JAK/STAT Pathways in Cytokine Signaling and Myeloproliferative Disorders

A Basic Overview of Cytokine Receptors

Cytokines are defined as a group of growth factors that bind to their cognate receptors and trigger intracellular signaling events that result in the modulation of gene expression. Most cytokine receptors are comprised of a multi-subunit complex containing a unique and specific ligand binding subunit and a signal transducing subunit, with the latter sometimes being structurally similar to other members of the cytokine receptor superfamily^1-4 (Fig. 1). The average extracellular domain is comprised of approximately 210 amino acids and contains one or more conserved cysteine (C) residues. A second conserved sequence of tryptophan-serine-X-tryptophan-serine (W-S-X-W-S) is also present in the carboxy-terminus.⁵ Structurally, the C-terminus is composed of 2 fibronectin type III modules that are connected by a hinge region, and also contains the W-S-X-W-S motif that is predicted to function as a ligand interaction domain. The cytoplasmic domains of cytokine receptors share a limited level of similarity in their membrane proximal regions, particularly in regions termed the box 1 or the proline-rich motif and the box 2 motif. These portions of the receptor are critical for proper function of the receptor and mediating mitogenic signals. In contrast, the membrane distal region, in which the box 1 and 2 motifs are located, is required for differentiation. Functions of regions within the cytoplasmic domains that do not contain box 1 or box 2 motifs vary from receptor to receptor (reviewed in Ihle et al.⁶ and Vainchenker et al.⁷).

There are 2 cytokine superfamilies that can be further subdivided into multiple subgroups of cytokine receptors based on the nature of shared subunits, and all are devoid of catalytic activity (reviewed in Baker et al.⁸). Nevertheless, interaction of a cytokine with its receptor rapidly induces tyrosine phosphorylation of the receptor and a variety of cellular proteins. In the early 1990s, genetic complementation studies in an interferon-alpha (IFN-α) unresponsive cell line showed that TYK2, an “orphan” tyrosine kinase, could rescue the mutant phenotype.⁹ Within a year, several additional studies were published that demonstrated that other members of the Janus kinase (JAK) family of tyrosine kinases mediated the signals triggered by cytokines.^10-13

Janus Kinases

There are 4 members of the JAK family: JAK1, JAK2, JAK3, and TYK2. Of these, TYK2 was the first to be discovered using a degenerate oligonucleotide-based screen aimed at isolating novel tyrosine kinases.¹⁴ Shortly thereafter, cDNAs encoding JAK1,¹⁵ JAK2,^11,16 and JAK3^17-19 were isolated using various cloning strategies.

The unique structure of the JAK kinases clearly distinguishes them from other members of the protein tyrosine kinase family (Fig. 2). The amino-terminal half of JAKs contains 2 motifs, a Src homology-2 (SH2) domain and a band four-point-one, ezrin, radixin, moesin (FERM) domain. The SH2 domain encompasses what was originally defined as the JH3 and part of the JH4 domains. However, in spite of the homology to SH2 sequences, this region does not appear to bind to phosphotyrosine residues.^20-22 Furthermore, mutation of a residue that is normally detrimental to SH2 domain function does not affect cytokine receptor activation. These studies suggest that the JAK family SH2 domains do not function as traditional SH2 domains and may, in fact, play roles as scaffolds.²³

The regions previously termed the JH5 to JH7 (as well as part of the JH4) domains comprise the FERM domain, which regulates catalytic activity and mediates association with receptors and other proteins. An intact FERM domain is required for activated mutants of JAK1 to support type I IFN signaling.²⁴ In addition, mutation of tyrosine 913 in the JAK2 FERM domain has also been shown to result in constitutive activation of the kinase in the absence of cytokine stimulation.²⁵ Patient-derived JAK3 proteins with mutations in the FERM domain illustrate the significance of this domain in the protein’s function. These proteins were shown to be devoid of kinase activity (partly due to the fact that the kinase and FERM domains interact) and also failed to associate with receptors.²⁶ Conversely, chimeric proteins that contain only the JAK3 FERM domain associate with the common gamma (γ_c) subunit.²⁷ Residues located in the JH7 region have been shown to mediate the binding of JAKs to the box 1/proline-rich region of cytokine receptors,^28-30 and this interaction ultimately regulates receptor localization and turnover.^31-33

