Surface Expression of Mpl: Aberrant Mpl Trafficking In Myeloproliferative Neoplasms Is Linked to Insufficient Expression of Wild Type Jak2
Authors: Cleyrat Cédric , Vilaine Mathias , Boissinot Marjorie , Girodon Francois , Steinkamp Mara P. , Dobo Irène , Lippert Éric , Corre Isabelle , Wilson Bridget S. , Hermouet Sylvie 
Published: October 2019

Abstract
Abstract 2820

Background:
Mpl, the receptor for thrombopoietin (Tpo), activates Jak2. Mpl and Jak2 are expressed both on platelets and on hematopoietic progenitors, where they initiate critical cell survival and proliferation signals in response to Tpo. Thus, interfering with the “Mpl/Jak2 couple” is likely to alter myelopoiesis. Indeed mutations in JAK2 or MPL are characteristic of myeloproliferative neoplasms (MPN). However, platelet Mpl expression is frequently low in MPN, including MPN with no mutation of JAK2.

Aim:
To determine whether expression of wild type Jak2 (Jak2wt) is altered in MPN, and whether changes in expression of Jak2wt affect Mpl expression.

Methods:
Mpl and Jak2 protein expression was analysed by immunoblotting in platelet and cell lysates. Expression of JAK2 wt and JAK2 V617F was also studied at the mRNA level using quantitative RT-PCR. Transient transfections of HEL and UKE-1 cells were used to study the effects of Jak2wt on Mpl and of MPL on Jak2wt and Jak2V617F. We also studied the cellular localization of Jak2wt, Jak2V617F and Mpl, expressed as chimeric proteins fused to Visible Fluorescent Proteins (VFP, derivatives of GFP) or detected by immunofluorescence methods.

Results:
Following SDS-PAGE and western blotting of normal platelet proteins, Mpl was typically found in two isoforms: the long, mature 84 kDa isoform (Golgi-processed, glycosylated, functional, Tpo-binding form expressed at the cell surface) and the short, 74 kDa isoform (immature Mpl). Four subgroups of MPN patients were defined according to platelet Mpl patterns: pattern 1, mature and immature Mpl expressed at comparable levels, typical of healthy donors and found in 37% of MPN tested; pattern 2, mature Mpl only, typical of reactive states, associated with high JAK2 wt expression and observed in 15% of MPN; pattern 3: immature Mpl only; and pattern 4: no Mpl. Patterns 3 and 4 were observed in 48% of MPN and were associated with low JAK2 wt levels (<200 copies/100 ABL copies). JAK2 wt mRNA levels varied widely in MPN and Mpl pattern was correlated with levels of JAK2 wt, but not JAK2 V617F, mRNA. Blotting studies performed on cell lysates from cultured lines homozygous for JAK2V617F (HEL, UKE-1) detected mainly the 74 kDa immature form of Mpl.
Transfection of JAK2wt in HEL and UKE-1 cells, alone or together with MPL, increased the yield of mature Mpl. Fluorescence studies of HEL cells revealed that both endogenous Mpl and the Mpl-VFP chimeric proteins were largely restricted to an intracellular pool, without significant co-localization with Jak2V617F. In contrast, significant levels of transfected Mpl-VFP reach the plasma membrane in K562 cells that express only Jak2wt; co-localization of Jak2 and Mpl in these cells was significant.

Conclusion:
JAK2 wt and Mpl levels are linked and both are frequently low in MPN, including JAK2 V617F-negative MPN. Co-localization of Mpl and Jak2V617F is poor whereas Mpl and Jak2wt are strongly co-localized, consistent with constitutive association and indicating a role for Jak2wt as chaperone along the exocytic or recycling pathways. Future work will seek to determine the intracellular location of aberrantly trafficked Mpl and establish if other mutations found in MPN are linked to defects in the expression or trafficking of Mpl.

Disclosures:
No relevant conflicts of interest to declare.