Ongoing Clinical Trials in Myeloproliferative Neoplasms (Research Issues and Perspectives) Part 1

Introduction

The classic Philadelphia-negative (Ph-negative) myeloproliferative neoplasms (MPNs) are a group of neoplastic disorders characterized by variable degrees of peripheral blood (PB) cell proliferation (i.e., erythrocytosis, and/or leukocytosis, and/or thrombocytosis), splenomegaly, bone marrow (BM) fibrosis, and an increased risk of thrombotic episodes and transformation to acute myeloid leukemia (AML) (Swerdlow et al. 2008). Classic Ph-negative MPNs include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) (Swerdlow et al. 2008). MF can also develop secondary to disease transformation from PV and ET. The prognosis of these Ph-negative MPNs is quite heterogeneous, as some patients can have a disease with indolent course and median survival of 15-20+ years (PV, ET), while in other cases, the disease can follow a very aggressive course, as in MF where the median survival ranges from 5 to 7 years only (Cervantes et al. 2009).

Currently, treatment options for patients with Ph-negative MPNs are few and far between.Patients with PV and ET are usually managed with anti-platelet agents and cytoreductive drugs (e.g., hydroxyurea) when their risk of thrombosis is deemed to be elevated (e.g., older age, previous thrombotic episodes). Treatment of MF is palliative and directed to alleviation of symptoms caused by cytopenias and massive splenomegaly. Allogeneic stem cell transplantation (SCT) is potentially curative, but only a minority of patients with MF can benefit from the procedure due to issues with age and comorbidities (Kroger et al. 2009) .

Fig. 18.1 Chemical structures of selected drugs in current development for classic Ph-negative MPNs. Ruxolitinib (JAK2 inhibitor), everolimus (mTOR inhibitor), pomali-domide (immunomodulatory drug), panobinostat (HDAC inhibitor)

Fortunately, the scenario for patients with Ph-negative MPNs seems to be changing. Over the past 5 years, many new drugs have been in development for treatment of patients with Ph-negative MPNs. This surge in new drugs is mainly due to a greater understanding of the pathophysiology of Ph-negative MPNs. It is now known that most patients with Ph-negative MPNs harbor activating mutations in the hematopoietic cytokines signaling pathways, such as JAK2 V617F (PV ~90%, MF ~50%, ET ~50%), JAK2 exon 12 mutations (PV ~5%) and MPL W515K/L (ET ~5%, MF ~10%) (Baxter et al. 2005; James et al. 2005; Kralovics et al. 2005; Levine et al. 2005; Pikman et al. 2006; Scott et al. 2007). Mutations in other genes have also been described, including TET2, ASXL1, EZH2, LNK, IDH. and IZKF1. and their role in pathogenesis is currently being investigated.

There are several different classes of compounds in clinical trials for patients with Ph-negative MPNs at present (Fig. 18.1). In this topic, we will review current clinical results in studies of JAK2 inhibitors (Table 18.1) and the most important other drugs (Table 18.2).

JAK2 Inhibitors

Rationale

Since the discovery of the JAK2 V617F mutation by four independent groups in 2005, the prospect of using enzyme inhibitors to target this mutated kinase, similar to imatinib in CML, has been alluring investigators (Baxter et al. 2005; James et al. 2005; Kralovics et al. 2005; Levine et al. 2005). JAK2 tyrosine kinases (TKs) are part of the JAK family of TKs that associate with the intracellular portion of cytokine receptors that do not possess intrinsic TK activity (e.g., erythropoietin receptor [EPOR], granulocyte colony-stimulating factor [G-CSF] receptor [GCSFR], thrombopoietin receptor [MPL]) (Leonard and O’Shea 1998).

Table 18.1 Complete and ongoing clinical trials with JAK2 inhibitors for patients with classic Ph-negative MPNs

CI clinical improvement, ET essential thrombocythemia, GI gastrointestinal, MF myelofibrosis, NA not available, PV polycythemia vera

Table 18.2 Clinical trials with drugs other than JAK2 inhibitors for patients with classic Ph-negative MPNs

CI clinical improvement, ET essential thrombocythemia, MF myelofibrosis, NA not available, PV polycythemia vera

When the putative ligand binds to the receptor, JAK TKs are activated and cross-phosphorylate each other and the receptor, increasing their activity and permitting binding of secondary messengers to the cytokine receptor, such as STATs (signal transducers and activators of transcriptions) (Leonard and O’Shea 1998). STATs are latent cytoplasmic transcription factors which bind to phosphorylated tyrosine residues on the intracellular portion of cytokine receptors. This is followed by phosphory-lation and activation of the STAT molecule by activated JAK kinases, STAT homodimerization, translocation to the nucleus, and activation of transcription of target genes (Yu and Jove 2004). STAT3 and STAT5a/b are the STAT family members most commonly implicated in neoplastic disorders, including Ph-negative MPNs, and some of their target genes are CCND1 (cellular proliferation), VEGF (angiogenesis) and BCL2L1, and MCL1 (resistance to apoptosis) (Yu and Jove 2004). Other pathways activated by JAK2 TKs include the Ras-Raf-MAPK (mitogen-activated protein kinase) pathway (Winston and Hunter 1995; Mizuguchi et al. 2000) and the phosphatidylinositol-3-kinase (PI3K) of the PI3K-Akt-mTOR pathway (Al-Shami and Naccache 1999; Bouscary et al. 2003).

