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4517 Targeting the JAK2V617F Hematopoietic Stem Cell through Beta1 Integrin

Program: Oral and Poster Abstracts
Session: 631. Myeloproliferative Syndromes and Chronic Myeloid Leukemia: Basic and Translational: Poster III
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Biological therapies, Antibody Therapy, Translational Research, MPN, Chronic Myeloid Malignancies, hematopoiesis, Diseases, Therapies, Myeloid Malignancies, Biological Processes
Monday, December 11, 2023, 6:00 PM-8:00 PM

Shinobu Matsuura, PhD, DVM1, Siyang Long, MS2*, Iris Karkempetzaki2,3*, Aikaterini Karagianni, MS, PhD2,3*, Xiaosheng Yang, MS2*, Nasi Huang, MD2*, Yuchen Liu, PhD4*, Chao Zhang, MS, PhD4* and Katya Ravid, Dsc2

1Cardiovascular Section/Department of Medicine, Boston University Chobanian and Avedisian School of Medicine, Boston, MA
2Whitaker Cardiovascular Institute, Boston University Chobanian and Avedisian School of Medicine, Boston, MA
3Department of Internal Medicine, University of Crete, School of Medicine, Heraklion, Greece
4Section of Computational Biomedicine/Department of Medicine, Boston University Chobanian and Avedisian School of Medicine, Boston, MA

The major barrier to progress in treatment of primary myelofibrosis (PMF) is the lack of effective therapies targeting the malignant JAK2V617F hematopoietic stem cell (HSC), the source of disease progression and relapse. Expression of JAK2V617F induces abnormal proliferation and differentiation of HSC, affecting all blood lineages but most prominently the erythro-megakaryocytic lineage, leading to clonal megakaryocyte proliferation, erythrocytosis and thrombocytosis.

In our previously published work (Matsuura et al, Blood 2020) we identified elevated expression of the α5 subunit of the α5β1 integrin receptor as a driver of JAK2V617F megakaryocyte proliferation. Expression of β1 integrin subunit was similar in wild-type (WT) and JAK2V617F megakaryocytes, but activation of β1 integrin, detected by the conformation-specific 9EG7 antibody, was elevated in JAK2V617F megakaryocytes. Following up on those findings, we detected elevated β1 integrin activation in long-term hematopoietic stem cells (HSCLT, LSKCD48-CD150+) from JAK2V617F transgenic mice (Xing et al, Blood 2008) when compared to WT controls.

To determine whether inhibition of β1 integrin in vivo reduces the number of HSCLT in JAK2V617F transgenic mice, we examined HSCLT 24 hours after intravenous administration of 2mg/Kg of anti-β1 integrin inhibitory antibody HMβ1-1. This regimen led to a sharp reduction in number and percentage of HSCLT in JAK2V617F transgenic mice, to 45% of control antibody-treated animals. The effect on WT HSCLT was negligible. HSCST (LSKCD48-CD150-) and MPP2 (LSKCD48+CD150+) were also reduced, but the reduction was not statistically significant. MPP3/4 (LSKCD48+CD150-) and LSK were not affected. Megakaryocyte progenitors (LKCD41+CD150+) were significantly reduced. Inhibition of β1 integrin also sharply reduced peripheral blood platelet numbers in WT animals. The reduction in JAK2V617F transgenic mice was not statistically significant. A longer observation time of 7 days demonstrated that while WT animals were able to mount a robust regenerative response with slight but significant increases in HSCLT and normalization of platelet numbers, in JAK2V617F transgenic mice the number of HSCLT remained depressed, as did downstream stem/progenitor populations at various significance levels.

To gain insight into the mechanisms that lead to reduction in HSCLT, we examined apoptosis, cell cycle and HSC mobilization, all processes known to be affected by β1 integrin. Although basal expression of the apoptotic marker annexin V was decreased in JAK2V617F HSCLT compared to WT, no differences were detected upon administration of HMβ1-1 in both genotypes at 4 hours post-administration. Similarly, peripheral blood colony assays revealed a basally higher level of stem/progenitor mobilization in JAK2V617F, which did not significantly increase upon HMβ1-1 administration. In contrast, the cell cycle profile, examined at 4 hours post-administration to account for the earliest effects, demonstrated a decrease in G0 phase cells and increase in S/G2/M cells indicating cell cycle induction and exit from quiescence in both WT and JAK2V617F HSCLT. The results indicate that inhibition of β1 integrin disrupts the adhesion-mediated cell cycle suppression in both WT and JAK2V617F HSCLT leading to exit from quiescence. However, the downstream consequences of cell division in HSCLT differ significantly, with an apparent loss of regenerative potential in JAK2V617F HSCLT, at least at the observed timepoints. Longer observation times are required to determine whether continuous β1 integrin inhibition can lead to JAK2V617F HSCLT exhaustion. More sensitive assays to monitor mobilization are warranted to quantify possible minute amounts of mobilized HSCLT.

Our preclinical studies indicate that β1 integrin is an attractive target to specifically reduce the number of JAK2V617F HSCLT in vivo. The role of β1 integrin in HSCLT has not been thoroughly studied due to lack of phenotype in β1 integrin-deficient HSC (Brakebush et al, Immunity 2002). Our studies using the anti-β1 integrin antibody in WT HSCLT confirm previous findings. However, β1 integrin seems to be critical for maintenance of JAK2V617F HSCLT, offering a possible therapeutic window. Understanding how β1 integrin supports quiescence in HSCLT will bring us a step closer to understanding the mechanisms that drive malignancy and fitness in JAK2V617F HSCLT.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH