Leukemia (2014) 28, 1486–1493 & 2014 Macmillan Publishers Limited All rights reserved 0887-6924/14 www.nature.com/leu

ORIGINAL ARTICLE

Ruxolitinib leads to improvement of pulmonary hypertension in patients with myelofibrosis A Tabarroki1, DJ Lindner1, V Visconte1, L Zhang2, HJ Rogers3, Y Parker1, HK Duong4, A Lichtin4, ME Kalaycio4, MA Sekeres1,4, SE Mountantonakis5, GA Heresi6 and RV Tiu1,4 Pulmonary hypertension (PH) is a frequently under recognized complication of myelofibrosis (MF). The pathophysiology of PH in MF is unknown and no definitive therapies have been established. We studied 15 patients with MF-associated PH and compared their echocardiographic and PH relevant biomarkers (nitric oxide (NO), N-terminal pro-hormone of brain natriuretic peptide (NT-pro BNP), von Willebrand antigen (vWB), ristocetin-cofactor activity (RCA) and uric acid (UA)) pre- and postruxolitinib treatment. Ruxolitinib decreased the plasma levels of NT-pro BNP (73%; P ¼ 0.043), UA (60%), vWB (86%) and RCA (73%; P ¼ 0.036). Improvements in echocardiographic findings were also seen in 66% of patients (P ¼ 0.022). Furthermore, marked increase in NO compared with baseline (69.75 vs 40.1 picomolar (pM); P ¼ 0.001) was observed post-ruxolitinib therapy, whereas no changes were noted with conventional therapies. Treatment with ruxolitinib also resulted in the reduction of key cytokines (tumor necrosis factor alpha, interleukin-4 (IL-4), IL-6 and IL-8) and induction of interferon-gamma. Animal studies further supported the role of ruxolitinib in the induction of NO levels. In conclusion, aberrant Janus kinase (JAK)-signal transducer and activator of transcription signaling in MF may mediate PH through dysregulation of NO and cytokine levels, which can be restored by therapy with JAK inhibitors suggesting that inhibition of this pathway is a novel target for the management of patients with PH. Leukemia (2014) 28, 1486–1493; doi:10.1038/leu.2014.5 Keywords: myelofibrosis; pulmonary hypertension; ruxolitinib

INTRODUCTION Myelofibrosis (MF) is a Philadelphia chromosome-negative myeloproliferative neoplasm (MPN) characterized by aberrant clonal proliferation of myeloid precursors accompanied by stromal fibrosis in the bone marrow.1 Pulmonary hypertension (PH) is an indolent and progressive vascular lung disease that can complicate the clinical course of patients with MF, occurring in approximately 30% of MF patients.2,3 The presence of MF-associated PH is correlated with poor survival.4–7 PH manifests with nonspecific symptoms such as dyspnea on exertion and fatigue and can be detected by showing elevated right ventricular systolic pressure (RVSP)435 mm Hg, right atrium enlargement and tricuspid regurgitation velocity X2.5 m/s using echocardiography.8,9 As PH progresses, patients can develop peripheral edema, jugular vein distention, exertional dyspnea, chest discomfort and eventually right heart failure.10,11 According to the World Health Organization, PH can be further classified into five subtypes. PH in MF patients would be categorized as PH with unclear multi-factorial causes.7,12 Multiple pathophysiologic mechanisms have been implicated in the development of PH in MF patients, including pulmonary myeloid metaplasia, obstruction of pulmonary vessels by megakaryocytes or platelets, smooth muscle hyperplasia caused by increased levels of platelet-derived growth factor, fibrosis in the pulmonary vascular intima and thrombotic occlusion of the pulmonary vasculature.3,13

Hence, no single unifying mechanism has been identified. Therapies that have been used for PH in the context of MF include coumadin,14 whole lung external beam radiotherapy,15,16 epoprostenol17 and bosentan.18 The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway is essential in physiologic cytokine signaling and hematopoiesis.19 Dysregulation of JAK family kinases, specifically JAK1 and JAK2, contributes to the pathogenesis of MF.20 Aberrant JAK signaling has also been implicated in the pathogenesis of other non-hematologic diseases including connective tissue diseases such as rheumatoid arthritis,21 cardiac hypertrophy22 and inflammatory bowel diseases.23 In idiopathic PH, the proliferation of pulmonary artery endothelial cells has been linked to STAT3 activation.24 Ruxolitinib, an oral JAK1/JAK2 inhibitor, is the first drug approved by the Food and Drug Administration for the treatment of intermediate- to high-risk MF patients.25 Its clinical efficacy includes improvements in splenomegaly related and cytokinemediated symptoms.26,27 Recent data also suggest improvements in survival.28 Given the association between MF and PH and the possible pathophysiologic link mediated by JAK signaling, we followed the changes in PH clinical, laboratory and echocardiographic findings in symptomatic MF patients with PH treated with ruxolitinib.