The JAK2 V617F mutation leads to constitutive signaling of the JAK2 TK (James et al. 2005). The V617F mutation is located in the pseudokinase domain (JAK homology 2 [JH2] domain). The JH2 domain interacts with and inhibits activity of the true TK domain (JH1) since aberrant JAK enzymes lacking the JH2 domain possess an increased TK activity (Saharinen and Silvennoinen 2002) . The V617F mutation disrupts this important interaction between the JH2 domain and the JH1 domain, and it is believed that this is the molecular mechanism through which the V617F mutation leads to constitutive JAK2 activation (Lindauer et al. 2001; Dusa et al. 2010) . When JAK2 V617F is expressed in EPOR-positive BaF/3 cells, it confers EPO-hypersensitivity and EPO-independent growth and survival (Levine et al. 2005) . Intracellularly, there is increased phosphorylation and activation of STAT5 and other downstream targets, such as Akt (James et al. 2005). In mice expressing JAK2 V617F in hematopoietic stem cells (HSC), there is development of a disease clinically similar to human PV, with increased red cell mass, splenomegaly, and hypercellularity of the BM (Lacout et al. 2006; Wernig et al. 2006; Mullally et al. 2010). Some mice later develop fibrosis in the BM, akin to human MF (Lacout et al. 2006; Wernig et al. 2006). More recently, laboratory studies have revealed a non-canonical role for JAK2 besides activating intracellular signaling pathways related to cell proliferation and survival. Dawson et al. demonstrated that JAK2 is present in the nucleus of HSCs and that it phosphorylates histone H3 at residue Y41 (H3Y41), preventing binding ofthe Heterochromatin Protein 1-a (HP1a) (Dawson et al. 2009). HP1a is responsible for mediating gene silencing through epigenetic mechanisms (Bannister et al. 2001; Lachner et al. 2001), and indeed, increased expression of the oncogene LMO2 was detected in JAK2 V617F-positive cells. Inhibition of JAK2 was associated with reduced phosphorylation of H3Y41 and reduced expression of LMO2 (Dawson et al. 2009). In a similar manner, another report demonstrated that JAK2 phosphorylation of the histone arginine methyltransferase enzyme PRMT5 was associated with decreased enzyme activity, decreased arginine methylation at histone targets, increased erythroid colony formation and proliferation (Liu et al. 2011). Thus, it is becoming clearer that JAK2 V617F exerts its effects on hematopoietic cells both through canonical targets (e.g., JAKSTAT pathway) and non-canonical targets (e.g., epigenetic modulation through histone modifications), making JAK2 a central component of targeted therapy for classic Ph-negative MPNs.

Current Clinical Trials

Ruxolitinib in Myelofibrosis

Ruxolitinib (formerly known as INCB018424/ INC424; Incyte, Wilmington, DE) is a dual JAK1/ JAK2 inhibitor currently in clinical trials for both MF and PV. It is the JAK2 inhibitor which is most advanced in clinical development. Pre-clinical studies demonstrated that ruxolitinib inhibited both JAK1 (half-maximal inhibitory concentration [IC50] 3.3 nM) and JAK2 (IC50 2.8 nM) and had decreased activity versus JAK3 (IC50 322 nM) (Quintas-Cardama et al. 2010). Ruxolitinib inhibited phosphorylation of JAK2 downstream target STAT3 and reduced viability of cells expressing EPOR and JAK2 V617F. In an animal model of MPN, mice who received ruxolitinib had a decrease in splenomegaly and pro-inflammatory cytokines levels (Quintas-Cardama et al. 2010). Ruxolitinib also inhibited proliferation of erythroid progenitors from patients with PV to a greater degree than cells from normal donors (Quintas-Cardama et al. 2010).

