The Development and Use of Janus Kinase 2 Inhibitors for the Treatment of Myeloproliferative Neoplasms




Following the discovery of the JAK2V617F mutation, Janus kinase (JAK) 2 inhibitors were developed as rationally designed therapy in myeloproliferative neoplasms (MPNs). Although JAK2 inhibitors have clinical efficacy in MPN, they are not clonally selective for the JAK2V617F-mutant cells. Because activated JAK-signal transducer and activator of transcription (STAT) signaling is a common feature of MPN, JAK2 inhibitors are efficacious regardless of the specific MPN phenotypic driver mutation. The Food and Drug Administration approved the JAK1/JAK2 inhibitor, ruxolitinib, for the treatment of myelofibrosis and polycythemia vera. Additional JAK2 inhibitors are currently in advanced phased clinical trials.


Key points








  • Janus kinase (JAK) 2 inhibitors were developed as rationally designed therapy in myeloproliferative neoplasms (MPNs) following the discovery of the activating JAK2V617F mutation.



  • The oral JAK1/JAK2 inhibitor ruxolitinib is approved by the Food and Drug Administration for the treatment of intermediate and advanced phase myelofibrosis and in certain cases of polycythemia vera.



  • Activated JAK-signal transducer and activator of transcription (STAT) signaling is a central feature of MPN and, as a result, JAK2 inhibitors have clinical efficacy regardless of the type of MPN phenotypic driver mutation.



  • Although providing clinical benefit to MPN patients, JAK2 inhibitors are not strongly clonally selective for either JAK2V617F-mutant or CALR -mutant MPN cells.



  • Despite an absence of clonal selectivity for MPN cells and no difference in the rate of leukemic transformation, ruxolitinib seems to improve overall survival in myelofibrosis.






Introduction


The discovery of the JAK2V617F mutation in patients with myeloproliferative neoplasms (MPNs) launched a new era of rationally designed molecularly targeted therapy in BCR-ABL negative MPN. The JAK2V617F mutation, which activates Janus kinase (JAK)-2 signaling, is present in more than 95% of patients with polycythemia vera (PV), approximately 65% of patients with myelofibrosis (MF), and 55% of patients with essential thrombocythemia (ET). Improved understanding of the molecular biology of MPN has established activated JAK-signal transducer and activator of transcription (STAT) signaling, driven by JAK2V617F, MPLW515L/K, or mutant calreticulin ( CALR ) at the center of MPN pathogenesis, establishing the JAK-STAT pathway as a key therapeutic target in these diseases ( Fig. 1 ). The thrombopoietin receptor, MPL is mutated in between 1% and 5% of MPN cases, leading to cytokine independent growth and activated JAK-STAT signaling. More recently, somatic mutations were discovered in the gene calreticulin ( CALR ) in 20% to 25% of ET and MF patients. Calreticulin is a calcium-binding chaperone protein that localizes to the endoplasmic reticulum (ER) under normal conditions. More than 30 different mutations in CALR have been identified and all result in a +1 base pair frameshift that leads to the generation of a new C-terminal peptide in the mutant CALR protein that lacks the KDEL ER retention signal. Recent work has demonstrated that mutant CALR activates JAK-STAT signaling through physical interaction with MPL consistent with the observation that JAK2 , MPL , and CALR mutations are typically mutually exclusive in MPN.




Fig. 1


JAK2-STAT signaling pathway activation in MPN. ( A ) Normally, JAK2-STAT signaling pathway activation occurs through ligand binding to and active dimerization of type 1 cytokine receptors (eg, MPL, EPOR, or GM-CSFR). Activated STAT translocates to the nucleus, where it binds promoters upregulating proliferation and cell survival genes. ( B ) The activating mutation V617F in JAK2 leads to constitutive activation of JAK2-signaling independent of ligand binding. ( C ) Mutant CALR physically interacts with MPL to activate the MPL signaling pathway in a thrombopoietin-independent manner. ( D ) Mutation to MPL at amino acid 515 causes constitutively active MPL signaling. Note: Purple receptor denotes any type 1 cytokine receptor; blue receptors denote MPL.


In addition to the 3 main MPN phenotype driver mutations, a few other mutations have been identified that also activate JAK-STAT signaling. For example, mutations in negative regulators of JAK2 , such as CBL and LNK , have been described. There is also a small group of patients without identifiable MPN phenotype driver mutations, the so called triple-negative patients, and additional germline and somatic mutations in JAK2 or MPL were recently identified in approximately 19% of these cases.


