(2008) 22, 23–30 & 2008 Nature Publishing Group All rights reserved 0887-6924/08 $30.00 www.nature.com/leu SPOTLIGHT REVIEW

JAK2 inhibitor therapy in myeloproliferative disorders: rationale, preclinical studies and ongoing clinical trials

A Pardanani

Division of Hematology and Internal Medicine, Mayo Clinic, Rochester, MN, USA

The recent identification of somatic such as forming unit-erythroid) was also demonstrated in PV patients.5 JAK2V617F that deregulate (JAK)–signal trans- Erythroid progenitors were also shown to be hypersensitive to ducer and activator of transcription signaling has spurred other /growth factors, including (IL)-3,6 development of orally bioavailable small-molecule inhibitors 4 7 that selectively target JAK2 kinase as an approach to patho- -like -1, and granulocyte– 8 genesis-directed therapy of myeloproliferative disorders (MPD). macrophage colony stimulating factor. Unlike in hereditary In pre-clinical studies, these compounds inhibit JAK2V617F- erythrocytosis where mutations in the Epo receptor (EpoR) mediated cell growth at nanomolar concentrations, and in vivo cytoplasmic domain (or alternatively, in the von Hippel–Lindau therapeutic efficacy has been demonstrated in mouse models (VHL) ) may be evident (reviewed by Kralovics and of JAK2V617F-induced disease. In addition, ex vivo growth of Skoda9), similar studies failed to identify EpoR mutations in progenitor cells from MPD patients harboring JAK2V617F or 10,11 MPLW515L/K mutations is also potently inhibited. JAK2 patients with PV. Similarly, in early studies, endogenous inhibitors currently in clinical trials can be grouped into those colony growth did not appear to be associated designed to primarily target JAK2 kinase (JAK2-selective) and with mutations in c-mpl (MPL), the receptor, in those originally developed for non-MPD indications, but that ET or PMF patients.12 These data suggested that the molecular nevertheless have significant JAK2-inhibitory activity (non- defect underlying hypersensitivity in MPD occurred JAK2 selective). This article discusses the rationale for using JAK2 inhibitors for the treatment of MPD, as well as relevant downstream of the . Several lines of evidence aspects of clinical trial development for these patients. For have indicated that a constitutive activity is instance, which group of MPD patients is appropriate for initial important for PV pathogenesis. For instance, the mutant Phase I studies? Should JAK2V617F-negative MPD patients be oncoproteins BCR-ABL and v-Src were capable of supporting included in the initial studies? What are the likely conse- normal erythroid development of fetal progenitor cells from quences of ‘off-target’ JAK3 and wild-type JAK2 inhibition? EpoR(À/À) miceFthese data additionally that a unique tyrosine How should treatment responses be monitored? kinase activity may not be necessary for terminal erythroid Leukemia (2008) 22, 23–30; doi:10.1038/sj.leu.2404948; 13 published online 20 September 2007 differentiation. Furthermore, several kinase inhibitors, includ- Keywords: tyrosine kinase inhibitor; polycythemia; myelofibrosis; ing AG-490, a non-selective JAK inhibitor that is unsuitable for clinical development, have been shown to inhibit terminal erythroid differentiation in an Epo-free culture system that uses CD34 þ progenitor cells from PV patients.14 Similarly, mesylate (Gleevec) has been shown to inhibit EEC growth Rationale in vitro, although its molecular target for this effect has not been identified.15 A common link in the aforementioned data is JAK- Several lines of evidence point to the key role of constitutively 2, which is the predominant tyrosine kinase of the JAK family of active Janus kinase (JAK)–signal transducer and activator of kinases (JAK1, JAK2, JAK3 and TYK2) that is activated in transcription (STAT) signaling in the pathogenesis of BCR-ABL- response to Epo, granulocyte–macrophage colony stimulating negative myeloproliferative disorders (MPD), namely polycythe- factor, IL-3, IL-5, thrombopoietin, growth and prolac- mia vera (PV), essential thrombocythemia (ET) and primary tin-mediated signaling (reviewed by Ihle et al.16). Indeed, the myelofibrosis (PMF). phenotype of JAK2-deficient mice is very similar (albeit more SPOTLIGHT severe) to Epo- or EpoR-mutant mice, with embryonic lethality observed at day 12.5 due to absence of definitive erythropoi- Pre-JAK2V617F era esis.17,18 In addition to mediating EpoR signaling via its kinase Early studies showed that erythropoiesis in PV patients was activity, JAK2 also enhances the normally inefficient folding of hypersensitive to (Epo),1,2 and possibly indepen- EpoR in the endoplasmic reticulum, thereby facilitating - dent of this cytokine.3,4 Culture of PV bone marrow cells independent cell-surface expression of the latter.19 Similarly, produced colony forming unit-derived erythroid colonies in the JAK2 (and TYK2) significantly enhances the cell-surface expres- absence of Epo, termed endogenous erythroid colonies (EEC), sion of MPL through stabilization of the mature form of the that were 9–37% of the erythroid colony number obtained in the receptor.20 Cumulatively, these data indicated that JAK2 kinase presence of Epo.3 Subsequently, growth of endogenous ery- was an ideal candidate proto- in MPD. throid bursts from more primitive erythroid progenitors (burst