The results of a phase I/II clinical trial with ruxolitinib for patients with MF (PMF or post-PV/ET) were recently published (Verstovsek et al. 2010b). In this study, 153 patients received ruxolitinib at different dose schedules. An individualized dose schedule of 15 mg twice daily (10 mg twice daily if platelets <100 χ 109/L) with further escalation to 20-25 mg twice daily if no response was observed represented the best balance between efficacy and safety. Dose-limiting toxicity (DLT) was thrombocytopenia. According to the International Working Group on Myelofibrosis Research and Treatment (IWG-MRT) response criteria, 44% of patients with splenomegaly had a clinical improvement (CI) with a decrease >50% in spleen size. Response rate in the cohort receiving the optimized dose schedule was 52%. At 12 months, 73% of patients in the 15 mg twice daily cohort maintained response. Importantly, response rate was seen in patients with both wild-type and mutated JAK2 and also in patients with post-PV/ET MF, showing that ruxolitinib is effective in all subgroups of MF. Other observed significant beneficial effects included an improvement in systemic symptoms, exercise capacity, and quality of life in the majority of patients. At 1 month, the majority of patients reported a greater than 50% improvement in their Myelofibrosis Symptom Assessment Form (MFSAF) score. Improvement in cytokine profile (decrease in pro-inflammatory cytokines and increase in EPO and leptin) accompanied the improvement in systemic symptoms. There was no decrease in BM fibrosis, and patients with JAK2 V617F had a mean decrease in allele burden of only 13%. Hematological toxicity was the most common reported side effect seen with rux-olitinib and consisted mainly of new-onset anemia and thrombocytopenia. However, the rate of grade 3-4 myelosuppression was substantially improved in the cohort which started with the dose-adjusted schedule of 15 mg twice daily rather than at maximum dose of 25 mg twice daily (2.9% versus 23.4% [grade 3-4 thrombocytopenia]; 8.3% versus 26.7% [grade 3-4 anemia]). Other side effects included diarrhea (all grades, 5.9%; grade 3-4, 0%), fatigue (all grades, 4.3%; grade 3-4, 1.3%) and headache (all grades, 3.3%; grade 3-4, 0%). Even though the study was not randomized to detect an improvement in survival or disease transformation, only three patients transformed to AML (rate of 0.016 patients/year), less than expected based on historical cohorts (Barosi et al. 2007). Interestingly, a recent publication showed that patients with MF who present with increased levels of pro-inflammatory cytok-ines (e.g., interleukin-8 [IL-8], IL-2R) have a worse survival compared to other patients (median survival 17 versus 80 months; p < 0.0001).Since ruxolitinib normalizes the cytokine profile of patients with MF, it would be interesting to see with longer follow-up if that translates into improved survival for these patients.

The results of the phase I/II trial with ruxoli-tinib were exciting and led to the launching of two phase III trials with ruxolitinib in the USA and Europe. Both clinical trials (Controlled MyeloFibrosis study with ORal JAK inhibitor Treatment I and II [COMFORT-I/II]) enrolled patients with PMF or post-PV/ET MF who required therapy and had a high or intermediate-2 risk score by the MF International Prognostic Score System (IPSS). In COMFORT-I (www. clinicaltrials.govregisternumberNCT00952289), 309 patients were randomized (1:1) in a doubleblind fashion between ruxolitinib (initial doses 15-20 mg twice daily) and placebo. Primary endpoint was the rate of patients with >35% decrease in spleen volume, as assessed by magnetic resonance imaging (MRI) or computed tomography (CT) at 24 weeks (corresponds roughly to a 50% decrease in spleen size by physical examination). Secondary endpoint was the duration of maintenance of a >35% reduction from baseline in spleen volume among subjects initially randomized to receive ruxolitinib. A recent press release from Incyte revealed that the study primary endpoint was achieved (Incyte 2010), with 42% of patients receiving ruxolitinib having achieved the endpoint, versus <1% of placebo patients (p < 0.0001). The COMFORT-II trial (NCT00934544) is a phase III controlled, open-label, randomized (2:1) study comparing ruxolitinib (initial doses 15-20 mg twice daily) versus best available therapy for patients with MF requiring therapy and with an IPSS risk of intermediated or high. Primary endpoint was the rate of patients with >35% decrease in spleen volume, as assessed by MRI/CT at 48 weeks. Secondary endpoint was the duration of maintenance of a >35% reduction from baseline in spleen volume. A total of 219 patients have been randomized in nine countries. Preliminary data in a press release from Incyte have stated that ruxolitinib provided a statistically significant reduction in spleen size compared to best available therapy (Incyte 2011). Full data from these studies are expected in an upcoming medical meeting.

Two other ongoing clinical trials with ruxoli-tinib deservemention. Thefirsttrial (NCT01317875) is a phase IB study which will determine the maximum safe starting dose (MSSD) for patients with MF (PMF and post-PV/ET) who have baseline platelet counts <100 χ 109/L. This is important since as many as 30% of patients with MF may develop platelet counts below these levels at some point during disease course (Gangat et al. 2010). Patients will receive ruxolitinib at initial dose of 5 mg twice daily, with escalation in increments of 5 mg in successive cohorts until the MSSD is determined. Then additional patients will be treated with ruxolitinib at the MSSD to confirm activity and safety. The other clinical trial (NCT01340651) will explore the activity and toxicity of a sustained release formulation of ruxolitinib administered to 40 patients with PMF or post-PV/ET MF. After 16 weeks of therapy with the sustained release formulation, patients will transition to an equivalent regimen of immediate release, twice daily ruxolitinib.