JAK2V617F-Mutant Vascular Niche Promotes JAK2V617F Clonal Expansion in Myeloproliferative Neoplasms

Zhan, Huichun; Lin, Chi Hua Sarah; Segal, Yarden; Kaushansky, Kenneth

doi:10.1182/blood.V128.22.952.952

Abstract

Introduction

The myeloproliferative neoplasms (MPNs) are clonal stem cell disorders characterized by hematopoietic stem/progenitor cell (HSPC) expansion and overproduction of mature blood cells. The acquired mutation JAK2^V617F plays a central role in these disorders. The mechanisms responsible for MPN HSPC expansion are not fully understood, limiting the effectiveness of current treatments. Endothelial cells (ECs) carrying the JAK2^V617F mutation can be detected in patients with MPNs. In this work we test the hypothesis that the JAK2^V617F-mutant vascular niche promotes JAK2^V617F clonal expansion in MPNs.

Methods

JAK2^V617F Flip-Flop (FF1) mice (Radek Skoda, Switzerland) and Tie2-Cre mice (Mark Ginsberg, UC San Diego) were crossed to generate a strain in which human JAK2^V617F is expressed specifically in hematopoietic cells and ECs (Tie2/FF1). Animal experiments were performed in accordance with the Institutional Animal Care and Use Committee guideline.

Results

Primary lung ECs (CD45^-CD31⁺) were isolated from Tie2/FF1 mice (JAK2^V617F ECs) and age-matched littermate control mice (JAK2^WT EC). JAK2^V617F ECs proliferated to a greater extent than JAK2^WT ECs. A vascular tube formation assay was performed as a measure of in vitro angiogenesis and revealed that JAK2^V617F ECs had significantly increased angiogenesis compared to JAK2^WT EC. (Figure 1)

To examine the role of JAK2^WT and JAK2^V617F ECs in MPN hematopoiesis in vitro, marrow Lin^-cKit⁺ HSPCs were isolated from Tie2/FF1 and age-matched littermate controls and cultured on a feeder layer of JAK2^WT or JAK2^V617F ECs under serum-free conditions. While there was no difference between JAK2^V617F and JAK2^WT HSPC proliferation when co-cultured with JAK2^WT EC, the JAK2^V617F HSPC displayed a relative growth advantage over the JAK2^WT HSPC (1.24-fold, p = 0.041) when co-cultured on JAK2^V617F EC.

Next, the effects of JAK2^V617F-bearing vascular niche on MPN hematopoiesis were studied in vivo using marrow transplantation assays. First, wild-type CD45.1 marrow cells were transplanted into lethally irradiated (950cGy) 8-14 week old Tie2/FF1 mice or controls (CD45.2) (n=7 in each group). During a >8-month follow up, all wild-type recipients displayed full donor engraftment, while 4 of 7 Tie2/FF1 recipient mice displayed recovery of JAK2^V617F-mutant hematopoiesis (mixed donor/recipient chimerism), suggesting that the JAK2^V617F-mutant vascular niche may protect the JAK2^V617F-mutant HSPCs from the lethal irradiation. Although marrow and splenic hematopoietic progenitors were significantly increased in the Tie2/FF1 recipients, there was no difference in peripheral blood (PB) cell count or PB hematopoietic progenitor cell numbers between the Tie2/FF1 mice and control mice. (Figure 2)

Second, we performed a competitive marrow transplantation experiment in which 5 x 10⁵ CD45.2 donor marrow cells from Tie2/FF1 mice were injected intravenously together with 5 x 10⁵ competitor CD45.1 wild-type marrow cells intro lethally irradiated Tie2/FF1 mice or control mice (CD45.2) (n=5 in each group). During a 4-month follow up, Tie2/FF1 recipients displayed lower PB CD45.1 (i.e. wild-type donor) chimerism than the control mice (p = 0.025), suggesting that the mutant vascular niche in Tie2/FF1 promoted the JAK2^V617F clonal maintenance/expansion and/or inhibited the wild-type hematopoiesis. By 18 weeks post-transplant, Tie2/FF1 recipients developed a MPN phenotype with elevated WBC count (22.7 vs. 9.85 x 10⁹/L, p = 0.049), platelet count (5336 vs. 1029 x 10⁹/L, p = 0.033), increased splenomegaly, increased total marrow and splenic hematopoietic progenitors, and increased CD150⁺CD48^- E-SLAM cells (a highly enriched HSPC population) in both marrow (5-fold, p = 0.0004) and spleen (5.5-fold, p = 0.000004) compared to the control mice. To begin to understand the EC signals responsible for JAK2^V617F clonal expansion and MPN pathogenesis, qRT-PCR of purified marrow ECs identified up-regulation of MPL and CXCL12 in both arterial (CD45^-CD31⁺Sca1⁺) and sinusoidal (CD45^-CD31⁺Sca1^-) marrow ECs in Tie2/FF1 mice compared to controls.

Conclusions

Our study establishes that JAK2^V617F-bearing ECs form an important part of the MPN HSPC niche and contribute to mutant stem/progenitor cell expansion both in vitro and in vivo.