TMPRSS6 Antisense Oligonucleotide Therapy Reverses Inflammation in Jak2^V617F Mutant Mice

Polycythemia vera (PV) is characterized by erythrocytosis, inflammation, and a propensity to develop thromboses. PV patients present with iron deficiency (ID), and the mainstay of treatment, i.e. therapeutic phlebotomy, often worsens the degree of ID. ID is associated with not only symptoms related to anemia but also cognitive impairment and fatigue in the absence of anemia. PV patients often suffer from symptoms attributed to both inflammation and ID. Whether and how ID influences the inflammatory milieu in PV remains unknown. We previously reported that hepcidin-mimetic therapy reverses erythrocytosis and alleviates associated symptoms in PV patients [Kremyanskaya NEJM 2024]. We hypothesize that ID contributes to the inflammatory phenotype in PV by affecting iron-recycling macrophages and hepcidin-mimetic therapy reverses macrophage ID, consequently reducing inflammation. To test this hypothesis, we transplanted Jak2^V617F;Scl Cre (PV) or wild type (WT) mouse bone marrow into recipient C57BL/6 mice. Cohorts of mice were placed on 35 ppm (iron replete (IR)) or 2.5 ppm ID diets. In addition, PV mice were treated with 2.5 mg/kg or 5 mg/kg of a transmembrane protease serine 6 (TMPRSS6) antisense oligonucleotide (ASO) (hepcidin inducer) vs control ASO weekly for 6 weeks. Prior to treatment, the PV mice developed robust erythrocytosis (Hb 18.6 g/dl) and splenomegaly. ID diet and TMPRSS6 ASO therapy both significantly reduced Hb and MCV levels without impacting the degree of splenomegaly. As expected TMPRSS6 ASO induced liver Hamp1 expression in PV mice. Although total WBC counts were unaffected, circulating monocytes were decreased in response to ID diet but not TMPRSS6 ASO treatment. We then analyzed CD45+ bone marrow and F4/80+ spleen cells (i.e. macrophage populations) in PV mice to assess the effects of the ID diet and TMPRSS6 ASO on macrophage iron and pro- vs anti-inflammatory signatures. First, bone marrow CD45+ cells from WT mice on ID diet exhibit increased transferrin receptor 1 (Tfr1) and decreased ferritin (Fth1) expression, consistent with relatively greater iron restriction vs control WT mice. Conversely, bone marrow CD45+ cells from TMPRSS6 ASO-treated PV mice exhibit reversed iron restriction characteristics, namely decreased Tfr1 and increased Fth1 expression. Assessment of pro- and anti-inflammatory markers in bone marrow CD45+ cells and splenic macrophages from IR, ID, and TMPRSS6 ASO-treated PV mice demonstrate 1) elevated pro-inflammatory markers (Il6, Il1b, Cd80, Cd86, Tnfa, Nos2, Cxcl9, and Tlr2) and decreased anti-inflammatory markers (Arg1, Cd274, Il10, Mrc1, Tgfb, and Ym1) in unmanipulated PV relative to WT mice; 2) increased pro-inflammatory and reduced anti-inflammatory markers in WT but not PV mice on ID diet; and 3) 50% suppression of pro-inflammatory markers and 3-4 fold increased anti-inflammatory markers in TMPRSS6 ASO-treated PV mice. Furthermore, liver expression of serum amyloid A1 (Saa1) and serum levels of CXCL9 and CXCL10 are increased in PV, and in vitro TLR2 and TLR4 activity is increased in response to serum from PV vs. WT mice, consistent with increased systemic inflammation, and reversed by TMPRSS6 ASO. These findings demonstrate that TMPRSS6 ASO treatment induces endogenous hepcidin expression, which leads to reversal of inflammation in PV mice. Finally, we hypothesized that ID may also induce inflammation in PV in part as a result of enhanced intestinal leakiness and possibly bacterial translocation. We evaluated markers of intestinal iron absorption and integrity in WT and PV mice and identified 1) evidence of enhanced intestinal leakiness (decreased duodenal F11r) between WT and PV (relative to WT) mice; 2) increased duodenal iron absorbing capacity (increased erythroferrone (Erfe mRNA) and ferroportin (Fpn1 mRNA) expression) in WT mice on ID diet (relative to WT on IR diet) and in PV relative to WT mice; and 3) reduced intestinal leakiness (increased duodenal claudin 3 (Cldn3) and occludin (Ocln) mRNA expression) and iron absorbing capacity in response to TMPRSS6 ASO in PV mice. These findings provide compelling evidence that TMPRSS6 ASO treatment of PV mice leads to reversal of macrophage iron deficiency-induced inflammatory signature, decreased iron absorbing capacity, and may involve reversal of bacterial translocation; we speculate that a similar mechanism may be involved in decreased symptoms in hepcidin mimetic treated PV patients.

Dunbar:Morphosys: Consultancy. Rivella:Meria GTx: Membership on an entity's Board of Directors or advisory committees; Ionis: Membership on an entity's Board of Directors or advisory committees; La Jolla Pharmaceutical Company: Consultancy; Disc Medicine: Consultancy; Protagonist: Consultancy. Guo:Ionis: Current Employment. Levine:Auron: Membership on an entity's Board of Directors or advisory committees; Mana: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Membership on an entity's Board of Directors or advisory committees; Jubilant: Membership on an entity's Board of Directors or advisory committees; Scorpion: Membership on an entity's Board of Directors or advisory committees; Zentalis: Membership on an entity's Board of Directors or advisory committees; Bridge Medicines: Consultancy; Ajax: Membership on an entity's Board of Directors or advisory committees; Kurome: Membership on an entity's Board of Directors or advisory committees; Mission Bio: Membership on an entity's Board of Directors or advisory committees; Syndax: Consultancy; Bridge Bio: Consultancy; Imago: Consultancy; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Epiphanes: Membership on an entity's Board of Directors or advisory committees; Anovia: Consultancy; Bakx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Qiagen: Membership on an entity's Board of Directors or advisory committees. Hoffman:Kymera: Research Funding; Protagonist Therapeutics: Consultancy; Karyopharm therapetics: Research Funding; Cellenkos: Research Funding; Dexcel: Research Funding; Silence Therapeutics: Consultancy. Ginzburg:Bay Clinical: Consultancy; Ionis: Consultancy; Disc Medicine: Consultancy; Protagonist Therapeutics: Consultancy, Research Funding; Denali: Consultancy; Takeda: Consultancy.