Following the discovery of the JAK2V617F mutation in 2005, JAK2 inhibitors were rapidly developed and, in 2011, the Food and Drug Administration (FDA) approved the oral JAK1/2 inhibitor ruxolitinib for the treatment of intermediate and advanced phase MF. Since then, several other JAK inhibitors have entered advanced phase clinical trials. This article discusses the development and use of JAK inhibitors in MPN over the past decade.




Introduction


The discovery of the JAK2V617F mutation in patients with myeloproliferative neoplasms (MPNs) launched a new era of rationally designed molecularly targeted therapy in BCR-ABL negative MPN. The JAK2V617F mutation, which activates Janus kinase (JAK)-2 signaling, is present in more than 95% of patients with polycythemia vera (PV), approximately 65% of patients with myelofibrosis (MF), and 55% of patients with essential thrombocythemia (ET). Improved understanding of the molecular biology of MPN has established activated JAK-signal transducer and activator of transcription (STAT) signaling, driven by JAK2V617F, MPLW515L/K, or mutant calreticulin ( CALR ) at the center of MPN pathogenesis, establishing the JAK-STAT pathway as a key therapeutic target in these diseases ( Fig. 1 ). The thrombopoietin receptor, MPL is mutated in between 1% and 5% of MPN cases, leading to cytokine independent growth and activated JAK-STAT signaling. More recently, somatic mutations were discovered in the gene calreticulin ( CALR ) in 20% to 25% of ET and MF patients. Calreticulin is a calcium-binding chaperone protein that localizes to the endoplasmic reticulum (ER) under normal conditions. More than 30 different mutations in CALR have been identified and all result in a +1 base pair frameshift that leads to the generation of a new C-terminal peptide in the mutant CALR protein that lacks the KDEL ER retention signal. Recent work has demonstrated that mutant CALR activates JAK-STAT signaling through physical interaction with MPL consistent with the observation that JAK2 , MPL , and CALR mutations are typically mutually exclusive in MPN.




Fig. 1


JAK2-STAT signaling pathway activation in MPN. ( A ) Normally, JAK2-STAT signaling pathway activation occurs through ligand binding to and active dimerization of type 1 cytokine receptors (eg, MPL, EPOR, or GM-CSFR). Activated STAT translocates to the nucleus, where it binds promoters upregulating proliferation and cell survival genes. ( B ) The activating mutation V617F in JAK2 leads to constitutive activation of JAK2-signaling independent of ligand binding. ( C ) Mutant CALR physically interacts with MPL to activate the MPL signaling pathway in a thrombopoietin-independent manner. ( D ) Mutation to MPL at amino acid 515 causes constitutively active MPL signaling. Note: Purple receptor denotes any type 1 cytokine receptor; blue receptors denote MPL.


In addition to the 3 main MPN phenotype driver mutations, a few other mutations have been identified that also activate JAK-STAT signaling. For example, mutations in negative regulators of JAK2 , such as CBL and LNK , have been described. There is also a small group of patients without identifiable MPN phenotype driver mutations, the so called triple-negative patients, and additional germline and somatic mutations in JAK2 or MPL were recently identified in approximately 19% of these cases.


Following the discovery of the JAK2V617F mutation in 2005, JAK2 inhibitors were rapidly developed and, in 2011, the Food and Drug Administration (FDA) approved the oral JAK1/2 inhibitor ruxolitinib for the treatment of intermediate and advanced phase MF. Since then, several other JAK inhibitors have entered advanced phase clinical trials. This article discusses the development and use of JAK inhibitors in MPN over the past decade.




