Rationale for combination therapy in myelofibrosis

Best Practice & Research Clinical Haematology 27 (2014) 197e208

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Rationale for combination therapy in myelofibrosis John Mascarenhas, MD, Assistant Professor of Medicine * Myeloproliferative Disorder Program, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, One Gustave L Levy Place, Box 1079, New York, NY, USA

Keywords: combination therapy myelofibrosis ruxolitinib panobinostat decitabine LDE225 PRM-151

* Tel.: þ1 212 242 3417; Fax: þ1 212 876 5276. E-mail address: [email protected].

http://dx.doi.org/10.1016/j.beha.2014.07.009 1521-6926/© 2014 Elsevier Ltd. All rights reserved.

Agents targeting the JAK-STAT pathway have dominated the investigational therapeutic portfolio over the last five years resulting in the first and only approved agent for the treatment of patients with myelofibrosis (MF). However, chromatin modifying agents, anti-fibrosing agents, and other signaling pathway inhibitors have also demonstrated activity and offer the potential to improve upon the clinical success of JAK2 inhibition. Due to the complex pathobiological mechanisms underlying MF, it is likely that a combination of biologically active therapies will be required to target the MF hematopoietic stem cell in order to achieve significant disease course modification. Ruxolitinib in partnership with panobinostat, decitabine, and LDE225 are being evaluated in current combination therapy trials based on preclinical studies that provide strong scientific rationale. The rationale of combination of danazol or lenalidomide with ruxolitinib is mainly based on mitigation of anti-JAK2-mediated myelosuppression. Combination trials of ruxolitinib and novel anti-fibrosing agents such as PRM-151 represent an attempt to address therapeutic limitations of JAK2 inhibitors such as reversal of bone marrow fibrosis. Ruxolitinib is also being incorporated in novel treatment strategies in the setting of hematopoietic stem cell transplantation for MF. As the pathogenetic mechanisms are better understood, potential drug combinations in MF will increase dramatically and demonstration of biologic activity in effective preclinical models will be required to efficiently evaluate the most active combinations with least toxicity in future trials.

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This manuscript will address the proposed goals of combination therapy approach and review the state of the art in combination experimental therapy for MF. © 2014 Elsevier Ltd. All rights reserved.

Introduction The approval of ruxolitinib (Jakafi, Incyte) by the Food and Drug Administration (FDA) in 2011 has greatly changed the treatment landscape of myelofibrosis (MF). Many intermediate and high risk MF patients with either enlarged spleens and/or the presence of MF-related symptoms are now being treated with ruxolitinib. Although the exact numbers of such patients are unavailable, it is presumed that many of the ruxolitinib-treated MF patients achieve a degree of improvement in symptom burden and reduction in spleen size that is deemed successful by the patient and/or treating physician. Keeping this in mind, the MF clinical investigator, now more than ever, is forced to critically re-evaluate treatment goals in a given MF patient. Broadly speaking, treatment goals can vary from purely palliative to curative, and can be specific such as amelioration of anemia, or attempt to modify the disease course. Patient and disease specific features influence the decision to focus on a particular treatment goal. Although ruxolitinib is effective for many MF patients in palliating symptoms and reducing splenomegaly, and recent analysis suggest prolongation of survival, it has not been shown to definitively alter the natural history of MF and this remains the focus of current clinical research. Due to the integral role of hyperactive JAK-STAT signaling in MF pathogenesis and the demonstrable clinical activity of JAK2 inhibitors, a number of clinical trials in 2014 are evaluating the safety and efficacy of JAK2 inhibitor combination therapy. This manuscript will highlight the challenge of determining which MF patients are appropriate for combination therapy and will review the state of the art of combination therapy for MF with emphasis on the preclinical rationale. Patient case The following case will serve as a basis for discussion regarding the appropriate role of combination therapy for MF and the different approaches that are currently being evaluated, including the rationale supporting each approach. A 60 year old male with treatment naive MF diagnosed three years ago presents with worsening anemia and the emergence of red blood cell transfusion dependence, night sweats, weight loss, and progressive splenomegaly over the last several months. He is stratified as high risk by the Dynamic International Prognostic Scoring System (DIPSS) and is initially prescribed ruxolitinib with rapid resolution of constitutional symptoms and moderate reduction of splenomegaly [1]. However, he continues to require frequent red blood cell transfusions with the continued documentation of peripheral blood blasts in the range of 3e5%. Although ruxolitinib has improved aspects of the disease process, transfusion dependent anemia and the potential for progression and/or transformation to acute leukemia remain a valid concern. Therapeutic options for this individual include a) continued therapy with ruxolitinib until evidence of disease progression (enlarging spleen or increasing peripheral blood blast count), b) hematopoietic stem cell transplant (HSCT) if an appropriate donor is available, or c) experimental therapy. Experimental therapeutic options would include (but are not limited to) monotherapy with another JAK2 inhibitor (momelotinib, pacritinib, NS018, LY2784544, BMS911543), histone deacetylase inhibitor (panobinostat, pracinostat, givinostat, vorinostat), telomerase inhibitor (imetelstat), mTOR inhibitor (everolimus), hedgehog pathway inhibitor (LDE225, IPI-926), or antifibrosing agent (PRM-151, GS6624). Both the preclinical and clinical development of these agents as monotherapies are reviewed extensively elsewhere and are beyond the scope of this manuscript which will instead review combination therapy approaches under evaluation.

