Abivertinib inhibits megakaryocyte differentiation and platelet biogenesis

Abstract

Abivertinib, a third-generation tyrosine kinase inhibitor, is originally designed to target epidermal growth factor receptor (EGFR)-activating mutations. Previous studies have shown that abivertinib has promising antitumor activity and a well-tolerated safety profile in patients with non-small-cell lung cancer. However, abivertinib also exhibited high inhibitory activity against Bruton’s tyrosine kinase and Janus kinase 3. Given that these kinases play some roles in the progression of megakaryopoiesis, we speculate that abivertinib can affect megakaryocyte (MK) differentiation and platelet biogenesis. We treated cord blood CD34⁺ hematopoietic stem cells, Meg-01 cells, and C57BL/6 mice with abivertinib and observed megakaryopoiesis to determine the biological effect of abivertinib on MK differentiation and platelet biogenesis. Our in vitro results showed that abivertinib impaired the CFU-MK formation, proliferation of CD34⁺ HSC-derived MK progenitor cells, and differentiation and functions of MKs and inhibited Meg-01-derived MK differentiation. These results suggested that megakaryopoiesis was inhibited by abivertinib. We also demonstrated in vivo that abivertinib decreased the number of MKs in bone marrow and platelet counts in mice, which suggested that thrombopoiesis was also inhibited. Thus, these preclinical data collectively suggested that abivertinib could inhibit MK differentiation and platelet biogenesis and might be an agent for thrombocythemia.

Keywords

abivertinib / Btk inhibitor / platelet / megakaryocyte / megakaryopoiesis / thrombopoiesis

References

[1] Huang J, Li X, Shi X, Zhu M, Wang J, Huang S, Huang X, Wang H, Li L, Deng H, Zhou Y, Mao J, Long Z, Ma Z, Ye W, Pan J, Xi X, Jin J. Platelet integrin aIIbb3: signal transduction, regulation, and its therapeutic targeting. J Hematol Oncol 2019; 12(1): 26

[2] van der Meijden PEJ, Heemskerk JWM. Platelet biology and functions: new concepts and clinical perspectives. Nat Rev Cardiol 2019; 16(3): 166–179

[3] Zhang W, Zha C, Lu X, Jia R, Gao F, Sun Q, Jin M, Liu Y. Anti-b₂GPI/b₂GPI complexes induce platelet activation and promote thrombosis via p38MAPK: a pathway to targeted therapies. Front Med 2019; 13(6): 680–689

[4] Lefrançais E, Ortiz-Muñoz G, Caudrillier A, Mallavia B, Liu F, Sayah DM, Thornton EE, Headley MB, David T, Coughlin SR, Krummel MF, Leavitt AD, Passegué E, Looney MR. The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors. Nature 2017; 544(7648): 105–109

[5] Espasandin YR, Glembotsky AC, Grodzielski M, Lev PR, Goette NP, Molinas FC, Marta RF, Heller PG. Anagrelide platelet-lowering effect is due to inhibition of both megakaryocyte maturation and proplatelet formation: insight into potential mechanisms. J Thromb Haemost 2015; 13(4): 631–642

[6] Di Buduo CA, Currao M, Pecci A, Kaplan DL, Balduini CL, Balduini A. Revealing eltrombopag’s promotion of human megakaryopoiesis through AKT/ERK-dependent pathway activation. Haematologica 2016; 101(12): 1479–1488

[7] Wang J, Yi Z, Wang S, Li Z. The effect of decitabine on megakaryocyte maturation and platelet release. Thromb Haemost 2011; 106(2): 337–343

[8] Thiele J, Kvasnicka HM, Schmitt-Graeff A. Effects of anagrelide on megakaryopoiesis and platelet production. Semin Thromb Hemost 2006; 32(4 Pt 2): 352–361

[9] Xu X, Mao L, Xu W, Tang W, Zhang X, Xi B, Xu R, Fang X, Liu J, Fang C, Zhao L, Wang X, Jiang J, Hu P, Zhao H, Zhang L. AC0010, an irreversible EGFR inhibitor selectively targeting mutated EGFR and overcoming T790M-induced resistance in animal models and lung cancer patients. Mol Cancer Ther 2016; 15(11): 2586–2597

[10] Ma Y, Zheng X, Zhao H, Fang W, Zhang Y, Ge J, Wang L, Wang W, Jiang J, Chuai S, Zhang Z, Xu W, Xu X, Hu P, Zhang L. First-in-human phase I study of AC0010, a mutant-selective EGFR inhibitor in non-small cell lung cancer: safety, efficacy, and potential mechanism of resistance. J Thorac Oncol 2018; 13(7): 968–977

[11] Benbarche S, Strassel C, Angénieux C, Mallo L, Freund M, Gachet C, Lanza F, de la Salle H. Dual role of IL-21 in megakaryopoiesis and platelet homeostasis. Haematologica 2017; 102(4): 637–646

