Short-term administration of JAK2 inhibitors reduces splenomegaly in mouse models of b-thalassemia intermedia and major

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Published Ahead of Print on November 2, 2017, as doi:10.3324/haematol.2017.181511. Copyright 2017 Ferrata Storti Foundation.

1 Published Ahead of Print on November 2, 2017, as doi: /haematol Copyright 2017 Ferrata Storti Foundation. Short-term administration of JAK2 inhibitors reduces splenomegaly in mouse models of b-thalassemia intermedia and major by Carla Casu, Vania Lo Presti, Paraskevi Rea Oikonomidou, Luca Melchiori, Osheiza Abdulmalik, Pedro Ramos, and Stefano Rivella Haematologica 2017 [Epub ahead of print] Citation: Casu C, Lo Presti V, Oikonomidou PR, Melchiori L, Abdulmalik O, Ramos P, and Rivella S. Short-term administration of JAK2 inhibitors reduces splenomegaly in mouse models of b-thalassemia intermedia and major. Haematologica. 2017; 102:xxx doi: /haematol Publisher's Disclaimer. E-publishing ahead of print is increasingly important for the rapid dissemination of science. Haematologica is, therefore, E-publishing PDF files of an early version of manuscripts that have completed a regular peer review and have been accepted for publication. E-publishing of this PDF file has been approved by the authors. After having E-published Ahead of Print, manuscripts will then undergo technical and English editing, typesetting, proof correction and be presented for the authors' final approval; the final version of the manuscript will then appear in print on a regular issue of the journal. All legal disclaimers that apply to the journal also pertain to this production process.

2 Title: Short-term administration of JAK2 inhibitors reduces splenomegaly in mouse models of ß-thalassemia intermedia and major Running title: JAK2 inhibitors for the treatment of ß-thalassemia Carla Casu 1, Vania Lo Presti 1, Paraskevi Rea Oikonomidou 1,2, Luca Melchiori 3, Osheiza Abdulmalik 1, Pedro Ramos 4 and Stefano RIvella 1. 1 Department of Pediatrics, Division of Hematology, Children s Hospital of Philadelphia (CHOP), Philadelphia, PA, USA 2 Penteli's Children's Hospital, Pediatric Department, Athens, Greece 3 Adaptimmune Therapeutics Ltd., Abingdon, UK 4 Novartis Pharma AG, Basel, Switzerland Correspondence: Stefano Rivella, PhD, Professor of Pediatrics, Department of Pediatrics, Division of Hematology, Children s Hospital of Philadelphia, 3615 Civic Center Blvd, Room 302B, Philadelphia, PA, 19104; rivellas@ .chop.edu. Phone: ; Fax: Word count: 1197 Figures: #2 Supplemental file: #2

3 Acknowledgments: This work is supported by grants from the National Institutes of Health (NIDDK-R01DK and NIDDK-R01DK to S.R.). Blood samples were analyzed at Institutional Clinical and Translational Science Award Research Center NIH/NCATS (National Center for Advancing Translational Sciences), Grant UL1TR The authors gratefully acknowledge the generous support by the Jean and Di Gaetano families and the Children s Hospital of Philadelphia Foundation. Beta-Thalassemia is one of the most common congenital red blood cell (RBC) disorders characterized by limited β-globin synthesis and ineffective erythropoiesis.(1) Based on the requirements for blood transfusion the disease can be classified as non-transfusion dependent thalassemia (NTDT), or transfusion dependent thalassemia (TDT).(2) We previously demonstrated that in β-thalassemia, in response to anemia and hypoxia, high levels of erythropoietin (EPO) are associated with increased activation of the JAK2/STAT5 pathway and number of erythroid progenitors, especially in the spleen.(3) The extramedullary hematopoiesis (EMH) leads to hepatosplenomegaly that enhances the entrapment of RBCs in the spleen and exacerbates the anemia, further worsening the hepatosplenomegaly. Although splenectomy has now become almost obsolete in patients with TDT, the procedure is still more frequently used in patients with NTDT, because it may improve anemia and avoid the need for transfusions. (4) Unfortunately, this procedure remarkably increase the rate of thromboembolic complications. (5) Clinical studies in patients affected by myeloproliferative disorders, characterized by activating JAK2 mutations (such as polycythemia vera, PV), suggest that JAK2 inhibitors (JAK2i) are an effective treatment for splenomegaly.(6-10) Given the central role of the EPO/JAK axis in β-thalassemia, we hypothesized that targeting JAK2 could be effective in reversing splenomegaly, and therefore be used as an alternative to splenectomy. In fact, preliminary studies showed that the use of a JAK2i improved splenomegaly in a mouse model of β-thalassemia intermedia or NTDT (Hbb th3/+ ).(3) In this subsequent study, we administered the JAK2i INCB (Ruxolitinib) and TG (Fedratinib, SAR302503) for 10 days to Hbb th3/+ mice, and to mice requiring chronic blood transfusions for survival (TDT). TDT mice (C57-FLC th3/th3 ) were generated by transplanting fetal liver cells (FLC) from Hbb th3/th3 embryos into C57BL/6 congenic animals. One month following engraftment, C57-FLC th3/th3 mice exhibit prominent