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Combination therapy rationale and identification of unmet clinical needs Understanding the rationale for the pursuit of combination therapy for the treatment of MF requires a brief review of unmet clinical needs and the acknowledgment that unlike chronic myelogenous leukemia (CML), which is universally driven by a sole oncogenic protein that is effectively treated by a number of targeted monotherapies, MF is a myeloproliferative neoplasm (MPN) with a complex genetic and epigenetic pathogenesis that will likely require polytherapy. In the absence of a unifying, targetable, disease-initiating molecular abnormality, combinations of therapies with non-overlapping toxicities and both overlapping mechanisms of action that may produce synergistic biological effects and non-overlapping mechanisms that may be complementary are the focus of next generation MF clinical trials. Similarly, the majority of both lymphoid and myeloid hematologic malignancies require combination therapy which is frequently more effective than monotherapy. Unmet clinical needs in MF treatment include a) amelioration of symptomatic anemia and elimination of red blood cell transfusion dependence, b) improvement in treatment limiting thrombocytopenia, c) reversal of bone marrow fibrosis and restoration of normal hematopoiesis, and d) achievement of cytogenetic and molecular remissions. The first two unmet needs are clinical issues that have direct impact on patient care and quality of life and successful correction of cytopenias can result in an immediate positive impact for an MF patient. Achieving the last two unmet needs would presumably (although not yet proven) correlate with improved progression free survival, but may not necessarily have an immediate impact on the patient. The definition of “disease modification” remains vague in the context of MF treatment. For example, JAK2 inhibitors have been documented to dramatically reduce heightened inflammatory cytokine expression in treated MF patients and this has been linked to the improvement in constitutional symptoms that patients frequently experience with ruxolitinib treatment [2]. In fact, certain cytokine expression profiles have been linked to MF clinical phenotype and cytokine profile patterns may also may have prognostic influence [3]. It would then appear that JAK2 inhibitor mediated down-regulation of the elevated inflammatory cytokine state in MF would be a form of disease modification. On the other hand, even in MF patients responding clinically to JAK2 inhibitor therapy, elimination of JAK2V617F as detected by polymerase chain reaction (PCR) of peripheral blood granulocytes is not obtained [4e6]. Therefore, JAK2 inhibitors thus far have not proven to have disease modifying potential as it relates to surrogate molecular markers of disease burden. Outside of HSCT, medicinal therapies evaluated in clinical trials for patients with MF have only occasionally resulted in disease modification in the form of elimination of bone marrow histopathological features and associated chromosomal/molecular abnormalities [7,8]. Although currently available and experimental therapies have the potential to modify certain aspects of the disease process, a therapeutic approach, other than HSCT, that will modify the chronic and progressive MF disease course resulting in improved overall survival is as of yet unavailable. Ruxolitinib in combination with hematopoietic stem cell transplantation Currently, restoration of normal polyclonal hematopoiesis with reversal of bone marrow fibrosis and elimination of clonal evidence of MF has only been reproducibly demonstrated in the setting of HSCT [9]. Reduced intensity conditioning (RIC) HSCT has proven to be a potentially curative therapeutic option for intermediate-2/high risk MF patients with a suitable donor and current transplant trials in MF are further evaluating this approach with modifications of the conditioning regimen and incorporation of pre-conditioning treatments such as JAK2 inhibitors (NCT01790295) [10,11]. Many clinical variables influence HSCT outcome. Splenomegaly has been implicated in causing poor engraftment in MF, presumably as a consequence of splenic sequestration of donor CD34þ cells [12,13]. Poor performance status, which is common in MF patients, as a result of disease related constitutional symptoms and cachexia is also associated with increased transplant related mortality (TRM) [14]. The potential role of inflammatory cytokines in graft versus host disease (GVHD) has more recently been investigated [15]. The rationale for the combination of either induction or consolidation JAK2 inhibitor therapy with HSCT is based on a hypothesis that JAK2 inhibitor mediated reduction in splenomegaly, downregulation of inflammatory cytokine expression, and improvement in performance status would lead to improvement in donor cell engraftment, and reduced TRM and GVHD.