Nevertheless, the most intriguing and unique feature of these proteins is the presence of 2 Jak homology (JH) domains, JH1 and JH2, which have extensive homology to tyrosine kinase domains. The presence of the 2 kinase domains is actually the basis for the name of the protein family, being named after Janus, the Roman god with 2 faces.¹⁵ Although the JH1 domain is a functional tyrosine kinase domain with a requisite YY motif in the activation loop,^34-36 the JH2 domain, despite harboring most of the conserved amino acids that are a characteristic of functional kinases, lacks any observable tyrosine kinase activity due to the absence of residues that are required for catalytic activity and nucleotide binding. However, it is now clear that this kinase-like domain plays a significant regulatory role in both the activity of JAK family proteins and cytokine-induced signaling. An early theoretical model of the JH1 and JH2 domains strongly suggested that the 2 domains interact with each other and that the JH2 domain exerts a negative effect on the kinase activity of the JH1 domain.³⁷ Subsequent biochemical studies demonstrated that both the JAK2 and JAK3 JH2 domains negatively regulate the proteins’ kinase activities.^38,39

The importance of the JH2 domain in the regulation of JAK activity has been underscored in patients suffering from myeloproliferative neoplasms (MPNs), in particular, those whose cells express the mutant JAK2^V617F protein.^40-43 This mutation disrupts the inhibitory role that the JH2 domain has on JH1, whereby the activation loop of JH1 adopts a conformation such that it can be phosphorylated by an adjacent JAK2^V617F molecule. The consequences of this mutation at the molecular level and its clinical significance are discussed in greater detail in subsequent sections of this article.

Cytokine Receptor Signaling via JAKs

JAKs mediate signals from a variety of cytokines and growth factors. In general, receptor dimerization/oligomerization due to ligand binding results in the juxtapositioning of the JAKs, either through homodimeric or heterodimeric interactions. The recruitment of JAKs results in their phosphorylation, either via autophosphorylation and/or transphosphorylation by other JAKs or other families of tyrosine kinases. This activation results in increased JAK kinase activity. Activated JAKs then phosphorylate receptors on target tyrosine residues that serve as docking sites that allow the binding of other SH2 domain–containing signaling molecules such as STATs, Src kinases, protein phosphatases, and other adaptor signaling proteins such as Shc, Grb2, and PI-3 kinase (reviewed in Baker et al.⁸) (Fig. 3).

The above model is supported by several studies in which investigators have demonstrated the ability of cytokines to rapidly induce receptor oligomerization leading to JAK2 activation.^10,13,14 Furthermore, studies using chimeric receptors with various combinations of extracellular ligand binding and cytoplasmic domains also favor this model,^44-46 which readily applies to both single chain receptors such as those for EPO, PRL, GH, and G-CSF as well as multichain receptors such as those for IL-3, IL-5, and GM-CSF. Cytokine binding results in the association of the JAKs with one of the receptor subunits. The receptor-associated JAK kinase can then either process the signal or, subsequent to receptor oligomerization, can recruit other JAKs in the vicinity. Homodimerization or heterodimerization of the JAKs, followed by their activation upon phosphorylation, finally results in the propagation of the initial signal and ultimately leads to the activation of transcription factors.

Signal Transducers and Activators of Transcription

Signal transducers and activators of transcription, or STATs, are transcription factors that were originally described by Darnell et al.^47,48 as ligand-induced transcription factors in IFN-treated cells. Subsequent studies by a number of groups showed that STATs play critical roles in signal transduction pathways triggered by several cytokines and growth factors (reviewed in Murray⁴⁹). To date, 7 mammalian STAT-encoding genes have been identified, and alternative splicing or posttranslational proteolytic cleavage generates additional forms of STATs 1 and 3.⁵⁰ STAT-4 also exists in 2 forms, termed STAT-4α and STAT-4β, and 2 STAT-5 isoforms, termed STAT-5a and STAT-5b, are encoded by separate genes that are linked in tandem.^51,52