1 Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA; 2Division of Hematology and Medical Oncology, Department of Medicine, UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA; 3Department of Clinical Pathology, Cleveland Clinic, Cleveland, OH, USA; 4Leukemia Program, Department of Hematologic Oncology and Blood Disorders, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA; 5Department of Cardiology, North Shore University Hospital, Manhasset, NY, USA and 6Department of Medicine, Pulmonary, Allergy and Critical Care Medicine, Respiratory Institute, Cleveland Clinic, Cleveland, OH, USA. Correspondence: Dr RV Tiu, Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH 44195, USA. E-mail: [email protected] Received 12 December 2013; accepted 16 December 2013; accepted article preview online 10 January 2014; advance online publication, 24 January 2014

Ruxolitinib and pulmonary hypertension A Tabarroki et al

1487 MATERIALS AND METHODS Patients We studied 15 patients with MF who received treatment with ruxolitinib in the outpatient MPN clinic. Samples and clinical parameters of the patients were obtained with written informed consent in accordance with the Declaration of Helsinki and all the protocols were approved by the Institutional Review Board of the Cleveland Clinic. Clinical data collected included age, sex, date of diagnosis, date of last follow-up, constitutional symptoms, spleen size, requirement for blood transfusion for at least 8 weeks, prognostic risk grouping (International Prognostic Scoring System (IPSS), Dynamic IPSS (DIPSS), DIPSS-Plus), hematologic and bone marrow parameters, and transthoracic echocardiographic (TTE) findings.

Echocardiographic studies The gold standard in the diagnosis of PH is by right heart catheter29 ization. However, echocardiography is the most useful imaging modality to assess PH and to exclude any underlying cardiac diseases.30 PH in these studies were defined by a TTE showing a RVSP X40 mm Hg in the presence of RV dysfunction and right atrium enlargement or even RVSP of measurements of B430 mm Hg.31 Echocardiographic parameters relevant to PH including RVSP, right atrium diameter and tricuspid regurgitation velocity were collected pre- and post-ruxolitinib therapy.

PH serum biomarkers Pre- and post-treatment plasma/serum samples were available for all patients. Serum biomarkers relevant to PH including N-terminal prohormone of brain natriuretic peptide (NT-pro BNP), uric acid (UA), von Willebrand antigen (vWB) and ristocetin-cofactor activity (RCA) were measured pre- and post-treatment with ruxolitinib.

Nitric oxide measurement In vivo, the half-life of nitric oxide (NO), a free radical, is measured in seconds; it is rapidly converted to nitrates and nitrites. To measure nitrates/ nitrites in serum/plasma, samples were reacted with VCl3/HCl, converting them back to NO, which yielded a chemiluminescent signal when it reacts with ozone. Chemiluminescent NO analysis of serum samples was performed using a GE Analytical Instruments Sievers NOA 280 (Boulder, CO, USA). A 50 ml glass impinger vial containing 30 ml VCl3 (0.4 M in 1 M HCl) þ 0.5 ml Antifoam Emulsion C (both from Sigma-Aldrich, St Louis, MO, USA) was equilibrated in a water bath at 95 1C. Helium (inert carrier) gas (10 psig) flowed through the impinger vial, a condenser, a water trap containing 10 ml NaOH to neutralize any HCl vapor carried from the sample impinger vial, and was delivered to the NOA. NOA settings were: cooler temperature –12 1C, cell pressure 12 torr, O2 supply pressure 6.4 psi. The baseline NOA output stabilized at 15 mV (high sensitivity scale) after 10 min. The system was purged for 30 min before running 100 mM KNO3 standards (1–500 ml) and serum samples. Standard curve was generated (Liquid Program software v 3.22 PNN, Sievers) and used to calculate fmol NO released by each injected sample (100 ml human serum or 20 ml mouse serum).

Enzyme-linked immunosorbent assay for cytokine analysis Peripheral blood from patients receiving either ruxolitinib or other non-JAK inhibitor conventional therapies like hydroxyurea was collected in heparin tubes. Plasma was removed by centrifugation (1500 r.p.m. for 5 min) and stored at  80 1C. In all, 50–100 ml of undiluted plasma were tested on a Multi-Analyte ELISArray Kits (SABiosciences, Valencia, CA, USA). Plasma concentrations for interleukins (ILs) including IL-1A, IL-1B, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17A, interferon-gamma (IFN-g, tumor necrosis factor alpha (TNFa and granulocyte-monocyte colony-stimulating factor (GMCSF) were measured pre- and post-treatment following the manufacturer’s instructions. Optical density was measured at 450 and 570 nm using a Wallac1420 Victor 3 plate reader (Perkin Elmer, Gaithersburg, MD, USA).

