Research ArticleHematologyOncology Open Access | 10.1172/JCI159638

Xu Han,1,2 Yang Mei,1,2 Rama K. Mishra,3 Honghao Bi,1,2 Atul D. Jain,4 Gary E. Schiltz,2,4,5 Baobing Zhao,1,2 Madina Sukhanova,1,2 Pan Wang,1 Arabela A. Grigorescu,6 Patricia C. Weber,7 John J. Piwinski,7 Miguel A. Prado,8 Joao A. Paulo,8 Len Stephens,9 Karen E. Anderson,9 Charles S. Abrams,10 Jing Yang,1,2 and Peng Ji1,2

1Department of Pathology, Feinberg School of Medicine,

2Robert H. Lurie Comprehensive Cancer Center,

3Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine,

4Department of Chemistry, and

5Department of Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

6Department of Molecular Biosciences, Weinberg College of Arts & Sciences, Northwestern University, Evanston, Illinois, USA.

7Harrington Discovery Institute, Cleveland, Ohio, USA.

8Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.

9Signaling Programme, The Babraham Institute, Cambridge, United Kingdom.

10Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Peng Ji, Department of Pathology, Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Ward 3-230, Chicago, Illinois 60611, USA. Phone: 312.503.3191; Email: peng-ji@fsm.northwestern.edu.

Find articles by Han, X. in: JCI | PubMed | Google Scholar |

1Department of Pathology, Feinberg School of Medicine,

2Robert H. Lurie Comprehensive Cancer Center,

3Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine,

4Department of Chemistry, and

5Department of Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

6Department of Molecular Biosciences, Weinberg College of Arts & Sciences, Northwestern University, Evanston, Illinois, USA.

7Harrington Discovery Institute, Cleveland, Ohio, USA.

8Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.

9Signaling Programme, The Babraham Institute, Cambridge, United Kingdom.

10Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Peng Ji, Department of Pathology, Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Ward 3-230, Chicago, Illinois 60611, USA. Phone: 312.503.3191; Email: peng-ji@fsm.northwestern.edu.

Find articles by Mei, Y. in: JCI | PubMed | Google Scholar |

1Department of Pathology, Feinberg School of Medicine,

2Robert H. Lurie Comprehensive Cancer Center,

3Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine,

4Department of Chemistry, and

5Department of Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

6Department of Molecular Biosciences, Weinberg College of Arts & Sciences, Northwestern University, Evanston, Illinois, USA.

7Harrington Discovery Institute, Cleveland, Ohio, USA.

8Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.

9Signaling Programme, The Babraham Institute, Cambridge, United Kingdom.

10Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Peng Ji, Department of Pathology, Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Ward 3-230, Chicago, Illinois 60611, USA. Phone: 312.503.3191; Email: peng-ji@fsm.northwestern.edu.

Find articles by Mishra, R. in: JCI | PubMed | Google Scholar

1Department of Pathology, Feinberg School of Medicine,

2Robert H. Lurie Comprehensive Cancer Center,

3Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine,

4Department of Chemistry, and

5Department of Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

6Department of Molecular Biosciences, Weinberg College of Arts & Sciences, Northwestern University, Evanston, Illinois, USA.

7Harrington Discovery Institute, Cleveland, Ohio, USA.

8Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.

9Signaling Programme, The Babraham Institute, Cambridge, United Kingdom.

10Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Peng Ji, Department of Pathology, Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Ward 3-230, Chicago, Illinois 60611, USA. Phone: 312.503.3191; Email: peng-ji@fsm.northwestern.edu.

Find articles by Bi, H. in: JCI | PubMed | Google Scholar

1Department of Pathology, Feinberg School of Medicine,

2Robert H. Lurie Comprehensive Cancer Center,

3Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine,

4Department of Chemistry, and

5Department of Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

6Department of Molecular Biosciences, Weinberg College of Arts & Sciences, Northwestern University, Evanston, Illinois, USA.

7Harrington Discovery Institute, Cleveland, Ohio, USA.

8Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.

9Signaling Programme, The Babraham Institute, Cambridge, United Kingdom.

10Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Peng Ji, Department of Pathology, Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Ward 3-230, Chicago, Illinois 60611, USA. Phone: 312.503.3191; Email: peng-ji@fsm.northwestern.edu.

