Targeting pleckstrin-2/Akt signaling reduces proliferation in myeloproliferative neoplasm models
Xu Han, Yang Mei, Rama K. Mishra, Honghao Bi, Atul D. Jain, Gary E. Schiltz, Baobing Zhao, Madina Sukhanova, Pan Wang, Arabela A. Grigorescu, Patricia C. Weber, John J. Piwinski, Miguel A. Prado, Joao A. Paulo, Len Stephens, Karen E. Anderson, Charles S. Abrams, Jing Yang, Peng Ji
Xu Han, Yang Mei, Rama K. Mishra, Honghao Bi, Atul D. Jain, Gary E. Schiltz, Baobing Zhao, Madina Sukhanova, Pan Wang, Arabela A. Grigorescu, Patricia C. Weber, John J. Piwinski, Miguel A. Prado, Joao A. Paulo, Len Stephens, Karen E. Anderson, Charles S. Abrams, Jing Yang, Peng Ji
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Research Article Hematology Oncology

Targeting pleckstrin-2/Akt signaling reduces proliferation in myeloproliferative neoplasm models

Abstract

Myeloproliferative neoplasms (MPNs) are characterized by the activated JAK2/STAT pathway. Pleckstrin-2 (Plek2) is a downstream target of the JAK2/STAT5 pathway and is overexpressed in patients with MPNs. We previously revealed that Plek2 plays critical roles in the pathogenesis of JAK2-mutated MPNs. The nonessential roles of Plek2 under physiologic conditions make it an ideal target for MPN therapy. Here, we identified first-in-class Plek2 inhibitors through an in silico high-throughput screening approach and cell-based assays, followed by the synthesis of analogs. Plek2-specific small-molecule inhibitors showed potent inhibitory effects on cell proliferation. Mechanistically, Plek2 interacts with and enhances the activity of Akt through the recruitment of downstream effector proteins. The Plek2-signaling complex also includes Hsp72, which protects Akt from degradation. These functions were blocked by Plek2 inhibitors via their direct binding to the Plek2 dishevelled, Egl-10 and pleckstrin (DEP) domain. The role of Plek2 in activating Akt signaling was further confirmed in vivo using a hematopoietic-specific Pten-knockout mouse model. We next tested Plek2 inhibitors alone or in combination with an Akt inhibitor in various MPN mouse models, which showed significant therapeutic efficacies similar to that seen with the genetic depletion of Plek2. The Plek2 inhibitor was also effective in reducing proliferation of CD34-positive cells from MPN patients. Our studies reveal a Plek2/Akt complex that drives cell proliferation and can be targeted by a class of antiproliferative compounds for MPN therapy.

Authors

Xu Han, Yang Mei, Rama K. Mishra, Honghao Bi, Atul D. Jain, Gary E. Schiltz, Baobing Zhao, Madina Sukhanova, Pan Wang, Arabela A. Grigorescu, Patricia C. Weber, John J. Piwinski, Miguel A. Prado, Joao A. Paulo, Len Stephens, Karen E. Anderson, Charles S. Abrams, Jing Yang, Peng Ji

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Figure 6

Plek2 inhibitors are effective in reducing myeloproliferation in vivo.

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Plek2 inhibitors are effective in reducing myeloproliferation in vivo. (...
(A) Total BM cells from 2-month-old JAK2V617F-knockin mice (CD45.2+) were transplanted into lethally irradiated recipient mice (CD45.1+). One month after transplant, recipient mice were treated with 25 mg/kg NUP-17d or vehicle control once every 2 days for 1 month. CBCs were performed after treatment. WT littermate control mice were used for comparison. WT, n = 10; n = 9 in the other 2 groups. (B) Spleen weight in mice from A. n = 5 in each group. (C) Representative H&E staining of spleens from indicated mice after treatment in A. Bottom figures are magnified parts of the figures above. Arrows point to megakaryocytes. Scale bars: 100 μm. (D) Representative H&E staining of lungs from indicated mice after treatment in A. Inserted figures are magnified parts. Scale bars: 100 μm. (E) Western blot analyses of indicated proteins from TER119+ BM cells purified from mice in A. (F) Same as A except 2-month-old JAK2V617F-knockin mice or WT littermates (CD45.2+) together with equal amounts (50:50) of WT BM cells (CD45.1+) were transplanted. CBCs were performed after treatment. WT (untreated control mice), n = 20; JAK2V617F+veh, n = 18; JAK2V617F+17d, n = 20. Data were combined from 2 independent experiments. (G) Spleen weight in mice from F. JAK2V617F+Veh, n = 9; n = 10 in the other 2 groups. (H) CD45.2+ ratio in peripheral blood from indicated mice from F. (I–K) Absolute CD45.2+ cell numbers of indicated HSPCs from BM from indicated mice in F. MEP, megakaryocyte-erythrocyte progenitor; CMP, common myeloid progenitor; GMP, granulocyte-monocyte progenitor; WT, n = 10; JAK2V617F+veh, n = 10; JAK2V617F+17d, n = 9. (L) Three-month-old JAK2V617F-knockin mice were treated with 25 mg/kg NUP-17d once every week. Same-sex JAK2V617F-knockin littermates were treated with vehicle as the control. Body weight was monitored. n = 7 in each group. (M) Survival data of indicated mice from I. Linked 2 points refer to same-sex JAK2V617F-knockin littermates. Error bars represent SEM of the mean. Comparisons between 2 groups were evaluated with 2-tailed t test, and comparisons among multiple groups were evaluated with 1-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001.