4 splenomegaly and severe ineffective erythropoiesis, rapidly becoming dependent on chronic blood transfusion for survival.(11) Hbb th3/+ mice treated with either JAK2i showed a mild reduction in hemoglobin (Hb) levels, (8-10%), and RBC (8-9%) when compared to vehicle-treated animals (Figure 1A and B). This mild worsening of anemia, however, was sufficient to increase serum EPO levels compared to placebo-treated Hbb th3/+ controls (up to 84%) (Figure 1C). Both JAK2i significantly reduced spleen weight (60% for INCB and 31% for TG101348; Figure 1D) and reticulocyte numbers (Supplementary Figure 1A). Compared to placebo-treated mice, flow cytometry studies revealed that animals receiving JAK2i exhibited a reduction in the number of erythroid nucleated cells in the spleen but not in the bone marrow (Figure 1E and Supplementary Figure 1B).(12) This suggests that JAK2i might target preferentially rapidly proliferating erythroid progenitors in the spleen, as in extramedullary stress erythropoiesis. No differences were observed in serum HEPCIDIN levels (Supplementary figure 2A) or other iron parameters (Supplementary Figure 2B-F). As the reduction in splenomegaly was associated with mild anemia, we investigated whether co-administration of a JAK2i with transfusion could further reduce the splenomegaly while preventing the anemia. We first tested this hypothesis by transfusing RBCs from transgenic animals expressing GFP into NTDT Hbb th3/+ mice. GFP animals were utilized in order to discriminate by flow cytometry the relative contribution of endogenous versus donor RBCs in circulation at the end of treatment. Weekly transfusions started one week prior to initiating treatment with JAK2i and continued throughout the experiment (for a total of three weeks). As expected blood transfusions reduced splenomegaly (28%) compared to non-transfused controls (Figure 2A). When the transfusion was combined with the administration of JAK2i for 10 days, the spleen weight was further reduced by 64% for INCB and 45% for TG versus animals that received transfusion alone (Figure 2A). This observation was supported by flow cytometry studies, which showed reduction in the proportion of erythroid nucleated cells in the spleen (Figure 2B and Supplementary Figure 3A). We did not observe any difference in Hb levels between transfused animals in presence or absence of JAK2i, although a trend towards lower Hb levels was noted in mice treated with both transfusion and JAK2i (Supplementary Figure 3B). The contribution of endogenous GFP - RBCs to the total number of RBCs was smaller in JAK2i treated mice compared to the placebo treated group, although the total number of transfused GFP + RBCs did not significantly differ among groups (Figure 2C).

