Leukemia (2014), 1–9 & 2014 Macmillan Publishers Limited All rights reserved 0887-6924/14 www.nature.com/leu

REVIEW

Novel myelofibrosis treatment strategies: potential partners for combination therapies BL Stein1,2, R Swords3, A Hochhaus4 and F Giles2 Of the myeloproliferative neoplasms (MPNs), myelofibrosis (MF) is associated with the greatest symptom burden and poorest prognosis and is characterized by constitutional symptoms, cytopenias, splenomegaly and bone marrow fibrosis. A hallmark of MF is dysregulation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway that has led to the development of JAK inhibitors targeting this pathway. Calreticulin gene mutations have recently been identified in JAK2 mutationnegative patients with MF. Identification of JAK inhibitor resistance and broad contributions to MF disease pathogenesis from epigenetic deregulators, pathways that work in concert with JAK/STAT (that is, mammalian target of rapamycin/AKT/ phosphoinositide 3-kinase, RAS/RAF/MEK, PIM kinase), fibrosis-promoting factors and the MF megakaryocyte, suggest that numerous options may be partnered with a JAK inhibitor. Therefore, we will discuss logical and potential partners for combination therapies for the treatment of patients with MF. Leukemia advance online publication, 1 July 2014; doi:10.1038/leu.2014.176

KEY ISSUES Myelofibrosis (MF) is the most symptomatic of the myeloproliferative neoplasms and is characterized by debilitating constitutional symptoms, cytopenias, splenomegaly and bone marrow fibrosis.  A hallmark of MF is dysregulation of the Janus kinase (JAK)/ signal transducer and activator of transcription signaling pathway, most notably associated with the JAK2 V617F mutation, present in 450% of patients with MF; calreticulin gene mutations were recently described in JAK2 mutation-negative patients with MF.  Consequently, a number of JAK inhibitors have entered clinical trials, and one has been approved for the treatment of intermediate- and high-risk MF.  Because of the molecular complexity and heterogeneity of MF, it is likely that the optimal use of JAK inhibitors will be in combination with other MF-related therapies. 

INTRODUCTION Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), including essential thrombocythemia (ET), polycythemia vera (PV) and myelofibrosis (MF), are a diverse group of clonal stem cell disorders derived from hematopoietic myeloid progenitors.1 Although all MPNs can result in a pronounced symptom burden, treatment needs have been typically unmet for those with MF, which carries the most adverse prognosis among the three. Historically, therapies for MF have included cytoreductive treatments such as hydroxyurea that control proliferation but often result in only transient responses and myelosuppression. However, studies in patients with MF have shown that

conventional treatments, such as hydroxyurea, may be no better than placebo for treating symptoms and splenomegaly.2 Hematopoietic stem cell transplantation is a more aggressive therapy but is an option for only a select minority of patients with MF, in part because of its considerable morbidity and mortality;3 however, benefits can include regression of bone marrow fibrosis, stabilization or elimination of the mutant clone, reduction in allele burden in Janus kinase 2 (JAK2) V617F-positive patients and possibly cure.4,5 The identification of Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling dysregulation in MF ushered in a new era of therapy. In 2005, the JAK2 V617F mutation was identified and is now recognized in B50 to 60% of patients with MF.6–9 In addition to the prevalent JAK2 V617F mutation, which results in the loss of negative regulation, JAK/STAT signaling can be dysregulated through acquisition of additional mutations in MPN-related genes, including CBL, LNK, MPL and JAK2 exon 12. More recently, calreticulin (CALR) gene mutations have been reported in patients with MF (and ET) who lack JAK2 V617F or MPL mutations. Klampfl et al.10 identified CALR mutations that caused a frameshift to the normal reading frame in 88% of patients with MF. When compared with JAK2 V617F-positive patients, patients with CALR mutations had a longer survival. In addition, the most common CALR mutation was shown to activate STAT5, resulting in cytokine-independent growth. Nangalia et al.11 also identified a high prevalence of CALR mutations in JAK2/MPL-negative patients with MF. Among 31 samples that underwent exome sequencing, 26 (84%) were found to have CALR mutations. In a follow-up cohort, 56% of patients with JAK2 V617F/MPL-negative MF had CALR mutations. Mutations were found to occur in multipotent progenitors and were associated with lower hemoglobin (Hgb) level, higher platelet count and perhaps a higher rate of post-ET MF (in those with ET). These results were confirmed in another

1 Northwestern University Feinberg School of Medicine, Chicago, IL, USA; 2Northwestern Medicine Developmental Therapeutics Institute, Robert H Lurie Comprehensive Cancer Center of Northwestern University, Chicago, IL, USA; 3Sylvester Comprehensive Cancer Center, Division of Hematology/Oncology, Miller School of Medicine, University of Miami, Miami, FL, USA and 4Klinik fu¨r Innere Medizin II, Ha¨matologie/Onkologie, Universita¨tsklinikum Jena, Jena, Germany. Correspondence: Dr BL Stein, Northwestern University Feinberg School of Medicine, 645 N Michigan Avenue, Suite 1020, Chicago 60611, IL, USA. E-mail: [email protected] Received 17 February 2014; revised 25 April 2014; accepted 7 May 2014; accepted article preview online 3 June 2014

Combination therapy partners for JAK inhibitors BL Stein et al

2 recent report of 256 patients with MF, among whom 74% of patients without JAK2 or MPL mutations had CALR mutations and a favorable prognosis.12 Apart from STAT5 activation, it is not yet clear how CALR mutations lead to MPN. However, in vitro and clinical evidence suggests that CALR mutations confer some sensitivity to JAK inhibition.10,13 These observations and supportive preclinical in vitro and in vivo evidence, along with clinical trial data, provide rationale for the use of JAK inhibitors. Table 1 summarizes the results from clinical trials of these agents in patients with MF. However, JAK inhibition has certain inherent challenges. First, rather than treating cytopenias, JAK inhibition is commonly associated with myelosuppression that is generally manageable. Second, symptoms and splenomegaly return within weeks of drug discontinuation (ruxolitinib). Third, these agents do not lead to complete disease remission nor do they eliminate the disease-initiating clone. Fourth, despite chronic JAK inhibition, persistent JAK/STAT signaling continues because of heterodimer formation with other JAK family members, such as JAK1 or tyrosine kinase 2 (TYK2). Finally, unlike neoplasms such as chronic myelogenous leukemia that are the consequence of a single molecular abnormality, the pathophysiology of MF is heterogeneous, not exclusively attributable to JAK/STAT signaling dysregulation, and therefore is more molecularly complex (Figure 1).14 Although these challenges temper enthusiasm for JAK inhibition, recent data support dependence of MPN cells on JAK2.15 In this study, using MPN murine models and primary samples, deletion of JAK2 in vivo and after JAK inhibitor exposure led to improvement in blood counts, organomegaly and mutation burden. These data suggest that

Table 1.

JAK2 remains an important target, but it is unlikely that monotherapy alone can sufficiently address the disease burden for most patients with MPN. In this review, we discuss candidate agents that may be combined with JAK inhibitors with a goal of either offsetting adverse effects (anemia) or, importantly, further altering the natural history of MPN (Table 2). Practical considerations: agents to treat anemia Androgens. Danazol may ameliorate the anemia associated with JAK inhibitors, although the efficacy as a single agent is largely restricted to retrospective study. One of the retrospective investigations of 30 patients with MF reported responses in 11, including 8 that were complete.16 A lack of transfusion dependence and a higher Hgb level before starting therapy predicted response. In another retrospective study of 39 patients with MF taking danazol, 17 responded, including 8 (21%) with an Hgb improvement of X1.5 g/dl, an Hgb 410 g/dl and transfusion independence for X8 weeks.17 Unlike prior studies, there were no identifiable patient characteristics, such as transfusion dependency, baseline Hgb level or cytogenetic results that influenced outcome. Toxicities attributable to danazol therapy include liver function test abnormalities, headache and virilization. Danazol in combination with ruxolitinib is currently being evaluated in a phase 2 study (NCT01732445). Shared toxicities with ruxolitinib may include headache. Although offsetting anemia could positively impact the MF symptom burden, and allow for dose escalation of ruxolitinib to maximize reduction in splenomegaly, this combination is not intended to induce disease remission.