Like most transcription factors, STATs exhibit a modular structure with 7 well-defined domains, including an N-terminal conserved domain, a coiled-coil domain, a DNA binding domain, a linker region, an SH2 domain, a tyrosine activation, and a C-terminal transactivation domain (Fig. 4). The amino-terminal region of STATs is well conserved among family members and is critical for STAT function, as small deletions in this region have been shown to eliminate the ability of STATs to be phosphorylated. This domain also functions in nuclear import, export, receptor binding and cooperates with the DNA binding domain. The amino-terminal region also regulates dimerization of STATs in their inactive state⁵³ (reviewed in Schindler et al.⁵⁴). The coiled-coil domain adopts an α-helical conformation, functions in receptor binding, and associates with regulatory proteins (reviewed in Kisseleva et al.⁵⁵). The DNA binding domain is also highly conserved among the STATs, and all STAT homodimers with the exception of STAT-2 differentially bind more than 10 related γ-activated sequence (GAS) elements that are characterized by the consensus sequence, TTNCNNNAA.^56,57 A complex comprised of STAT1, STAT2, and IFN regulatory factor (IRF) 9 binds to the IFN-α/β–stimulated response element (ISRE) (AGTTN₃TTTC) (reviewed in O’Shea et al.⁵⁸). The linker domain functions as a spacer to maintain proper conformation between the dimerization and DNA binding domains. The SH2 domain, which is the most highly conserved domain among the STATs, plays a very important role in STAT signaling, being critical for the recruitment of STATs to activated receptor complexes and for the interaction with JAK and Src kinases. In addition, this domain is required for STAT homodimerization and heterodimerization, which in turn appears to be critical for nuclear localization and DNA binding activities. The transactivation domain varies among family members and, as the name implies, modulates the transcriptional activation of target genes. C-terminally truncated isoforms of STATs 3, 4, and 5 are missing portions of their transactivation domains and reportedly behave as dominant-negative proteins (reviewed in Schindler and Strehlow⁵⁹).

In an unstimulated cell, STATs are inactive, cytosolic proteins that exist in an unphosphorylated state. Cytokine stimulation induces phosphorylation of tyrosine residues on the receptor that serve as docking sites for STATs via their SH2 domains. Once bound to the receptor, all members of the STAT family become tyrosine phosphorylated in response to cytokine stimulation at a conserved carboxy-terminal tyrosine, Y694, for example, in the case of STAT-5. Phosphorylation of this tyrosine residue appears to be achieved by growth factor receptors as well as JAK and Src kinases, depending on the cell type and the nature of the ligand/receptor interactions. This form of phosphorylation induces reorientation of the STAT proteins and homodimerization and heterodimerization via the interaction of the SH2 domain of one STAT molecule with the phosphotyrosine residue of another. Once phosphorylated, the dimerized STATs translocate to the nucleus. In addition to tyrosine phosphorylation, all STATs, with the exception of STAT-2, are regulated by serine phosphorylation at a conserved PSMP motif that is located in the transactivation domain. C-terminal serine phosphorylation is stimulated by several cytokines and is mediated by serine/threonine kinases including, but not limited to, ERK, p38, JNK, mTOR, NLK, CaMKII, IKKε, and PKCδ and positively regulates the transactivation potential of these proteins (reviewed in Schindler et al.⁵⁴ and Khwaja⁶⁰).

Cytokine Signaling and STAT Activation by JAKs and Src Family Kinases

Because the cytokine receptor-ligand interactions result in the activation of JAK kinases that often exist in association with cytokine receptors, and because this activation is obligatory for the activation of STATs, it is widely accepted that STATs are substrates for JAK kinases. However, activated JAK kinases do not seem to exhibit specificity for a particular STAT, as different receptors activate a common STAT even though they activate distinctively different JAKs.^48,61 In addition, chimeric receptor molecules that harbor different JAK binding sites but identical STAT binding sites can activate the same STAT protein(s).^61,62 Thus, the specificity for STAT phosphorylation appears to be dictated by the docking sites for STATs that are present in the receptors themselves.

The notion that STATs are activated by kinases other than JAKs was at first demonstrated by studies aimed at investigating the molecular mechanisms associated with Src-mediated transformation. v-Src–transformed NIH3T3 cells constitutively express tyrosine-phosphorylated STAT-3,^63,64 and in vitro studies have shown that v-Src can bind to and phosphorylate STAT-3.⁶⁴ Similarly, v-Src–transformed 32Dcl3 myeloblastic cells constitutively express phosphorylated forms of STAT-1, -3, and -5 in the absence of cytokine.⁶⁵ In this model, STAT-3 activation is blocked by a dominant-negative mutant of Src, but not that of JAK-2.⁶⁶ These events mirror the signaling events induced by IL-3 stimulation, whereby the same STATs are activated and endogenous c-Src associates with and mediates the activation of STAT-3.

