Targeting Pleckstrin-2 for the Treatment of Myeloproliferative Neoplasms

Myeloproliferative neoplasms are a group of hematopoietic malignancies that are characterized with the activated JAK2-STAT5 signaling and uncontrolled myeloid cell proliferation. Current first line therapies remain suboptimal with ongoing risks for thrombosis, hemorrhage, impaired quality of life, and risk of transformation to acute leukemia. More recent FDA-approved JAK inhibitors are associated with poor tolerance due to the key roles of JAK2 in normal hematopoiesis. There are unmet medical needs to develop novel targeted therapies to treat the disease.

Pleckstrin-2 (Plek2) is a widely expressed PH domain-containing protein that binds to phosphoinositide with unclear functions. We previously showed that Plek2 is overexpressed in JAK2^V617F mutation positive myeloproliferative neoplasms (MPNs). We identified that Plek2 is a downstream effector of the JAK2-STAT5 pathway. Furthermore, through a mouse genetic approach, we discovered that knockout of Plek2 significantly ameliorated the MPN phenotypes and reverted lethality and thrombosis in JAK2^V617F knock-in mice. These studies demonstrate that Plek2 is critical for the pathogenesis of MPNs with the activated JAK2-STAT5 pathway, and form a strong foundation for the development of Plek2 inhibitors for the treatment of MPNs. Importantly, our published study shows that Plek2 knockout mice do not develop anemia or cytopenia, indicating Plek2’s proto-oncogenic function is mainly manifested in the disease background, which makes Plek2 inhibitors less likely to cause severe adverse effects compared to JAK inhibitor ruxolitinib.

Based on these studies, we used an in silico approach to screen for putative Plek2 binding small molecules and identified hit compounds that bind to the DEP domain of Plek2. Further medicinal chemistry studies identified lead compound NUP-17d that inhibited proliferation of the hyperproliferative hematopoietic cells with potency comparable to ruxolitinib. In addition, biochemical assays showed that NUP-17d inhibited Akt phosphorylation. Further mechanistic studies revealed that Plek2 promoted Akt-PtdIns(3,4)P2 binding via direct interaction with Akt and enhanced Akt activation; while NUP-17d inhibited this process. In addition to the biochemical assays, we also demonstrated Plek2’s function through a mouse genetic approach using a Pten hematopoietic specific knockout mouse model. Knockout of Plek2 significantly reverted the myeloproliferative phenotype in these mice and markedly extended their survival. Therefore, NUP-17d blocks hematopoietic cell proliferation through the disruption of the Plek2 complex and inhibition of the PI3K-Akt pathway. Indeed, treatment of erythropoietin-induced myeloproliferative mouse model and JAK2^V617F knock-in model with NUP-17d significantly ameliorated MPN phenotypes including increase in CBC and splenomegaly.

These studies establish Plek2 as a oncoprotein mediating JAK2-STAT and PI3K-Akt signaling pathways in myeloproliferative neoplasms. In addition to hematologic malignancies, Plek2 is also found to be highly upregulated with an associated worse prognosis in many solid tumors (data from oncomine, kmplot). Therefore, targeting Plek2 could have a broad impact in cancer therapy, especially in cancers with upregulated JAK2-STAT or PI3K-Akt pathway.