Find articles by Jain, A. in: JCI | PubMed | Google Scholar

1Department of Pathology, Feinberg School of Medicine,

2Robert H. Lurie Comprehensive Cancer Center,

3Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine,

4Department of Chemistry, and

5Department of Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

6Department of Molecular Biosciences, Weinberg College of Arts & Sciences, Northwestern University, Evanston, Illinois, USA.

7Harrington Discovery Institute, Cleveland, Ohio, USA.

8Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.

9Signaling Programme, The Babraham Institute, Cambridge, United Kingdom.

10Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Peng Ji, Department of Pathology, Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Ward 3-230, Chicago, Illinois 60611, USA. Phone: 312.503.3191; Email: peng-ji@fsm.northwestern.edu.

Find articles by Schiltz, G. in: JCI | PubMed | Google Scholar |

1Department of Pathology, Feinberg School of Medicine,

2Robert H. Lurie Comprehensive Cancer Center,

3Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine,

4Department of Chemistry, and

5Department of Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

6Department of Molecular Biosciences, Weinberg College of Arts & Sciences, Northwestern University, Evanston, Illinois, USA.

7Harrington Discovery Institute, Cleveland, Ohio, USA.

8Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.

9Signaling Programme, The Babraham Institute, Cambridge, United Kingdom.

10Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Peng Ji, Department of Pathology, Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Ward 3-230, Chicago, Illinois 60611, USA. Phone: 312.503.3191; Email: peng-ji@fsm.northwestern.edu.

Find articles by Zhao, B. in: JCI | PubMed | Google Scholar |

1Department of Pathology, Feinberg School of Medicine,

2Robert H. Lurie Comprehensive Cancer Center,

3Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine,

4Department of Chemistry, and

5Department of Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

6Department of Molecular Biosciences, Weinberg College of Arts & Sciences, Northwestern University, Evanston, Illinois, USA.

7Harrington Discovery Institute, Cleveland, Ohio, USA.

8Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.

9Signaling Programme, The Babraham Institute, Cambridge, United Kingdom.

10Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Peng Ji, Department of Pathology, Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Ward 3-230, Chicago, Illinois 60611, USA. Phone: 312.503.3191; Email: peng-ji@fsm.northwestern.edu.

Find articles by Sukhanova, M. in: JCI | PubMed | Google Scholar |

1Department of Pathology, Feinberg School of Medicine,

2Robert H. Lurie Comprehensive Cancer Center,

3Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine,

4Department of Chemistry, and

5Department of Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

6Department of Molecular Biosciences, Weinberg College of Arts & Sciences, Northwestern University, Evanston, Illinois, USA.

7Harrington Discovery Institute, Cleveland, Ohio, USA.

8Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.

9Signaling Programme, The Babraham Institute, Cambridge, United Kingdom.

10Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Peng Ji, Department of Pathology, Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Ward 3-230, Chicago, Illinois 60611, USA. Phone: 312.503.3191; Email: peng-ji@fsm.northwestern.edu.

Find articles by Wang, P. in: JCI | PubMed | Google Scholar

1Department of Pathology, Feinberg School of Medicine,

2Robert H. Lurie Comprehensive Cancer Center,

3Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine,

4Department of Chemistry, and

5Department of Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

6Department of Molecular Biosciences, Weinberg College of Arts & Sciences, Northwestern University, Evanston, Illinois, USA.

7Harrington Discovery Institute, Cleveland, Ohio, USA.

8Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.

9Signaling Programme, The Babraham Institute, Cambridge, United Kingdom.

10Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Peng Ji, Department of Pathology, Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Ward 3-230, Chicago, Illinois 60611, USA. Phone: 312.503.3191; Email: peng-ji@fsm.northwestern.edu.

Find articles by Grigorescu, A. in: JCI | PubMed | Google Scholar

1Department of Pathology, Feinberg School of Medicine,

2Robert H. Lurie Comprehensive Cancer Center,

3Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine,

4Department of Chemistry, and

5Department of Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

6Department of Molecular Biosciences, Weinberg College of Arts & Sciences, Northwestern University, Evanston, Illinois, USA.

7Harrington Discovery Institute, Cleveland, Ohio, USA.

8Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.

9Signaling Programme, The Babraham Institute, Cambridge, United Kingdom.

10Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Peng Ji, Department of Pathology, Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Ward 3-230, Chicago, Illinois 60611, USA. Phone: 312.503.3191; Email: peng-ji@fsm.northwestern.edu.