5 This finding, together with the reduction of nucleated erythroid cells in the spleen (Figure 1E, 2B, Supplementary 1A and 3A), indicates that the combination of JAK2i and transfusion further suppresses extramedullary erythropoiesis in the spleen. However, in this setting, the loss of endogenous RBC was only partially balanced by the transfused RBCs (Figure 2C). Next, we tested the JAK2i in TDT C57-FLC th3/th3 mice. Animals that received transfusions showed 35% reduction in spleen weight when compared with non-transfused animals (Figure 2D). The combination of blood transfusion with administration of JAK2i for 10 days further reduced spleen weight (84% for INCB and 79% for TG101348; Figure 2D) when compared with untransfused animals, with neither detrimental nor incremental effects on Hb levels and number of RBCs in circulation (Supplementary Figure 3C and D). Our first observation is that combined administration of JAK2i and transfusion can further reduce the splenomegaly. This would be very helpful in those patients that are unable to receive transfusion and iron-chelation therapy, as well as those with clinically symptomatic splenomegaly or hypersplenism.(13) Based on our data, we can only speculate that this reduction may prevent exacerbation of the anemia in the long term. However, our data also indicate that this combination further suppressed endogenous erythropoiesis and RBC production. For this reason, potential clinical trials using these inhibitors should likely aim for a reduction of splenomegaly, rather than increasing the levels of total RBCs (endogenous + donor) in circulation. Our second observation pertains to safety. Long-term use of JAK2i has proven to be effective for the treatment of myeloproliferative disorders such as Polycythemia Vera.(10, 14) However, in these studies, the positive effect on splenomegaly was associated with thrombocytopenia due to the inhibitory effect of JAK2i on megakaryopoiesis.(15) In this study, we administered two JAK2i for only 10 days and noted that the spleen weight was almost normalized when transfusion was combined with the JAK2i. This short treatment was not associated with thrombocytopenia (not shown), though could be a possibility if duration of treatment was prolonged. Our third major observation is related to the variation in the reduction of splenomegaly comparing INCB vs. TG101348, in absence or presence of transfusion (Figure 1D and 2A). These differences could be due to the distinct activity of the two compounds. INCB is a selective JAK1/JAK2 inhibitor (respectively with IC nm and IC nm) while TG is a potent inhibitor of JAK2 (IC50 3 nm) with 30-

6 fold less selectivity for JAK1.(10, 14) Our data showed that amelioration of splenomegaly observed using TG is not as effective as INCB018424, suggesting a role of JAK1 in modulating extramedullary hematopoiesis and ineffective erythropoiesis. Further studies need to be performed in order to underline the role of Jak1 in the pathophysiology of β-thalassemia. In conclusion, we showed that short term suppression of JAK2, and eventually also JAK1, in presence of transfusion can effectively reduce splenomegaly by limiting the number of nucleated erythroid cells, with no detrimental effect on other lineages or anemia. This approach could be utilized as an alternative to splenectomy in thalassemic patients.

7 References 1. Rivella S. The role of ineffective erythropoiesis in non-transfusion-dependent thalassemia. Blood Rev. 2012;26 Suppl 1:S Weatherall DJ. Pathophysiology of thalassaemia. Baillieres Clin Haematol. 1998;11(1): Libani IV, Guy EC, Melchiori L, et al. Decreased differentiation of erythroid cells exacerbates ineffective erythropoiesis in beta-thalassemia. Blood. 2008;112(3): Musallam KM, Taher AT, Rachmilewitz EA. beta-thalassemia intermedia: a clinical perspective. Cold Spring Harb Perspect Med. 2012;2(7):a Crary SE, Buchanan GR. Vascular complications after splenectomy for hematologic disorders. Blood. 2009;114(14): Jamieson C, Hasserjian R, Gotlib, et al. Effect of treatment with a JAK2-selective inhibitor, fedratinib, on bone marrow fibrosis in patients with myelofibrosis. J Transl Med. 2015;13: Mesa RA. Ruxolitinib, a selective JAK1 and JAK2 inhibitor for the treatment of myeloproliferative neoplasms and psoriasis. IDrugs. 2010;13(6): Pasquier F, Cabagnols X, Secardin L, Plo I, Vainchenker W. Myeloproliferative neoplasms: JAK2 signaling pathway as a central target for therapy. Clin Lymphoma Myeloma Leuk. 2014;14 Suppl:S Verstovsek S, Kantarjian H, Mesa RA, et al. Safety and efficacy of INCB018424, a JAK1 and JAK2 inhibitor, in myelofibrosis. N Engl J Med. 2010;363(12): Wernig G, Kharas MG, Okabe R, et al. Efficacy of TG101348, a selective JAK2 inhibitor, in treatment of a murine model of JAK2V617F-induced polycythemia vera. Cancer Cell. 2008;13(4): Rivella S, May C, Chadburn A, Riviere I, Sadelain M. A novel murine model of Cooley anemia and its rescue by lentiviral-mediated human beta-globin gene transfer. Blood. 2003;101(8): Casu C, Oikonomidou PR, Chen H, et al. Minihepcidin peptides as disease modifiers in mice affected by beta-thalassemia and polycythemia vera. Blood. 2016;128(2): Taher A, Vichinsky E, Musallam K, Cappellini MD, Viprakasit V. In: Weatherall D, ed. Guidelines for the Management of Non Transfusion Dependent Thalassaemia (NTDT). Nicosia, Cyprus, 2013.

