Hepcidin Mimetics in Polycythemia Vera: Resolving the Irony of Iron deficiency and Erythrocytosis

Abstract

Purpose of review:

Development of hepcidin therapeutics has been a ground-breaking discovery in restoring iron homeostasis in several hematological disorders. The hepcidin mimetic, rusfertide, is in late-stage clinical development for treating polycythemia vera (PV) patients with a global phase 3 trial [NCT05210790] currently underway. Rusfertide serves as the first possible non-cytoreductive therapeutic option to maintain hematocrit control and avoid phlebotomy in PV patients. In this comprehensive review, we discuss the pathobiology of dysregulated iron metabolism in PV, provide the rationale for targeting the hepcidin-ferroportin axis, and elaborate on the pre-clinical and clinical trial evidence supporting the role of hepcidin mimetics in PV.

Recent findings:

Recently, updated results from two phase 2 clinical trials [NCT04057040 & NCT04767802] of rusfertide (PTG300) demonstrate that the drug is highly effective in eliminating the need for therapeutic phlebotomies, normalizing hematological parameters, repleting iron stores and relieving constitutional symptoms in patients with PV. In light of these findings, additional hepcidin mimetic agents are also being evaluated in PV patients.

Summary:

Hepcidin agonists essentially serve as a “chemical phlebotomy” and are poised to vastly improve the quality of life for phlebotomy requiring PV patients.

Introduction

Philadelphia chromosome negative myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis, are characterized by a unique gene expression profile resulting in upregulation of the JAK-STAT pathway [1–4]. Although considered an indolent disease, PV is associated with a significantly increased incidence of thrombosis (26%) and fibrotic progression (16%) over a 20 year follow up period [5]. Moreover, in 3–5% patients, PV transforms into a type of acute leukemia, called MPN-blast phase, which is associated with a universally dismal prognosis [6]. The clinical phenotype of PV is marked by excessive erythrocytosis resulting in burdensome constitutional symptoms such as fatigue, problems with concentration, pruritis, and microvascular symptoms. PV patients are frequently iron deficient at the time of diagnosis [7–9], and this is further exacerbated by therapeutic phlebotomies administered with the goal of maintaining hematocrit (Hct) below 45% to decrease thrombotic risk [10]. PV patients, however, likely spend substantial time with Hct levels above 45% owing to the significant time intervals between patient visits.

Repeated phlebotomies may in part dampen erythropoiesis by inducing iron deficiency but also potentially contribute to PV-associated systemic symptoms due to the depletion of iron stores in non-hematopoietic tissues [11]. Recent analysis of PV patients treated with ruxolitinib, a JAK1/2 inhibitor, suggests that symptom improvement is at least partly attributable to reversal of systemic iron deficiency [12]. PV as compared to secondary forms of erythrocytosis is associated with relative suppression of hepcidin potentially due to a greater degree of expanded erythropoiesis and iron depletion [13]. Moreover, erythrocytosis in PV continues despite iron deficiency [14], suggesting that disturbed iron metabolism is central to the disease biology, providing a rationale for hepcidin mimetic use in PV. Herein, we review the current understanding of dysregulated iron utilization in PV and how targeting the hepcidin-ferroportin axis can help restore more physiological systemic iron distribution. We further build upon this knowledge to assimilate the pre-clinical and clinical trial evidence supporting the therapeutic efficacy of hepcidin mimetics in PV.

