 and Oct4. (G) Schematic of proposed mechanism.

Figure 6.

Selinexor treatment inhibits TPO signaling in vitro. D1 cells derived from murine fetal livers were incubated with selinexor at indicated doses. (A) Apoptosis was measured by annexin V positivity in all cells by FACS on D4; n = 4. (B) TPO was added at 1× (70 ng/mL) or 10× on D1. In addition, selinexor was added at indicated doses and was either washed out after 6 hours or kept in culture continuously. MK number on D4 was quantified by FACS. Values were normalized to 1× TPO control for each biological replicate, and then replicates averaged; n = 3. (C) Immunofluorescence of pSTAT3 was visualized in MKs treated with 500 nM selinexor or vehicle control. pSTAT3 is shown in green, and nuclear staining (Hoechst) is shown in blue. Bars represent 10 μm. To quantify nuclear fluorescence, the nuclear area was determined by thresholding in ImageJ (white outline), and the nuclear area and average, minimum, and maximum fluorescent intensities of pSTAT3 staining in the nucleus (as gated by Hoechst) were quantified in ImageJ (D). n = 12 cells per group; 3 biological replicates; ***P < .001. (E) MKs were serum starved in TPO-free media and treated with 500 nM selinexor or vehicle control for 6 hours. TPO was then added for 15 minutes to probe TPO signaling, and then MKs were separated into nuclear (N) and cytoplasmic (C) fractions. Fractions were probed for STAT3 and pSTAT3. The loading controls were as follows: actin (total protein), histone H3 (nuclear protein), and GAPDH (cytoplasmic protein). (F) Positive selection of CD41+ cells was performed on D1 of maturation to isolate a population of predominantly MK progenitors. After selection, the cells were treated with TPO or TPO and selinexor. On D4, quantitative polymerase chain reaction was performed as described to measure the levels of Klf4 and Oct4. (G) Schematic of proposed mechanism.

Figure 7.

Coordinated dosing of TPO agonists overcomes selinexor-induced thrombocytopenia in patients. (A-B) TPO knockout mice were treated with vehicle control, selinexor (15 mg/kg every other day, for 3 doses per week), TPO (300 ng/mL, single treatment), or selinexor and TPO, and platelet counts were measured on D0 (before treatment) (A) and D6 (B). *P < .05 compared with vehicle control; n = 3 mice per group. (C-F) Representative human patient data showing the recovery effects of dosing interruption ± TPO agonists romiplostim or eltombopag on patients with selinexor-induced grade 4 thrombocytopenia. Dashed lines mark periods of selinexor and/or TPO mimetic treatment.