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Rationale for specific combination therapies As noted above, it is reasonable to assume based on the current pathobiological understanding and clinical experience with single agent treatment, combination therapy will be required to achieve significant therapeutic success in MF. Due to increased understanding of the acquired cellular and (epi) genetic alterations implicated in MPN pathogenesis, and the development of new agents in several novel classes, combination therapy trials given either concurrently or sequentially are becoming increasingly common. Pre-clinical studies support the rationale of combining various agents in ongoing and planned clinical trials. Given the fact that ruxolitinib represents the first in class small molecule inhibitor of the JAK-STAT signaling pathway, has proven clinical activity in MF, and is the only FDAapproved therapeutic for patients with MF, the first wave of combination therapy trials incorporate ruxolitinib with a variety of agents often predicated on supporting pre-clinical rationale (see Table 1). Combination of JAK2 inhibitor and chromatin modifying agent Panobinostat (LBH589, Novartis) is an oral pan deacetylase inhibitor capable of enhancing acetylation of lysine residues on histones (and non-histone proteins) resulting in changes in chromatin structure and up-regulation of tumor suppressor gene transcription promoting apoptosis of MPN hematopoietic progenitor cells (HPCs) [16]. Panobinostat has been shown to induce apoptosis in the JAK2V617F-expressing human erythroleukemia HEL92.1.7 cell line and Ba/F3-JAK2V617F cells [17]. Interestingly, panobinostat treatment of primary JAK2V617F-positive MPN cells has been shown to result in reduction of JAK2V617F protein levels through induction of hyperacetylation of heat shock protein 90 (HSP90), in which JAK2V617F is a client protein rendered susceptible to proteosomal degradation [17]. Oral panobinostat has been evaluated as a single agent in two trials in patients with intermediate/ high risk MF, irrespective of JAK2V617F status. In a single center, phase I dose escalation study, thrombocytopenia was found to be the dose limiting toxicity (DLT) and 25 mg thrice weekly was determined to be the recommended phase II dose (RPTD) [7]. A total of 18 MF patients were treated on this protocol and five were evaluable for response after six cycles with thee achieving clinical improvement (CI) by spleen criteria and two stable disease (SD) using response criteria by the International Working Group for Myelofibrosis Research and Treatment (IWG-MRT) [18]. Most importantly it was demonstrated that two patients treated for over 12 cycles achieved improvement in anemia, elimination of leukoerythroblastosis, and significant reduction in bone marrow reticulin fibrosis. In a multi-centered phase II study of oral panobinostat at starting doses of 40 mg thrice weekly, clinical activity was limited by excessive toxicity in the form of thrombocytopenia and diarrhea [19]. Nevertheless, correlative studies from this trial demonstrated expected on target effects with decreased downstream targets of JAK-STAT signaling, reduced inflammatory cytokines levels, and decreased JAK2V617F allele burden. Collectively, these two trials showed that panobinostat is best tolerated at low doses and when given continuously over a prolonged period can achieve optimum results.