Biology of Hepcidin

Hepcidin, a tightly folded polypeptide hormone produced by the hepatocytes, is the primary regulator of iron homeostasis [15–17]. Hepcidin negatively regulates iron availability via occlusion and degradation of ferroportin, the iron exporter channel. Ferroportin is expressed on all cells involved in iron homeostasis, i.e. duodenal enterocytes, macrophages, and hepatocytes. Thus, hepcidin excess results in decreased intestinal iron absorption and intracellular sequestration of iron within the reticuloendothelial system [18–19], whereas a low hepcidin state is essential for recovery from iron deficiency. Hepcidin expression is in turn modulated by iron in a feedback loop. Hepatocytes upregulate hepcidin transcription by sensing the iron abundance in circulation- either directly via the cell surface expression of transferrin receptor 2 (TfR2) and HFE (homeostatic iron regulator) or indirectly, via iron-stimulated expression of specific bone morphogenic proteins (BMPs) [20, 21]. In a low-iron state, transferrin receptor 1 (TfR1) expression is increased and HFE and TfR1 exist as a complex. As circulatory iron levels increase, HFE gets displaced to make room for iron transporter transferrin to bind with TfR1 [22, 23]. When iron is abundant in the circulation, TfR2 expression exceeds that of TfR1 [24,25]. HFE, now acting as an iron-sensor, preferentially binds to TfR2, resulting in a series of downstream effects [24,26]. This TfR2:HFE complex engages with hemojuvelin, which serves as a co-receptor for the BMP family of signaling proteins and activates the BMP-SMAD pathway [27]. BMP2 and 6 are the key ligands that bind to the BMP receptor in the liver, inducing phosphorylation and activation of SMAD1/5/8 which, in complex with SMAD4, translocate to the nucleus to induce hepcidin transcription [28,29].

This iron-hepcidin feedback loop is further influenced by inflammation and erythropoiesis. IL-6, and to some degree IL-22 and IL-1β, induce JAK-STAT3 signaling in cooperation with the BMP-SMAD pathway to induce hepcidin synthesis [30–33]. Since the majority of iron is utilized during hemoglobin production, erythropoiesis is a key third party moderator of hepcidin and iron metabolism. It is well established that hepcidin is suppressed in iron deficiency anemia, resulting in a functional increase in intestinal iron absorption and iron release from sites of iron recycling (i.e. splenic macrophages) and storage (i.e. hepatocytes). Intriguingly, the signal for hepcidin suppression to meet the iron demands of stress erythropoiesis is not mediated directly by high erythropoietin (EPO) levels, the state of anemia per se or subsequent tissue hypoxia but is a consequence of expanded erythropoiesis itself [34,35]. Recent evidence implicates erythroferrone (ERFE), a peptide hormone secreted by erythroblasts, in both pathologic and physiologic regulation of hepcidin [36]

Pathophysiology of PV involves dysregulation of iron metabolism

PV is a clonal hematopoietic stem cell disorder driven by EPO hypersensitive signaling of the JAK2-STAT5 pathway resulting in excess proliferation of erythroid precursors [1–4, 37–39]. The vast majority (~95%) of PV patients exhibit the acquired JAK2V617F mutation in their stem cells and approximately 3% have acquired mutations in exon 12 of the JAK2 gene [40–41]. Interestingly, exon 12 mutations exclusively present with isolated erythrocytosis and significantly higher Hct levels relative to PV patients with JAK2V617F [42–43]. Moreover, iron deficiency is much more common in patients with exon 12 mutation suggesting that the dominant erythroid phenotype in exon 12 mutant PV is driven by differential iron regulation by specific regions of the JAK2 gene that allow for an even greater utilization of iron towards RBC production as compared to JAK2V617 positive PV [44].

In addition, we previously demonstrated that JAK2 mutant PV patients experience a greater degree of iron deficiency as compared to JAK2 wild type patients with secondary erythrocytosis [13]. This is evidenced by significantly lower MCVs, serum iron and ferritin concentrations, and transferrin saturation; this systemic iron deficiency in PV patients does not resolve despite elevated ERFE with consequent hepcidin suppression. Hepcidin suppression would be expected to result in enhanced intestinal iron absorption and mobilization of intracellular recycled and stored iron, resulting in iron influx into the circulation and recovery from iron deficiency. However, this is not the case in PV, where a low hepcidin state is insufficient to replenish iron stores, implying dysregulated iron homeostasis. We hypothesize that ineffective recovery from iron deficiency despite relative hepcidin suppression in PV may result from the combined effects of concurrent inflammation, insufficiently elevated ERFE with insufficiently suppressed hepcidin, and / or aberrant hypoxia signaling in the intestine preventing recovery from iron deficiency (Figure 1) [45–47]. A recent report suggests that ERFE may have a diminished role relative to that of inflammation in regulating hepcidin expression in PV patients [48]. In this study, deletion of Erfe in PV mice did not alter hepcidin levels or disease severity, resulting in unchanged Hct levels or RBC numbers. The authors further explored the hypothesis that inflammation associated with PV leads to hepcidin upregulation. Using a human hepatocyte cell line, HepG2 cells, the authors demonstrated that the increased hepcidin expression was induced by PV plasma but not plasma from normal controls and was normalized by blocking IL-6 binding to its receptor [48]. These findings suggest that inflammatory cytokines in PV may be crucial to disordered iron utilization. In addition, persistent erythropoiesis despite iron deficiency in PV may also occur as a consequence of aberrantly iron hypersensitive erythropoiesis preventing physiological mechanisms that normally coordinate iron supply with erythropoietic output (Figure 1) [14, 49–50]. A review on dysregulated iron metabolism in PV was recently published [13].