In vivo animal studies Serum NO levels can be influenced by many clinical factors including smoking, dietary intake and comorbidities (iron deficiency anemia, atherosclerosis, hypertension and diabetes mellitus).32 In addition, NO can also be affected by medications such as sildenafil and HMG-CoA reductase inhibitors.33–39 To evaluate the effects of ruxolitinib on NO levels in a pure system not affected by these factors and to ensure that the serum & 2014 Macmillan Publishers Limited

NO changes observed were not influenced by other factors, we performed NO studies in normal and PH Caveolin-1 mice. Eight normal (CD1) and six Caveolin-1 outbred mice (Jackson Laboratory, Bar Harbor, ME, USA) were treated with ruxolitinib (50 mg/kg per mouth, daily for 5 days and three consecutive cycles with 14 days interval between each cycle). Caveolin-1 (CAV-1  /  ) mice are genetically manipulated mice harboring a homozygous mutation of the CAV-1 gene, which is crucial for cell proliferation and signal transduction.40 Histologically, CAV-1  /  mice have thickened alveolar septa, increased number of endothelial cells, reduced alveolar spaces, increased density of basement membranes and reticulin fibers similar to PH and right ventricular hypertrophy. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Cleveland Clinic. Blood was collected by retro-orbital puncture at baseline and at the end of each treatment cycle. White blood cells, hemoglobin and platelets were measured by a Hemavet 500 (Drew Scientific Incorporation, Dallas, TX, USA). Plasma was extracted by centrifugation (1500 r.p.m. for 5 min) and NO level was measured in duplicate samples as described in the methodology on NO measurement.

Statistical analysis Categorical data were summarized as frequency counts and percentages, continuous data were presented as medians and ranges and the comparison between two groups was performed by two-sample Wilcoxon signed rank test. Spearmen rank test was used to evaluate the correlations between the continuous variables. Cut-points for laboratory data for PH plasma biomarkers were also tested using a recursive partitioning algorithm. Statistical analyses were performed using R (www.r-project.org). Data were considered statistically significant if the P-value was p0.05.

RESULTS Baseline characteristics of the MF patients treated with ruxolitinib Among 15 patients, 7 had primary MF, 4 post-essential thrombocytosis and 4 post-polycythemia vera MF. In this cohort, seven were females and eight were males. The median age of the cohort was 70.5 years (50–83 years). Twelve patients were JAK2 V617F positive and three were wild type. Using the DIPSS-Plus risk grouping, four cases were intermediate-1 (INT-1), five were intermediate-2 (INT-2) and six were high risk. The median dose of ruxolitinib was 10 mg BID (two times per day) (5 mg QOD (every other day)–20 mg BID). Median duration of disease was 67 months (6–191 months). Median duration of treatment with ruxolitinib was 10.5 months (4–18 months). Median duration of follow-up was 15.7 months (6–28 months). Of note, all the patients are still alive (Table 1). Ruxolitinib therapy induces changes in pulmonary parameters: serum PH biomarkers and echocardiographic findings Before the initiation of ruxolitinib treatment, NT-pro BNP levels, a surrogate biomarker for PH, were measured and found to be elevated in 93% (14/15) of patients. Other biomarkers associated with PH including plasma UA, vWB and RCA levels were all elevated in 60% (9/15), 40% (6/15) and 20% (3/15) of patients at baseline, respectively. The strongest correlation among serum biomarkers with PH was plasma vWB levels (r2 ¼  0.97, Po0.001). Echocardiographic findings by TTE pre- and post-ruxolitinib therapy were available in all the patients. All 15 patients had documented PH with a mean RVSP of 50.62 mm Hg (42–75) (normal pressure p30 mm Hg; Table 2). Further analysis demonstrated that, RCA mainly correlated with improvement in PH by TTE findings (r2 ¼  0.17, P ¼ 0.065). Ruxolitinib therapy resulted in reductions in NT-pro BNP level in 73% (11/15; P ¼ 0.043), plasma UA levels in 60% (9/15), plasma vWB in 86% (13/15) and plasma RCA in 73% (11/15; P ¼ 0.036) of this cohort, respectively. Greater than 50% reductions in NT-pro BNP levels, plasma UA, vWB and RCA levels were observed in 26% (4/15), 13% (2/15), 26% (4/15) and 33% (5/15) of patients post-ruxolitinib therapy, respectively (Table 3). This translated to marked improvements in RSVP by TTE Leukemia (2014) 1486 – 1493