Table 1 Available combination therapy trials for patients with myelofibrosis. Class of agent 1

Agent 1

Class of agent 2

Agent 2

Phase of study

NCT identifier

JAK 1/2 inhibitor

Ruxolitinib

Chromatin modifying agents

Panobinostat

I/II Ib II I/II II Ib/II II II II I/II

NCT01693601 NCT01433445 NCT01787487 NCT02076191 NCT01375140 NCT01644110 NCT01732445 NCT01981850 NCT01369498 NCT01787552

Immunomodulatory agents Androgen Anti-fibrosing agent Hedgehog pathway inhibitor

Azacytidine Decitabine Lenalidomide Pomalidomide Danazol PRM-151 GS-6624 LDE225

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Preclinical studies of panobinostat and JAK2 inhibitor provide rationale for the combination therapy approach in MF patients. Cotreatment with panobinostat and the selective JAK2 inhibitor TG101209, led to enhanced apoptosis in both HEL and Ba/F3-JAK2V617F cells, as well as increased cytotoxicity of primary CD34þ MPN cells compared to normal CD34þ HPCs [17]. Panobinostat in combination with ruxolitinib was also shown to have synergistic activity in a murine bone marrow transplant model of JAK2V617F-driven MF leading to increased reduction of splenomegaly, and improved bone marrow and spleen histopathology compared to each drug given individually [20]. Additionally, the combination of panobinostat and ruxolitinib resulted in further reduction in phosphorylated STAT-5 levels and increased histone H3 acetylation suggesting both complementary and non-overlapping biological activity. Importantly, thrombocytopenia, the dose limiting toxicity of both agents was not found to be exacerbated in combination in this murine model. Currently, a phase Ib trial (Novartis sponsored, multi-institutional European study) and a phase I/I trial (investigator-initiated single center, United States study) of ruxolitinib in combination with panobinostat are being conducted (NCT01693601, NCT01433445). The primary objective of both trials is to define the safety and tolerability of combination ruxolitinib and panobinostat therapy in MF patients and to determine the optimal dosing schedule that can be administered for prolonged period of times. In order to overcome the potential overlapping toxicity of myelosuppression, lower starting doses and modified treatment schedules (intermittent weekly dosing of panobinostat) are being explored. Changes in spleen and symptom response will be assessed as well as changes in bone marrow histomorphologic abnormalities and degree of bone marrow fibrosis. Additional exploratory objectives include assessment of changes in molecular and cytogenetic markers of the malignant MF HSC. The results of the dose escalation portion of the Novartis-sponsored combination trial (NCT01433445) have been reported at the American Society of Hematology meeting in 2013 [21]. A total of 38 patients with MF have been treated on this protocol and the combination of these two agents was shown to be well tolerated with grade 4 thrombocytopenia (2 patients) and grade 3 nausea (1 patient) as dose limiting toxicities. At the time of data cutoff, 13 patients discontinued and the most common reasons were adverse event in 7 and disease progression in 4 patients. Approximately 50% of treated patients across all dose cohorts achieved a 50% or greater reduction in palpable splenomegaly at some point during the study. The recommended phase II doses were determined to be 15 mg twice daily for ruxolitinib and 25 mg thrice weekly, every other week for panobinostat. The dose expansion phase is currently ongoing and will provide further data on efficacy and safety. Reduced expression of CXCR4 expression by MF CD34þ cells is believed to contribute to abnormal hematopoietic stem cell trafficking in MF and homing to sites of extramedullary hematopoiesis (EMH) such as the spleen [22]. Sequential treatment with the demethylating agent azacytidine followed by the HDAC inhibitor trichostatin A was shown to restore MF CD34þ CXCR4 expression and correct abnormal trafficking of MF CD34þ to the bone marrow in a NOD/SCID mouse model [23]. A number of genes that play key regulatory functions in cell cycling, transcription, and intracellular signal transduction have been shown to be silenced by gene promoter site hypermethylation including P15INK4b/ P15INK4a, RARb2, CXCR4, and SOCS [24e27]. These findings provide scientific rationale for the evaluation of DNA methyltransferase (DNMT) inhibitors in MF. 5-azacytidine (Vidaza, Celgene) and decitabine (Dacogen, EISAI) have been evaluated in patients with MF. Reports of clinical activity of decitabine as a monotherapy in both chronic and blast phase MF have been published [28e30]. Decitabine given subcutaneously at a dose of 0.3 mg/kg/d on days 1e5 and 8e12 every 42 days was evaluated in 21 patients with chronic and blast phase (4 patients) MF in a phase II trial [31]. Myelosuppression was frequent as would be expected and clinical responses included CR (1 patient), PR (2 patients), and CI-anemia (2 patients) and CI-platelets (2 patients). Decitabine therapy was associated with a decrease in circulating CD34þ HPCs and the investigators proposed this as a potential novel biomarker to predict decitabine response in the treatment of MF. Decitabine given as an infusion at a dose of 20 mg/m2 for five consecutive days and repeated every four weeks was shown to result in a median overall survival that had not been reached at 9 months of follow up in six patients with MF in blast phase, which has a dismal prognosis and a reported median survival of approximately 3e5 months [29,32,33]. 5-azacytidine has also been reported to have clinical activity in patients with MF in blast phase. The median overall survival of 26 patients with MF blast phase