Correspondence: Dr A Pardanani, Division of Hematology and JAK2V617F era Internal Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN JAK2 was directly linked to MPD pathogenesis with the 55905, USA. E-mail: [email protected] identification of JAK2V617F, a recurring gain-of-function muta- 21–24 Received 14 August 2007; accepted 17 August 2007; published tion, in MPD patients. Involvement of JAK2 was consistent online 20 September 2007 with earlier studies that identified loss of heterozygosity (LOH) JAK2 inhibitor therapy in MPD A Pardanani 24 in 9p (9pLOH) as the commonest chromosomal activated and phosphatidylinositol 3 kinase/ alteration in PV (B33% cases).25 This cytogenetically occult protein kinase B (AKT) pathways, albeit in a cytokine- change results from mitotic recombination (that is, acquired independent fashion.21,23,24,49 The particular importance of ), wherein homozygosity of all (and the STAT pathway is underscored by studies with ETS mutations) contained within the 9pLOH interval reflects a translocation variant 6 (ETV6)-TEL- (JAK2), a duplication event, rather than loss of the wild-type allele, as is fusion oncoprotein identified in human leukemiasFthis de- seen with classical tumor suppressor genes (that is, hemizygos- regulated kinase leads to constitutive activation of STAT-5 in Ba/ ity). In this regard, PV patients exhibit homozygosity for the F3 cells, and induces a lethal hematopoietic disease in mice in a JAK2V617F allele more frequently than ET patients, in granu- BMT assay, a process that has been demonstrated to be STAT- locytes26 as well as in erythroid colonies.27 While JAK2V617F is 5a/b-dependent.50–52 Under cytokine-free conditions, coexpres- restricted to myeloid malignancies, it is not associated with a sion of JAK2WT reportedly competes with JAK2V617F in a dose- specific disease phenotypeFthe frequency of JAK2V617F using dependent fashion, which is reflected in the suppression of sensitive allele-specific assays is estimated to be 95% in PV, and JAK2V617F-mediated STAT-5 transcriptional activity in JAK2- 50% in ET and PMF.26,28 In contrast, p5% of patients with deficient g-2A cells.21 Although the molecular basis for this chronic myelomonocytic leukemia, myelodysplastic syndrome dominant-negative effect has not been elucidated (for example, or de novo acute myeloid leukemia (AML) harbor this particular competition for binding to EpoR or to downstream signaling .29,30 JAK2V617F has been identified in the hemato- molecules), it may explain the basis for selection of JAK2V617F- poietic stem cell compartment,31 and has been demonstrated in homozygous clones, since these are predicted to enjoy a non-myeloid cells including B and T ,32 as well as selective growth advantage relative to JAK2V617F-heterozygous SPOTLIGHT natural killer cells.33 JAK2V617F recapitulates some of the key clones. Although JAK2V617F, MPLW515L and JAK2 exon 12 in vitro biological characteristics of MPDFfor instance, it mutations appear to activate similar signaling pathways in vitro, increases the survival of cytokine-dependent cells cultured in their in vivo expression in murine BMT assays results in the absence of cytokines,21,23,24 and appears to promote ex vivo relatively distinct phenotypic featuresFfor instance, EEC growth from PV progenitor cells.21 Furthermore, in vivo JAK2V617F is associated with predominant erythrocytosis expression of JAK2V617F in a murine bone marrow transplant (neutrophilia is seen only in the Balb/C background); in contrast, (BMT) assay produces an MPD