Find articles by Weber, P. in: JCI | PubMed | Google Scholar

1Department of Pathology, Feinberg School of Medicine,

2Robert H. Lurie Comprehensive Cancer Center,

3Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine,

4Department of Chemistry, and

5Department of Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

6Department of Molecular Biosciences, Weinberg College of Arts & Sciences, Northwestern University, Evanston, Illinois, USA.

7Harrington Discovery Institute, Cleveland, Ohio, USA.

8Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.

9Signaling Programme, The Babraham Institute, Cambridge, United Kingdom.

10Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Peng Ji, Department of Pathology, Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Ward 3-230, Chicago, Illinois 60611, USA. Phone: 312.503.3191; Email: peng-ji@fsm.northwestern.edu.

Find articles by Piwinski, J. in: JCI | PubMed | Google Scholar

1Department of Pathology, Feinberg School of Medicine,

2Robert H. Lurie Comprehensive Cancer Center,

3Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine,

4Department of Chemistry, and

5Department of Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

6Department of Molecular Biosciences, Weinberg College of Arts & Sciences, Northwestern University, Evanston, Illinois, USA.

7Harrington Discovery Institute, Cleveland, Ohio, USA.

8Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.

9Signaling Programme, The Babraham Institute, Cambridge, United Kingdom.

10Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Peng Ji, Department of Pathology, Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Ward 3-230, Chicago, Illinois 60611, USA. Phone: 312.503.3191; Email: peng-ji@fsm.northwestern.edu.

Find articles by Prado, M. in: JCI | PubMed | Google Scholar |

1Department of Pathology, Feinberg School of Medicine,

2Robert H. Lurie Comprehensive Cancer Center,

3Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine,

4Department of Chemistry, and

5Department of Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

6Department of Molecular Biosciences, Weinberg College of Arts & Sciences, Northwestern University, Evanston, Illinois, USA.

7Harrington Discovery Institute, Cleveland, Ohio, USA.

8Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.

9Signaling Programme, The Babraham Institute, Cambridge, United Kingdom.

10Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Peng Ji, Department of Pathology, Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Ward 3-230, Chicago, Illinois 60611, USA. Phone: 312.503.3191; Email: peng-ji@fsm.northwestern.edu.

Find articles by Paulo, J. in: JCI | PubMed | Google Scholar |

1Department of Pathology, Feinberg School of Medicine,

2Robert H. Lurie Comprehensive Cancer Center,

3Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine,

4Department of Chemistry, and

5Department of Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

6Department of Molecular Biosciences, Weinberg College of Arts & Sciences, Northwestern University, Evanston, Illinois, USA.

7Harrington Discovery Institute, Cleveland, Ohio, USA.

8Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.

9Signaling Programme, The Babraham Institute, Cambridge, United Kingdom.

10Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Peng Ji, Department of Pathology, Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Ward 3-230, Chicago, Illinois 60611, USA. Phone: 312.503.3191; Email: peng-ji@fsm.northwestern.edu.

Find articles by Stephens, L. in: JCI | PubMed | Google Scholar

1Department of Pathology, Feinberg School of Medicine,

2Robert H. Lurie Comprehensive Cancer Center,

3Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine,

4Department of Chemistry, and

5Department of Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

6Department of Molecular Biosciences, Weinberg College of Arts & Sciences, Northwestern University, Evanston, Illinois, USA.

7Harrington Discovery Institute, Cleveland, Ohio, USA.

8Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.

9Signaling Programme, The Babraham Institute, Cambridge, United Kingdom.

10Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Peng Ji, Department of Pathology, Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Ward 3-230, Chicago, Illinois 60611, USA. Phone: 312.503.3191; Email: peng-ji@fsm.northwestern.edu.

Find articles by Anderson, K. in: JCI | PubMed | Google Scholar

1Department of Pathology, Feinberg School of Medicine,

2Robert H. Lurie Comprehensive Cancer Center,

3Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine,

4Department of Chemistry, and

5Department of Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

6Department of Molecular Biosciences, Weinberg College of Arts & Sciences, Northwestern University, Evanston, Illinois, USA.

7Harrington Discovery Institute, Cleveland, Ohio, USA.

8Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.

9Signaling Programme, The Babraham Institute, Cambridge, United Kingdom.

10Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Peng Ji, Department of Pathology, Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Ward 3-230, Chicago, Illinois 60611, USA. Phone: 312.503.3191; Email: peng-ji@fsm.northwestern.edu.