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treated with 5-azacytidine was shown to be 8 months at a median follow up of 20 months by the Groupe Francophone des Myelodysplasies (GFM) [34]. However, 5-azacytidine was not found to have significant clinical activity in a single center phase II trial of 34 MF patients [35]. In this study, despite the finding of global DNA hypomethylation, a single patient obtained a PR and seven achieved CI for a modest overall response rate of 24%. Due to the excessive myelosuppression that can be seen with 5azacytidine in this setting, and the inconvenience of seven day dosing, a trial of a 5 day schedule at the conventional dose was also conducted in ten MF patients [36]. Due to poor tolerance resulting in a low median number of treatment cycles, no clinical responses were seen in this study. Ruxolitinib has also been tested in patients with MF in blast phase. In a phase II trial of ruxolitinib at dose of 25e50 mg twice daily in patients with acute leukemia, 18 with MF blast phase were enrolled and three of these achieved a CR or CR with incomplete blood count recovery [37]. Overall, ruxolitinib given at higher doses in advanced stages of MF was well tolerated and only four patients experienced grade 3/4 drug related toxicity. Laboratory evidence of the acquisition of mutations in genes encoding for epigenetic modifiers such as TET2, IDH1/2, and ASXL1 appear to contribute to disease progression and evolution of MF to blast phase disease which often has a distinct mutational profile compared to de novo acute myeloid leukemia [38,39]. In addition, in vitro drug studies utilizing bone marrow cells from a JAK2V617F-driven transplant murine model of blast phase MF have demonstrated that exposure to decitabine or ruxolitinib inhibits colony formation in a methylcellulose colony-forming assay [34]. Importantly, the combination of decitabine and ruxolitinib in this assay significantly reduces colony formation when compared to either drug alone, thus providing pre-clinical evidence for the combination study in blast phase MF. The Myeoloproliferative Disorders Research Consortium (MPD-RC) is currently conducting a multicenter phase I/II trial of combination ruxolitinib and decitabine in patients with MF in advanced forms (accelerated and blast phase) [NCT02076191]. Decitabine is administered as a five consecutive day infusion at 20 mg/m2 and ruxolitinib is given continuously at increasing doses in a dose cohort escalation schema. The primary objective of the phase I trial is to determine the safety and tolerability of these two agents in combination and in the phase II trial is to determine the response rate by conventional and proposed MF blast phase criteria [40]. Correlative studies will assess the change in global DNA methylation status of leukemic blasts after combination therapy and evaluate this marker for predictive potential of therapeutic response. Additionally, the mutational status of a panel of candidate genes in patients with MF blast phase will be analyzed in order to better elucidate the genetic and epigenetic alterations that contribute to leukemic transformation of an MPN. 5-azacytdine in combination with ruxolitinib is currently being evaluated in a single institution phase II trial involving patients with intermediate/high risk MF or myelodysplastic/myeloproliferative overlap syndrome [NCT01787487]. Ruxolitinib will be dosed at either 15 or 20 mg twice daily depending on the baseline platelet count and given continuously, while 5-azacytidine will be given subcutaneously at a dose of 25 mg/m2 for five consecutive days at the start of cycle four. The primary objective of this trial is to determine the overall response rate by IWG-MRT criteria after six cycles of therapy. Combination of JAK2 inhibitor and alternative signaling pathway inhibitors Increased activity of the JAK-STAT3/5, RAS/RAF/MAPK, and PI3K/AKT/mTOR pathways confer a proliferative and survival advantage to MF HPCs [41,42]. The dual PI3K/AKT and mTOR inhibitor BEZ235 has been shown to reduce PI3K/AKT and mTOR signaling, and induce cell-cycle growth arrest and apoptosis in HEL cells as well as MF CD34þ HPCs in culture [42]. Co-treatment with BEZ235 and a JAK2 inhibitor (either TG101209 or SAR302503) induced synergistic killing of HEL cells and MF CD34þ HPCs but not normal CD34þ HPCs. These laboratory studies strongly support the clinical trial evaluation of the dual PI3K/mTOR inhibitor in combination with a JAK2 inhibitor in patients with MF. This is a combination approach that represents a potential clinical trial application and Fig. 1 summarizes other potential approaches that would need to be tested in the clinic. The hedgehog (Hh) signaling pathway is a key regulator of cell growth and differentiation during embryonic development as well as maintenance and expansion of somatic stem cells [43]. Reactivation

