Inhibition of Megakaryocyte Differentiation by Antibody-Drug Conjugates (ADCs) is Mediated by Macropinocytosis: Implications for ADC-induced Thrombocytopenia

Mol Cancer Ther. 2017 Sep;16(9):1877-1886. doi: 10.1158/1535-7163.MCT-16-0710. Epub 2017 Jun 27.

Abstract

Thrombocytopenia is a common adverse event in cancer patients treated with antibody-drug conjugates (ADC), including AGS-16C3F, an ADC targeting ENPP3 (ectonucleotide pyrophosphatase/phosphodiesterase-3) and trastuzumab emtansine (T-DM1). This study aims to elucidate the mechanism of action of ADC-induced thrombocytopenia. ENPP3 expression in platelets and megakaryocytes (MK) was investigated and shown to be negative. The direct effect of AGS-16C3F on platelets was evaluated using platelet rich plasma following the expression of platelet activation markers. Effects of AGS-16C3F, T-DM1, and control ADCs on maturing megakaryocytes were evaluated in an in vitro system in which human hematopoietic stem cells (HSC) were differentiated into MKs. AGS-16C3F, like T-DM1, did not affect platelets directly, but inhibited MK differentiation by the activity of Cys-mcMMAF, its active metabolite. FcγRIIA did not appear to play an important role in ADC cytotoxicity to differentiating MKs. AGS-16C3F, cytotoxic to MKs, did not bind to FcγRIIA on MKs. Blocking the interaction of T-DM1 with FcγRIIA did not prevent the inhibition of MK differentiation and IgG1-mcMMAF was not as cytotoxic to MKs despite binding to FcγRIIA. Several lines of evidence suggest that internalization of AGS-16C3F into MKs is mediated by macropinocytosis. Macropinocytosis activity of differentiating HSCs correlated with cell sensitivity to AGS-16C3F. AGS-16C3F was colocalized with a macropinocytosis marker, dextran-Texas Red in differentiating MKs. Ethyl isopropyl amiloride (EIPA), a macropinocytosis inhibitor, blocked internalization of dextran-Texas Red and AGS-16C3F. These data support the notion that inhibition of MK differentiation via macropinocytosis-mediated internalization plays a role in ADC-induced thrombocytopenia. Mol Cancer Ther; 16(9); 1877-86. ©2017 AACRSee related article by Zhao et al., p. 1866.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antineoplastic Agents, Immunological / adverse effects
  • Antineoplastic Agents, Immunological / pharmacology*
  • Blood Platelets / drug effects
  • Blood Platelets / metabolism
  • Cell Differentiation / drug effects*
  • Hematopoietic Stem Cells / cytology
  • Hematopoietic Stem Cells / drug effects
  • Hematopoietic Stem Cells / metabolism
  • Humans
  • Immunoconjugates / adverse effects
  • Immunoconjugates / pharmacology*
  • Megakaryocytes / cytology*
  • Megakaryocytes / drug effects*
  • Megakaryocytes / metabolism
  • Pinocytosis*
  • Platelet Activation / drug effects
  • Protein Transport
  • Receptors, IgG / metabolism
  • Thrombocytopenia / blood
  • Thrombocytopenia / chemically induced

Substances

  • Antineoplastic Agents, Immunological
  • Fc gamma receptor IIA
  • Immunoconjugates
  • Receptors, IgG