Find articles by Abrams, C. in: JCI | PubMed | Google Scholar

1Department of Pathology, Feinberg School of Medicine,

2Robert H. Lurie Comprehensive Cancer Center,

3Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine,

4Department of Chemistry, and

5Department of Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

6Department of Molecular Biosciences, Weinberg College of Arts & Sciences, Northwestern University, Evanston, Illinois, USA.

7Harrington Discovery Institute, Cleveland, Ohio, USA.

8Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.

9Signaling Programme, The Babraham Institute, Cambridge, United Kingdom.

10Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Peng Ji, Department of Pathology, Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Ward 3-230, Chicago, Illinois 60611, USA. Phone: 312.503.3191; Email: peng-ji@fsm.northwestern.edu.

Find articles by Yang, J. in: JCI | PubMed | Google Scholar

1Department of Pathology, Feinberg School of Medicine,

2Robert H. Lurie Comprehensive Cancer Center,

3Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine,

4Department of Chemistry, and

5Department of Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

6Department of Molecular Biosciences, Weinberg College of Arts & Sciences, Northwestern University, Evanston, Illinois, USA.

7Harrington Discovery Institute, Cleveland, Ohio, USA.

8Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.

9Signaling Programme, The Babraham Institute, Cambridge, United Kingdom.

10Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Peng Ji, Department of Pathology, Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Ward 3-230, Chicago, Illinois 60611, USA. Phone: 312.503.3191; Email: peng-ji@fsm.northwestern.edu.

Find articles by Ji, P. in: JCI | PubMed | Google Scholar |

Published January 31, 2023 - More info

Published in Volume 133, Issue 6 on March 15, 2023
J Clin Invest. 2023;133(6):e159638. https://doi.org/10.1172/JCI159638.
© 2023 Han et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published January 31, 2023 - Version history
Received: February 23, 2022; Accepted: January 25, 2023 View PDF 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.

Graphical Abstract Introduction

Philadelphia chromosome–negative (Ph-negative) myeloproliferative neoplasms (MPNs) are a group of BM diseases featuring excessive production of myeloid cells and increased risk of evolving to acute myeloid leukemia. JAK2V617F mutation is the leading cause of Ph-negative MPNs that include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) (1–5). Unlike with many effective therapies for Ph-induced MPNs, developing effective therapies for Ph-negative MPNs is in its nascence. The FDA-approved JAK inhibitor ruxolitinib reduces spleen size and blood cell counts in MPN patients (6–8). However, it is associated with marked long-term side effects, including anemia and thrombocytopenia, due to the essential role of JAK2 in hematopoiesis. Therefore, new therapeutic strategies are needed for treating Ph-negative MPNs, such as compounds that target JAK/STAT effectors that may cause fewer side effects.

Pleckstrin-2 (Plek2) is a membrane protein and paralog of Plek1. Plek2 is widely expressed and involved in actin rearrangement (9). Overexpression of Plek2 induces lamellipodia and peripheral ruffle formation (10). We previously showed that Plek2 is important in the regulation of actin cytoskeleton, cell differentiation, and apoptosis in terminal erythroid differentiation (11). Subsequent studies revealed that Plek2 is a downstream target of the JAK2/STAT5 pathway and overexpressed in JAK2V617F-positive patients with MPNs. In addition to the erythroid lineage, Plek2 is also upregulated in the myeloid and megakaryocytic lineages induced by JAK2V617F mutation. The critical role of Plek2 in the pathogenesis of MPNs was manifested by a JAK2V617F-knockin mouse model in which loss of Plek2 largely rescued the lethality of the JAK2 mutant mice (12). Plek2 deficiency not only ameliorated myeloproliferative phenotypes in the JAK2V617F mutant mice; it also markedly reduced thrombosis in many organ systems. Notably, Plek2-KO mice exhibited only minor age-related anemia without other severe phenotypes, which indicates that Plek2’s proproliferative function is tumor specific (12).