phenotype resembling PV in MPLW515L is associated with marked thrombocytosis (and these animals.34–37 The degree of JAK2V617F overexpression increased bone marrow reticulin fibrosis).21,34–37,39 This con- relative to endogenous wild-type JAK2 (JAK2WT) is relatively trasts with expression of most other activated tyrosine kinases modest (2- to 3-fold) in this model.36 Although the murine BMT (for example, BCR-ABL p210, TEL-JAK2, FLT3-ITD or FIP1L1- model suggests that JAK2V617F may be sufficient for PV PDGFRA) in a similar assay, which results in a polyclonal MPD expression, it does not address other key issues, including the of short latency, that is characterized by a predominant extent to which differences in JAK2V617F dosage overproduction of maturing .51,53–55 determine which MPD is expressed (for example, PV or ET with higher or lower JAK2V617F levels, respectively).26,38 While JAK2V617F is the predominant disease-associated allele in JAK2 inhibitor development MPD, it is becoming clear that other alleles may also be At present, several groups are actively developing orally involved. In general, the novel allele, when present, is found in bioavailable, small-molecule inhibitors of JAK2 kinase. Broadly MPD cases that are JAK2V617F negative (for example, speaking, JAK2 inhibitors may be classified as ‘JAK2-selective’ MPLW515L or MPLW515K),39 although in rare cases, these (Class I) or ‘non-JAK2 selective’ (Class II) depending on whether alleles may occur in conjunction with JAK2V617F (for example, or not the particular inhibitor was developed to primarily target JAK2D620E, MPLW515L/K).40–42 A similar co-occurrence of JAK2 kinase (including JAK2V617F). Class II inhibitors were BCR-ABL and JAK2V617F alleles has also been described in primarily developed for non-MPD indications but are still MPD patientsFthe clinical phenotype in these patients (that is, considered to have therapeutic potential in MPD given their chronic myeloid leukemia (CML) versus PV, for instance) may significant ‘off-target’ JAK2 kinase-inhibitory activity. For Class I depend on which mutant clone is dominant.43,44 The MPLW515 inhibitors, one strategy has been to refine an existing tyrphostin mutations are restricted to patients with JAK2V617F-negative (tyrosine phosphorylation inhibitor) scaffold based on available PMF (B10%) or JAK2V617F-negative ET (1%). More recently, a molecular structural data for JAK2 and JAK3 kinase series of novel somatic mutations in JAK2 exon 12 were domains,56,57 to rationally design compounds that selectively identified in rare cases of JAK2V617F-negative PV (representing bind to the JAK2 (relative to JAK3) kinase catalytic site at low B3% of all PV cases), particularly in those exhibiting a nanomolar concentrations.58,59 Such a strategy yields com- relatively isolated erythrocytosis, rather than the panmyelosis pounds that are potent inhibitors of both wild-type (JAK2WT) that typically characterizes PV.45,46 Other activating alleles of and mutant (JAK2V617F) alleles. There are efforts underway, JAK kinases have also been described recently in acute however, to develop cell-based screens that will identify allele- megakaryoblastic leukemia cell lines, namely JAK2T875N47 specific inhibitors of JAK2V617FFthese have the potential for a and JAK3A572V.48 While these particular alleles have not been more optimal therapeutic window since only the disease allele detected in patients as yet, they illustrate the potential for will be inhibited. additional activating alleles involving JAK family members.

