Nitric Oxide As a Mediator of Bone Marrow Fibrosis in Patients with Myelofibrosis
Authors: Tabarroki Ali , Lindner Daniel , Visconte Valeria , Papandantonakis Nikolaos , Ai Jing , Sekeres Mikkael A. , Advani Anjali S. , Kalaycio Matt E. , Cinalli Tracy , Colaluca Kristin , Rupp Gina , Desamito John , Rogers Heesun J. , Tiu Ramon V. 
Published: October 2019

Abstract
Bone marrow (BM) fibrosis is a key pathomorphologic feature of patients (pts) with primary myelofibrosis (PMF) and the fibrotic phases of essential thrombocythemia (post-ET MF) and polycythemia vera (post-PV MF). The degree of BM fibrosis appears to correlate with survival. Indeed worse survival has been associated with increased BM fibrosis. The BM stromal microenvironment is important in the pathogenesis of BM fibrosis. Cellular components (fibroblasts, macrophages, endothelial cells, adipocytes), structural fibrils (collagen, reticulin) and extracellular matrix components are all forming elements of the BM stroma. Increased stromal fibrosis has been linked to abnormalities in the number/ function of megakaryocytes and platelets in hematologic diseases. Several cytokines like Platelet Derived Growth Factor (PDGF) and Transforming Growth Factor-Beta (TGF-b) have been also linked to the pathophysiology of BM fibrosis. PDGF has been shown to increase fibroblast growth in megakaryocytes and platelets although increased PDGF did not correlate with increased production of either reticulin or collagenous fibrosis. Moreover, PMF pts have increased TGF-b levels in platelets, megakaryocytes, and monocytes. Nitric Oxide (NO) is a ubiquitous gas important in physiologic processes particularly vasodilatation. Dysregulation of NO levels has been implicated in pulmonary hypertension (PH), hemoglobinopathies, and cardiovascular diseases. In Peyronie’s disease, a localized fibrosis of the penile tunica albuginea, increased NO production by expression of iNOS decreases collagen deposition by neutralization of profibrotic reactive oxygen species and decreased myofibroblast formation. Aside from its role in maintaining normal vascular tone, NO also plays a role in fibroblast formation and collagen biosynthesis. We previously reported that ruxolitinib, a JAK1/2 inhibitor restores NO levels leading to improvement of PH in MF pts (Tabarroki et al., Leukemia 2014). We now hypothesize that plasma/serum NO level is a key regulator of BM fibrosis in MF and that ruxolitinib treatment (Tx) leads to improvement of BM fibrosis by NO modulation. Using a Sievers 280i NO analyzer we measured the plasma/serum NO level of a large cohort (n=75) of pts with myeloid and myeloproliferative neoplasms (MPN) [MDS, RARS/RCMD=8; MPN, ET=8, PV=8, MF=24, Mastocytosis=7; MDS/MPN, CMML=11, MDS/MPN-U, RARS-T=9]. Healthy subjects (n=10) were used as a control. MPN pts had low NO (nM) levels among the pts studied with the lowest level found in MF pts: MF=30.31±11.8, PV=39.0±16.1, ET=36±20.3, RARS=74.6±41.7 (P=.01), CMML=84.4±89.2 (P=.04), RCMD=163.4±103.8 (P<.001), RARS-T=131.1±99.8 (P<.001). In total, NO levels were lower in classic MPN (n=40, 35.3±16.6) compared to MDS (n=8, 119±62.8; P=.001) and MDS/MPN (n=20, 105±94.6; P=.008). When we looked at the correlation between NO levels and BM fibrosis grade we found that there is an inverse correlation between NO levels and worsening BM fibrosis grade from grade MF1 to MF3. NO levels in normal (n=10) vs MF1 (n=3) were 53.3 vs 39.1, P=.025; normal vs MF2 (n=7) were 53.3 vs 37, P=.021; normal vs MF3 (n=12) were 53.3 vs 34.4, P=.006. A total of 8 pts who were treated with ruxolitinib and had at least 1 pre and 1 post Tx (≥3 months from initiation of ruxolitinib) were tested for NO levels. Among the 8 pts, 4 pts who demonstrated improvement in BM scores had a trend towards improved NO levels after ruxolitinib Tx [NO pre vs post; pt #1: 6 vs 10.5; pt#2: 4.3 vs 6.4; pt#3 49.7 vs 52.1; pt#4 36 vs 41.3; P=.02] while 4 had worsening or had no change in BM fibrosis grade and had a minimal change or decline in the NO (pt#5: 18.4 vs 23, pt#6: 14.29 vs 12.1, pt#7: 32.7 vs 32.1, pt#8: 110.9 vs 40.4). One pt who had improvement in BM fibrosis grade after ruxolitinib Tx had increased iNOS expression by Western blotting (pt#1) while no iNOS expression (pt#5) was noted in the pt who did not have improvement in BM fibrosis. Of note, multi-analytic cytokines profile also showed an overall decrease in cytokines especially a 2.8 fold-decrease in IL8 levels post-Tx in the pt with improvement in BM fibrosis. In conclusion, NO is decreased in MPN particularly in MF and may be a key mediator of BM fibrosis in MF. Pharmacologic therapies such as JAK inhibitors may mediate improvement of BM fibrosis by modulation of NO levels in MF.

Disclosures
Tiu: Gilead: Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; Incyte : Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.