Figure 1: Multiple proposed factors involved in ineffective recovery from systemic iron deficiency in PV:

Decreased gut hypoxia signaling reduces oral iron absorption, hyperactive JAK-STAT signaling induces IL-6 production which co-ordinates with BMP-SMAD pathway to upregulate hepcidin synthesis, lower than expected ERFE levels with consequent insufficient hepcidin suppression and finally, aberrant iron hypersensitive erythropoiesis allows for preferential iron utilization for hemoglobin synthesis at the expense of other cellular requirements, depleting iron stores.

Current standard of care for PV

Current treatment strategies for PV are aimed at reducing the risk of thrombosis and alleviating systemic symptoms. Unfortunately, none of the currently available treatment options definitively demonstrate effectiveness in preventing disease progression to myelofibrosis or MPN-blast phase. The European Leukemia Network (ELN) guidelines are based on a two-tiered risk categorization with low risk PV patients defined as those younger than 60 years of age with no prior history of thrombosis and high risk PV patients as those older than 60 years or those with any history of thrombotic event [51].

For high risk PV patients, consensus guidelines recommend cytoreductive therapy with supplemental phlebotomies and daily low dose aspirin [52]. Hydroxyurea (HU) has the longest track record of use in this setting. A 2017 report on the long term outcomes of patients from the ECLAP database reported a significant reduction in the cumulative rate of cardiovascular events, hematologic transformation, and overall mortality in the concurrently HU with phlebotomy treated group of PV patients relative to those treated with phlebotomy alone [53]. A similar benefit was not observed in the low risk PV patient group owing to the occurrence of a limited number of thrombotic or fatal events. Peg-IFN and recently FDA approved Ropeginterferon α2b (Ropeg-IFN) are the two interferon α formulations that are also used as front line therapy in high risk PV patients. They demonstrate delayed but eventually greater hematological response rates relative to HU with long term treatment [54–56]. In recent PROUD-PV/CONTINUATION PV studies, Ropeg-IFN demonstrated a greater decrease in JAK2V617F allele burden over time compared to standard therapy, with 20% of patients achieving <1% allele frequency after 6 years of treatment. Similar effects with greater decrease in JAK2V617F allele burden over time compared to HU was seen with Peg-IFN in MPN-RC 112 study.

An individualized approach dictates the best first line therapy for each PV patient with both HU and IFN serving as excellent cytoreductive options. Despite these well-accepted, effective therapeutic options, more than 35% of PV patients continued to require phlebotomies to maintain Hct control within the first year of treatment with HU or peg-IFN as evidenced by the MPN-RC 112 trial [56]. Ruxolitinib is FDA approved for PV patients who are intolerant or refractory to HU based on two large RCTs. Ruxolitinib has shown significant clinical benefit in controlling Hct levels, reducing spleen size, and improving systemic symptoms [57,58]. Despite this, approximately 20% of PV patients continued to require concurrent phlebotomy during treatment with ruxolitinib [57].

Recommended standard of care for low risk PV includes once daily low dose aspirin and periodic phlebotomies to maintain Hct levels below 45%; this approach stems from well-accepted evidence for reduction of cardiovascular events and mortality with maintenance of Hct levels below 45% [10, 59]. Cytoreductive therapy is not typically indicated in low risk PV patients, however, may occasionally be utilized in those patients when high phlebotomy requirements lead to worsening constitutional symptoms from systemic iron deficiency or intractable pruritus [60]. In some low risk PV patients, increasing leukocytosis and thrombocytosis may also prompt initiation of cytoreductive therapy although this approach remains relatively controversial owing to the lack of evidence that reversal of asymptomatic leukocytosis and thrombocytosis results in effective reduction of the thrombotic risk of PV patients. Ropeg-IFN has a longer half-life relative to Peg-IFN and has been suggested as front line therapy option even in low risk phlebotomy dependent PV patients. However, there is currently no evidence from long-term phase 3 trials to justify these recommendations [61].