Ruxolitinib and pulmonary hypertension A Tabarroki et al

1488 (mean: 35.62, range: 55–30 mm Hg) with average reduction of 11.4 mm Hg (17–7 mm Hg in 66% (10/15) of patients; P ¼ 0.022) post-ruxolitinib therapy (Table 4). Ruxolitinib therapy increases plasma NO levels NO measurements were available for all patients’ pre- and posttreatment with ruxolitinib. The baseline mean NO level (before initiation of ruxolitinib) in the MPN cohort was 40.1 pM (range 5–68). This was at the lower limit of normal individuals with the NO levels set at 453 pM. Ruxolitinib therapy resulted in an increase in NO levels in 80% (12/15) of patients with a mean of 69.75 pM (P ¼ 0.001). A 450% increase was observed in 46% (7/15)

Table 1. Baseline characteristics of the myelofibrosis patients with pulmonary hypertension treated with ruxolitinib Values (N ¼ 15)

Baseline characteristics of the patients Median age (range)—years Male sex—% of patients Myelofibrosis subtype—% of patients Primary myelofibrosis Post-polycythemia vera myelofibrosis Post-essential thrombocytosis myelofibrosis

70.5 (50–83) 53.3 47 27 27

DIPSS-Plus risk stratifications—% of patients Low Intermediate 1 Intermediate 2 High

0 27 33 40

Previous treatment—% of patients Cytoreductive agents IMiDs IFN Danazol/prednisone Hypomethylating agents JAK2V617F mutant—% of patients

66 18 18 12 6 80

Metaphase cytogenetics risk grouping (IPSS-Plus) —% of patients Favorable 47 Normal 33 Unfavorable 20 No growth 13 BM blast—% of patients No blast X1 but o10%

20 80

Transfusion dependency (RBC/platelets) —% of patients Before ruxolitinib After ruxolitinib

47 53

Abbreviations: BM, bone marrow; DIPSS-Plus, Dynamic International Prognostic Scoring System-Plus; IFN, interferon; IMiDs, immunomodulatory agents; IPSS, International Prognostic Scoring System; RBC, red blood cell.

Table 2.

of patients. No improvements in NO levels were observed in three patients post-ruxolitinib therapy (patients #4, 6 and 11; Table 3). However, no clinical deterioration in pulmonary symptoms was noted. To validate these findings, NO levels were measured preand post-therapy with non-JAK inhibitor treatment modalities in a subset of patients. Serum samples of patients treated with hydroxyurea (N ¼ 6) and immunomodulatory agents (thalidomide, N ¼ 3 and lenalidomide, N ¼ 1) were obtained. No changes in NO levels were observed post-treatment with these two agents (P ¼ 0.6; Figure 1). No differences in RVSP were observed by TTE in MF patients pre- and post-treatment with hydroxyurea (47.75 vs 45.25 mm Hg, P ¼ 0.2). Of note, we observed that increase in NO levels resulted in improvements in some of the patients’ symptoms like dyspnea and chest discomfort. In one patient, oxygen requirement also diminished from 5 to 3 L/min (patient #14; Table 3). Ruxolitinib treatment modulates cytokine and ferritin levels To better understand the impact of ruxolitinib on alteration of the cytokine profile and the possible role of these cytokines in induction of NO, pre- and post-ruxolitinib plasma/serum samples were tested for cytokine profiling. Treatment with ruxolitinib resulted in reduction of IL-1b (16%), IL-2 (17%), IL-4 (32%), IL-6 (38%), IL-8 (38%), IL-12 (3%), TNF-a (65%) and GM-CSF (81%), whereas IFN-g (41%), IL-1a (10%), IL-17A (22%) and IL-10 (17%) levels increased (Figure 2a). The reduction in IL-4, IL-6, IL-8, TNF-a and GM-CSF levels by ruxolitinib was statistically significant (for IL-4, TNF-a, GM-CSF, P ¼ 0.01, and for IL-6 and IL-8, P ¼ 0.05). In contrast, therapy with other non-JAK inhibitor-based agents like hydroxyurea did not result in a statistically significant reduction in any of the disease related cytokines (Figure 2b). Hypoxia can alter iron metabolism through hypoxia-inducible factor mediated pathways.41 In 15 patients, pre- and postruxolitinib therapy ferritin levels were available in 11 patients of which 8 were requiring red blood cell transfusion. Among the non-red blood cell transfusion-dependent patients (patients #1, 8 and 12), serum ferritin levels decreased post-ruxolitinib (223 vs 99.3 ng/ml; Table 3). Ruxolitinib treatment induces an increase in NO levels in normal CD1 and CAV-1  /  mice To exclude the effects of concomitant medications and comorbid conditions of patients on NO levels pre- and post-ruxolitinib therapy, we also performed NO measurements in normal CD1 and PH CAV-1  /  mice. NO levels, white blood cells, hemoglobin and platelets from normal CD1 and CAV-1  /  mice were measured pre- and post-administration of ruxolitinib in three different cycles of treatment. All the blood lineages dropped compared with baseline after initiation of ruxolitinib (Figures 3a and c). In both mice models, after each cycle of ruxolitinib, NO levels were higher compared with baseline, consistent with the pattern observed in patients (Figures 3b and d). In normal CD1 mice, the measurements in end of each cycle showed a trend of increase in NO levels compared with baseline at cycle 2 (P ¼ 0.25) and a

