Hepcidin is elevated in primary and secondary myelofibrosis and remains elevated in patients treated with ruxolitinib

Abstract

Hepcidin, a peptide hormone produced by hepatocytes, is a key regulator of iron homeostasis in mammals and an important mediator in the innate immune system.1, 2 Pathologically elevated hepcidin levels can cause iron-restricted erythropoiesis and anaemia.3 Myelofibrosis (MF) is a Philadelphia-chromosome-negative myeloproliferative neoplasm (MPN) that is characterized by bone-marrow fibrosis.4 Anaemia is a common problem in MF and is associated with inferior quality-of-life measures and poor prognosis.5, 6 To further assess the relationship between hepcidin and anaemia in MF, we measured serum hepcidin levels in 99 MF patients including 49 primary (PMF) and 50 secondary MF (SMF) patients. Twenty-four of these patients received ruxolitinib therapy, a frequently prescribed JAK1/2 inhibitor for treatment of MF. We also evaluated the relationship between hepcidin and haemoglobin levels, inflammatory cytokines, serum iron markers and erythroferrone. MF patient peripheral-blood (PB) or bone-marrow (BM) samples and healthy donor control PB samples were obtained with written consent according to a protocol approved by the Washington University Human Studies Committee (WU no. 01–1014). Normal control samples were extracted directly from PB. Clinical and genetic information for patients included in the study was obtained through retrospective chart review by the authors Amy Zhou, Maggie J. Allen, and Stephen T. Oh. Statistical analysis was performed using GraphPad Prism (GraphPad Software, La Jolla, CA, USA). The Mann–Whitney U test and Spearman correlations were performed as indicated. Patient characteristics are summarized in Table 1. Hepcidin levels (ng/ml) were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) assay from Intrinsic Lifesciences, La Jolla, CA, USA (Intrinsic Hepcidin IDx™ ELISA) in 99 MF patient plasma samples and nine normal controls. C-reactive protein (CRP), transferrin saturation and ferritin were measured on plasma samples using standard laboratory methods at our institution. Hepcidin was significantly elevated in MF patients compared to normal controls (Figure 1A; median 81.1 vs 8.5, p < 0.0001). There was no difference in hepcidin levels between PMF and SMF patients (Figure 1B; median 103.3 vs 80.8, p = 0.38). A trend between higher hepcidin levels and lower haemoglobin was observed but was not statistically significant (p = 0.719). No correlation was found between hepcidin levels and CRP, transferrin saturation, or ferritin. Hepcidin expression did not differ in samples obtained from patients on treatment with ruxolitinib versus those who were not (median 105.1 vs 80.2, respectively, p = 0.98) (Figure 1C). Erythroferrone (ng/ml) concentrations were quantified using a sandwich ELISA protocol.7 Initially, 96-well plates were coated with a monoclonal capture antibody, washed and blocked. After a 1-h incubation at 25°C with agitation (350 rpm), the plate was washed and incubated for 1 h with 100 μl of biotinylated detection antibody per well (1 μg/ml). The plate was then washed, incubated for 45 min with Neutravidin–horseradish peroxidase conjugate 1/5000 (100 μl per well), and developed with TMB Substrate System for ELISA (ThermoScientific, Waltham, MA, USA) at room temperature for 10 min. The reaction was stopped by adding 50 μl of 2 N sulphuric acid, and the plates were read on a Spectramax 250 (Molecular Devices, San Jose, CA, USA) at 450 nm. Erythroferrone levels were significantly higher in MF patients compared to normal controls (Figure S1A), but no correlation was found between hepcidin and erythroferrone (Figure S1B). When separated by mutational status, no significant difference was observed between hepcidin in JAK2- and CALR-mutated patients, but in non-transfused patients, hepcidin was significantly elevated in CALR- and JAK2-mutated patients compared to triple-negative patients (Figure S1C). Concentrations of 13 cytokines (IFN, IL-10, IL-2, IL-2Rα, IL-6, IL-8, MIP-1α, MIP-1β, TNFα, VEGF, TGF-β1, TGF-β2, TGF-β3) were measured using multiplex cytokine analysis (Meso Scale Discovery, Rockville, MD, USA). No significant correlation was observed between any of the cytokines measured and hepcidin, including IL-6 (Table S1). IL-6 was elevated in MF compared to normal controls and remained elevated in ruxolitinib-treated patients (Figure S2A). Ferritin was elevated in patients on treatment with ruxolitinib compared to those who were not (Figure S2B,C). Our results are consistent with previous studies demonstrating that hepcidin is significantly elevated in MF compared to normal controls.8, 9 We found no significant correlation between any of the cytokines evaluated including IL-6 and IL-2, which was also observed previously in the study by Pardanani et al.8 This differs from the findings by Birgegard et al., where a significant positive correlation between hepcidin and IL-2, IL-6 and TNF-α was observed.9 