Taken together, these findings provide evidence of the unmet need requiring targeted novel therapeutic options to treat both low and high risk PV patients.

Pre-Clinical evidence for use of hepcidin mimetics in PV

Evidence from studies of anemia of chronic inflammation, a condition in which inflammation-mediated increases in hepcidin expression leads to iron sequestration and iron restricted erythropoiesis, predicts that increased hepcidin in PV may also enable suppression of erythropoiesis. Hepcidin elevation would be expected to sequester recycled and stored iron and prevent iron absorption, resulting in reduced iron availability for erythropoiesis and replenishing iron stores within liver and splenic macrophages, thus aiding in recovery from systemic iron deficiency (Figure 2) [13,62–63].

Figure 2: Rationale for using hepcidin mimetic in PV:

Hepcidin mimetic such as rusfertide blocks iron export from storage sites by blocking and degrading ferroportin, thus sequestering iron within the reticuloendothelial compartment and limiting iron availability for unchecked erythropoiesis.

Initially, pre-clinical studies demonstrated proof-of-principle for this approach using minihepcidins, engineered peptides with the necessary functional ferroportin binding domain [64]. Use of minihepcidin in Jak2^V617F mice, a well-established PV mouse model [65], resulted in a significant dose-dependent decrease in RBC count, Hct and splenomegaly [66]. In addition, minihepcidin resulted in increased iron in the splenic red pulp of PV mice, consistent with the expectation that hepcidin mimetics would lead to sequestration of recycled iron. More recently, administration to Jak2^V617F mice of another hepcidin mimetic agent—transmembrane protease 6 (TMPRSS6) antisense oligonucleotide, leading to the downregulation of TMPRSS6 gene product which prevents the degradation of HJV, yielding an increase in endogenous hepcidin expression in the liver—also resulted in decreased RBC counts and Hct levels as well as suppression of bone marrow erythroblast numbers [67]. Similar findings were also recently demonstrated using a parenteral synthetic hepcidin [68] and an orally bioavailable ferroportin inhibitor [69]. Together, these pre-clinical studies provide significant support for the rationale of targeting the hepcidin-ferroportin axis in restoring iron homeostasis to ameliorate erythrocytosis (Figure 2) and possibly eliminate phlebotomy requirement in PV patients.

Clinical trials of PTG-300 (Rusfertide) in PV

In the last few years, significant progress has been made towards translating the use of hepcidin mimetics for treatment of PV patients. Preliminary results from two phase 2 trials, REVIVE (NCT04057040) and PACIFIC (NCT04767802), evaluating the safety and efficacy of hepcidin mimetic rusfertide (PTG-300) in phlebotomy-requiring PV patients treated with phlebotomy alone or with concurrent cytoreductive agents demonstrate a virtual elimination of phlebotomy requirements, control of erythrocytosis, increase in systemic iron stores, and a potential decrease in systemic symptoms [70–72]. The REVIVE study evaluated rusfertide in 70 PV patients requiring at least 3 phlebotomies in the 6 month period prior to study enrollment [70]. A dramatic reduction in the need for phlebotomies was seen during the first 28 weeks of treatment, with 84% of the subjects achieving phlebotomy-independence. Efficacy was demonstrated in both low and high risk PV patients, including patients treated with phlebotomy alone as well as those treated with concurrent HU, IFN or ruxolitinib. Hct control was sustained in patients treated for up to 2 years on the study.

The PACIFIC trial enrolled 20 PV patients in Asia, where practice patterns differ from those in the US and Europe. As a consequence, patients had a higher baseline Hct of greater than 48% [71]. Mean Hct level prior to the initiation of rusfertide was 50.7%, which rapidly improved to the goal Hct below 45% within an average of 4.7 weeks. In both of these phase 2 trials, iron stores vastly improved with normalization of ferritin, transferrin saturation and MCV. Rusfertide was very well tolerated with mostly grade 1–2 adverse events, most frequent being transient injection site reactions which were managed by local therapies alone.