Hematologic and cardiac parameters pre- and post-ruxolitinib treatment

Basic characteristics Nitric oxide (pM) Right ventricular systolic pressure (mm Hg) (trans thoracic echocardiography) NT-pro BNP (pg/dl) Uric acid (mg/dl) vWB antigen (IU/dl) Ristocetin-cofactor activity

Normal values

Pre-treatment (mean)

Post-treatment (mean)

P-value o0.5

453 15–30

40.1 50.62

69.75 35.62

0.049 0.0005

o125 2–7 50–173 35–153

981 7.8 183 127

654 6.2 146 105

0.043 0.05 0.036 0.036

Abbreviations: NT-pro BNP, N-terminal pro-hormone of brain natriuretic peptides; vWB, Von Willebrand antigen.

Leukemia (2014) 1486 – 1493

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Ruxolitinib and pulmonary hypertension A Tabarroki et al

1489 Table 3.

Pulmonary hypertension serum/plasma biomarkers pre- and post-ruxolitinib therapy Ruxolitinib dose (Initial)

Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient

#1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 #12 #13 #14 #15

Nitric oxide(pM)

15 mg BID 5 mg QOD 5 mg BID 20 mg BID 15 mg BID 10 mg BID 10 mg BID 10 mg BID 5 mg BID 5 mg BID 15 mg BID 10 mg BID 10 mg BID 5 mg BID 5 mg BID

NT-pro BNP(pg/dl)

Uric acid (mg/dl)

vWB antigen

Ristocetincofactor activity

Ferritin (ng/ml)

Pre-Tx

Post-Tx

Pre-Tx

Post-Tx

Pre-Tx

Post-Tx

Pre-Tx

Post-Tx

Pre-Tx

Post-Tx

Pre-Tx

Post-Tx

18.3 14.1 5.9 14.7 29.6 41.2 56 40.9 46.9 68.1 62.5 31.9 62.53 58.69 50.24

24.7 15.2 10.4 12.1 31 30.2 180.1 111 73 105 61 40 68.11 90.48 156.2

1044 204 3228 129 254 211 380 1166 276 NA 60 157 179 3308 3140

412 190 2374 98 190 368 345 609 272 NA 67 57 NA 656 2678

6.2 10.1 11.6 5 6.3 5.6 8.5 9.2 8.7 11.2 4.7 7.5 4.3 9.4 8.7

7.1 6.7 7.2 5.8 4.6 3.1 6.7 6.5 8 6.7 5.6 7.2 4.7 8.4 NA

115 105 284 141 287 215 96 125 154 198 373 91 255 162 146

79 96 272 26 111 127 46 147 100 173 465 47 241 141 120

36 46 151 127 265 109 81 66 67 147 258 130 216 84 135

29 41 173 16 98 39 23 103 74 134 448 51 169 80 98

26a 69.3 1018 136.2 65 2492 354 62a 225.1 56.1 575 581a 204.3 NA 361

18 NA 1301 NA 117 3537 2225 46 945.5 NA 1620 234 1850.1 98.3 125

Abbreviations: BID, two times per day; NA, not applicable; NT-pro BNP, N-terminal pro-hormone of brain natriuretic peptides; QOD, every other day; TX, treatment; vWB, Von Willebrand antigen . aPatients not receiving blood transfusions.

Table 4.

TTE parameters of the myelofibrosis cohort RVSP(mm Hg)

Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient

#1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 #12 #13 #14 #15

Pre-Tx

Post-Tx

68 45 53 37 63 40 42 45 47 NA 43 47 NA 43 47

55 32 44 30 56 42 30 30 NA 36 32 NA 36 32 NA

Tricuspid regurgitation velocity (m/s)

RA pressure (mm Hg)

Valvular abnormality

Ejection fraction (%)

3.9 3.2 3.4 2.8 3.7 2.7 3 3.1 3 NA 3 3 NA 3 3

5 4 5 4 8 10 5 5 10 5 5 10 5 5 10

None None MR (1 þ ), PR (2 þ ), TR(1 þ ) MR (1 þ ), TR (1 þ ) MR (2 þ ), TR (2 þ ) TR (1 þ ), PR (1 þ ) TR (1 þ ), MR (1 þ ) TR (1 þ ) TR (3 þ ), AR (1 þ ) None None TR (3 þ ), AR (1 þ ) None None TR (3 þ ), AR (1 þ )