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Fig. 1. Potential combination therapies for patients with myelofibrosis (MF). Each class of agent listed has biological rationale and in some cases demonstrated pre-clinical activity in combination with agents from other classes listed. The decision to combine two agents may be influenced by an approach designed to capitalize on non-overlapping mechanisms of action or minimize treatment related toxicity of the partner agent. Pre-clinical studies demonstrating activity of combinations of agents in murine models of MF and primary MF hematopoietic cells often provide the rationale supporting early phase clinical trials in patients with MF. Myelofibrosis, mTOR mammalian target of rapamycin, IMiD immunomodulator, DAC deacetylase, DNMT DNA methyltransferase, SMO smoothened, HSP90 heat shock protein 90.

of this pathway in cancer stem cells has also been implicated in the pathogenesis of myeloid malignancies [44]. Hyperactivity of the Hh pathway and resultant up-regulation of target genes has been observed in primary MPN cells as well demonstrated in a murine bone marrow transplant model of MF [45]. LDE225 (sonidegib, Novartis) is an orally bioavailable selective small molecule inhibitor of SMO, which leads to decreased Hh pathway expression of target genes [46]. Combination therapy of ruxolitinib and LDE225 in the preclinical murine model of MF resulted in improved reduction of leukocytosis, thrombocytosis, splenomegaly, bone marrow fibrosis, and JAK2V617F allele levels compared to ruxolitinib treatment alone [45]. This preclinical data forms the basis and rationale for a combination study in MF. A multi-center, phase Ib/II combination therapy study of LDE225 and ruxolitinib in patients with intermediate-1 or higher risk MF is currently recruiting participants in order to assess safety and determine the recommended phase II doses for these agents in combination. There is a growing appreciation of the potential combinations of signaling pathway inhibitors to either produce synergistic or complementary biological effects in MPN cells and these pre-clinical studies often provide rationale for translational research in patients with MF. The combinations of ruxolitinib with the PIM inhibitor LGH447 or the specific PI3K inhibitor BKM120 have also shown synergistic activity in murine models of MPN providing pre-clinical rationale for the evaluation of these two approaches in clinical trial [47,48]. Recently, an ex-vivo model using flow cytometry to detect markers of apoptosis to identify drugs with synergistic activity in combination with ruxolitinib in both a JAK2V617F-driven cell line and primary MF cells was reported [49]. This novel system would potentially allow for the evaluation of synergistic activity of combinations of drugs with differing mechanisms of action involved in cell proliferation, differentiation, survival, cell cycle, chromatin modifications and immune response. Such a high throughput screening tool could streamline the clinical trial evaluation of combinations of agents so as to selectively pursue those that offer the highest likelihood of synergistic activity, thereby expediting the process of clinical trial evaluation, reducing the overall costs, and diminishing unnecessary exposure of toxic agents to patients.

