Possible Association between GRP78 Expression and the Risk of Thromboembolism in JAK2V617F-Positive PV and ET Patients

Glucose-regulated protein 78 (GRP78) is a chaperone protein that plays a crucial role in the endoplasmic stress response. Recently, we reported that GRP78 expression is upregulated in cell lines derived from myeloproliferative neoplasms (MPNs) harboring JAK2V617F (Nakajima K et al. Leukemia Research 2023). Treatment with ruxolitinib diminished GRP78 expression in these cells. We also found that GRP78 was secreted into the culture media by these cells and stimulated the JNK pathway in human stromal cell lines when they were cocultured with MPN cells via a transwell system. Elevated GRP78 expression was also confirmed in the bone marrow cells of patients with primary myelofibrosis and post-polycythemia vera (PV) or essential thrombocythemia (ET) myelofibrosis.

Interestingly, recent publications have suggested that GRP78 is involved in thromboembolic events (TEs), as shown in a variety of diseases, including diabetes mellitus, atherosclerosis and COVID-19. Furthermore, GRP78 interacts with several coagulation factors, including tissue factor or factor VIII, to stabilize and activate these proteins.

On the basis of these findings, we speculated that GRP78 may contribute to TEs in MPN. To this end, we initially evaluated the expression levels of GRP78 in JAK2V617F-positive PV (n=16) and ET (n=14) patients and analyzed the correlations between the expression levels of GRP78 and clinical parameters. GRP78 expression levels in bone marrow samples obtained at the time of diagnosis were analyzed via immunohistochemistry, and the ratio of the GRP78 expression area to the total cellular area was calculated. We found that the GRP78 expression area was significantly greater in PV patients than in ET patients. (13.9% vs. 7.0%, p=0.003). The ratio of the GRP78 expression area was correlated with the JAK2V617F allele burden (AB) (Pearson r=0.4, p=0.03), white blood cell count (r=0.58, p=0.0008), neutrophil count (r=0.46, p=0.01), monocyte count (r=0.46, p=0.01) and hemoglobin level (r=0.36, p=0.049). Next, we analyzed the serum GRP78 levels in JAK2V617F-positive MPN patients (PV=10, ET=9 and myelofibrosis = 3) via ELISA. We found a significant correlation between the serum GRP78 level and the JAK2V617F allele burden (Pearson r=0.47, p=0.03). In addition, the serum GRP78 level was positively correlated with the hemoglobin concentration (r=0.503, p=0.02)hematocrit level (r=0.558, p=0.009) and monocyte count (r=0.435, P=0.049). Among the 22 patients, 17 patients were treated with cytoreductive agents (hydroxyurea = 14, anagrelide = 1 and ruxolitinib = 2), and the remaining 5 patients received only antiplatelet therapy at the time of sample collection. Serum GRP78 levels did not differ between patients who received cytoreductive therapy and those who did not receive these agents.

In accordance with our previous in vitro observations that GRP78 levels are regulated by JAK2V617F activity, we confirmed positive correlations between JAK2V617F AB and GRP78 expression area in bone marrow biopsy samples and serum GRP78 levels.

Recent studies revealed that high JAK2V617F AB is a risk factor for thromboembolic events both in PV and ET patients. In addition, increased white blood cell counts are associated with an increased incidence of thromboembolic events in MPN patients.

Together with previous findings, our present observations that GRP78 levels in bone marrow cells and serum are associated with high JAK2V617F AB and high white blood cell counts suggest that GRP78 may be correlated with the risk of thromboembolism in MPN patients. Further studies with large cohorts of patients are needed.

Kirito:GSK: Honoraria; PharmaEssentia Japan: Other: Lecture fees.