Plek2 is not only highly expressed in the hematopoietic cells; recent studies demonstrated that it is also overexpressed in many solid tumors and associated with worse prognosis (13–16). Specifically, Plek2 gene upregulation was predicted in 2 independent studies to result in a shorter progression-free survival and poor prognosis in lung adenocarcinoma (14, 15). Plek2’s high expression was associated with poor overall survival in gastric cancer (17). The mechanisms of Plek2 in tumorigenesis can be tissue specific, as Plek2 is reported to promote the invasion and metastasis of gallbladder cancer through binding and activation of EGFR (13). Plek2 could also be negatively regulated by miR-873. Reduced expression of miR-873 in pancreatic cancer stem cell led to upregulation of Plek2 and activation of the PI3K pathway (16). The possible role of Plek2 in the PI3K pathway was further indicated through bioinformatic analyses in osteosarcoma (18). However, the mechanisms of Plek2 in driving cell proliferation through PI3K signaling are unknown.

Plek2 contains 2 pleckstrin homology (PH) domains on the amino and carboxyl termini of the protein, which flank a dishevelled, Egl-10, and pleckstrin (DEP) domain. Previous loss-of-function studies showed that deletion of the DEP domain induced the most significant functional defects of Plek2 (10, 19). In this study, we aimed to reveal the mechanism of Plek2 in MPN pathogenesis and identify small-molecule compounds that target Plek2 and block its functions in MPNs. To this end, we first took an in silico approach to screening small molecules that bind to the DEP domain of Plek2 and inhibit its function in driving proliferation. These inhibitors were then used to explore the roles of Plek2 in MPNs. We found that Plek2 functioned as a regulatory protein to directly interact with Akt and recruit PI3K effector proteins as well as heat shock protein Hsp72, which led to the activation and stabilization of Akt. The functions of Plek2 in Akt activation were further demonstrated using a Pten hematopoietic-specific KO mouse model. We further revealed the therapeutic potentials of the Plek2 inhibitors in various MPN models. These studies establish Plek2’s critical role in driving cell proliferation when overexpressed. Thus, the small-molecule Plek2 inhibitors have important implications for treating MPNs and possibly other cancers with activated Akt signaling.

Results

Development of Plek2 small-molecule inhibitors that block cell proliferation. To screen small molecules that bind to Plek2 and potentially inhibit its function, we chose to focus on the DEP domain instead of the PH domains that can be found in many proteins. The DEP domain of Plek2 is highly conserved among different species (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/JCI159638DS1). As there are no reported crystal structures, we built a homology model of Plek2 using prime module (20) incorporated in the Schrödinger platform (https://www.schrodinger.com/products/prime), considering the primary amino acid sequence of the DEP domain as the query, and identified relevant template structures by homology search using BLAST and PSI-BLAST engines. The Plek2 model was then subjected to MolProbity validation, and the model scored 95% or more (21), indicating its suitability for carrying out further in silico screening. We then performed a virtual high-throughput screening (vHTS) using a 3-tier Glide platform implemented in Schrödinger suite (22). We screened approximately 100,000 drug-like small-molecule compounds for those that could bind to the Plek2 DEP domain. From this set, we identified 28 hit compounds that potentially bind to Plek2 (Supplemental Figure 1, B and C, and Supplemental Table 1).

The hit compounds were subsequently screened using a mouse BM erythroid progenitor culture system in erythropoietin-containing (EPO-containing) medium (Figure 1A) (11, 23, 24). These cells underwent rapid proliferation with supraphysiological levels of EPO and closely mimicking accelerated erythropoiesis in MPNs. In these cells, Plek2 was also highly expressed, especially in the late stages of the culture (Supplemental Figure 1D). We tested each of the 28 hits in this system and found compound 17 (NUP-17) to have the most significant inhibitory effects on erythroid proliferation and enucleation (Supplemental Figure 1, E and F). The IC50 in blocking cell proliferation was 56 μM. NUP-17 also had a significant inhibitory effect on enucleation, but minimal effects on cell differentiation (Supplemental Figure 2A).

Figure 1

Development of Plek2 small-molecule inhibitors that block cell proliferation. (A) Schematics of HSPC in vitro culture system for the secondary screen of the hit compounds. Graph is generated using BioRender. (B) The chemical structure of NUP-17d. (C) BM lineage–negative cells were cultured in EPO-containing medium with different amounts of NUP-17d added at the beginning of culture. Cell proliferation was analyzed at 48 hours in culture. IC50 was calculated. (D and E) Same as C except cell differentiation, enucleation in D, and apoptosis in E were analyzed using flow cytometry. (F) ITC analyses demonstrate a direct interaction of NUP-17d with full-length Plek2. (G) 1 × 105 BM lineage–negative cells were cultured with EPO medium as in C or medium containing stem cell factor (SCF). NUP-17d (10 μM) was added at the beginning of cell culture. Cell proliferation was analyzed at 48 hours in culture. (H) BM lineage–negative cells purified from the indicated mice were cultured in SCF medium as in G. NUP-17d (10 μM) was added at the beginning of cell culture. Cell proliferation was analyzed at 48 hours in culture. (I) BM lineage–negative cells transduced with Plek2 shRNA or control scrambled shRNA were cultured in EPO medium and treated with NUP-17d as in G. Cell proliferation was analyzed at 48 hours in culture. Western blotting assay demonstrates the downregulated Plek2 levels. 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 < 0.01; ***P < 0.001.