[12] Yamashita Y, Miyazato A, Shimizu R, Komatsu N, Miura Y, Ozawa K, Mano H. Tec protein-tyrosine kinase is involved in the thrombopoietin/c-Mpl signaling pathway. Exp Hematol 1997; 25(3): 211–216

[13] Avraham H, Price DJ. Regulation of megakaryocytopoiesis and platelet production by tyrosine kinases and tyrosine phosphatases. Methods 1999; 17(3): 250–264

[14] Sangkhae V, Saur SJ, Kaushansky A, Kaushansky K, Hitchcock IS. Phosphorylated c-Mpl tyrosine 591 regulates thrombopoietin-induced signaling. Exp Hematol 2014; 42(6): 477–486.e4

[15] Wang ML, Blum KA, Martin P, Goy A, Auer R, Kahl BS, Jurczak W, Advani RH, Romaguera JE, Williams ME, Barrientos JC, Chmielowska E, Radford J, Stilgenbauer S, Dreyling M, Jedrzejczak WW, Johnson P, Spurgeon SE, Zhang L, Baher L, Cheng M, Lee D, Beaupre DM, Rule S. Long-term follow-up of MCL patients treated with single-agent ibrutinib: updated safety and efficacy results. Blood 2015; 126(6): 739–745

[16] Yan X, Zhou Y, Huang S, Li X, Yu M, Huang J, Wang J, Ma Z, Jin J, Pan J, Li C, Li F, Jin J. Promising efficacy of novel BTK inhibitor AC0010 in mantle cell lymphoma. J Cancer Res Clin Oncol 2018; 144(4): 697–706

[17] Huang S, Yu M, Shi N, Zhou Y, Li F, Li X, Huang X, Jin J. Apigenin and abivertinib, a novel BTK inhibitor synergize to inhibit diffuse large B-cell lymphoma in vivo and vitro. J Cancer 2020; 11(8): 2123–2132

[18] Huang S, Li C, Zhang X, Pan J, Li F, Lv Y, Huang J, Ling Q, Ye W, Mao S, Huang X, Jin J. Abivertinib synergistically strengthens the anti-leukemia activity of venetoclax in acute myeloid leukemia in a BTK-dependent manner. Mol Oncol 2020; 14(10): 2560–2573

[19] Huang S, Pan J, Jin J, Li C, Li X, Huang J, Huang X, Yan X, Li F, Yu M, Hu C, Jin J, Xu Y, Ling Q, Ye W, Wang Y, Jin J. Abivertinib, a novel BTK inhibitor: anti-leukemia effects and synergistic efficacy with homoharringtonine in acute myeloid leukemia. Cancer Lett 2019; 461: 132–143

[20] Yang M, Qian J, Huang J, Jiao Y, Tang W, Xu X, Xu W, Luo FR, Jin J. A phase I study of the BTK inhibitor abivertinib (AC0010) in patients with relapsed or refractory B-cell lymphoma. HemaSphere 2019; 3(S1): 210 doi: 10.1097/01.HS9.0000560160.25056.77

[21] Ogura M, Morishima Y, Ohno R, Kato Y, Hirabayashi N, Nagura H, Saito H. Establishment of a novel human megakaryoblastic leukemia cell line, MEG-01, with positive Philadelphia chromosome. Blood 1985; 66(6): 1384–1392

[22] Hearn JI, Green TN, Chopra M, Nursalim YNS, Ladvanszky L, Knowlton N, Blenkiron C, Poulsen RC, Singleton DC, Bohlander SK, Kalev-Zylinska ML. N-methyl-D-aspartate receptor hypofunction in Meg-01 cells reveals a role for intracellular calcium homeostasis in balancing megakaryocytic-erythroid differentiation. Thromb Haemost 2020; 120(4): 671–686

[23] Su X, Mi J, Yan J, Flevaris P, Lu Y, Liu H, Ruan Z, Wang X, Kieffer N, Chen S, Du X, Xi X. RGT, a synthetic peptide corresponding to the integrin b3 cytoplasmic C-terminal sequence, selectively inhibits outside-in signaling in human platelets by disrupting the interaction of integrin aIIbb3 with Src kinase. Blood 2008; 112(3): 592–602 doi:10.1182/blood-2007-09-110437

[24] Yin H, Litvinov RI, Vilaire G, Zhu H, Li W, Caputo GA, Moore DT, Lear JD, Weisel JW, Degrado WF, Bennett JS. Activation of platelet αIIbβ3 by an exogenous peptide corresponding to the transmembrane domain of αIIb. J Biol Chem 2006; 281(48): 36732–36741