Based on these results, a second model of STAT activation has been proposed, where JAK kinases may be more crucial to the phosphorylation of cytokine/growth factor receptors. Moreover, JAK-mediated phosphorylation may create docking sites on the receptors for binding of SH2-containing proteins such as STATs, Src kinases, and other signaling intermediates (reviewed in Baker et al.⁸). JAKs or Src kinases, depending on the nature of the STAT that is being activated, then induce tyrosine phosphorylation and activation of STAT proteins (Fig. 3). These observations suggest that 2 independent pathways can mediate STAT activation, one that is dependent on JAKs and the other that is dependent on other tyrosine kinases, such as Src family kinases (SFKs). Recent studies using targeted inhibitors/therapies support this model. SRC inhibition by either shRNA or dasatinib results in JAK-dependent phosphorylation of STAT3. Sustained SRC inhibition (by drugs such as dasatinib) has also been shown to result in compensatory activation of JAK kinase activity and JAK-STAT3 binding that permits proliferation and survival, even in the absence of SRC activity.^67,68

The notion that different STATs might be phosphorylated by other tyrosine kinases under different conditions is also supported by studies with other tyrosine kinases such as v-ABL and BCR-ABL. Examination of the molecular mechanisms associated with v-Abl–mediated transformation shows that B cells transformed by this oncogene exhibit constitutively activated forms of JAK1 and JAK3 as well as STAT-1, -3, and -5.^69,70 Activated JAK1 in these cells was found to be associated with the v-Abl protein. Similarly, the BCR-ABL oncogene constitutively activates STAT-1 and STAT-5 in a variety of hematopoietic cell systems in vitro.^71-74 While some studies have shown that BCR-ABL has very little if any effect on the activation of JAKs,^71-74 others indicate that the oncogene is required for the activation of JAK2 in hematopoietic cells that are transformed by oncogenic variants of ABL.^75,76

These studies highlight the role of JAKs, JAK2, in particular, in the development of hematological malignancies. Recent discoveries of oncogenic JAK2 translocations and activating mutations further demonstrate its role in these diseases.

Chromosomal Translocations Involving the JAK2 Locus

Rearrangements of the JAK2 gene that lead to constitutively activated tyrosine kinase activity with oncogenic properties have been known for more than a decade. These translocations result in a variety of JAK2 chimeric transcripts, and expression of their resultant fusion proteins often leads to the development of leukemias of both myeloid and lymphoid origins (Fig. 5).

Activating Mutations of the JAK2 Gene

The vast majority of chromosomal translocations of the JAK2 gene lead to leukemias and lymphomas. Similarly, all activating point mutations, deletions, and insertions in this gene lead to myeloproliferative neoplasms that can further develop into leukemias and MDS upon the acquisition of additional genetic lesions (Fig. 6).

Other Mechanisms of JAK2 Activation

Activation of the JAK-STAT pathway has also been observed in diseases with defects in proteins that signal upstream of the Janus kinases. One such example is the constitutive activation of JAK2 and STAT1 in cells derived from monosomy 7 MDS patients that arises due to aberrant cytokine receptor signaling.^108,109 Monosomy 7 cells show increased expression of a differentiation-defective GCSFR isoform (IV) that fails to internalize following GCSF binding, as would normally occur for the full-length receptor. This receptor variant is also defective in facilitating phosphorylation of STAT-3, but its ability to signal phosphorylation of STAT-1 and -5 is unimpaired.^109,110 Consequently, the ability of these cells to differentiate is limited, although their proliferation via JAK-2 remains unhindered.

The genetic aberrations in JAK2 reviewed above have opened new avenues for diagnosing and classifying patients with myeloproliferative neoplasms. These findings have also identified activated JAK2 as an attractive molecular target for small molecule inhibitors.

Development of Small Molecule JAK2 Inhibitors

The discovery of genetic lesions leading to the activation of JAK2 kinase activity in leukemia and lymphomas along with the obligatory association of MPN (e.g., PV) with activating JAK2 mutations has generated tremendous enthusiasm for the development of JAK2 inhibitors for the treatment of these hematological indications. Consequently, a large number of chemotypes have been identified that possess Janus kinase inhibitory activity. These molecules range from substrate-competitive inhibitors inspired by the structure of the canonical JAK2 inhibitor, tyrphostin AG490, to ATP-competitive pyridones and pyrimidine analogs¹¹¹ (Fig. 7). The majority of these compounds were intentionally developed as JAK2 inhibitors and are designated as class I inhibitors. Class II inhibitors were initially developed as inhibitors of other target kinases and were later found to possess JAK2 inhibitory activity. While patents have been filed for a large number of JAK2 inhibitors by a host of pharmaceutical companies and medical institutions, only a fraction of these inhibitors have entered into clinical trials. The therapeutic outcomes of these compounds are currently being assessed. These drugs have been reviewed extensively since the 51st Annual American Society of Hematology Meeting (ASH 2009),^112-115 and we have summarized and discussed the chemical identity (where available), preclinical findings, and current status in clinical trials of these compounds below.