57 58 50 55 50 60 60 57 56 59 60 56 59 60 56

Right ventricular systolic function

Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal

Abbreviations: AR, aortic regurgitation; MR, mitral regurgitation; NA, not applicable; PR, pulmonic regurgitation; RA, right atrium; RVSP, right ventricular systolic pressure; TR, tricuspid regurgitation; TTE, transthoracic echocardiography; Tx, treatment.

considerable and significant increase at cycle 3 (P ¼ 0.04) of treatment (Figure 3b). Furthermore, a consistent and statistically significant increase in NO levels was observed after each cycle of ruxolitinib in a PH mouse model, CAV-1  /  mice. In comparison with baseline, after first, second and third cycles of ruxolitinib therapy, NO levels increased by 2.5-fold (9.9 vs 24.9 pM, P ¼ 0.032), by 2-fold (18.7 vs 38.1 pM, P ¼ 0.037) and by 2.8-fold (19.8 vs 45.2 pM, P ¼ 0.013), respectively. DISCUSSION PH is a progressive and fatal lung disease that can complicate the clinical course of MF patients. In this cohort of MF patients, serum biomarkers, including NT-pro BNP, plasma vWB, UA and RCA, implicated in the pathophysiology of idiopathic PH, were helpful both in the diagnosis of PH in MF patients, and in following PH treatment response. These biomarkers are frequently included in & 2014 Macmillan Publishers Limited

studies evaluating PH in patients.42 Several studies using various NT-pro BNP cutoff points have been shown to have good specificity and positive predictive value for the diagnosis of PH. Ataga et al.43 showed that a NT-pro BNP level of 4304 pg/ml has a specificity of 90% for PH while Machado et al.44 found that NT-pro BNP levels of X160 pg/ml had a 78% positive predictive value for the diagnosis of PH. Among hematologic patients with PH including those with beta-thalassemia and sickle cell anemia, NT-pro BNP levels of X153 pg/ml was established as a valuable diagnostic marker for PH with a sensitivity of 485% and a specificity of 94%.45,46 Furthermore high levels of UA are associated with worse outcomes in patients with PH.47 UA levels are also one of the biomarkers measured in clinical trials that investigate new agents for the management of PH.48,49 Elevated UA levels also contribute to depletion of NO levels in pulmonary artery endothelial cells.50 Plasma vWB has also been evaluated in PH patients and in a limited study, it was shown that vWB could be a prognostic marker for PH patients.51 These biomarkers have Leukemia (2014) 1486 – 1493

Ruxolitinib and pulmonary hypertension A Tabarroki et al

1490

Nitric Oxide (pM)

not been studied in the context of MF patients with PH. These prior studies support the use of the aforementioned biomarkers and TTE findings in PH in the context of MF.

90 80 70 60 50 40 30 20 10 0

P = 0.049

The use of the following biomarkers to evaluate response indicated that plasma vWB factor is the most predictive marker of PH associated with MF in our study while NT-pro BNP is the most predictive marker in non-MF-associated PH with levels X180 pg/ml associated with decreased OS.26,52 In our study, 80% of patients had a significant reduction of NT-pro BNP posttreatment with ruxolitinib. Among them, 20% had NT-pro BNP reduction below 180 pg/ml, whereas in 26% of patients NT-pro BNP levels decreased by 450%. This translated to improvements in PH by TTE. Aside from NT-pro BNP, other PH biomarkers including plasma vWB, UA and RCA were helpful in the assessment of therapeutic response in PH and correlated well with the improvements in PH and PH-related symptoms particularly dyspnea in our patient cohort. High UA is a known indicator of PH.53 It can increase as a direct result of tissue ischemia/necrosis and can be considered as an indicator of vasoconstriction in the pulmonary vasculature.53 In addition, high levels of UA can activate angiogenesis by modulating hypoxia-induced factor 1.54 At baseline and during treatment course, none of the patients were on xanthine oxidase inhibitor therapy. In this study, 60% of our cases showed reduction in UA levels post-ruxolitinib treatment with a 450% reduction in five patients. Furthermore, prior studies have shown that lowering UA levels can lead to improve PH.55 High plasma level of vWB factor is another biomarker of PH.56 High vWB is an indicator of endothelial damage and hypoxic stress

P = 0.6

*

Pre-R

Post-R

Pre-C

R=Ruxolitinib

Post-C

C=Conventional therapies

Figure 1. Differences in NO measurements pre- and post-treatment with ruxolitinib and conventional therapies. NO levels were measured in 100 ml of serum or plasma samples derived from MF patients untreated and treated with ruxolitinib (N ¼ 15) and conventional therapies (hydroxyurea, N ¼ 6 and immunomodulatory drugs, N ¼ 4) using a Sievers 280i NOA. Bar graphs represent the mean of the concentration of NO levels expressed as pM in pre-treatment (black bars) and post-treatment (white bars) with ruxolitinib (a) and conventional therapies (b). Error bars denote the s.d. Each NO measurement was done in duplicates. *Pp0.05 is considered statistical significant. C, conventional therapies; R, ruxolitinib.

