Clofarabine induces ERK/MSK/CREB activation through inhibiting CD99 on Ewing sarcoma cells

PLoS One. 2021 Jun 16;16(6):e0253170. doi: 10.1371/journal.pone.0253170. eCollection 2021.

Abstract

Clofarabine, an FDA approved purine analog, is used in the treatment of relapsed or refractory acute lymphoblastic leukemia. Clofarabine acts by inhibiting DNA synthesis. We demonstrated that clofarabine may have a novel function though inhibiting CD99, a transmembrane protein highly expressed on Ewing Sarcoma (ES) cells. CD99 is a validated target in ES whose inhibition may lead to a high therapeutic index for patients. Here we present additional data to support the hypothesis that clofarabine acts on CD99 and regulates key signaling pathways in ES. Cellular thermal shift assay indicated a direct interaction between clofarabine and CD99 in ES cell lysates. Clofarabine induced ES cell death does not require clofarabine's conversion to its active form by deoxycytidine kinase. A phosphokinase array screen with clofarabine and a CD99 blocking antibody identified alterations in signaling pathways. CD99 inhibition with clofarabine in ES cells caused rapid and sustained phosphorylation of ERK, MSK, and CREB. However, activation of this pathway did not correlate with clofarabine induced ES cell death. In summary, we demonstrated that clofarabine may activate ERK, MSK, and CREB phosphorylation through CD99 within minutes, however this paradoxical activation and subsequent ES cell death requires additional investigation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 12E7 Antigen / antagonists & inhibitors*
  • Antimetabolites, Antineoplastic / pharmacology*
  • Blotting, Western
  • CREB-Binding Protein / metabolism*
  • Cell Line, Tumor
  • Clofarabine / pharmacology*
  • Humans
  • MAP Kinase Signaling System / drug effects*
  • Phosphorylation
  • Ribosomal Protein S6 Kinases, 90-kDa / metabolism*
  • Sarcoma, Ewing / drug therapy
  • Sarcoma, Ewing / metabolism*
  • Signal Transduction / drug effects*

Substances

  • 12E7 Antigen
  • Antimetabolites, Antineoplastic
  • CD99 protein, human
  • Clofarabine
  • CREB-Binding Protein
  • CREBBP protein, human
  • Ribosomal Protein S6 Kinases, 90-kDa

Grant support

We are grateful to Children’s Cancer Foundation, St. Baldrick's Foundation, Hyundai Hope On Wheels, Alan B. Slifka Foundation for providing peer reviewed grant support for the studies included in this manuscript.