ATP-Competitive JAK2 Inhibitors

INCB018424 (ruxolitinib; Incyte, Wilmington, DE) is an orally available pyrrolo [2,3-d]pyrimidine analog that exhibits subnanomolar affinity for JAK2 and JAK1 and a markedly lower activity against JAK3. The drug exhibits nanomolar growth inhibitory activity against patient cells harboring JAK2^V617F and also inhibits JAK2/STAT5 signaling in JAK2 mutant cells in vitro and in a murine model of MPN. INCB018424 has successfully completed phase II clinical trials in PV, ET, and primary and secondary MF patients and caused reductions in splenomegaly, constitutional symptoms, pruritis, cachexia, and erythrocytosis. However, patients also experienced dose-limiting toxicity (DLT) due to thrombocytopenia, anemia, and cytokine rebound. Whereas INCB018424 markedly improves the quality of life, it does not reduce JAK2^V617F allele burden or improve bone marrow histopathology. Phase III clinical trials are ongoing.

TG101348 (TargeGen, San Diego, CA) is a biaryl-meta-pyrimidine analog that exhibits low nanomolar activity against wild-type and V617F-mutated JAK2. This compound also inhibits FLT3 and RET kinase activity but shows appreciable selectivity for JAK2 over other members of the JAK family. TG101348 has demonstrated therapeutic efficacy in a JAK2^V617F-induced bone marrow transplantation mouse model of PV, with dose-dependent reductions in splenomegaly, hematocrit, extramedullary hematopoiesis, and endogenous erythroid colony formation. Among the clinical benefits are reductions in splenomegaly, constitutional symptoms, pruritis, leukocytosis, thrombocytosis, and JAK2 allele burden (50%) in a third of the patients, with a minor improvement in bone marrow cellularity and reticulin fibrois with extended treatment.¹¹⁴ Side effects include increased amylase, lipase, and transaminase levels; diarrhea; nausea; vomiting; thrombocytopenia; and anemia. Patients with JAK2^V617F-induced MPN are currently enrolled in phase I/II clinical trials.

CEP-701 (lestaurtinib; Cephalon, Frazer, PA) is a staurosporine (indolocarbazole) analog initially developed as an orally available ATP-competitive FDA-approved FLT3 inhibitor for the treatment of AML. A decade after it was first patented, CEP-701 was pulled out of phase III trials because its efficacy against CML could not be demonstrated. CEP-701 has recently been found to be a low nanomolar class II inhibitor of JAK2 with the ability to inhibit the growth of JAK2^V617F-expressing cells in the nanomolar range. Benefits of the drug include reduction in splenomegaly, pruritis, and anemia, whereas side effects include diarrhea, nausea, vomiting, thrombocytosis, leukocytosis, thrombocytopenia, and thrombosis in patients with PV. This compound is currently in phase II trials for the treatment of primary myelofibrosis (PMF) and post-PV/ET MF. While there is no evidence that treatment with lestaurtinib causes positive changes in bone marrow fibrosis or cytogenetic response, an ongoing multicenter phase I/II clinical trial suggests that CEP-701 partially decreases the mutant allelic burden in MF patients.¹¹⁶

CYT387 (Cytopia, Melbourne, Australia) is a phenyl-amino-pyridine derivative that potently inhibits JAK1 and JAK2 and exhibits 10-fold lower activity against JAK3. This molecule is effective in blocking signaling via the JAK/STAT pathway in cells harboring the JAK2^V617F mutation and also inhibits the growth of these cells in the low micromolar range. CYT387 was shown to be efficacious in a subcutaneous xenograft MPN model and inhibits in vitro endogenous erythroid colony (EEC) formation by cells isolated from PV patients. This drug is in ongoing phase I/II clinical trials for patients with myelofibrosis. Clinical outcomes have yet to be reported.

XL019 (Exelixis, South San Francisco, CA) is a potent low nanomolar JAK family inhibitor with reasonable selectivity for JAK2 over other Janus kinases. After successfully completing phase I clinical trials in PMF patients and showing reduction in splenomegaly, anemia, and pruritis, clinical trials were discontinued due to neuropathy (DLT). Notably, this is the only reported anti-JAK2 agent that demonstrated a reduction in circulating blasts in 10% to 20% of patients.¹¹³

SB1518 (S*Bio, Singapore; licensed to Onyx [Emeryville, CA] as ONX0803) is an orally bioavailable, potent, and selective JAK2 inhibitor with undisclosed structure (probably macrocyclic). While this agent causes a reduction in splenomegaly, side effects include gastrointestinal symptoms (DLT), diarrhea, nausea, and thrombocytopenia. Phase I/II clinical trials are ongoing for patients with chronic idiopathic myelofibrosis. As is true for other JAK2 inhibitors, treatment with SB1518 did not cause any reduction in tumor burden or decrease in bone marrow pathology.