JAK2 inhibitors in clinical trials for patients with MF Anti-JAK2 IC50

Other targets

Current phase of evaluation

Clinical benefits reported

Adverse events

Ruxolitinib

2.8

JAK1

3 (completed), FDA approved

Myelosuppression, headache, dizziness, easy bruising

79–81

Momelotinib (CYT387)

18

JAK1, TYK2, CDK2, JNK1, RICJ2

Splenomegaly, MF symptom burden, improved overall survival, potential stabilization of fibrosis Splenomegaly, MF symptoms, improvement in anemia

82–84

Fedratinib (SAR302503, formerly TG101348)

3

FLT3, RET

Pacritinib (SB1518)

23

FLT3

3

Increase in amylase/lipase (DLT), headache (DLT), thrombocytopenia, headache, peripheral neuropathy, elevated transaminases Elevated amylase/lipase (DLT), nausea, diarrhea, anemia, thrombocytopenia, elevated transaminases; cases of Wernicke encephalopathy reported Gastrointestinal symptoms (DLT), diarrhea, nausea, thrombocytopenia

IC50 ¼ 0.055 mM for JAK2 V617F vs 2.26 mM for WT JAK2 o0.5

Selective for JAK2 V617F

1

1/2 1/2a 1/2

Drug

Gandotinib (LY2784544) AZD1480 BMS911543

1.1

Aurora-A, FLT4, FGFR1, TRKA JAK2

NS-018

o1

SRC, FLT3, ABL

3

3 (completed; program suspended)

Splenomegaly, MF symptoms, possible JAK2 V617F allele burden reduction Splenomegaly, MF symptoms without significant myelosuppression Splenomegaly, MF symptoms, potential bone marrow fibrosis NA Splenomegaly (JAK inhibitor naive and failure patients) NA

Reference(s)

85–87

88,89

Elevated creatinine, tumor lysis

90

NA

NA

Myelosuppression, nausea, amylase and lipase increase

91

NA

NA

Abbreviations: CDK2, cyclin-dependent kinase 2; DLT, dose-limiting toxicity; FDA, Food and Drug Administration; FGFR1, fibroblast growth factor receptor 1; IC50, half-inhibitory maximal concentration; JAK, Janus kinase; MF, myelofibrosis; NA, not available; TYK2, tyrosine kinase 2; WT, wild type.

Leukemia (2014) 1 – 9

& 2014 Macmillan Publishers Limited

Combination therapy partners for JAK inhibitors BL Stein et al

3

Predisposition allele

Figure 1.

HSC

JAK/STAT dysregulation JAK2 V617F, MPL, CBL, LNK mutations CALR mutation mTOR/PI3K/AKT, RAS/RAF/MEK,PIM activation HSP90 Cytokine overexpression and neoangiogenesis Host modifiers Aurora Kinase A

MF/MF acceleration

The pathophysiology of MF is heterogenous and molecularly complex.

Immunomodulatory agents Lenalidomide and pomalidomide. Immunomodulatory drugs (IMiDs) possess antiangiogenic and anti-inflammatory activities, supporting investigation for MF treatment. The earliest IMiD studied in MF was thalidomide that provided modest improvement in anemia and thrombocytopenia rather than a reduction of splenomegaly. The adverse events (AEs) of thalidomide therapy include constipation, sedation and neuropathy, limiting its longterm use.18 Lenalidomide, a second-generation IMiD, has been evaluated in phase 2 studies. In one trial involving 48 patients with MF, 10 mg daily with prednisone led to only modest improvement, as 19% and 10% of patients met criteria for improvement in anemia and splenomegaly, respectively.19 No reduction in marrow fibrosis or angiogenesis was reported. As with myelodysplastic syndromes, lenalidomide may be effective in patients with MF with deletions of 5q.19,20 In a pooled analysis, lenalidomide (particularly with prednisone) was more effective than thalidomide: response rates were 34–38% vs 16%.21 Myelosuppression was the most commonly observed AE of lenalidomide treatment. This AE could limit the safety and efficacy when combined with ruxolitinib, which is also myelosuppressive, and will be an important consideration as a phase 2 trial of lenalidomide and ruxolitinib accrues (NCT01375140). Pomalidomide, a more potent IMiD, was evaluated in a multicenter, double-blind phase 3 study versus placebo (NCT01178281).22 However, the study failed to meet the primary end point of anemia response, with an equal proportion of patients with MF in the pomalidomide (n ¼ 152) and placebo (n ¼ 77) arms achieving a response (16% vs 16%, P ¼ 1). This result tempers enthusiasm for the phase 1b/2 study of ruxolitinib and pomalidomide (NCT01644110); however, earlier studies have shown responses in a subgroup of MF patients with JAK2 mutations and modest rather than marked splenomegaly. Therefore, perhaps amelioration of splenomegaly with JAK inhibition may enhance the anemia response to pomalidomide. Treatment of low-risk MF: interferon-a Interferon suppresses hematopoietic progenitors, bone marrow fibroblast progenitors and platelet-derived growth factor. The advent of a pegylated version of interferon has renewed interest in this class, perhaps for use early on in the course of ET or PV, with a goal of preventing or delaying fibrosis.23 Long-term followup of 62 French and Belgian patients with MF treated with interferon-a was recently reported; 46% experienced an improvement in splenomegaly, 82% experienced an improvement in MF symptoms, and 73%, 64% and 78% of patients experienced an improvement in anemia, leukocytosis and thrombocytosis, respectively.24 Complementing this study was a prospective trial of 32 lower-risk patients with early MF treated with recombinant or pegylated interferon.25 An overall response & 2014 Macmillan Publishers Limited

rate of 78% was observed, with 3 complete remissions (9.4%), 12 partial remissions (37.5%), 3 clinical improvements (9.4%) and 7 with (22%) stable disease. Follow-up bone marrow biopsy results were available for 22 patients. Twelve patients had a reduction in cellularity that occurred after a median treatment duration of 2 years. Three patients, all of whom also experienced reductions in splenomegaly, had significant improvements in megakaryocyte morphology, marrow architecture and reductions of reticulin and collagen fibrosis (grade 3 to 1). Accordingly, a study will compare pegylated interferon with watchful waiting in patients with early MF (NCT01758588). Interferon may reduce the JAK2 V617F allele burden and impact marrow histology, suggesting a novel concept and possible partner with a JAK inhibitor.26 However, the impact of interferon may be best demonstrated in early MF/patients with low-risk MF—the role of JAK inhibitors has not yet been defined in this patient population. Myelosuppression has been shown in prior interferon studies in patients with MF;23 also, it is possible that an initial negative impact on quality of life attributed to interferon side effects could offset improvement in the MF symptom burden seen with JAK inhibitors. Addressing epigenetic dysregulation Restoring epigenetic regulation represents another treatment strategy for MF patients. Somatic mutations of TET2, DNMT3A, EZH2, ASXL1 and IDH1/2, which participate in epigenetic regulation, have been identified in patients with MPN, particularly those with MF. Of these, ASXL1 and IDH1/IDH2 mutations may independently and adversely affect prognosis and leukemia-free survival.27 Recognition of the impact of these mutations on disease pathogenesis and evolution provides a basis for the investigation of epigenetic therapies such as histone deacetylase inhibitors and hypomethylating agents.28,29 Histone deacetylase inhibitors. Four different histone deacetylase inhibitors have been evaluated in MF patients. Results from a phase 1 study of panobinostat (LBH589) have been published; clinical responses were evaluated in 5 of 18 patients with MF receiving more than 6 cycles of therapy.30 Spleen size normalized in 3 of 5 patients, and disease stabilized in 2 others. Two patients experienced an improvement in Hgb levels; 1 patient had a near complete remission, and another experienced resolution of bone marrow fibrosis. MF symptom improvement was noted as well. The dose-limiting toxicity was thrombocytopenia, and 39% and 17% had grade 3/4 anemia and neutropenia, respectively. Fatigue and musculoskeletal AEs were noted in 33% of patients. In another phase 2 study that evaluated an increased dose (40 mg times per week (t.i.w.)), the correlative arm supported the anti-MPN effects of panobinostat, including decreases in JAK/STAT signaling, JAK2 V617F allele burden and inflammatory cytokine levels; however, only 1 patient (3%) had a clinical response.31 In addition, only Leukemia (2014) 1 – 9

