Defective Autophagy Process and Increased ROS Formation in Primary Myelofibrosis

Introduction: We and others have reported markedly increased ROS formation in primary myelofibrosis (MF) including post-ET or PV-MF when compared with essential thrombocytosis (ET), polycythemia vera (PV) and normal controls. A growing body of evidence illustrates the interdependency between the Keap1-Nrf2 pathway and p62-mediated selective autophagy, are playing an important role in major cellular defense mechanisms against oxidative and electrophilic stress ; dysregulation of p62-Keap1-Nrf2 axis has been implicated in tumor development. Therefore, we studied p62 mediated autophagy and Nrf2 pathways in patients with MF ,ET and PV.

Materials and Methods:33 patients including 15 patients with MF, 7 patients with PV, 11 patients with ET and 10 normal controls were studied. Cellular ROS measurement: Cellular ROS was determined by a dichlorofluorescein (DCF) assay. The oxidation of H2DCFDA was measured by flow cytometry. Real-time PCR assay: Total RNA was extracted from normal control or patient mononuclear cells using RNeasy Kits from Qiagen (Germantown, MD). Real-time PCR was performed using SsoAdvanced™ Universal SYBR^® Green Supermix (Bio-Rad, Hercules, CA) on Bio-Rad iQ5 Multicolor Real-Time PCR Detection System. Western blotting: Total cell lysates were obtained from normal control or patient mononuclear cells. Primary antibodies against Beclin-1, LC3 and p62 were purchased from Cell Signaling Technology (Danvers, MA).

Results: Fig.1a shows significantly elevated ROS in MF (2127±261.6) when compared with PV (1090 ± 163.8) or ET (1031 ± 140.9) or controls (549.1 ± 52.50). Fig.1b. shows that Nrf2 protein levels (ratio to controls) was significantly reduced in MF (41.84 ± 29.33), PV (14.42 ± 2.96), ET (14.05 ± 3.02), than the controls ( 101.3 ± 7.64). Fig.1c shows that the RT-PCR values of p62 was significantly increased in MF (1.34 ± 0.06) and grouped PV (1.21 ± 0.07), ET (1.14 ± 0.08) and MF as MPN (1.27 ± 0.04) than controls (1.00 ± 0.05),( P<0.05); ET or PV were not significantly elevated than controls.

Conclusions: Patients with MF had increased levels of ROS when compared with PV and ET while Nrf2 levels were significantly reduced in all three( ET,PV, MF) which implies defective detoxification process in MPNs. P62, a marker for defective autophagy was significantly increased in MF than controls . This implies that there is a defective autophagy process in MF, contributing further to the formation of P62 and ROS. There is also a defective detoxification mechanism which further leads to more ROS formation and further DNA damage. Further studies are in progress on other autophagy markers which will enlighten more about the pathophysiology of MF.

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