Pre-clinical studies Pre-clinical studies There scant data if any in the public domain pertaining to the pre-clinical testing of JAK2 inhibitors, particularly for the Class I JAK2V617F-activated signal transduction inhibitors that have recently entered clinical trials. Currently JAK2V617F appears to activate the same downstream signaling available data for JAK2 inhibitors are summarized in Table 1. pathways as does JAK2WT in response to Epo stimulation, Early studies on the effects of JAK2V617F inhibition made use including STAT, extracellular signal-regulated kinase/mitogen- of tool compounds (for example, AG-490, JAK inhibitor I)65 that

Leukemia JAK2 inhibitor therapy in MPD A Pardanani 25 Table 1 Pre-clinical testing of JAK2 inhibitors

Compound/drug JAK2 IC50 (nM) HEL IC50 (nM) JAK2/JAK3 IC50 Mouse xenograft MPD primary cells MPD patients

MK-045760 190 295 ÀÀ++ CEP-701 ()61 1 30–100 À + +(PV, ET, PMF, AML) + a p100 nM (Tarceva)62 4000 2000 X5xb À +(PV) À c 5000 nM a 10 000–50 000 nM AT928363 1.2 + À + À + TG10120958 6 152 28x + +(PV, PMF) À a 300–600 nM Go¨ 697664 130 500–1000b 1–2xb À +(AML) À d 1000 nM