We observed that hepcidin levels remain markedly elevated in patients treated with ruxolitinib, suggesting that suppression of JAK/STAT signalling alone (downstream of IL-6) by ruxolitinib is not sufficient to normalize hepcidin production. This is consistent with the clinical observation that ruxolitinib does not improve anaemia in MF.10, 11 Momelotinib, another JAK1/2 inhibitor, has been shown to improve anaemia via inhibition of ACVR1/ALK2, decreasing the production of hepcidin in hepatocytes.12, 13 This suggests that targeting hepcidin may be beneficial in treating anaemia in MF. To our knowledge, our study is the first to evaluate differences in hepcidin levels between patients with different driver mutations in MF (JAK2-mutated, CALR-mutated, and triple-negative). We observed that JAK2-mutated and CALR-mutated patients had a significantly higher hepcidin level than patients who were triple-negative, although it is difficult to draw conclusions based on the small sample size of CALR-mutated (N = 11) and particularly triple-negative patients (N = 4) and warrants further investigation in larger cohorts. Our study is also the first to evaluate erythroferrone levels in MF. Erythroferrone levels were significantly elevated in MF, but did not correlate with hepcidin levels, demonstrating a complex relationship between erythropoietic signalling and hepcidin in MF. In conclusion, our findings indicate that abnormal hepcidin production is a hallmark of both PMF and SMF and that hepcidin remains elevated in patients treated with ruxolitinib. Although anaemia in MF is complex, multifactorial and incompletely understood, the impressive pathologic increase in hepcidin concentrations in this condition make hepcidin an attractive therapeutic target. This work was supported by NIH grants R01HL134952 (Stephen T. Oh) and T32HL007088 (Jared S. Fowles). Support for patient sample collection and processing was provided by NIH grant P01CA101937. CRP and Iron studies were run by the Core Laboratory for Clinical Studies (CLCS) at Washington University in St. Louis which is supported in part by the Washington University Diabetes Research and Training Centre (DRTC) and the Washington University Institute of Clinical and Translational Sciences (ICTS). Technical support was provided by the Alvin J. Siteman Cancer Centre Tissue Procurement Core Facility and Immunomonitoring Laboratory, which are supported by NCI Cancer Centre Support Grant P30CA91842. The Immunomonitoring Laboratory is also supported by the Andrew M and Jane M Bursky Centre for Human Immunology and Immunotherapy Programs. The authors thank D. Bender for assistance with multiplex cytokine measurement. Amy Zhou is the principal author and designed the study, performed most of the data collection and analysis, made some of the figures, and wrote the majority of the manuscript. Tim Kong performed most of the statistical analysis, assisted with data analysis, made many of the figures, and assisted with the writing of the manuscript. Jared S. Fowles assisted with performing the hepcidin measurements and data analysis. Chun-Ling Jung performed the erythroferrone measurements and assisted with data analysis. Maggie J. Allen assisted with clinical data collection. Daniel A.C. Fisher assisted with analysis of the cytokine data. Mary Fulbright assisted with performing the cytokine measurements. Elizabeta Nemeth and Tomas Ganz both assisted with data analysis and the writing of the manuscript. Stephen T. Oh helped to develop the study design, assisted with data analysis and interpretation, and assisted with the writing of the manuscript. Amy Zhou has served as a consultant for Disc Medicine and receives research support from Rigel. Stephen T. Oh has served on scientific advisory board/consultant for Disc Medicine, Blueprint Medicines, PharmaEssentia, Constellation, Geron, Abbvie, Sierra Oncology, Incyte, Kartos Therapeutics, CTI BioPharma, Celgene/Bristol Myers Squibb, Gilead Sciences and Novartis. Tomas Ganz receives grant/research support from NIH, Akebia/Keryx and Sierra Oncology. He serves on the scientific advisory board/consultant/board of directors for ADARx, Alnylam, Akebia/Keryx, Ambys, American Regent, Disc Medicine, Global Blood Therapeutics, Gossamer Bio, Incyte, Intrinsic LifeSciences, Ionis, Pharmacosmos, Rockwell, Sierra Oncology and Silarus Pharma. He is a stock shareholder in Intrinsic LifeSciences and Silarus Pharma. Elizabeta Nemeth receives grant/research support from NIH. She serves on the scientific advisory board/consultant/board of directors for Disc Medicine, Intrinsic LifeSciences, Ionis Pharmaceuticals, Vifor Pharma, Protagonist Therapeutics, AstraZeneca/Fibrogen and Silarus Pharma. She is a stock shareholder in Intrinsic LifeSciences and Silarus Pharma. All other authors have no disclosures. Original data can be requested by contacting the corresponding authors. Figure S1 Figure S2 Table S1 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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