8 14. Quintas-Cardama A, Vaddi K, Liu P, et al. Preclinical characterization of the selective JAK1/2 inhibitor INCB018424: therapeutic implications for the treatment of myeloproliferative neoplasms. Blood ;115(15): Al-Ali HK, Stalbovskaya V, Gopalakrishna P, Perez-Ronco J, Foltz L. Impact of ruxolitinib treatment on the hemoglobin dynamics and the negative prognosis of anemia in patients with myelofibrosis. Leuk Lymphoma. 2016;57(10):

9 Figure Legends Figure 1. JAK2i significantly improved splenomegaly in a mouse model of β- thalassemia intermedia. Short term treatment with the JAK2i INCB or TG in a mouse model of β-thalassemia intermedia (NTDT-Hbb th3/+ ) resulted in very mild exacerbation of anemia (A) and reduction in RBC number (B). This effect was associated with expected increase in serum EPO level (C). The significant reduction in splenomegaly suggests that JAK2i may target preferentially rapidly proliferating erythroid progenitors in the spleen, limiting their expansion and thus allowing the reduction in the spleen size (D). Some expected variability in spleen size was observed in controls animals receiving the placebo which can be attribute to the diversity among mice. This variability seemed to be blunted by the administration of the inhibitors. Representative flow cytometry analysis (showing the different erythroid populations) indicated the reduction of nucleated erythroid cells in the spleen (depicted by number 1 through 4; while population 5 represents RBC) after treatment with JAK2i. (E). Analysis was performed using One-way ANOVA with Tukey multiple comparison adjustment. Results represent mean ± SD, ****P <0.0001, ***P < 0.001, **P < 0.01, *P < Figure 2. Co-administrated of JAK2i and transfusion further improved extramedullary hematopoiesis. Administration of transfusion (indicated with TXF in the graphs) and JAK2i in a mouse model of β-thalassemia intermedia (NTDT-Hbb th3/+ ) almost normalized spleen size compared to wild type (WT) values (A). In the same mice, a reduction in the proportion of nucleated erythroid cells in the spleen was detected by flow cytometry (B). When the administration of a JAK2i was combined with transfusion, suppression of erythropoiesis was more pronounced, as indicated by a decreased number of endogenous RBC (C). Splenomegaly was also improved in a mouse model of β-thalassemia major (TDT-C57-FLC th3/th3 ) when JAK2i and transfusion were administered simultaneously (D). Analysis was performed using One-way ANOVA with Tukey multiple comparison adjustment or Two-way ANOVA with Sidak s multiple comparison adjustment. Results represent mean ± SD, ****P <0.0001, ***P < 0.001, **P < 0.01.