Abbreviations: AML, acute myeloid leukemia; ET, essential thrombocythemia; IC50, inhibitor concentration at which 50% decrease in kinase activity or cell proliferation is observed; n/a, not available; JAK2, Janus kinase-2; MPD, myeloproliferative disorders; nM indicates nanomolar; PMF, primary myelofibrosis; PV, polycythemia vera. aWith cytokines. bApproximateFextrapolated from data. cWithout cytokines. d6975% decrease in proliferation for primary AML blasts harboring FLT3-ITD (internal tandem duplication) mutations. inhibit JAKs non-selectivelyFthese compounds, although suffi- In a mouse xenograft model with Ba/F3-V617F cells, cient for limited in vitro experiments that provide proof-of- administration of TG101209 (200 mg dayÀ1) by oral gavage principle, have no potential for further development as bona fide rescued the animals from a fully penetrant, rapidly fatal ‘drugs’ for the treatment of MPD. hematopoietic malignancy.58 A marked decrease in STAT-5 In general, laboratory studies with JAK2 inhibitors have phosphorylation was evident in splenic tumors following employed the following experimental systems: (i) cell lines administration of a single dose (100 mg kgÀ1) of TG101209, known to harbor JAK2V617F (for example, human erythroleu- and in vivo therapeutic efficacy was correlated with a significant kemia cells) or that are engineered to express the mutant kinase decrease in the circulating tumor burden in these animals. In an (for example, Ba/F3-V617F); (ii) culture of hematopoietic independent murine BMT model of PV, the continuous progenitor cells from JAK2V617F-positive MPD patients; and parenteral administration of AG-490 by subcutaneous infusion (iii) in vivo studies using murine models of MPDFeither a over a 2-week period resulted in a significant decrease in the xenograft model or JAK2V617F expression in a murine retroviral hematocrit and reticulocyte count in recipients of JAK2V617F- bone marrow transduction and transplantation assay. (but not JAK2WT) transduced bone marrow.36 Interestingly, a The growth of cell lines harboring JAK2V617F or MPLW515L similar effect was seen with imatinib, but not with is inhibited at nanomolar concentrations by the JAK2-selective (Sprycel), administered by oral gavage in this model, suggesting inhibitors TG101209 and TG101348.58 The JAK2/JAK3 selec- that Src kinase-directed therapies may not be efficacious for the tivity of these inhibitors has been assessed using leukemic cell treatment of PV. While the aforementioned studies are lines that harbor either JAK2- or JAK3-activated allelesFCHRF informative, they do not provide a detailed characterization of (CHRF-288-11) cells (JAK2T875N) are observed to be more JAK2 inhibitor effects in mouse MPD models, particularly with potently inhibited than CMK cells (JAK3A572V) in such regard to disease-relevant end points including blood counts, experiments. organomegaly, extramedullary hematopoiesis, bone marrow The effect on primary cells has been assessed by culturing pathology and progenitor-cell function, although it is expected progenitor cells in the presence of JAK2 inhibitors. In experi- that such data will be forthcoming in the near future. ments with TG101209 and/or TG101348, we have observed the following:58 (i) progenitors from MPD patients may be more sensitive to JAK2 inhibition than those from healthy controls, Clinical trials consistent with a greater dependence on JAK2 signaling in the SPOTLIGHT former; (ii) EEC growth is inhibited at nanomolar concentrations, Clinical trials with small-molecule JAK2 inhibitors that are with preliminary data suggesting that some mutant alleles are ongoing or in development are summarized in Table 2. All trials more sensitive to inhibition than others (MPLW515L/KDJAK2 with Class I (JAK2-selective) inhibitors are first-in-human exon 12 mutation4JAK2V617F) (A Pardanani, unpublished studiesFthe following issues are pertinent from the standpoint observations). The corresponding Epo-supported erythroid of clinical trials with this group of inhibitors: colonies are less potently inhibited, reflecting the generally lower prevalence of JAK2 mutations in these colonies relative to EECs; (iii) EEC growth is also suppressed in patients who do not Which of the MPD patient groups is most suited for harbor a known MPD-associated mutation, indicating that the initial (Phase I) studies with JAK2 inhibitors? Should abnormal MPD clone in such cases is as dependent on eligibility be restricted to those patients who are deregulated JAK–STAT signaling as those with known JAK2 or JAK2V617F positive? MPL mutations; and (iv) in at least some cases, exposure to Until the toxicity profile including dose-limiting toxicities inhibitor results in the selective suppression of mutation- become apparent from ongoing Phase I studies, it is reasonable harboring coloniesFin these cases, it is possible however that to restrict use of newer JAK2-selective inhibitors in the a fraction of the surviving colonies (JAK2WT) reflect persistence investigational setting to patients with ‘high-risk’ MPD. Such of a ‘pre-JAK2’ MPD clone that is less sensitive to JAK2 novel therapies are particularly appealing for patients with inhibition. ‘intermediate-risk’ or ‘high-risk’ PMF (by standard criteria, for

Leukemia JAK2 inhibitor therapy in MPD A Pardanani 26 Table 2 Clinical trials with JAK2 inhibitors

Inhibitor Company Phase in Indication Route Primary target development

JAK2-selective INCB018424 Incyte Phase I/II (open) PMFa Oral JAK2 XL019 Exelixis Phase I (open) PMFa Oral JAK2 TG101348 TargeGen Phase I/II PMFa Oral JAK2 (pending)

Non-JAK2 selective MK-0457 (VX680) Merck Phase I (open) Relapsed/refractory hematological malignancies, i.v. Aurora kinases Phase II (open) including JAK2V617F-positive MPD (A, B, C) CML or Ph+ ALL with T315I mutation CEP-701 Cephalon Phase II (open) PMFa Oral FLT3 (Lestaurtinib) AT9283 Astex Phase I/II (open) Acute , CML, high-risk MDS or PMF i.v. Aurora kinases Therapeutics Abbreviations: ALL, acute lymphoblastic leukemia; CML, chronic myelogenous leukemia; EGFR, epidermal ; i.v., intravenous; JAK2, Janus kinase-2; MDS, myelodysplastic syndrome; MPD, myeloproliferative disorder; Ph, ; PMF, primary SPOTLIGHT myelofibrosis. aRelapsed/refractory PMF, if newly diagnosed, then must have intermediate or high-risk PMF (Mayo Clinic or Dupriez criteria),66,67 or symptomatic splenomegaly X10 cm below costal margin.

