S127 CHRONIC MYELOID LEUKEMIA IS ASSOCIATED WITH INCREASED NEUTROPHIL EXTRACELLULAR TRAP FORMATION THAT IS AUGMENTED BY PONATINIB – A POTENTIAL CONTRIBUTOR TO VASCULAR TOXICITY?
Authors: Telerman A. , Granot G. , Shacham‐Abulafia A. , Ellis M.H. , Yarchovsky‐Dolberg O. , Raanani P. , Wolach O. 
Published: June 2019

Background:

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm (MPN) driven by the occurrence of the bcr‐abl1 fusion gene. Cardiovascular (CVS) and thrombotic complications are increasingly reported with the use of certain tyrosine kinase inhibitors (TKI's) used to treat CML such as ponatinib. The mechanism underlying these CVS adversities is not fully understood but may involve off‐target effects that alter the function of vascular endothelial cells as well as other cells involved in the pathogenesis of thrombosis.

More than a decade ago Brinkmann and collegues (Science, 2004) described the occurance of neutrophil extracellular traps (NETs). In response to various stimulli, neutrophils can expell extracellular strands of decondensed DNA in complex with histones and other neutrophil granular proteins. These structures have the ability to ensnare and kill microbes but are also implicated in the pathogenesis of autoimmunity and thrombosis. Previous studies demonstrated that NETs have a role in the increased thrombotic tendency associated with Philadelphia‐negative MPNs (Wolach et al, STM 2018).

Aims:

To study NET formation in CML and evaluate the possible effect of the different TKI's on NET formation.

Methods:

Neutrophils were isolated from patients with CML (n = 7) and from age and gender matched controls (n = 7) by Ficoll gradient. NET formation was analyzed in‐vitro following neutrophil stimulation with phorbol 12‐myristate 13‐acetate (PMA), ionomycin and lipopolysaccharides (LPS) and assessed by the NETosis Assay Kit (Cayman chemical) as well as by immunofluorescence (IF) studies. PAD4 and citrullinated H3 (H3cit) expression was determined by Western blot. Membrane expression of CD11b and reactive oxygen species (ROS) production were analyzed by flow cytometry. NET formation and ROS production were assessed in resting and stimulated conditions and after ex‐vivo pre‐treatment of neutrophils with clinically relevant concentrations of various TKI's.

Results:

Neutrophils from patients with CML had increased NET formation at baseline (p = 0.03) and after stimulation with PMA (p = 0.002). Pretreatment of neutrophils with ponatinib significantly augmented PMA‐induced NET formation as compared to DMSO, imatinib and dasatinib treated neutrophils (p = 0.05 for all comparisons). ROS generation was significantly enhanced in CML patient samples compared to controls (p < 0.01). Ponatinib treatment also led to an increase of ROS levels in neutrophils from patients with CML. PAD4 and H3cit expression was higher in neutrophil lysates form patients with CML (n = 3) as compared to controls (n = 3), (p < 0.05, p < 0.01, respectively).

Summary/Conclusion:

CML is associated with an increase in NET formation which is further augmented by ponatinib. Further studies are needed to assess the possible role of increased NET formation in CML on the CVS and thrombotic complications associated with ponatinib exposure.

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