12 Material and Methods Animal models. All animals (2.5 to 3.5-month old male all in C57BL/6 background) were bred at the mouse facility of the Children s Hospital of Philadelphia. We used Hbb th3/+ mice (Jackson Laboratories) as a model of β-thalassemia intermedia and C57-FLC th3/th3 animals as model for β-thalassemia-major. C57-FLC th3/th3 were generated by transplanting Hbb th3/th3 fetal liver cells (FLC) from embryos collected at 14.5 to 15.5 days of gestation into irradiated WT (C57BL/6) 2-month old syngeneic animals. Transfusions started 1-month after transplant, when the phenotype was fully established. Based on previous experiments, we did not expect any difference between genders treated with JAK2i. For this study only males were utilized. Drug preparation and administration. INCB (Ruxolitinib) and TG (Fedratinib, SAR302503) were purchased from Chemietek and were administered for 10 days, twice daily by oral gavage, at a dose of 180 and 120 mg/kg respectively. Stock solution was in DMSO. Transfusion. Hbb th3/+ and C57-FLC th3/th3 mice were infused weekly, for a total of three weeks (starting one week before the JAK2i administration) via retro-orbital venous plexus with 300 µl freshly harvested blood from normal healthy GFP-C57BL/6 animals. GFP blood was collected from the retro-orbital plexus into acid citrate dextrose (7 volumes of blood for 1 volume of acid citrate dextrose), under anesthesia. Measurement of tissue iron content and serum iron parameters. Serum parameters (iron, Transferrin saturation) were measured using the Iron/TIBC Reagent Set (BioPacific Diagnostic Inc). Serum EPO was analyzed using a Mouse Erythropoietin Quantikine Elisa Kit (R&D Systems) following manufacturer s instructions. Serum HEPCIDIN was measured using the Hepcidin-Murine-Compete kit (Intrinsic Lifesciences) following manufacturer s instructions. Liver and spleen iron content was determined by non-heme iron analysis. Perls Prussian Blue staining on liver and spleen sections were prepared at the Pathology core laboratories of the Children s Hospital of Philadelphia.

13 Images were captured using a Leica DM4000B upright scope paired with a Spot RT/ SE Slider camera (10x/N.A objective) and then acquired using the Spot 5.1 software. FACS analysis: Single cell suspensions of BM and spleen cells (1*10 6 cells per sample) were incubated with anti-mouse CD71, anti-mouse Ter119 antibodies (BD Biosciences Pharmingen) and anti-mouse CD44 marker (BD Biosciences Pharmingen) to study the erythroid compartment. Analysis of the cells using CD44 and Ter119 allows the separation of erythroid cells into distinct populations (indicated as 1 to 5). For all analyses, cells were sorted using a FACSCalibur instrument (BD) and the results analyzed with FlowJo software (Tree Star Inc.). Statistics. Statistical analysis was performed using One-way ANOVA with Tukey multiple comparison adjustment or Two-way ANOVA with Sidak s multiple comparison adjustment. WT values data are displayed as a reference guide but are not included in the test comparisons. All data were analyzed using GraphPad Prism version 6 (Microsoft GraphPad Software, La Jolla California USA). Study approval. All the animal studies were conducted under protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the Children s Hospital of Philadelphia.

14 Supplementary Figure 1 A RETIC (x10^9 cells/l) WT (#4) Hbb th3/+ * Placebo (#13) INCB (#7) TG (#12) B Erythroid populations/absolute number Placebo (#5) Hbb th3/+ INCB (#5) TG (#5) P5 P4 P3 P2 P1 P3-Placebo vs. P3-INCB =**** P3-Placebo vs. P3-TG =** P4-Placebo vs. P4-INCB =**** P4-Placebo vs. P4-TG =**** P5-Placebo vs. P5-INCB =**** P5-Placebo vs. P5-TG =*** Supplementary Figure 1. Ineffective erythropoiesis in the spleen was improved after Jak2i treatment. Reduction in spleen size after JAK2i administration was associated with a significant decrease in reticulocyte numbers (A) and erythropoiesis in the spleen (B) when compared with placebo. The different splenic erythroid populations were analyzed by taking in consideration the absolute number of cells. Individual sub-populations were compared among each group of animals. End point analysis revealed a substantial reduction for all of 5 populations, showing significance for population 3 and 4 which represent Polychromatic erythroblast (3) and Ortho/Retic (4). Analysis was performed using Two-way ANOVA with Tukey multiple comparison adjustment. Results represent mean ± SD, ****P <0.0001, ***P < 0.001, **P < 0.01,