Combination therapy partners for JAK inhibitors BL Stein et al

4 Table 2.

Selected logical partners for combination therapy with JAK inhibitors

Agent

Target/rationale

Potential relief of MF symptom burden

Unique results

Adverse effect

Clinical trials in combination with JAK inhibitor

Androgens

Stimulation of erythropoiesis through enhanced erythropoietin release or iron incorporation

Anemia response

Anemia responses in patients with normal cytogenetics

Ruxolitinib plus danazol (NCT01732445)

Erythropoietinstimulating agents

Erythropoietin levels inappropriately low in some patients with MF

Anemia response

Anemia responses in patients with reduced erythropoietin levels

Liver function test abnormalities, headache, virilization, prostate cancer monitoring Progressive splenomegaly, hypertension, thrombosis

IMiDs: pomalidomide and lenalidomide

Suppression of angiogenesis and inflammatory cytokines

Anemia response

Myelosuppression, peripheral neuropathy

Pomalidomide and ruxolitinib (NCT01644110) Lenalidomide and ruxolitinib (NCT01375140)

Interferon-a

Suppression of hematopoietic and fibroblast progenitors

Panobinostat

Epigenetic regulation

Hypomethylating agents: 5-azacytidine and decitabine HSP90 inhibition

Epigenetic regulation

Splenomegaly (variable), anemia response Splenomegaly, MF symptoms, anemia response Not yet clear

Pomalidomide: higher response rates in JAK2 V617F-positive patients without marked splenomegaly or significant increase in blasts Durable responses in phase 2 testing of lenalidomide with prednisone Improvement in marrow atypia and bone marrow fibrosis Improvement in marrow histology and JAK2 V617F allele burden Efficacy in accelerated MPN (post MPN AML or MDS) with 52% OR Preclinical studies suggest ability to overcome JAK inhibitor resistance Preclinical synergy with JAK inhibition

PI3K/mTOR inhibition Antifibrotic agents

HSP90 overexpression; modulates client proteins involved in JAK/STAT signaling Inhibition of PI3K/AKT/mTOR pathway activation in MPN Direct targeting of bone marrow fibrosis: Monoclonal antibody against TGF-b: fresolimumab (GC1008) Monoclonal antibody against LOX: simtuzumab (GS-6624)

Unknown

MF symptoms, splenomegaly (everolimus) Unclear

Less severe anemia when combined with ruxolitinib but no change in transfusion requirements

Myelosuppression, systemic and metabolic toxicity Thrombocytopenia

NCT01433445 and NCT01693601

Myelosuppression

5-Azacytidine followed by ruxolitinib (NCT01787487)

Myelosuppression, gastrointestinal

Ruxolitinib and buparlisib (BKM120; PI3K inhibitor): (NCT01730248) Simtuzumab, either alone or in combination with ruxolitinib (NCT01369498) PRM-151, an agent used in idiopathic pulmonary fibrosis will also be studied in patients with MF in phase 2, alone or with ruxolitinib

Abbreviations: AML, acute myeloid leukemia; HSP90, heat-shock protein 90; IMiD, immunomodulatory drug; JAK, Janus kinase; LOX, lysyl oxidase; MDS, myelodysplastic syndromes; MF, myelofibrosis; MPN, myeloproliferative neoplasms; mTOR, mammalian target of rapamycin; OR, overall response; PI3K, phosphatidylinositol 3-kinase; STAT, signal transducer and activator of transcription; TGF-b, transforming growth factor-b.

16 of 35 patients could complete more than two cycles of therapy; thrombocytopenia and diarrhea were common AEs. These studies suggest that a dosing regimen of o30 mg TIW may be ideal. Preclinical in vitro studies provide rationale for combining panobinostat with JAK inhibitors. In one study, panobinostat and fedratinib downregulated JAK2 signaling and increased cytotoxicity in MF cells to a greater extent than either agent alone.32 Furthermore, in preclinical mouse models of a JAK2driven MF-like disease, the combination of ruxolitinib and panobinostat resulted in synergistic efficacy with reduced clonal growth, decreased bone marrow hypercellularity and improvement in fibrosis.33 Therefore, combination therapy using Leukemia (2014) 1 – 9

ruxolitinib and panobinostat is currently being evaluated in patients with MF (NCT01433445 and NCT01693601). Results from the dose-escalation portion of one of these studies were presented in 2013.34 A total of 38 patients were evaluated in 6 dose cohorts ranging from ruxolitinib 5 mg twice daily (b.i.d.) with panobinostat 10 mg 3 times per week (t.i.w.) every other week (q.o.w.) to ruxolitinib 15 mg b.i.d. with panobinostat 25 mg t.i.w. q.o.w. The maximum tolerated dose was not reached; however, primarily findings of anemia and a potential drug–drug interaction at the highest doses resulted in selecting ruxolitinib 15 mg b.i.d. with panobinostat 25 mg t.i.w./q.o.w. as the dose for the expansion phase of the study. Overall, the combination was well & 2014 Macmillan Publishers Limited

Combination therapy partners for JAK inhibitors BL Stein et al

5 tolerated with low rates of grade 3/4 anemia and thrombocytopenia, and encouraging reductions in splenomegaly were observed. Results from combination studies involving histone deacetylase inhibitors and JAK inhibitors are intriguing, and mature results are greatly anticipated, given strength of preclinical data, preclinical synergy and single-agent efficacy demonstrated with both agents. Givinostat has been evaluated in a phase 2 multicenter pilot study of patients with MF (n ¼ 16), PV (n ¼ 12) and ET (n ¼ 1).35 Gastrointestinal side effects were common (62%), as was grade 1/2 anemia (21%) and thrombocytopenia (10%).35 Response rates were low in MF as only 3 patients (19%) had a clinical response in either anemia (n ¼ 1) or splenomegaly/symptoms (n ¼ 2). In another trial, vorinostat resulted in a 14% response rate in 14 patients with MF, but with frequent toxicity.36 Pracinostat has been evaluated in 22 patients with MF; 6 (27%) experienced a reduction in splenomegaly (median reduction, 3 cm) but did not meet the criteria for clinical improvement.37 Five with hepatomegaly experienced a median 3-cm decrease in liver size, and two had an anemia response based on International Working Group criteria. Fatigue was common, occurring in 91% of patients. Grade 3/4 neutropenia and thrombocytopenia were seen in 13% and 21% of patients, respectively. Twenty-one patients discontinued treatment, primarily because of lack of response or disease progression. Hypomethylating agents. In an earlier phase 2 study of 34 patients with MF, 5-azacytidine (7-day schedule) resulted in global hypomethylation, yet clinical responses were seen in only 8 patients (24%; 1 with partial remission and 7 with clinical improvement).38 The median duration of response was only 4 months, and myelosuppression was relatively common (grade 3/4 neutropenia, 29%). In a smaller study of 10 patients with MF, no patient improved when 5-azacytidine was administered on a 5-day schedule, and discontinuations were frequent.39 The most promising results using 5-azacytidine have been reported in those whose MPN accelerated to myelodysplastic syndromes or acute myeloid leukemia; in a French study, an overall response rate of 52% was reported, with patients often reverting back to their chronic-phase MPN.40 Finally, decitabine was evaluated in 21 patients with MF; 7 of 19 evaluable patients responded (1 complete remission, 2 partial remissions and 4 with hematologic improvement), although spleen size reduction was not reported. Grade 3/4 neutropenia and thrombocytopenia occurred in 95% and 52% of patients, respectively.41 A phase 3 combination (NCT01787487) of ruxolitinib and 5-azacytidine is underway, and from the perspective of safety, overlapping toxicity includes myelosuppression and gastrointestinal toxicity. Based on single-agent studies, the patient with accelerated MF may benefit most from this combination, as hypomethylation may delay blast progression and improve cytopenias, whereas JAK inhibition may address cytokine-associated symptoms and splenomegaly. It is possible that sequential use (ruxolitinib will be added after 3 cycles of 5-azacytidine) may reduce potential for severe cytopenias that can be seen with both agents. Overcoming JAK inhibitor resistance Heat shock protein 90 inhibitors. Heat shock proteins (HSPs) are a family of molecular chaperones that assist in the folding and stability of their client proteins. More specifically, HSP90 modulates the activity of nearly 200 proteins, and several of these participate in oncogenic signaling pathways, including JAK/ STAT.42 As previously mentioned, even in the presence of JAK inhibition, JAK2 remains persistently activated through heterodimer formation with other JAKs, including JAK1 and TYK2,43 that offers a mechanism for JAK inhibitor resistance. This finding has been observed in cell lines, murine models and patient & 2014 Macmillan Publishers Limited