The global, multicenter, randomized, placebo-controlled phase 3 VERIFY trial (NCT05210790) is currently underway [73] and will further clarify the potential role of rusfertide in management of patients with PV.

Conclusions and future directions

Due to the promising early results of the clinical trials using rusfertide in PV patients, several additional hepcidin mimetic agents are currently being explored for this indication (Table 1). Based on convincing pre-clinical evidence [67], a phase 2 trial using sapablursen (antisense oligonucleotide against TMPRSS6 mRNA) in monthly subcutaneous dosing is under way (NCT05143957) [74]. In addition, SLN124, a small interfering RNA against TMRSS6 targeted to the liver, currently in a phase 2 trial for β-thalassemia and myelodysplastic syndrome patients, is also being explored for PV patients [75]. Whether exogenous administration or stimulation of endogenous hepcidin expression will be equivalently effective in limiting iron availability for erythropoiesis in PV remains to be determined. Finally, vamifeport, is a first-in-class oral ferroportin inhibitor, which is currently being studied in a phase 2 trial in sickle cell disease patients, may also find application in PV as an oral alternative [76].

Table 1:

Hepcidin mimetics in clinical development

Drug/ Molecule name	Class	Mechanism of Action	Clinical Trials
Rusfertide	Synthetic hepcidin mimetic peptide	Contains functional ferroportin binding domain- directly binds to and degrades ferroportin	• Preliminary results from two phase 2 trials (NCT04057040, NCT04767802): Rusfertide is highly effective in eliminating phlebotomy, reverses iron deficiency, no major toxicities. • Phase 3 trial underway (NCT05210790)
Sapablursen	Tmprss6 ASO	Downregulation of TMPRSS6 gene product -prevents the degradation of HJV and increases endogenous hepcidin expression	• Ongoing Phase 2a trial in phlebotomy dependent PV patients (NCT05143957)
SLN124	Tmprss6 siRNA		• Phase 1/2 trial in PV (NCT05499013) planned to begin in 2023 • Ongoing phase 1 study in alpha/beta thalassemia and low risk MDS (NCT04718844)
Vamifeport	Ferroportin inhibitor	Orally bioavailable small molecular directly inhibits ferroportin	• Ongoing phase 2 trial (NCT04817670) in sickle cell disease • Pre-clinical evidence in PV67

Overall, the rusfertide trials provide encouraging data that hepcidin mimetics are highly efficacious in achieving phlebotomy independence and thus breaking the vicious cycle of repeated phlebotomy-induced systemic iron deficiency that further exacerbates symptom burden in PV patients. Although requiring many years of study, it remains to be seen whether a more consistent Hct control as achieved by rusfertide will translate into a long term reduction of thrombotic risk in PV patients and whether decreased iron availability can re-establish a proliferative benefit to normal rather than mutant hematopoietic stem cells in this disease. Albeit less well accepted, some data suggests that increasing leukocytosis may also be associated with increased thromboembolic risk and extreme thrombocytosis may be associated with excessive numbers of bleeding episodes [77]. As such, some PV patients may benefit from a myelosuppressive agent in combination with rusfertide. Moreover, the expanding tool kit of potential therapies with differing mechanisms of action provide opportunities to target multiple sequelae of PV in parallel.

Taken together, a deeper understanding of disordered iron metabolism in several hematological disorders has ushered in an era of hepcidin therapeutics with the potential to fulfill the unmet need in a gamut of disease entities, ranging from iron loading anemias to PV, and possibly additional novel clinical applications on the horizon.

Key points.

1. Pathophysiology of PV is marked by dysregulated iron metabolism as evidenced by systemic iron deficiency and aberrant iron hypersensitive erythropoiesis.

2. Hepcidin agonists can restore a more physiological iron utilization in PV by redistributing iron away from the hemoglobin compartment and into storage sites.

3. Phase 2 trials demonstrate that rusfertide, a hepcidin mimetic, is highly effective in achieving and maintaining hematocrit below 45%, eliminating the need for therapeutic phlebotomies, repleting iron stores and relieving constitutional symptoms.

4. Additional strategies targeting the hepcidin-ferroportin axis are currently being explored in PV.