example, Mayo Clinic or Dupriez),66,67 given the significant common ancestral ‘pre-JAK2’ clone.76,77 Consequently, it disease-related morbidity in this setting (cachexia/other consti- appears unlikely at the present time that JAK2 inhibitors will tutional symptoms, hepatosplenomegaly, extramedullary hema- have a central role in treating JAK2V617F-negative AML that topoiesis, symptomatic anemia, and so on) and the poor evolves from pre-existing JAK2V617F-positive MPD, although it prognosis (median survival of 2–3 years) of these patients.66,67 should be pointed out that STAT-3 and STAT-5a/b activation in At present, there is virtually no effective treatment for these leukemic blasts has been described previously.78 patients, although recently introduced immunomodulatory drugs have shown promise68Fconventional therapies are palliative in intent (for example, prevention of thrombohemor- How should treatment responses be assessed in these rhagic complications), and do not alter the natural history of studies? Which correlative studies are likely to be the disease or prevent clonal evolution. Furthermore, only a most useful in this regard? minority of patients are eligible for potentially curative Treatment responses are assessed using a combination of (i) allogeneic stem cell transplants after either fully myeloabla- disease-specific clinical measures and (ii) biological surrogates tive69,70 or reduced-intensity conditioning.71,72 In contrast to that reflect inhibition of disease-related molecular signaling (that patients with PMF, those with ET or PV have a considerably is constitutive JAK2 kinase activity) and/or suppression of the longer life expectancy (median survival exceeds 20 years with abnormal hematopoietic clone. In the case of PMF, the recently conventional therapy, with some combination of phleboto- proposed International Working Group consensus criteria my7low-dose aspirin7hydroxyurea).73,74 Consequently, it is capture traditional measures of response for this diseaseFcli- difficult at present to justify the initial use of JAK2 inhibitors in nical (that is, resolution of disease-related symptoms and signs, unselected ET or PV patients until the safety and tolerability of including hepatosplenomegaly), hematologic (that is, normal- these agents at therapeutically active doses has been confirmed ization of peripheral blood counts, and resolution of leukoer- in higher risk MPD patients. It may be possible, however, to ythroblastosis) and histologic (that is, resolution of identify PV or ET patients at high risk for myelofibrotic and/or osteomyelofibrosis).79 Although not incorporated into the formal leukemic transformation as potential candidates for up-front response criteria, the International Working Group document JAK2 inhibitor therapy.75 emphasizes the importance of collecting information on Up to 50% of ET and PMF patients and 5% of PV patients are cytogenetic and/or molecular markers in PMF patients, if JAK2V617F negativeFalternative mutant alleles (some yet to be available, both at baseline and during follow-up. This supple- identified) constitutively activate JAK2 signaling to account for mentary information is likely to become increasingly important the myeloproliferation observed in these patients. Limited in the near future as the various biologic markers are validated in vitro data suggest that JAK2-selective inhibitors suppress in a prospective fashion to track clinically meaningful responses. hematopoietic colony growth even in the absence of Given the availability of reliable quantitative assays, monitor- JAK2V617FFin such assays, progenitors harboring ing of JAK2V617F mutation burden is feasible, and will be an MPLW515L/K mutations (or no identifiable mutation) appear important element of JAK2 inhibitor trials, to assess the depth of to be inhibited at least as potently as those harboring response, as well as the extent to which suppression of the JAK2V617F.58 These observations raise the possibility that most, mutant clone correlates with clinical responses. Such assays if not all, MPD patients in ‘chronic phase’ may ultimately qualify have already been used to assess the molecular response to for JAK2 inhibitor-based therapy, at least from the standpoint of treatment of PV to interferon80–82 or after allogeneic stem cell disease mechanism. In the case of leukemic transformation of transplantation.83 A similar response has been observed with MPD however, the leukemic blasts in most instances are found lenalidomide (Revlimid) therapy in a patient who concurrently to be JAK2V617 negative, which indicates that both the MPD harbored the del(5)(q31) cytogenetic abnormality.84 clone and the subsequently evolved AML clone (that are positive In patients without identifiable cytogenetic or molecular and negative for JAK2V617F, respectively) are derived from a abnormalities, other assays such as EEC growth or X-chromo-