Plasma Cytokine (pg/ml)

3.0

Pre-Ruxolitinib

Post-Ruxolitinib

2.5 P=0.01

2.0 1.5

P=0.05 P=0.05

P=0.01

1.0 P=0.01 0.5

GM-CSF

TNF

IFN-γ

IL 17A

IL 12

IL 10

IL 8

IL 6

Pre-Hydroxyurea

3.0 Plasma Cytokine (pg/mL)

IL 4

IL 2

IL 1B

IL 1A

0.0

Post-Hydroxyurea

2.5 2.0 1.5 1.0 0.5

GM-CSF

TNF

IFN-γ

IL 17A

IL 12

IL 10

IL 8

IL 6

IL 4

IL 2

IL 1B

IL 1A

0.0

Figure 2. Cytokine profiling pre- and post-treatment with ruxolitinib and conventional therapies. A panel of cytokines was measured on 50–100 ml of undiluted plasma using an enzyme-linked immunosorbent assay (Multi-Analyte ELISArray Kits; SABiosciences) pre- and post-ruxolitinib (a) and hydroxyurea therapy (b), respectively. Bar graphs represent mean of plasma cytokine expressed in pg/ml of pre-treatment (black bar) and posttreatment (white bar). Error bars represent s.d. for ruxolitinib (N ¼ 6) and hydroxyurea (N ¼ 3). Each measurement was done in duplicates. Leukemia (2014) 1486 – 1493

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Ruxolitinib and pulmonary hypertension A Tabarroki et al

1491 Normal Mice 12

Normal Mice

WBC (k/μl)

PLT(k/μL)

Hgb(g/dL)

10 Nitric Oxide (pM)

120

Units

8 6 4 2

Pre-ruxolitinib

Post-ruxolitinib P = 0.04 *

100 80 60 40 20 0

0 1

5

Cycle 1

21

26

Cycle 2

40

45

Cycle 1

Cycle 2

Cycle 3

Cycle 3

Days CAV-1-/-

CAV-1-/12

WBC (k/μl)

Hgb(g/dL)

PLT(k/μL)

120 Nitric Oxide(pM)

10 Units

8 6 4 2

Pre-ruxolitinib

Post-ruxolitinib

100 80 P = 0.03

60 40 20

P = 0.03

P = 0.01 *

*

*

0

0 1

5

21

Cycle 1

26

Cycle 1

Cycle 2

Cycle 3

Cycle 2 Days

Figure 3. Effect of ruxolitinib treatment in normal CD1 mice and CAV-1  /  mice. (a, c) Ruxolitinib was administrated to eight normal CD1 and six CAV-1  /  mice in dose of 50 mg/kg per mouth daily for 5 consecutive days for three cycles with 2 weeks interval between each cycle. Hematologic parameters (white blood cells (WBCs), hemoglobin (Hgb) level and platelets (PLT) count) were measured on 20 ml of blood derived by retro-orbital puncture before and after administration of ruxolitinib. Line graph shows mean of WBC (gray line), Hgb (black line) and PLT (dash line) in each cycle. The value of PLT was divided by 102. (b, d) NO levels were measured in 20 ml of plasma/serum from eight normal CD1 and six CAV-1  /  mice pre-treatment (black bar) and post-treatment (white bar) with ruxolitinib. Bar graphs show the mean NO concentration expressed in pM for each cycle. *Pp0.05 is considered statistically significant.