samples but is reversible with drug interruption. Of note, the JAK2 inhibitor-resistant cells were degradable by an HSP90 inhibitor, PU-H71. Recent preclinical data have also demonstrated improved efficacy when PU-H71 was combined with a JAK inhibitor.15 HSP90 inhibition with PU-H71 has also suppressed cell growth and signaling in JAK2 mutant cell lines, mouse models (ET and PV) and primary samples.44 The HSP90 inhibitor tanespimycin (17-AAG) has also been found to interrupt signaling in a homozygous JAK2 V617F cell line.45 Others have identified mutations (G935R, Y931C and E864K) that conferred resistance to a panel of JAK inhibitors; however, HSP90 inhibition (particularly with AUY922) led to degradation of both wild-type and mutant JAK2 in dependent cells.46 The HSP90 inhibitor ganetespib resulted in sustained depletion of JAK2, including JAK2 V617F, with loss of STAT activity in vitro and in vivo.47 With murine and human cell lines and primary MPN CD34 þ samples, AUY922 resulted in depletion of JAK2 V617F and downstream signaling proteins.48 In combination with a JAK inhibitor, there was synergistically enhanced apoptosis of the MPN CD34 þ cells; furthermore, JAK inhibitor-resistant cell lines retained sensitivity to the HSP90 inhibitors AUY922 and tanespimycin. Given the strength of preclinical data, ability to overcome two resistance mechanisms, more effective targeting of JAK2 and demonstrated synergy, HSP90 inhibitors represent quite logical partners to JAK inhibitor therapy and favorable combinations. Currently, a phase 2 study of AUY922 in patients with primary MF, post-PV and post-ET MF is underway and recruiting participants (NCT01668173). Targeting additional signaling abnormalities Hedgehog inhibition. The hedgehog (Hh) pathway is involved in the determination of stem cell fate and is critical for embryonic and organ development, including the development of the hematopoietic system.49 Abnormal Hh signaling is associated with a range of malignancies, including those of hematopoietic origin, such as chronic myelogenous leukemia and multiple myeloma.50 The role of Hh in MPN is less well defined, but abnormal activation of this pathway has been demonstrated and preclinical studies have suggested that JAK2 activity affects Hh signaling.50,51 Several Hh inhibitors have been developed, including PF04449913, saridegib (IPI-926) and erismodegib (LDE225). In a phase 1a dose-escalation study that included patients with hematologic malignancies, PF-04449913 showed efficacy in patients with MF. Five patients attained stable disease, and one patient achieved clinical improvement in splenomegaly.52 Therefore, a phase 2 trial has been initiated to investigate the use of PF-04449913 in patients with various hematologic malignancies, including MF, and is currently recruiting (NCT00953758). In addition, a phase 2 clinical trial of saridegib in MF has been completed, although the results have not been published (NCT01371617). Given the common downstream effects on gene regulation of JAK/STAT and Hh signaling, it is possible that inhibition of both pathways may exhibit synergistic therapeutic benefits for patients with MF. The recently reported preclinical results confirm this hypothesis in a mouse MPN model. Although erismodegib, an inhibitor of the Smoothened receptor that suppresses Hh pathway signaling, did not show efficacy alone, combination of erismodegib with ruxolitinib had increased efficacy over ruxolitinib monotherapy and marked reduced bone marrow fibrosis.53 Accordingly, a phase 1b/2 dose-finding study of the combination of ruxolitinib and erismodegib has been planned. Mammalian target of rapamycin and AKT inhibition. Pathway activation downstream of JAK/STAT, including the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin Leukemia (2014) 1 – 9

Combination therapy partners for JAK inhibitors BL Stein et al

6 (mTOR) pathway, has been demonstrated in MPN.54 The in vitro exposure to the mTOR inhibitor everolimus decreased the proliferation of cells with the JAK2 V617F mutation.54,55 This agent was also studied in 39 (30 evaluable) patients with MF in a multicenter phase 1/2 program.54 Six patients clinically improved (splenomegaly decreased in 5 patients, Hgb levels increased in 1 patient) and 1 patient achieved a partial remission; the remaining 23 patients had stable disease. Myelosuppression was noted, as 27% experienced grade 2/3 anemia and 7% and 3% experienced grade 2 neutropenia and thrombocytopenia, respectively. Mild stomatitis occurred in 70% of patients. Significant changes in the JAK2 V617F allele burden or levels of proinflammatory cytokines were not seen. Synergistic inhibition of the growth of MPN cells with the combination of PI3K/mTOR inhibitors and JAK1/2 inhibitors has been reported,56,57 and a phase 1b clinical trial is underway to evaluate the safety of ruxolitinib with buparlisib (BKM120, a PI3K inhibitor; NCT01730248). Finally, serine/threonine-specific protein kinase AKT also represents a therapeutic target in MPN.58 MK-2206, a nonmyelosuppressive AKT inhibitor, was shown to reduce hepatosplenomegaly and megakaryocyte burden in an MPLdriven murine model, reduce megakaryocyte colony formation in patient samples and synergistically inhibit growth of JAK2 V617FSET2 cells with ruxolitinib. The preclinical data showing synergy, along with the nonmyelosuppressive of nature of MK-2206 are intriguing (hopefully avoiding overlapping toxicity), but the clinical impact of this agent is unknown as it has not yet been tested in patients with MF. RAS/RAF/MEK inhibition. The RAS/RAF/MEK signaling pathway is involved in a vast array of cellular functions, including cell proliferation and migration, nuclear transport, mRNA processing and protein translation.59 MEK inhibition with trametinib (GSK1120212; NCT00920140) and pimasertib (AS703026; NCT00957580) is currently being investigated in patients with hematologic malignancies. Given the interplay between the JAK/ STAT signaling pathway and the RAS/RAF/MEK pathway,60,61 and the fact that the latter has been shown to be activated by the JAK2 V617F mutation,62 a further preclinical evaluation of the effect of a combination of a JAK inhibitor and an MEK inhibitor in MF is warranted before moving to clinical trials. Proviral integrations of Moloney virus kinase inhibition. Proviral integrations of Moloney virus (PIM) serine/threonine kinases regulate JAK/STAT and mTOR signaling and represent key players in both hematologic and solid cancers.63 PIM kinases are primarily associated with reducing myc expression and inhibiting cyclin-dependent kinase 2, but they also exhibit a regulatory role in JAK2/STAT5 signaling and were shown to mediate resistance to mTOR inhibition.64,65 Importantly, two of the family members, PIM1 and PIM2, have been found to be upregulated in MPN and are therefore possible targets, particularly in combination with PI3K/AKT/mTOR inhibitors.66 Synergistic activity has been observed for the combination of fedratinib and SGI-1776 in primary human MPN cells with the JAK2 V617F mutation.67 Combination of the PIM inhibitor LGH447 with ruxolitinib has also demonstrated synergistic activity in preclinical models.68 Targeting fibrosis. Transforming growth factor-b may have a role in promoting bone marrow fibrosis, and the enzyme lysyl oxidase is involved in megakaryocyte proliferation and generation of fibrosis, thus representing potential targets for therapy.69 Fresolimumab (GC1008; NCT01291784), a monoclonal antibody directed against transforming growth factor-b, is currently undergoing evaluation in a phase 1 study. In mouse models, a monoclonal antibody directed against lysyl oxidase, simtuzumab (GS-6624), demonstrated a decrease in organ fibrosis and is Leukemia (2014) 1 – 9