Leukemia JAK2 inhibitor therapy in MPD A Pardanani 27 some inactivation pattern analysis in informative females, which 60 months, only 4% of patients initially assigned to the imatinib are although more cumbersome and less reliable than treatment arm discontinued treatment due to an adverse event. JAK2V617F monitoring, may provide additional information These data suggest that significant mutant-over-wild-type regarding treatment-related effects on the abnormal MPD clone. selectivity may not be essential for therapeutic efficacy of Inhibition of JAK2 (and JAK2V617F) kinase-induced tyrosine small-molecule tyrosine kinase inhibitors. Recent in vitro studies phosphorylation can also be monitored in biological specimens with the JAK2-selective inhibitor TG101209 indicates a modest collected during the course of clinical trials with JAK2 inhibitors, ‘therapeutic window’, wherein progenitor cells from although such assays are more cumbersome than monitoring of JAK2V617F-positive MPD patients are observed to be more JAK2V617F allele burden. In this regard, it may be feasible to sensitive to TG101209’s inhibitory effects than progenitors from follow substrates of JAK2 kinase, akin to the monitoring of CRKL healthy controls.58 phosphorylation as a surrogate for BCR-ABL kinase activity MK-0457 (formerly, VX-680) is a small-molecule inhibitor of in neutrophils of CML patients undergoing treatment with the Aurora kinases (AUR A, B and C)92 that is also active imatinib.85 The degree to which STAT-5 and/or STAT-3 (nanomolar potency) against a small set of wild-type and mutant phosphorylation is inhibited in peripheral blood cells that are tyrosine kinases, including BCR-ABL/ABL,93 JAK260 and FLT3. stimulated ex vivo with an appropriate cytokine (for example, MK-0457 is currently being studied in patients with CML or IL-6, granulocyte-macrophage colony stimulating factor) can be Ph þ acute lymphocytic leukemia who harbor the T315I quantitatively monitored by phospho-flow analysis in patients mutation (Phase II), as well as in patients with relapsed/ treated with JAK2 inhibitors in ongoing clinical trials. refractory leukemias, including those with refractory or trans- formed JAK2V617F-positive MPD (Phase I).60,94,95 Although dose-dependent myelosuppression was seen at all dose levels What are the predicted class-specific toxicities of JAK2- studied (8–32 mg mÀ2 hÀ1 administered by a continuous 5-day selective inhibitors? What are the likely consequences intravenous infusion), no extramedullary XGrade 3 adverse of JAK3 inhibition?y. of wild-type JAK2 inhibition? events attributable to MK-0457 were noted in preliminary What have we learned from studies with non-JAK2 reports from these studies. selective inhibitors? CEP-701 (Lestaurtinib) is a small-molecule inhibitor of TRKA Detailed information regarding inhibitor effects either from that was originally developed for possible use in prostate . animal toxicology studies or from use in human subjects is It was subsequently recognized to have FLT3-inhibitory activity presently unavailable because such information is proprietary (nanomolar potency),96 and was consequently evaluated in a and/or preliminary. Until such information becomes publically Phase I/II study in heavily pre-treated patients with refractory/ available, concerns relating to the safety of JAK2-selective relapsed or poor-risk AML who harbored FLT3-activating inhibitors in MPD patients will persist. In considering potential mutations.97 In both this study and a subsequent Phase II study class-specific toxicities, these concerns relate primarily to in older AML patients,98 CEP-701 was relatively well tolerated potential untoward effects resulting from inhibition of (i) wild- (commonest reported adverse event was mild GI