15 Supplementary Figure 2 A Log 10 Serum HAMP (ng/ml) Hbb th3/+ WT (#6) Placebo (#5) INCB (#6) TG (#6) B Log 10 Serum Iron (ug/dl) WT (#5) Hbb th3/+ Placebo (#9) INCB018424(#7) TG (#7) C Log 10 Transferrin Saturation (%) WT (#5) Hbb th3/+ Placebo (#9) INCB018424(#7) TG (#7) D Hbb th3/+ E Hbb th3/+ Total Liver Iron (ug) WT (#5) Placebo (#6) INCB (#6) TG (#6) Total Spleen Iron (ug) WT (#5) Placebo (#6) INCB (#6) TG (#6)

Supplementary Figure 2 F WT Hbb th3/+ Placebo Hbb

levels (A) as well as other iron parameters such

Total liver and spleen iron content of animals

compared with mice receiving the placebo (D-E).

Blue staining of liver and spleen sections (F).

16 Supplementary Figure 2 F WT Hbb th3/+ Placebo Hbb th3/+ INCB Hbb th3/+ TG Liver Spleen Supplementary Figure 2. Iron parameters were unchanged after ten days of treatment with JAK2i. No differences were observed in serum HEPCIDIN levels (A) as well as other iron parameters such as serum iron and transferrin saturation (B-C). Total liver and spleen iron content of animals receiving the inhibitors did not change when compared with mice receiving the placebo (D-E). These findings were confirmed by Perls Prussian Blue staining of liver and spleen sections (F). Analysis was performed using One-way ANOVA with Tukey multiple comparison adjustment. Results represent mean ± SD.

17 A Erythroid populations/absolute number Placebo (#5) Hbb th3/+ TXF+Placebo (#7) TXF+INCB (#4) TXF+TG (#5) P5 P4 P3 P2 P1 Supplementary Figure 3 B Hbb th3/+ C C57-FLC th3/th3 Hb (g/dl) *** *** **** Hb (g/dl) *** **** *** 0 WT (#5) Placebo (#9) TXF+Placebo (#9) TXF+INCB (#8) TXF+TG (#9) 0 Not Transfused (#4) TXF+Placebo (#6) TXF+INCB (#6) TXF+TG (#6) C57-FLC th3/th3 D RBC (x10^6 cells/ul) Not Transfused (#4) **** *** TXF+Placebo (#6) ** TXF+INCB (#6) TXF+TG (#6)

18 Supplementary Figure 3 Supplementary Figure 3. JAK2i treatment combined with transfusion significantly improved ineffective erythropoiesis in the spleen without detrimental effect on anemia. NTDT mice that received co-administration of transfusion (indicated with TXF in the graphs) and JAK2i showed reduction in ineffective erythropoiesis. This is indicated by the same trend in reduction of erythroid progenitor cells observed in thalassemic animals that did not undergo transfusion (A). Individual sub-populations were compared among each group of animals. Analysis was performed using Two-way ANOVA with Tukey multiple comparison adjustment. Animals that received JAK2i associated with the transfusion did not show any significant differences in Hb level when compared with animals receiving transfusion alone (B). In a mouse model of TDT, combination of blood transfusion and JAK2i did not have any negative effect on the efficacy of the transfusion but did not result in any increase in Hb levels or RBC numbers. (C-D). Analysis was performed using One-way ANOVA with Tukey multiple comparison adjustment. Results represent mean ± SD, ****P <0.0001, ***P < 0.001, **P < 0.01.

Innovative therapies for ß-hemoglobinopathies

Innovative therapies for ß-hemoglobinopathies Stefano Rivella, PhD Dept. of Pediatrics, Division of Hematology at Children s Hospital of Philadelphia (CHOP) Cell and Molecular Biology Graduate Group (CAMB)