AZD-1480 (AstraZeneca, London, UK), a pyrazoyl pyrimidine, is the most potent JAK2 inhibitor in clinical trials with a picomolar IC₅₀ value and fair selectivity for JAK2 over JAK3. This compound blocks JAK/STAT signaling, inhibits proliferation, and induces apoptosis in the SET2 megakaryoblastic JAK2^V617F-positive cell line. AZD-1480 has also been shown to inhibit the growth of stem cells transfected with the mutant JAK2 gene in a murine model. Phase I/II clinical trials with MF patients are ongoing.

MK0457 (tozasertib; Vertex, Cambridge, MA and Merck, Whitehouse Station, NJ) is a class II JAK2 inhibitor that was originally developed as an Aurora kinase inhibitor. This compound entered the clinic as an antileukemic agent but was pulled out of phase I trials due to questions regarding its cardiac safety.

Substrate-Competitive Inhibitors of JAK2

LS104 (Hospital for Sick Children [Toronto, Canada], licensed to LymphoSign [Toronto, Canada], acquired by Aegera [Montreal, Canada]) is an analog of tyrphostin AG490 and is the only non–ATP-competitive JAK2 inhibitor in clinical trials. This molecule has also been shown to inhibit BCR-ABL kinase activity but does not inhibit other tyrosine kinases such as those of the Src family. In preclinical testing, LS104 displayed cytotoxicity against a variety of leukemic cell lines of myeloid and lymphoid origin and has recently entered phase II clinical trials for ALL therapy.

ON044580 (Onconova Therapeutics Inc., Newtown, PA) is an α-benzoyl styryl benzyl sulfide and has an AG490-like backbone. Interestingly, this inhibitor has properties that are similar to LS104, even though the 2 compounds represent different chemotypes. ON044580 is a dual JAK2 and BCR-ABL kinase inhibitor, is non-ATP competitive, and has a high degree of specificity as revealed by testing against a panel of 300 kinases.^117,118 Further, ON044580 is cytotoxic against cells overexpressing JAK2^V617F and BCR-ABL as well as ex vivo samples from CML patients regardless of disease stage or imatinib sensitivity. ON044580 has elicited favorable cytogenetic outcomes in Monosomy 7 MDS patient samples. The in vivo efficacy and safety of ON044580 have yet to be demonstrated.

Concluding Remarks

Whereas the preclinical results with JAK2 inhibitors for MPN therapy have been promising, these agents have not met with the same degree of success in the clinic. It is now widely accepted that JAK2 inhibitors as single oral agents significantly improve the quality of life of MPN patients by the palliation of debilitating disease-related symptoms such as palpable splenomegaly, pruritis, weight loss, early satiety, and erythrocytosis. This property makes them attractive options for patients with MPN either as primary or second-line therapies in those who have failed hydroxyurea and pegylated interferon. However, to date, these agents do not significantly effect bone marrow fibrosis, alter marrow histopathology, reverse cytopenias, reduce red cell transfusion requirements, or significantly reduce allele burden. Although some patients may have a reduction in allele burden, there are no data to suggest that these agents can eradicate the JAK2^V617F-bearing clone. This is in contrast to studies demonstrating that treatment with pegylated interferon can eradicate the JAK2^V617F clone and lead to molecular complete remissions and restore polyclonal hematopoiesis in a small percentage of patients with MPN.¹¹⁹ No phase III study has been completed, and thus, there is no evidence to suggest that these agents can alter the natural history of MPN in the clinic. These agents may palliate disease-related symptoms, but there is no evidence for a curative potential. While the safety profile of these JAK2 inhibitors is acceptable, their associated toxicities of anemia, thrombocytopenia, and gastrointestinal discomfort, although manageable, will require the prudent choosing of candidates. It is certain that JAK2 inhibitors will continue to play an important role in alleviating the debilitating symptoms associated with MPN. Future clinical trials will need to focus on combination targeted therapy as the nature and significance of the MPN stem cell population are further demonstrated at the clinical and molecular levels.¹²⁰