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Combination of JAK2 inhibitor and agents targeting anemia Ruxolitinib is associated with myelosuppression and in particular grade 3/4 anemia which was reported to occur at a rate of 45% in the COMFORT-1 and 42% in COMFORT-2 trials, regardless of change from baseline [4,5]. The anemia associated with ruxolitinib therapy is dose dependent and predictably nadirs in the first two to three months of therapy, remaining relatively stable at a 1 g/dL level below the baseline hemoglobin. Combining agents that have the potential to ameliorate ruxolitinib therapy associated anemia is the motivating rationale for ruxolitinib combination therapy with danazol (NCT01732445), immunomodulators (NCT01375140, NCT01644110), and recombinant erythropoietin [50]. Synthetic androgens have been used in to address MF associated anemia [51e53]. Although the exact mechanism of action leading to improvement in anemia with danazol is unknown, it has been hypothesized that a combination of increased stimulation of erythropoiesis and decreased clearance of red blood cells attributed to down regulation of monocyte Fcg receptor expression [54]. Danazol, a derivative of the synthetic steroid ethisterone, was reported to have an anemia response rate of approximately 40% in 33 MF patients with baseline anemia treated with a 600 mg daily dose and followed for a median of approximately 20 months [55]. A phase II trial of combination ruxolitinib and danazol is currently being conducted (NCT01732445). This open label study includes patients with intermediate/high MF and a baseline hemoglobin less than 10 g/dL or meeting criteria for transfusion dependence. The primary objective of this trial is to assess the overall response rate by IWG-MRT criteria. Immunomodulatory agents (IMiDs) such as thalidomide, lenalidomide, and most recently pomalidomide have been evaluated in multiple studies with reported anemia response rates that range from 20 to 40% and are reviewed extensively elsewhere [56]. Often these agents are given in combination with a prednisone taper, and this perhaps may have been the first combination therapy approach for MF. IMiDs, in particular thalidomide, can be associated with cumulative toxicity in the form of neuropathy, sedation, constipation, and thrombosis while myelosuppression is more common with lenalidomide and can often limit the duration of treatment. Pomalidomide, the most potent IMiD has recently completed a placebo controlled randomized phase III study in patients with transfusion dependent anemia, which failed to meet the primary endpoint of red blood cell transfusion independence [57]. Importantly, an earlier study appears to suggest MF patients with JAK2V617F and minimal splenomegaly are more likely to experience an anemia response with pomalidomide therapy, and this may not have been appreciated in the large phase III trial which did not exclude patients based on JAK2 mutational status or massive splenomegaly [58]. Ruxolitinib in combination with lenalidomide is currently being evaluated in a single center phase II trial [NCT01375140]. The primary objective of this trial is to determine the overall response rate by IWG-MRT criteria after three cycles of combination therapy. Ruxolitinib will be dosed continuously at 15 mg twice daily and lenalidomide will be given concurrently at a daily dose of 5 mg for 21 days of each 28 day cycle. Prednisone taper starting at 30 mg daily can be added for those patients not achieving response by cycle 4. Whether a combination of ruxolitinib and pomalidomide in a selected group of patients with JAK2V617F-positive MF without massive splenomegaly would prove effective would require similar clinical trial evaluation. The potential for erythropoiesis stimulating agents (ESAs) to improve MF related anemia has been tested in several trials with responses ranging from 20 to 60% [59e61]. Clinical variables associated with higher likelihood of anemia response to ESA therapy include transfusion naivety, higher baseline hemoglobin, and an endogenous serum erythropoietin level below 125 U/l. Of note, in an analysis of risk factors for leukemic transformation in patients with MF, a prior history of ESA and danazol therapy were noted to have a positive correlation in multivariate analysis [62]. A summary of responses from a subset of 13 patients from COMFORT-II who received ruxolitinib in combination with ESA support has been presented [50]. In this analysis, anemia responses by IWG-MRT criteria were not appreciated, although a decrease in grade of anemia after six weeks of combination therapy was reported. Importantly, ESA treated patients in this subgroup analysis also attained spleen responses with ruxolitinib treatment and did not experience additional toxicity. The combination of an ESA and ruxolitinib should be tested in a prospective trial prior to the general use in the community setting.