[25] Liu D, DeGrado WF. De novo design, synthesis, and characterization of antimicrobial b-peptides. J Am Chem Soc 2001; 123(31): 7553–7559

[26] Perdomo J, Yan F, Leung HHL, Chong BH. Megakaryocyte differentiation and platelet formation from human cord blood-derived CD34⁺ cells. J Vis Exp 2017; 130(130): e56420

[27] Huang J, Huang S, Ma Z, Lin X, Li X, Huang X, Wang J, Ye W, Li Y, He D, Yang M, Pan J, Ling Q, Li F, Mao S, Wang H, Wang Y, Jin J. Ibrutinib suppresses early megakaryopoiesis but enhances proplatelet formation. Thromb Haemost 2021;121(2):192–205

[28] Salzmann M, Hoesel B, Haase M, Mussbacher M, Schrottmaier WC, Kral-Pointner JB, Finsterbusch M, Mazharian A, Assinger A, Schmid JA. A novel method for automated assessment of megakaryocyte differentiation and proplatelet formation. Platelets 2018; 29(4): 357–364

[29] Li X, Yin X, Wang H, Huang J, Yu M, Ma Z, Li C, Zhou Y, Yan X, Huang S, Jin J. The combination effect of homoharringtonine and ibrutinib on FLT3-ITD mutant acute myeloid leukemia. Oncotarget 2017; 8(8): 12764–12774

[30] Du X, Yin S, Zhou F, Du X, Xu J, Gu X, Wang G, Li J. Reduction-sensitive mixed micelles for selective intracellular drug delivery to tumor cells and reversal of multidrug resistance. Int J Pharm 2018; 550(1-2): 1–13

[31] Yang Y, Akada H, Nath D, Hutchison RE, Mohi G. Loss of Ezh2 cooperates with Jak2V617F in the development of myelofibrosis in a mouse model of myeloproliferative neoplasm. Blood 2016; 127(26): 3410–3423

[32] Zheng J, Wang SS, Shen KP, Huang XW, Li M, Chen L, Peng X, An HM, Hu B. Ursolic acid potentiated oxaliplatin to induce apoptosis in colorectal cancer RKO cells. Pharmazie 2020; 75(6): 246–249 doi: 10.1691/ph.2020.0417

[33] Belmokhtar CA, Hillion J, Ségal-Bendirdjian E. Staurosporine induces apoptosis through both caspase-dependent and caspase-independent mechanisms. Oncogene 2001; 20(26): 3354–3362

[34] Zhang W, Lu Y, Zhen T, Chen X, Zhang M, Liu P, Weng X, Chen B, Wang Y. Homoharringtonine synergy with oridonin in treatment of t(8;21) acute myeloid leukemia. Front Med 2019; 13(3): 388–397

[35] Miyashita N, Onozawa M, Yokoyama S, Hidaka D, Hayasaka K, Kunishima S, Teshima T. Anagrelide modulates proplatelet formation resulting in decreased number and increased size of platelets. Hemasphere 2019; 3(4): e268

[36] Lopez JJ, Palazzo A, Chaabane C, Albarran L, Polidano E, Lebozec K, Dally S, Nurden P, Enouf J, Debili N, Bobe R. Crucial role for endoplasmic reticulum stress during megakaryocyte maturation. Arterioscler Thromb Vasc Biol 2013; 33(12): 2750–2758

[37] Thorp BC, Badoux X. Atrial fibrillation as a complication of ibrutinib therapy: clinical features and challenges of management. Leuk Lymphoma 2018; 59(2): 311–320

[38] Burger JA, Tedeschi A, Barr PM, Robak T, Owen C, Ghia P, Bairey O, Hillmen P, Bartlett NL, Li J, Simpson D, Grosicki S, Devereux S, McCarthy H, Coutre S, Quach H, Gaidano G, Maslyak Z, Stevens DA, Janssens A, Offner F, Mayer J, O’Dwyer M, Hellmann A, Schuh A, Siddiqi T, Polliack A, Tam CS, Suri D, Cheng M, Clow F, Styles L, James DF, Kipps TJ; RESONATE-2 Investigators. Ibrutinib as initial therapy for patients with chronic lymphocytic leukemia. N Engl J Med 2015; 373(25): 2425–2437

[39] Treon SP, Tripsas CK, Meid K, Warren D, Varma G, Green R, Argyropoulos KV, Yang G, Cao Y, Xu L, Patterson CJ, Rodig S, Zehnder JL, Aster JC, Harris NL, Kanan S, Ghobrial I, Castillo JJ, Laubach JP, Hunter ZR, Salman Z, Li J, Cheng M, Clow F, Graef T, Palomba ML, Advani RH. Ibrutinib in previously treated Waldenström’s macroglobulinemia. N Engl J Med 2015; 372(15): 1430–1440