currently being evaluated in MF, either alone or in combination with ruxolitinib (NCT01369498). In addition, PRM-151, an agent used in idiopathic pulmonary fibrosis, has been studied in phase 2 in patients with MF, either alone or with ruxolitinib.70 Results regarding safety and efficacy will be needed before critically evaluating this strategy. Targeting the abnormal megakaryocyte. MF megakaryocytes displayed impaired polyploidization and maturation and may elaborate profibrotic cytokines, and therefore represent an important therapeutic target. A prior study identified small molecules, including dimethylfasudil and MLN8237 (alesertib), that resulted in polyploidization, arrest of proliferation and apoptosis of malignant megakaryocytes in acute megakaryocytic leukemia.71 The target of these agents, aurora kinase A, also plays a role in MF pathogenesis. Recent data show that inhibiting aurora kinase A with these agents resulted in polyploidization, arrested proliferation and apoptosis of JAK2 V617F megakaryocytic SET2 cell line, as well as megakaryocytes derived from MF patient samples.72 Moreover, synergy was shown with ruxolitinib, and polyploidy inducers resulted in growth arrest of JAK inhibitor persistent cells. Polyploid induction also resulted in a decrease in liver and spleen weight, leukocytosis, thrombocytosis, bone marrow fibrosis and transforming growth factor-b levels in MF murine models. Synergy with ruxolitinib was also noted using this system. This combination is of particular interest given the strength of the preclinical data and targeting of a perhaps fundamental aspect of MF disease pathogenesis, the abnormal megakaryocyte. These data provide rationale for exploring aurora kinase inhibitors with JAK inhibitors in clinical trials; in another setting, myelosuppression has been observed with alisertib, suggesting a potential overlapping toxicity.73 CONCLUSION Conventional therapies have insufficiently addressed the needs of patients with MPN, particularly those with MF. Scientific advances, including the discovery of JAK/STAT dysregulation, have improved our understanding of disease pathogenesis and led to the development of the first class of medications specifically approved for MF. JAK inhibitors have shown great promise in relieving disease-related symptoms and splenomegaly. Unique features have been reported with many JAK inhibitors, including the ability to modify cytokine levels, stabilize or improve bone marrow fibrosis, ameliorate anemia and prolong survival. Given the complexity of disease pathogenesis, it appears likely that a single oral agent will not sufficiently address the burden experienced by patients with MF. Therefore, in this next era of MF treatment, the challenge lies in the identification of rational combinations involving JAK inhibitors, without overlapping toxicity compromising patient safety. Identification of JAK inhibitor resistance and broad contributions to MF disease pathogenesis (described in this review) suggest that numerous options may be partnered with a JAK inhibitor. As each option is evaluated, patient needs/goals (for example, offsetting treatmentemergent anemia), disease status (early versus accelerated MF) and demonstrable synergy are important considerations (Figure 2). A practical approach includes partnering JAK inhibitors with agents that have the potential to relieve anemia that can be exacerbated by JAK inhibitors—options such as androgens and IMiDs have been described. Erythropoietin-stimulating agents may be an additional consideration as a post hoc analysis evaluated the combination of ruxolitinib with erythropoietin-stimulating agents in 13 patients and found it to be well tolerated, without impairment of splenic response, although there was no change in transfusion rates.74 In addition, in this regard, a novel erythropoietic agent is being evaluated in MF (sotatercept & 2014 Macmillan Publishers Limited

Combination therapy partners for JAK inhibitors BL Stein et al

7 JAKi prior to HSCT

IFN + JAKi

Early/Low Risk MF

High-Risk and/or Accelerating MF HMT followed by JAKi

Ameliorating the burden of anemia

JAKi with: Danazol IMIDS Epo Stim agents Sotatercept

Selected Novel MF treatment strategies

Selected Unique Targets

Synergistic combinations

Telomerase CALR Abnormal spliceosome machinery

JAK-inhibitors +: HSP90i HDACi mTOR/PI3K/AKTi Aurora Kinase A i

Figure 2.

Selected novel MF treatment strategies.

(ACE-011); NCT01712308). A strategy for a patient with low-risk MF may involve the use of interferon, in combination with a JAK inhibitor, yet the role of each as a single agent has not been well defined in this population. Patients with accelerating MF may benefit from strategies that combine JAK inhibition with hypomethylating therapies. In those preparing for stem cell transplantation, JAK inhibition before this procedure represents a novel strategy, with a goal of improving functional status and splenomegaly (NCT01790295). Two studies suggested an improvement in symptoms and splenomegaly before transplant, without rebound effects upon discontinuation, or adverse early outcomes. Mature data are needed, but the strategy appears promising.75,76 In this review, several combinations are intriguing, based on an ability to address heterogeneous aspects of pathogenesis, strong preclinical data and synergy; maturing results from clinical trials of JAK inhibitors with HSP90 inhibitors, histone deacetylase inhibitors, mTOR/PI3K/AKT and aurora kinase A inhibitors are eagerly awaited. The treatment landscape may be further augmented by addressing unique mechanisms of disease pathogenesis, including overexpression of telomerase. Telomerase inhibition with imetelstat appears promising (NCT01731951), as 4 complete remissions and 1 partial remission were recently reported among 18 patients; myelosuppression was the most common AE.77 Mutations involving the spliceosome machinery, such as SRSF2, are prognostically adverse, and may represent an additional target for therapy.78 The discovery of CALR mutations in patients with MF also presents a new potential therapeutic target for patients with JAK2 V617F-negative MF. With the current pace of discovery and translation, and the increasing availability of novel agents and rational therapeutic combinations, it is hoped that MF patients will soon achieve even better control of their disease and more frequent remissions.

CONFLICT OF INTEREST BLS was previously a member of Speakers Bureau, Incyte Corporation and has participated in Advisory Boards for Incyte Corporation and Sanofi Oncology. RS has declared no conflict of interest. AH has received honoraria and research funding from

& 2014 Macmillan Publishers Limited

Novartis, Bristol-Myers Squibb and Pfizer; and FG has received honoraria and research funding from Novartis.

ACKNOWLEDGEMENTS Financial support for medical assistance was provided by Novartis Pharmaceuticals. We thank Daniel Hutta and Matthew Hoelzle for their medical editorial assistance with this manuscript.

AUTHORSHIP All authors drafted and approved this manuscript.