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Combination of JAK2 inhibitor and anti-fibrosing agents Advanced grade of bone marrow fibrosis in patients with MF has been shown to correlate with poorrisk clinico-hematologic features such as anemia, thrombocytopenia, leukopenia, splenomegaly, and presence of peripheral blood blasts, and may have prognostic significance for survival [63,64]. It is presumed that reticulin fibrosis of the bone marrow in MF is a secondary consequence of cytokine activated mesenchymal-derived fibroblasts, however JAK2 inhibitors have not consistently demonstrated the ability to eliminate bone marrow fibrosis. Most recently, detection of JAK2V617F and chromosomal abnormalities from fibroblasts isolated from the bone marrow of patients with MF provides evidence of clonal involvement of fibroblasts (personal communication from Srdan Verstovsek). Additionally, serum levels of pentraxin-2 (PTX2) which is known to have a regulatory function in scaring and healing was found to be reduced in patients with MF in comparison to healthy controls. PRM-151 is a recombinant form of human PTX2, which inhibits monocytic differentiation into fibroblasts and pro-fibrotic macrophages, while promoting their differentiation into regulatory macrophages [65]. This first-in-class modulator of the fibrosis pathway has demonstrated the ability to reduce pre-existing fibrosis in preclinical models of fibrotic disease such as bleomycin induced lung fibrosis, renal fibrosis, radiation induced oral mucositis and offers an innovative approach to the treatment of pathological bone marrow fibrosis [66e68]. A phase II trial with four treatment arms of infusional PRM-151 provided weekly or every four weeks either alone or in combination with ruxolitinib is currently underway (NCT01981850). This trial which enrols patients with intermediate-1 or higher risk MF and requires at least grade 2 bone marrow fibrosis according to the European Consensus on Grading of Bone Marrow Fibrosis has completed stage 1 and results from the interim analysis are anticipated this year. The results of the first stage of this innovative approach will determine which treatment arm(s) will be further evaluated in stage II based on overall response rates by IWG-MRT and toxicity profiles. Interferon-a The role of interferon-a for the treatment of MF remains unclear and presently poorly defined [69]. The potential role of low dose pegylated interferon in combination with JAK 1/2 inhibitors or statins is of theoretical interest, remains untested, and is addressed expertly elsewhere [70]. Summary The discovery of JAK2V617F and the subsequent appreciation of aberrant over-activity of the JAKSTAT pathway as a disease target reinvigorated translational research efforts in MPNs and resulted in the first and only FDA-approved therapeutic for patients with MF. Further advances in the understanding of the complex pathobiological mechanisms underlying MF have also led to the evaluation of various novel therapeutics based on scientific rationale. Targeting disease initiating/supporting pathways that will result in selective deletion of the malignant MPN HSC remain the focus of MPN research. By effectively eliminating the MPN HSC, the possibility of true disease process modification and potential cure may one day be attainable. Although no single monotherapy has definitively proven to extend progression free survival, several agents have demonstrated a signal of clinical activity and preclinical studies support the evaluation of these agents in combination therapy approach. Combination therapy trials will attempt to target multiple MF-related pathobiological processes, including impaired signaling pathways and alterations in the genome and epigenome that appear to coexist and contribute to the complexity and molecular heterogeneity of this MPN. It is important that clinical research and combination therapy trials, in particular, continue to move beyond the current available monotherapies as we evaluate innovative therapeutic approaches that may one day offer all MF patients the reality of improved survival. Conflict of interest Clinical research funding paid to my institution from Incyte, Novartis, and Roche.

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