Ibrutinib Suppresses Early Megakaryopoiesis but Enhances Proplatelet Formation

Abstract

Ibrutinib, an irreversible inhibitor of Bruton's tyrosine kinase, has a favorable safety profile in patients with B cell-related malignancies. A primary adverse effect of ibrutinib is thrombocytopenia in the early stages of treatment, but platelet counts increase or recover as treatment continues. Currently, the effects of ibrutinib on megakaryopoiesis remain unclear. In this study, we investigated the mechanism by which ibrutinib induces thrombocytopenia using cord blood CD34+ hematopoietic stem cells (HSCs), a human megakaryoblastic cell line (SET-2), and C57BL/6 mice. We show that treatment with ibrutinib can suppress CD34+ HSC differentiation into megakaryocytes (MKs) and decrease the number of colony-forming unit-MKs (CFU-MKs). The ibrutinib-dependent inhibition of early megakaryopoiesis seems to mainly involve impaired proliferation of progenitor cells without induction of apoptosis. The effects of ibrutinib on late-stage megakaryopoiesis, in contrast to early-stage megakaryopoiesis, include enhanced MK differentiation, ploidy, and proplatelet formation in CD34+ HSC-derived MKs and SET-2 cells. We also demonstrated that MK adhesion and spreading, but not migration, were inhibited by ibrutinib. Furthermore, we revealed that integrin αIIbβ3 outside-in signaling in MKs was inhibited by ibrutinib. Consistent with previous clinical observations, in C57BL/6 mice treated with ibrutinib, platelet counts decreased by days 2 to 7 and recovered to normal levels by day 15. Together, these results reveal the pathogenesis of ibrutinib-induced transient thrombocytopenia. In conclusion, ibrutinib suppresses early megakaryopoiesis, as evidenced by inhibition of MK progenitor cell proliferation and CFU-MK formation. Ibrutinib enhances MK differentiation, ploidy, and proplatelet formation, while it impairs integrin αIIbβ3 outside-in signaling.

Multifaceted Immunomodulatory Effects of the BTK Inhibitors Ibrutinib and Acalabrutinib on Different Immune Cell Subsets – Beyond B Lymphocytes

“…Consistent with the clinical observation, ibrutinib treatment impairs the proliferation of megakaryocyte progenitor cells during early stage megakaryopoiesis and decreases the number of colony-forming units of megakaryocytes (CFU-MKs) derived from human cord blood CD34+ hematopoietic stem cells (HSCs) or a human megakaryoblastic cell line SET-2 in vitro (Huang et al, 2021). On the other hand, ibrutinib enhances the differentiation and ploidy of megakaryocytes as well as proplatelet formation during late-stage megakaryopoiesis (Huang et al, 2021). Ibrutinib also impairs megakaryocyte adhesion and spreading on immobilized fibrinogen by inhibiting the integrin αIIbβ3 outside-in signaling in megakaryocytes (Huang et al, 2021).…”

“…However, ibrutinib treatment is associated with an increased risk of major bleeding (grade 3-4), which is much reduced with acalabrutinib treatment (Byrd et al, 2016;Wang et al, 2018;Pellegrini et al, 2021). After initiation of ibrutinib therapy, the majority of CLL patients show a small decrease in platelet counts on day 2, which is followed by a rapid increase in platelet counts several days later (Lipsky et al, 2015;Huang et al, 2021). Platelets from ibrutinib-treated CLL patients exhibit reduced surface levels of GPIb-IX complex and αIIbβ3 integrin (also known as GPIIb/IIIa), higher membrane fluidity, lower resting membrane potential and higher level of ROS production compared to those derived from untreated CLL patients and healthy volunteers (Dobie et al, 2019;Popov et al, 2020).…”

“…On the other hand, ibrutinib enhances the differentiation and ploidy of megakaryocytes as well as proplatelet formation during late-stage megakaryopoiesis (Huang et al, 2021). Ibrutinib also impairs megakaryocyte adhesion and spreading on immobilized fibrinogen by inhibiting the integrin αIIbβ3 outside-in signaling in megakaryocytes (Huang et al, 2021). In vivo administration of ibrutinib in C57BL/6 mice results in thrombocytopenia in the bone marrow associated with a decrease in platelet counts at day 2 to day 7, which is recovered to normal levels by day 15 after treatment (Huang et al, 2021).…”

“…Ibrutinib also impairs megakaryocyte adhesion and spreading on immobilized fibrinogen by inhibiting the integrin αIIbβ3 outside-in signaling in megakaryocytes (Huang et al, 2021). In vivo administration of ibrutinib in C57BL/6 mice results in thrombocytopenia in the bone marrow associated with a decrease in platelet counts at day 2 to day 7, which is recovered to normal levels by day 15 after treatment (Huang et al, 2021). Thus, the complex effects of ibrutinib on platelet counts are mediated at least partially by its differential modulation of megakaryocyte differentiation and function at different stages of megakaryopoiesis (Table 5), although the underlying molecular mechanisms remain unclear and require further investigation.…”

“…Thus, the complex effects of ibrutinib on platelet counts are mediated at least partially by its differential modulation of megakaryocyte differentiation and function at different stages of megakaryopoiesis (Table 5), although the underlying molecular mechanisms remain unclear and require further investigation. Bruton's tyrosine kinase is expressed in megakaryocytes and platelets, but XLA patients do not show a bleeding phenotype (Busygina et al, 2019;Huang et al, 2021). Bleedings observed in CLL and MCL patients that receive ibrutinib or acalabrutinib therapy are mainly attributable to the drug-induced platelet dysfunctions (Bye et al, 2017;Chen et al, 2018;Nicolson et al, 2018;Denzinger et al, 2019;Dmitrieva et al, 2020;Ninomoto et al, 2020).…”