REFERENCES 1 Mesa RA, Green A, Barosi G, Verstovsek S, Vardiman J, Gale RP. MPN-associated myelofibrosis (MPN-MF). Leuk Res 2011; 35: 12–13. 2 Mesa RA, Kiladjian JJ, Verstovsek S, Al-Ali HK, Gotlib JR, Gisslinger H et al. Comparison of placebo and best available therapy for the treatment of myelofibrosis in the phase 3 COMFORT studies. Haematologica 2013; 99: 292–298. 3 Tefferi A. How I treat myelofibrosis. Blood 2011; 117: 3494–3504. 4 Alchalby H, Badbaran A, Zabelina T, Kobbe G, Hahn J, Wolff D et al. Impact of JAK2V617F mutation status, allele burden, and clearance after allogeneic stem cell transplantation for myelofibrosis. Blood 2010; 116: 3572–3581. 5 Gupta V, Hari P, Hoffman R. Allogeneic hematopoietic cell transplantation for myelofibrosis in the era of JAK inhibitors. Blood 2012; 120: 1367–1379. 6 James C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, Lacout C et al. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature 2005; 434: 1144–1148. 7 Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR et al. A gain-offunction mutation of JAK2 in myeloproliferative disorders. N Engl J Med 2005; 352: 1779–1790. 8 Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, Swanton S et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 2005; 365: 1054–1061. 9 Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ et al. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell 2005; 7: 387–397. 10 Klampfl T, Gisslinger H, Harutyunyan AS, Nivarthi H, Rumi E, Milosevic JD et al. Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J Med 2013; 369: 2379–2390.

Leukemia (2014) 1 – 9

Combination therapy partners for JAK inhibitors BL Stein et al

8 11 Nangalia J, Massie CE, Baxter EJ, Nice FL, Gundem G, Wedge DC et al. Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2. N Engl J Med 2013; 369: 2391–2405. 12 Tefferi A, Lasho TL, Finke CM, Knudson RA, Ketterling R, Hanson CH et al. CALR vs JAK2 vs MPL-mutated or triple-negative myelofibrosis: clinical, cytogenetic and molecular comparisons. Leukemia 2014; 28: 1472–1477. 13 Passamonti F, Caramazza D, Maffioli M. JAK inhibitor in CALR-mutant myelofibrosis. N Engl J Med 2014; 370: 1168–1169. 14 Garber K. JAK2 inhibitors: not the next imatinib but researchers see other possibilities. J Natl Cancer Inst 2009; 101: 980–982. 15 Bhagwat N, Koppikar P, Keller M, Marubayashi S, Shank K, Rampal R et al. Improved targeting of JAK2 leads to increased therapeutic efficacy in myeloproliferative neoplasms. Blood 2014; 123: 2075–2083. 16 Cervantes F, Alvarez-Larran A, Domingo A, Arellano-Rodrigo E, Montserrat E. Efficacy and tolerability of danazol as a treatment for the anaemia of myelofibrosis with myeloid metaplasia: long-term results in 30 patients. Br J Haematol 2005; 129: 771–775. 17 Shimoda K, Shide K, Kamezaki K, Okamura T, Harada N, Kinukawa N et al. The effect of anabolic steroids on anemia in myelofibrosis with myeloid metaplasia: retrospective analysis of 39 patients in Japan. Int J Hematol 2007; 85: 338–343. 18 Thapaliya P, Tefferi A, Pardanani A, Steensma DP, Camoriano J, Wu W et al. International working group for myelofibrosis research and treatment response assessment and long-term follow-up of 50 myelofibrosis patients treated with thalidomide-prednisone based regimens. Am J Hematol 2011; 86: 96–98. 19 Mesa RA, Yao X, Cripe LD, Li CY, Litzow M, Paietta E et al. Lenalidomide and prednisone for myelofibrosis: Eastern Cooperative Oncology Group (ECOG) phase2 trial E4903. Blood 2010; 116: 4436–4438. 20 Tefferi A, Lasho TL, Mesa RA, Pardanani A, Ketterling RP, Hanson CA. Lenalidomide therapy in del(5)(q31)-associated myelofibrosis: cytogenetic and JAK2V617F molecular remissions. Leukemia 2007; 21: 1827–1828. 21 Jabbour E, Thomas D, Kantarjian H, Zhou L, Pierce S, Cortes J et al. Comparison of thalidomide and lenalidomide as therapy for myelofibrosis. Blood 2011; 118: 899–902. 22 Tefferi A, Passamonti F, Barbui T, Barosi G, Begna K, Cazzola M et al. Phase 3 study of pomalidomide in myeloproliferative neoplasm (MPN)-associated myelofibrosis with RBC-transfusion-dependence. Blood 2013; 122: abstract 394. 23 Kiladjian JJ, Chomienne C, Fenaux P. Interferon-alpha therapy in bcr-abl-negative myeloproliferative neoplasms. Leukemia 2008; 22: 1990–1998. 24 Ianotto JC, Boyer-Perrard F, Gyan E, Laribi K, Cony-Makhoul P, Demory JL et al. Efficacy and safety of pegylated-interferon alpha-2a in myelofibrosis: a study by the FIM and GEM French cooperative groups. Br J Haematol 2013; 162: 783–791. 25 Silver RT, Lascu E, Feldman EJ, Ritchie E, Roboz GJ, De Sancho MT et al. Recombinant interferon alpha (rIFN) may retard progression of early myelofibrosis by reducing splenomegaly and by decreasing marrow fibrosis. Blood 2013; 122: abstract 4053. 26 Hasselbalch HC. Perspectives on the impact of JAK-inhibitor therapy upon inflammation-mediated comorbidities in myelofibrosis and related neoplasms. Expert Rev Hematol 2014; 7: 203–216. 27 Vannucchi AM, Lasho TL, Guglielmelli P, Biamonte F, Pardanani A, Pereira A et al. Mutations and prognosis in primary myelofibrosis. Leukemia 2013; 27: 1861–1869. 28 Vannucchi AM, Biamonte F. Epigenetics and mutations in chronic myeloproliferative neoplasms. Haematologica 2011; 96: 1398–1402. 29 Abdel-Wahab O, Pardanani A, Bernard OA, Finazzi G, Crispino JD, Gisslinger H et al. Unraveling the genetic underpinnings of myeloproliferative neoplasms and understanding their effect on disease course and response to therapy: proceedings from the 6th International Post-ASH Symposium. Am J Hematol 2012; 87: 562–568. 30 Mascarenhas J, Lu M, Li T, Petersen B, Hochman T, Najfeld V et al. A phase I study of panobinostat (LBH589) in patients with primary myelofibrosis (PMF) and postpolycythaemia vera/essential thrombocythaemia myelofibrosis (post-PV/ET MF). Br J Haematol 2013; 161: 68–75. 31 DeAngelo DJ, Mesa RA, Fiskus W, Tefferi A, Paley C, Wadleigh M et al. Phase II trial of panobinostat, an oral pan-deacetylase inhibitor in patients with primary myelofibrosis, post-essential thrombocythaemia, and post-polycythaemia vera myelofibrosis. Br J Haematol 2013; 162: 326–335. 32 Wang Y, Fiskus W, Chong DG, Buckley KM, Natarajan K, Rao R et al. Cotreatment with panobinostat and JAK2 inhibitor TG101209 attenuates JAK2V617F levels and signaling and exerts synergistic cytotoxic effects against human myeloproliferative neoplastic cells. Blood 2009; 114: 5024–5033. 33 Baffert F, Evrot E, Ebel N, Rowlli C, Andraos R, Qian Z et al. Improved efficacy upon combined JAK1/2 and pan-deacetylase inhibition using ruxolitinib (INC424) and panobinostat (LBH589) in preclinical mouse models of JAK2 V617F-driven disease. Blood 2011; 118: abstract 798. 34 Ribrag V, Harrison CN, Heidel FH, Kiladjian J, Acharyya S, Mu S et al. A phase Ib, dose-finding study of ruxolitinib plus panobinostat in patients with primary