of endothelium, causing stimulation of hypoxia-induced factor 1 production and inhibition of stromal-derived cell factor-1, resulting in vasoconstriction and thickening of pulmonary vascular endothelium.57 vWB levels diminished after treatment with ruxolitinib in 86% of patients. Furthermore, vWB may have a potential role in interaction between megakaryocytes and fibroblasts in bone marrow and thus may promote fibrosis.58 In addition, there is an integral association between hypoxia-induced factor 1-a, hypoxia and serum iron levels. Hypoxia induces the inhibition of prolyl hydroxylase-mediated hydroxylation of proline residues in hypoxia-induced factor 1-a preventing its ubiquitination and subsequent proteasomal degradation. Of interest, the average serum ferritin levels in three non-red blood cell transfusion-dependent MF patients decreased post-ruxolitinib therapy. Patients with improvements in PH post-ruxolitinib therapy had associated increase in their plasma NO levels, which may be one of the key underlying mechanism leading to improvement in the PH relevant echocardiographic and biomarkers in MF patients. This was further supported by similar findings in normal CD1 and CAV-1  /  mice treated with ruxolitinib, which excludes patientrelated factors (unrelated to MF) that may influence serum NO levels. NO, or endothelium relaxing factor, is an endogenous substance secreted by endothelial cells that induces relaxation via reduction in the tone of smooth muscle vasculature.59 NO is also important in the reduction of platelet aggregation and leukocyte adhesion to the endothelium leading to stable organ perfusion.60 The same mechanism may modulate thrombosis risk in MPNs, an important problem in this group of diseases. In addition, physiologic NO secretion in response to hypoxia is compromised in PH patients, resulting in augmentation of vascular tone.61,62 An explanation for the increase in NO levels & 2014 Macmillan Publishers Limited

may be due to alterations in cytokine levels post-ruxolitinib therapy. Prior studies have suggested that IFN-g can directly trigger inducible NO synthase, and hence increase NO production,63,64 whereas, other cytokines such as TNF-a, IL-4 and IL-10 can inhibit production of NO.65 We observed an increase in NO inducers and reduction in NO inhibitors post-ruxolitinib therapy. Similar changes were not observed in MF patients with PH treated with non-JAK inhibitor therapy. Based on our findings, NO levels increased in 80% of patients’ post-ruxolitinib treatment in association with improvement in patients’ symptoms (dyspnea at rest/exertion and chest discomfort). NO is a ubiquitous gas that affects many physiologic and pathogenic processes. For example, increase in NO levels has been implicated in the resolution of thrombosis in mouse models by affecting platelet activity.66,67 Therefore, it is conceivable that the elevation in NO levels brought about by JAK inhibitor therapy may lead to reduced risk of thrombosis and contribute to other beneficial pleiotropic effects in MF and possibly other MPNs. Patients with untreated PH regardless of cause who are 450 years old have projected median overall survival of 90%, 76%, 57% and 44% after 1, 3, 5 and 7 years, respectively.68 Aside from an allogeneic hematopoietic cell transplant, JAK inhibitor therapy is the first pharmacologic therapy shown to improve survival outcomes in some patients with MF.69 Cytokine dysregulation has been associated with a poor prognosis in MF.70 The modulation of cytokine levels by ruxolitinib may partly explain the survival improvements in MF, perhaps through reversal of cachexia, and improvement in weight; additionally, this same modulation may improve PH-related echocardiographic changes and biomarkers leading to improve patient outcomes for those with MF that also suffer from this complication. This study demonstrates that biomarkers such as NT-pro BNP, NO, vWB and Leukemia (2014) 1486 – 1493

Ruxolitinib and pulmonary hypertension A Tabarroki et al

1492 RCA are modulated by this drug and may become useful parameters to evaluate the severity and follow therapeutic response in MF-associated PH. Furthermore, it also suggests that aberrant JAK-STAT signaling in MF mediates PH by dysregulation of NO, and cytokine levels can be restored by therapy with ruxolitinib. This study also suggests that the clinical and biological effects of ruxolitinib therapy may be mediated through modulation of NO levels. It is possible that similar therapeutic benefits can be derived by patients with other subtypes of PH including idiopathic PH. This suggests that the benefits of JAK inhibition in MF may go beyond its ability to control cytokinemediated symptoms and splenomegaly and can lead to robust improvement in PH. Clinical trials that will include a larger number of MF patients with PH and taking into account more robust diagnostic modalities of PH including pulmonary catheterization and 6- min walk test will be helpful to further validate these results. CONFLICT OF INTEREST RVT is on the Speaker’s bureau of INCYTE. The remaining authors declare no conflict of interest.

ACKNOWLEDGEMENTS This work was supported in full or partially by Cleveland Clinic Seed Support, American Cancer Society, Scott Hamilton CARES grant (RVT) and Athymic Animal and Xenograft Core Facility (NIH/NCI P30 CA043703-23 (DL)).

AUTHOR CONTRIBUTIONS AT conducted experiments, analyzed the data and wrote the manuscript; DJL provided scientific advice and measured the NO levels; VV performed experiments and edited the manuscript; LZ analyzed statistical data; HJR performed the vWB and RCA measurements and edited the manuscript; YP assisted with mice experiments; HKD contributed patients and edited the manuscript; AL, MEK, MAS contributed patients and edited the manuscript; SEM provided expertise on echocardiographic findings and edited the manuscript; GAH provided expertise on PH, edited the manuscript and provided scientific advice; RVT conceived and designed the study, analyzed the data, and wrote the manuscript.

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