Based on similarity searching from the in silico screening, we further identified a series of analogs of NUP-17 and found that compound NUP-17d had significantly improved potency (Supplemental Figure 2, B and C, and Figure 1B). The IC50 of NUP-17d was 7.8 μM in the erythroid in vitro proliferation assay (Figure 1C). NUP-17d also significantly inhibited enucleation and differentiation (at higher dosage) of the cultured erythroid cells (Figure 1D) and induced apoptosis (Figure 1E). NUP-17d bound to Plek2 and Plek2-DEP with high affinity (KD = 190 and 305 nM, respectively) in an isothermal titration calorimetry (ITC) assay (Figure 1F and Supplemental Figure 2D). NUP-17d reduced cell proliferation more significantly when the BM lineage–negative cells were cultured in EPO-containing medium compared with those cultured in medium containing stem cell factor to maintain their progenitor status (Figure 1G). We also tested erythroblasts from JAK2V617F-knockin mice with NUP-17d, which showed that JAK2V617F-positive erythroblasts were more sensitive to the compound treatment (Figure 1H). The effect of NUP-17d was Plek2 specific, since no inhibitory effect was observed when Plek2 was acutely depleted by shRNA (11) (Figure 1I).

In addition, we also overexpressed Plek2 in Cos-7 cells, which induced prominent lamellipodia formation, as previously reported (9). NUP-17, and to a greater extent NUP-17d, dramatically reverted lamellipodia in Plek2-overexpressed cells (Supplemental Figure 2, E and F). We next constructed a biotin-conjugated NUP-17d (Supplemental Figure 3A) and confirmed NUP-17d binding to Plek2 and Plek2-DEP in in vitro binding assays (Supplemental Figure 3, B and C). Addition of free NUP-17d reduced the binding of biotin–NUP-17d to Plek2 (Supplemental Figure 3D). Cellular thermal shift assay (CETSA) further revealed specific interaction of NUP-17d with Plek2 in vivo (Supplemental Figure 3E). These studies establish NUP-17d as an effective Plek2 inhibitor.

To further characterize the specificity of the binding between NUP-17d and Plek2 DEP, we applied NUP-17d to the docking model and revealed lysine 156, serine 162, and arginine 194 to be critical for binding (Supplemental Figure 4A). Sequence alignment of the DEP domain in various proteins showed that these residues are not conserved and are highly specific to Plek2 (Supplemental Figure 4B). We next mutated these residues to asparagine, alanine, and glycine, respectively and obtained a recombinant Plek2 DEP 3M protein. An in vitro ITC experiment showed that NUP-17d completely lost its binding to DEP 3M (Supplemental Figure 4C). We further generated a full-length Plek2 3M construct and transduced it into Cos-7 cells. Compared with the WT Plek2, the Plek2 3M mutant failed to increase lamellipodia formation (Supplemental Figure 4, D and E). The mutant also failed to promote proliferation of HEL cells, a hematopoietic cell line harboring a JAK2 mutation (Supplemental Figure 4F). These studies indicate that the inhibitor-binding residues of Plek2 are critical for its function.

Plek2 inhibitors block Plek2-mediated Akt activation. Plek2 is associated with membranes via its PH domains, and that association is dependent on D3 phosphoinositide upon the activation of PI3K (10). The mechanisms of Plek2 in driving cell proliferation remain unclear. We performed an RNA-Seq analysis in the cultured BM erythroblasts treated with NUP-17d. Compared with what occurred in the mock-treated cells, genes in several pathways, including PI3K/AKT/mTOR signaling and mTORC1 signaling, were enriched (Figure 2A), which was confirmed by gene set enrichment analyses (GSEAs) (Supplemental Figure 5A). Consistently, treatme