intolerance); type JAK2 and/or (ii) JAK3. however, only modest clinical efficacy was observed in these The JAK family members mediate signaling from enzymati- patients. More recently, CEP-701 has been reported to inhibit cally deficient type I/II cytokine receptors, which bind over 50 JAK2 kinase (IC50 ¼ 1nM in kinase assays) and also inhibit the different cytokines that regulate a broad range of physiologic proliferation of progenitor cells from MPD patients in vitro.61 An processes in mammals ranging from body growth to adiposity, ongoing Phase II study is examining the efficacy of CEP-701 in and include hematopoiesis and innate/adaptive immune re- PMF patients. sponses (reviewed by O’Shea et al.86). The cytokines are CP-690,550 is an orally bioavailable JAK3 inhibitor with subdivided based on their selective use of shared receptor nanomolar potency that is currently in clinical trials in renal subunits, including the (gc) subunit transplant patients for prevention of acute rejection and in (includes IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21) and gp130 as a novel immunosuppressive agent. It was subunit (includes IL-6, IL-11, , G-CSF and so on). shown previously to prolong survival in animal models of heart Within cells, the JAKs associate with specific cytokine receptors and allotransplantation.99 In animal studies, dose- (for example, JAK3 with gc, and JAK1 with gc or gp130 receptor dependent anemia was commonly seen, which is consistent subfamilies, respectively) once cytokines have bound to their with its known ‘off-target’ JAK2-inhibitory activity (20-fold less cognate receptors. While JAK1, JAK2 and TYK2 are ubiquitously JAK2 inhibition as compared to JAK3)99Finterestingly, it was SPOTLIGHT expressed, the expression of JAK3 appears to be restricted to possible to overcome the suppressive effect on erythropoiesis by hematopoietic cells. The generation of knockout mice has administering exogenous Epo in cynomolgus monkeys that were helped elucidate JAK functionsFJAK1-deficient mice are viable exposed to lower levels of the drug.100 Chronic exposure to but die in the early post-natal period due to failure to nurse;87 CP-690,550 has been reported to cause changes in immune JAK2 deficiency is embryonically lethal due to failure of cell subsets in rodent101 and monkey allotransplant models,102 definitive hematopoiesis;17,18 JAK3-deficient mice are viable an effect that is reflected primarily in reduced numbers and fertile but display a severe combined immunodeficiency of natural killer cells and CD8 þ T lymphocytes; in contrast, phenotype.88 B- numbers may be relatively preserved. Indeed, there were concerns during the early development of Data from the aforementioned studies suggest that dose- imatinib mesylate (Gleevec) for the treatment of CML that ‘off- dependent cytopenias may be commonly observed, and could target’ inhibition of non-BCR-ABL tyrosine kinases (c-ABL, represent the dose-limiting toxicity in Phase I studies with JAK2- PDGFR, KIT, ARG) may lead to undue toxicity89Fin this selective inhibitors. Such a finding would not be surprising given regard, it was known for instance that c- knockout mice the known importance of JAK2WT signaling in normal exhibit decreased neonatal viability as well as lymphopenia.90 hematopoiesis. It may turn out however, as underscored by Clinical experience with imatinib however reveals it to be a the experience with imatinib in CML, that significant mutant- remarkably safe agentFin their latest update of IRIS study over-wild-type selectivity may not be necessary for therapeutic patients, Druker et al.91 report that after a median follow-up of efficacy with this class of kinase inhibitors. Moreover, the Class I

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