[40] Gremmel T, Frelinger AL 3rd, Michelson AD. Platelet physiology. Semin Thromb Hemost 2016; 42(3): 191–204

[41] Wen Q, Goldenson B, Crispino JD. Normal and malignant megakaryopoiesis. Expert Rev Mol Med 2011; 13: e32

[42] Bianchi E, Norfo R, Pennucci V, Zini R, Manfredini R. Genomic landscape of megakaryopoiesis and platelet function defects. Blood 2016; 127(10): 1249–1259

[43] Songdej N, Rao AK. Hematopoietic transcription factor mutations: important players in inherited platelet defects. Blood 2017; 129(21): 2873–2881

[44] Roweth HG, Parvin S, Machlus KR. Megakaryocyte modification of platelets in thrombocytopenia. Curr Opin Hematol 2018; 25(5): 410–415

[45] Akinleye A, Chen Y, Mukhi N, Song Y, Liu D. Ibrutinib and novel BTK inhibitors in clinical development. J Hematol Oncol 2013; 6(1): 59

[46] Busygina K, Denzinger V, Bernlochner I, Weber C, Lorenz R, Siess W. Btk inhibitors as first oral atherothrombosis-selective antiplatelet drugs? Thromb Haemost 2019; 119(8): 1212–1221

[47] Atkinson BT, Ellmeier W, Watson SP. Tec regulates platelet activation by GPVI in the absence of Btk. Blood 2003; 102(10): 3592–3599

[48] Futatani T, Watanabe C, Baba Y, Tsukada S, Ochs HD. Bruton’s tyrosine kinase is present in normal platelets and its absence identifies patients with X-linked agammaglobulinaemia and carrier females. Br J Haematol 2001; 114(1): 141–149

[49] Pasquet JM, Quek L, Stevens C, Bobe R, Huber M, Duronio V, Krystal G, Watson SP. Phosphatidylinositol 3,4,5-trisphosphate regulates Ca²⁺ entry via btk in platelets and megakaryocytes without increasing phospholipase C activity. EMBO J 2000; 19(12): 2793–2802

[50] Bobe R, Wilde JI, Maschberger P, Venkateswarlu K, Cullen PJ, Siess W, Watson SP. Phosphatidylinositol 3-kinase-dependent translocation of phospholipase Cγ2 in mouse megakaryocytes is independent of Bruton tyrosine kinase translocation. Blood 2001; 97(3): 678–684

[51] Markham A, Dhillon S. Acalabrutinib: first global approval. Drugs 2018; 78(1): 139–145

[52] Series J, Garcia C, Levade M, Viaud J, Sié P, Ysebaert L, Payrastre B. Differences and similarities in the effects of ibrutinib and acalabrutinib on platelet functions. Haematologica 2019; 104(11): 2292–2299

[53] Bye AP, Unsworth AJ, Vaiyapuri S, Stainer AR, Fry MJ, Gibbins JM. Ibrutinib inhibits platelet integrin aIIbb3 outside-in signaling and thrombus stability but not adhesion to collagen. Arterioscler Thromb Vasc Biol 2015; 35(11): 2326–2335

[54] Lipsky AH, Lozier JN, Wiestner A. Response to comment on incidence and risk factors of bleeding-related adverse events in patients with chronic lymphocytic leukemia treated with ibrutinib. Haematologica 2016; 101(3): e124–e125

[55] Kriegsmann K, Kriegsmann M, Witzens-Harig M. Acalabrutinib, a second-generation Bruton’s tyrosine kinase inhibitor. Recent Results Cancer Res 2018; 212: 285–294

[56] Balduini A, Badalucco S, Pugliano MT, Baev D, De Silvestri A, Cattaneo M, Rosti V, Barosi G. In vitro megakaryocyte differentiation and proplatelet formation in Ph-negative classical myeloproliferative neoplasms: distinct patterns in the different clinical phenotypes. PLoS One 2011; 6(6): e21015

Acknowledgements

The authors thank all the laboratory members for helpful discussion. We thank Dr. Huan Deng in Department of Pathology, the Fourth Affiliated Hospital of Nanchang University for technical assistance on spleen and bone marrow histology. This work was supported by grants from National Natural Science Foundation of China (Nos. 82070118 and 81820108004) and the Zhejiang Provincial Natural Science Foundation of China (Nos. LY20H080008 and Y19H080009).

Compliance with ethics guidelines

Jiansong Huang, Xin Huang, Yang Li, Xia Li, Jinghan Wang, Fenglin Li, Xiao Yan, Huanping Wang, Yungui Wang, Xiangjie Lin, Jifang Tu, Daqiang He, Wenle Ye, Min Yang, and Jie Jin declare no conflict of interest. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975 as revised in 2000. Informed consent was obtained from all patients included in the study. All institutional and national guidelines for the care and use of laboratory animals were followed.

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