Leukemia (2014) 1 – 9

35

36

37

38

39

40

41

42 43

44

45

46

47

48

49 50 51 52

53

54

55

56

57

myelofibrosis (PMF), post–polycythemia vera mf (PPV-MF), or post–essential thrombocythemia mf (PET-MF): identification of the recommended phase II dose. Blood 2013; 122: abstract 4045. Rambaldi A, Dellacasa CM, Finazzi G, Carobbio A, Ferrari ML, Guglielmelli P et al. A pilot study of the Histone-Deacetylase inhibitor Givinostat in patients with JAK2V617F positive chronic myeloproliferative neoplasms. Br J Haematol 2010; 150: 446–455. Andersen C, Mortensen N, Vestergaard H, Bjerrum O, Klausen THasselbalch H. A phase II study of vorinostat (MK-0683) in patients with primary myelofibrosis (PMF) and post-polycythemia vera myelofibrosis (PPV-MF). Haematologica 2013; 98: abstract P279. Quintas-Cardama A, Kantarjian H, Estrov Z, Borthakur G, Cortes J, Verstovsek S. Therapy with the histone deacetylase inhibitor pracinostat for patients with myelofibrosis. Leuk Res 2012; 36: 1124–1127. Quintas-Cardama A, Tong W, Kantarjian H, Thomas D, Ravandi F, Kornblau S et al. A phase II study of 5-azacitidine for patients with primary and post-essential thrombocythemia/polycythemia vera myelofibrosis. Leukemia 2008; 22: 965–970. Mesa RA, Verstovsek S, Rivera C, Pardanani A, Hussein K, Lasho T et al. 5-Azacitidine has limited therapeutic activity in myelofibrosis. Leukemia 2009; 23: 180–182. Thepot S, Itzykson R, Seegers V, Raffoux E, Quesnel B, Chait Y et al. Treatment of progression of Philadelphia-negative myeloproliferative neoplasms to myelodysplastic syndrome or acute myeloid leukemia by azacitidine: a report on 54 cases on the behalf of the Groupe Francophone des Myelodysplasies (GFM). Blood 2010; 116: 3735–3742. Odenike O, Godwin J, van Besien K, Huo D, Sher D, Burke P et al. Phase II trial of low dose, subcutaneous decitabine in myelofibrosis. Blood 2008; 112: abstract 2809. Jhaveri K, Modi S. HSP90 inhibitors for cancer therapy and overcoming drug resistance. Adv Pharmacol 2012; 65: 471–517. Koppikar P, Bhagwat N, Kilpivaara O, Manshouri T, Adli M, Hricik T et al. Heterodimeric JAK-STAT activation as a mechanism of persistence to JAK2 inhibitor therapy. Nature 2012; 489: 155–159. Marubayashi S, Koppikar P, Taldone T, Abdel-Wahab O, West N, Bhagwat N et al. HSP90 is a therapeutic target in JAK2-dependent myeloproliferative neoplasms in mice and humans. J Clin Invest 2010; 120: 3578–3593. Bareng J, Jilani I, Gorre M, Kantarjian H, Giles F, Hannah A et al. A potential role for HSP90 inhibitors in the treatment of JAK2 mutant-positive diseases as demonstrated using quantitative flow cytometry. Leuk Lymphoma 2007; 48: 2189–2195. Weigert O, Lane AA, Bird L, Kopp N, Chapuy B, van Bodegom D et al. Genetic resistance to JAK2 enzymatic inhibitors is overcome by HSP90 inhibition. J Exp Med 2012; 209: 259–273. Proia DA, Foley KP, Korbut T, Sang J, Smith D, Bates RC et al. Multifaceted intervention by the Hsp90 inhibitor ganetespib (STA-9090) in cancer cells with activated JAK/STAT signaling. PLoS One 2011; 6: e18552. Fiskus W, Verstovsek S, Manshouri T, Rao R, Balusu R, Venkannagari S et al. Heat shock protein 90 inhibitor is synergistic with JAK2 inhibitor and overcomes resistance to JAK2-TKI in human myeloproliferative neoplasm cells. Clin Cancer Res 2011; 17: 7347–7358. Merchant AA, Matsui W. Targeting Hedgehog — a cancer stem cell pathway. Clin Cancer Res 2010; 16: 3130–3140. Irvine DA, Copland M. Targeting hedgehog in hematologic malignancy. Blood 2012; 119: 2196–2204. Fiskus W, Ganguly S, Kambhampati S, Bhalla KN. Role of additional novel therapies in myeloproliferative neoplasms. Hematol Oncol Clin North Am 2012; 26: 959–980. Jamieson C, Cortes JE, Oehler V, Baccarani M, Kantarjian HM, Papayannidis C et al. Phase 1 dose-escalation study of PF-04449913, an oral hedgehog (Hh) inhibitor, in patients with select hematologic malignancies. Blood 2011; 118: abstract 424. Bhagwat N, Keller MD, Rampal R, Koppikar P, Shank K, De Stanchina E et al. Improved efficacy of combination of JAK2 and hedgehog inhibitors in myelofibrosis. Blood 2013; 122: abstract 666. Guglielmelli P, Barosi G, Rambaldi A, Marchioli R, Masciulli A, Tozzi L et al. Safety and efficacy of everolimus, a mTOR inhibitor, as single agent in a phase 1/2 study in patients with myelofibrosis. Blood 2011; 118: 2069–2076. Vannucchi AM, Bogani C, Bartalucci N, Guglielmelli P, Tozzi L, Antonioli E et al. The mTOR inhibitor, RAD001, inhibits the growth of cells from patients with myeloproliferative neoplasms. Blood 2009; 114: abstract 2914. Vannucchi A, Bogani C, Bartalucci N, Tozzi L, Martinelli S, Guglielmelli P et al. Inhibitors of PI3K/Akt and/or mTOR inhibit the growth of cells of myeloproliferative neoplasms and synergize with JAK2 inhibitor and interferon. Blood 2011; 118: abstract 3835. Fiskus W, Verstovsek S, Manshouri T, Smith JE, Peth K, Abhyankar S et al. Dual PI3K/AKT/mTOR inhibitor BEZ235 synergistically enhances the activity of JAK2 inhibitor against cultured and primary human myeloproliferative neoplasm cells. Mol Cancer Ther 2013; 12: 577–588.

