Eosinophil Differentiation and Viability in Hypereosinophilia Is Impaired By Genetic and Pharmacologic Targeting of LCP1 and PKC Beta
Authors: Ma Guangxin , Gezer Deniz , Herrmann Oliver , Reiter Andreas , Jawhar Mohamad , Schemionek Mirle , Brümmendorf Tim H. , Koschmieder Steffen , Chatain Nicolas 
Published: October 2019

Hypereosinophilia (HE) is caused by a variety of disorders, ranging from parasite infections to autoimmune diseases and cancer. Massive overproduction of hyperactive eosinophils leads to enhanced tissue infiltration and, in some cases, severe organ damage. Clonal malignancies with HE involve the FIP1L1-PDGFRA (F/P)-positive neoplasms and other neoplasms harboring PDGFRA and PDGFRB rearrangements, all of which are sensitive to imatinib. However, most subtypes do not carry a defined genetic aberration and, therefore, lack specific treatment.

In eosinophils, modulation of the actin cytoskeleton is mandatory for priming and migratory function. In this study, we aimed to target the actin-binding protein LCP1, also termed L-plastin, to reduce eosinophilic burden in HE. LCP1 is a direct target of the serine/threonine kinase PKCß, which phosphorylates LCP1 at serine 5, essential for its full activity. We confirmed high LCP1 expression and Ser5 phosphorylation dependent on F/P activity in the HES cell line Eol-1 and 32D-F/P cells. The PKCβ selective inhibitor enzastaurin led to dephosphorylation of LCP1 in Eol-1 cells, and this was associated with significantly reduced proliferation (p < 0.0001), metabolic activity (p < 0.0001), and colony formation (11.8 fold reduced) as well as enhanced apoptosis (p < 0.0001) and impaired migration to SDF-1 (p < 0.001). Apoptosis was associated with ß-Catenin and c-Jun protein stabilization and increased expression of p73. Reduced surface expression of CD11b was detected (1.5 fold reduction; p=0.0043), which may explain the impaired migration capacity to SDF-1. While enzastaurin did not alter F/P-induced STAT3, STAT5, and ERK1/2 phosphorylation, it specifically inhibited STAT1Tyr701 and AKTSer473 but not AKTThr308 phosphorylation, and shRNA knock-down experiments confirmed that this process was mediated by LCP1.

The mTORC2 complex triggers AKTSer473 phosphorylation, and PDK1 is the main Ser/Thr kinase responsible for AKTThr308 phosphorylation. Knockdown of LCP1 protein or reduction of its phosphorylation by enzastaurin treatment resulted in mTORC2 activity loss, while PDK1 activity remained unchanged, suggesting differential phosphorylation of AKTSer473 and AKTThr308. Importantly, AKT substrate activity was significantly reduced upon loss ofAKTSer473 phosphorylation, and SIN1T86 phosphorylation as an indicator of mTORC2 activity was dependent on LCP1 protein, suggesting that targeting of LCP1 reduces mTORC2 activity.

Biologically, both enzastaurin treatment and LCP1 knockdown led to impaired IL5-induced eosinophil differentiation of HoxB8-immortalized murine bone marrow progenitor cells (10.1 fold decrease; p=0.021). Moreover, enzastaurin reduced eosinophil differentiation (1.38 fold decrease; p=0.005) and survival (1.6 fold decrease; p=0.0078) of primary peripheral blood-derived samples from HE patients in vitro.

In conclusion, our data demonstrate that HE involves active LCP1, which triggers mTORC2 activity and eosinophil migration, and that the PKCβ inhibitor enzastaurin may provide a novel treatment approach for hypereosinophilic disorders.

Disclosures

Reiter: Incyte: Consultancy, Honoraria. Brümmendorf:Takeda: Consultancy; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Merck: Consultancy; Janssen: Consultancy.