& 2014 Macmillan Publishers Limited

Combination therapy partners for JAK inhibitors BL Stein et al

9 58 Khan I, Huang Z, Wen Q, Stankiewicz MJ, Gilles L, Goldenson B et al. AKT is a therapeutic target in myeloproliferative neoplasms. Leukemia 2013; 27: 1882–1890. 59 Santarpia L, Lippman SM, El-Naggar AK. Targeting the MAPK-RAS-RAF signaling pathway in cancer therapy. Expert Opin Ther Targets 2012; 16: 103–119. 60 Steelman LS, Abrams SL, Whelan J, Bertrand FE, Ludwig DE, Basecke J et al. Contributions of the Raf/MEK/ERK, PI3K/PTEN/Akt/mTOR and Jak/STAT pathways to leukemia. Leukemia 2008; 22: 686–707. 61 McCubrey JA, Steelman LS, Abrams SL, Bertrand FE, Ludwig DE, Basecke J et al. Targeting survival cascades induced by activation of Ras/Raf/MEK/ERK, PI3K/ PTEN/Akt/mTOR and Jak/STAT pathways for effective leukemia therapy. Leukemia 2008; 22: 708–722. 62 Oku S, Takenaka K, Kuriyama T, Shide K, Kumano T, Kikushige Y et al. JAK2 V617F uses distinct signalling pathways to induce cell proliferation and neutrophil activation. Br J Haematol 2010; 150: 334–344. 63 Brault L, Gasser C, Bracher F, Huber K, Knapp S, Schwaller J. PIM serine/threonine kinases in the pathogenesis and therapy of hematologic malignancies and solid cancers. Haematologica 2010; 95: 1004–1015. 64 Lin YW, Beharry ZM, Hill EG, Song JH, Wang W, Xia Z et al. A small molecule inhibitor of Pim protein kinases blocks the growth of precursor T-cell lymphoblastic leukemia/lymphoma. Blood 2010; 115: 824–833. 65 Hammerman PS, Fox CJ, Birnbaum MJ, Thompson CB. Pim and Akt oncogenes are independent regulators of hematopoietic cell growth and survival. Blood 2005; 105: 4477–4483. 66 Wernig G, Gonneville JR, Crowley BJ, Rodrigues MS, Reddy MM, Hudon HE et al. The Jak2V617F oncogene associated with myeloproliferative diseases requires a functional FERM domain for transformation and for expression of the Myc and Pim proto-oncogenes. Blood 2008; 111: 3751–3759. 67 Fiskus W, Manepalli RR, Balusu R, Bhalla KN. Synergistic activity of combinations of JAK2 kinase inhibitor with PI3K/mTOR, MEK or PIM kinase inhibitor against human myeloproliferative neoplasm cells expressing JAK2V617F. Blood 2010; 116: abstract 798. 68 Saci A, Pinzon-Ortiz M, Wang D, Rong X, Growney J, Squier P et al. The combination of JAK inhibitor, ruxolitinib, and PIM inhibitor, LGH447, in preclinical models of myeloproliferative neoplasia. Blood 2013; 122: abstract 4100. 69 Papadantonakis N, Matsuura S, Ravid K. Megakaryocyte pathology and bone marrow fibrosis: the lysyl oxidase connection. Blood 2012; 120: 1774–1781. 70 Dillingh MR, van den Blink B, Moerland M, van Dongen MG, Levi M, Kleinjan A et al. Recombinant human serum amyloid P in healthy volunteers and patients with pulmonary fibrosis. Pulm Pharmacol Ther 2013; 26: 672–676. 71 Wen Q, Goldenson B, Silver SJ, Schenone M, Dancik V, Huang Z et al. Identification of regulators of polyploidization presents therapeutic targets for treatment of AMKL. Cell 2012; 150: 575–589. 72 Goldenson B, Malinge S, Stein BL, Lasho TL, Breyfogle L, Schultz R et al. Aurora A kinase is a novel therapeutic target in the myeloproliferative neoplasms. Blood 2013; 122: 109–109. 73 Friedberg JW, Mahadevan D, Cebula E, Persky D, Lossos I, Agarwal AB et al. Phase II study of alisertib, a selective Aurora A kinase inhibitor, in relapsed and refractory aggressive B- and T-cell non-Hodgkin lymphomas. J Clin Oncol 2014; 32: 44–50. 74 McMullin MF, Harrison CN, Niederwieser D, Demuynck H, Jakel N, Sirulnik A et al. The use of erythropoietic-stimulating agents (ESAs) with ruxolitinib in patients with primary myelofibrosis (PMF), post-polycythemia vera myelofibrosis (PPV-MF), and post-essential thrombocythemia myelofibrosis (PET-MF). Blood 2012; 120: abstract 2838. 75 Stu¨big T, Alchalby H, Ditschkowski M, Wolf D, Wulf G, Zabelina T et al. JAK inhibition with ruxolitinib as pretreatment for allogeneic stem cell transplantation

& 2014 Macmillan Publishers Limited

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

in primary or post-ET/PV myelofibrosis. Leukemia 2014; e-pub ahead of print 26 February 2014; doi:10.1038/leu.2014.86. Jaekel N, Behre G, Behning A, Wickenhauser C, Lange T, Niederwieser D et al. Allogeneic hematopoietic cell transplantation for myelofibrosis in patients pretreated with the JAK1 and JAK2 inhibitor ruxolitinib. Bone Marrow Transplant 2014; 49: 179–184. Tefferi A, Begna K, Laborde RR, Patnaik MM, Lasho TL, Zblewski D et al. Imetelstat, a telomerase inhibitor, induces morphologic and molecular remissions in myelofibrosis and reversal of bone marrow fibrosis. Blood 2013; 122: abstract 662. Lasho TL, Jimma T, Finke CM, Patnaik M, Hanson CA, Ketterling RP et al. SRSF2 mutations in primary myelofibrosis: significant clustering with IDH mutations and independent association with inferior overall and leukemia-free survival. Blood 2012; 120: 4168–4171. Verstovsek S, Mesa RA, Gotlib J, Levy RS, Gupta V, DiPersio JF et al. A double-blind, placebo-controlled trial of ruxolitinib for myelofibrosis. N Engl J Med 2012; 366: 799–807. Harrison C, Kiladjian JJ, Al-Ali HK, Gisslinger H, Waltzman R, Stalbovskaya V et al. JAK inhibition with ruxolitinib versus best available therapy for myelofibrosis. N Engl J Med 2012; 366: 787–798. Verstovsek S, Kantarjian H, Mesa RA, Pardanani AD, Cortes-Franco J, Thomas DA et al. Safety and efficacy of INCB018424, a JAK1 and JAK2 inhibitor, in myelofibrosis. N Engl J Med 2010; 363: 1117–1127. Pardanani A, Gotlib J, Gupta V, Roberts AW, Wadleigh M, Sirhan S et al. Update on the long-term efficacy and safety of momelotinib, a JAK1 and JAK2 inhibitor, for the treatment of myelofibrosis. Blood 2013; 122: abstract 108. Pardanani A, Gotlib J, Gupta V, Roberts AW, Wadleigh M, Sirhan S et al. An expanded multicenter phase I/II study of CYT387, a JAK-1/2 inhibitor for the treatment of myelofibrosis. Blood 2011; 118: 1645–1646 (abstract 3849). Pardanani AD, Caramazza D, George G, Lasho TL, Hogan WJ, Litzow MR et al. Safety and efficacy of CYT387, a JAK-1/2 inhibitor for the treatment of myelofibrosis. J Clin Oncol 2011; 29(Suppl): abstract 6514. Pardanani A, Harrison CN, Cortes JE, Cervantes F. Results of a randomized, double-blind, placebo-controlled phase III study (JAKARTA) of the JAK2-selective inhibitor fedratinib (SAR302503) in patients with myelofibrosis. Blood 2013; 122: abstract 393. Pardanani A, Gotlib J, Jamieson C, Cortes JE, Talpaz M, Stone R et al. SAR302503: interim safety, efficacy and long-term impact on JAK2 V617F allele burden in a phase I/II study in patients with myelofibrosis. Blood 2011; 118: abstract 3838. Gotlib J, Pardanani A, Jamieson C, Cortes J, Talpaz M, Stone R et al. Long-term follow up of a phase 1/2 study of SAR302503, an oral JAK2 selective inhibitor, in patients with myelofibrosis (MF). Haematologica 2012; 97(s1): 145 (abstract 0361). Komrokji RS, Wadleigh M, Seymour JF, Roberts AW, To LB, Zhu HJ et al. Results of a phase 2 study of pacritinib (SB1518), a novel oral JAK2 inhibitor, in patients with primary, post-polycythemia vera, and post-essential thrombocythemia myelofibrosis. Blood 2011; 118: 130 (abstract 282). Verstovsek S, Dean JP, Cernohous P, Komrokji RS, Seymour JF, Mesa RA et al. Pacritinib, a dual JAK2/FLT3 inhibitor: an integrated efficacy and safety analysis of phase II trial data in patients with primary and secondary myelofibrosis (MF) and platelet counts r100,000/ml. Blood 2013; 122: abstract 395. Verstovsek S, Mesa RA, Salama ME, Giles JLK, Pitou C, Zimmermann AH et al. Phase I study of LY2784544, a JAK2 selective inhibitor, in patients with myelofibrosis (MF), polycythemia vera (PV), and essential thrombocythemia (ET). Blood 2013; 122: abstract 665. Pardanani A, Roberts AW, Seymour JF, Burbury K, Verstovsek S, Kantarjian HM et al. BMS-911543, a selective JAK2 inhibitor: a multicenter phase 1/2a study in myelofibrosis. Blood 2013; 122: abstract 664.

Leukemia (2014) 1 – 9