Preclinical development of Janus kinase inhibitors


Following the discovery of the JAK2V617F mutation, the effects of the mutation on hematopoiesis were quickly modeled in mice. Using a retroviral bone marrow transplant (BMT) model, it was shown that JAK2V617F alone was sufficient to engender MPN in mice, thus validating the mutation as a key molecular target in MPN. In parallel, JAK2 inhibitors were rapidly developed by several pharmaceutical companies and were soon shown to be safe and efficacious in the treatment of JAK2V617F-driven MPN in mice and, subsequently, in MPLW515L retroviral BMT MPN mouse models. Later, using a genetic Jak2V617F knockin mouse model, it was demonstrated that JAK2 inhibitors, although effective at reducing blood counts and splenomegaly, did not effectively target disease-propagating MPN stem cells, a finding that was subsequently validated in clinical trials, in which JAK2 inhibitors have been disappointing in their ability to induce molecular remissions in MPN subjects. More recently, the JAK inhibitor ruxolitinib was demonstrated to be efficacious in a transgenic model of mutant CALR, although effects on mutant CALR allele burden were not measured. Genetic knockout of Jak2 in a retroviral MPLW515L MPN mouse model was shown to be superior to JAK2 inhibitor treatment, suggesting that more potent JAK2 inhibition could enhance clinical efficacy in MPN patients. However, JAK2 signaling is also required for normal hematopoietic stem cell (HSC) function, as shown in a series of studies in which severe cell-intrinsic defects in HSC function, impaired hematopoiesis, and reduced survival were demonstrated following hematopoietic-specific conditional genetic deletion of Jak2 in adult mice. These results have raised concerns regarding the potential for on-target hematological toxicity from more potent JAK2 inhibitors and reinforced efforts to develop JAK2V617F mutant-specific inhibitors. Combining ruxolitinib therapy with heat shock protein 90 (HSP90) inhibition was also shown to be efficacious in preclinical MPN mouse models. This resulted in a phase II clinical trial of the HSP90 inhibitor, AUY922 in subjects with MF, which was terminated early due to excess gastrointestinal toxicity. However, despite the toxicity, all 6 subjects treated in the trial experienced at least a partial response and 1 subject remained on study for longer than 1 year.




Clinical development of Janus kinase inhibitors


Ruxolitinib for Myelofibrosis


Ruxolitinib is a JAK1/2 inhibitor approved for use in patients with intermediate-II and high risk MF. Ruxolitinib was approved based on 2 randomized phase III studies: Controlled Myelofibrosis Study with Oral JAK Inhibitor Treatment (COMFORT) I and II. COMFORT I compared ruxolitinib to placebo and COMFORT II compared ruxolitinib it to best available therapy (BAT). The primary endpoint of these studies was spleen volume reduction of greater than or equal to 35% at 24 weeks for the COMFORT I study and at 48 weeks for the COMFORT II study. Secondary endpoints included durability of response, improvement in symptoms as measured by the MF symptom assessment form (SAF), and overall survival.


In both studies, ruxolitinib led to durable symptom and spleen volume reduction. The MPN SAF total symptom score (TSS) was used to monitor changes in symptoms in the COMFORT I study. In the COMFORT II study, several quality of life and symptom measurement tools were used, including the European Organization for Research and Treatment of Cancer (EORTC) quality-of life questionnaire core model (QLQ-C30) and the Functional Assessment of Cancer Therapy-Lymphoma (FACT-Lym) scale. In COMFORT I, 45.9% of subjects experienced a greater than 50% improvement in the MPN SAF TSS at 24 weeks, compared with 5.3% of subjects receiving placebo in COMFORT I. Significant and consistent improvements in symptoms were also found in the COMFORT II study in subjects receiving ruxolitinib compared with worsening symptoms in subjects receiving best available therapy (BAT). Based on the remarkable benefit of ruxolitinib in alleviating MF-related symptoms, the revised International Working Group (IWG) for Myelofibrosis Research and Treatment and the European LeukemiaNet incorporated the term “clinical improvement” to the response criteria.


Three-year pooled follow-up of these studies has shown a statistically significant improvement in overall survival. Five-year pooled follow-up of the COMFORT II study showed that, among patients achieving a greater than or equal to 35% spleen volume reduction, the probability of maintaining response was 0.48 (95% confidence interval, 0.35–0.60) at 5 years (median 3.2 years). The median overall survival was not reached in ruxolitinib treated subjects and was 4.1 years in BAT subjects. No new adverse events were reported with longer follow-up.


At the final 5-year follow-up of the COMFORT studies, the mean spleen volume reduction for subjects who remained on ruxolitinib was 37.6% at 264 weeks, at this time 18.5% of subjects randomized to ruxolitinib maintained a greater than or equal to 35% reduction in spleen volume compared with baseline. Median duration of this response was 168.3 weeks.


The most common toxicities hematologic toxicities of ruxolitinib are thrombocytopenia and anemia. The initial drop in platelets and hemoglobin is generally most pronounced in the first month of therapy and improves or stabilizes thereafter. The starting dose for ruxolitinib is based on platelet count. Subjects with a platelet count of greater than 200 × 10 9 /L start at 20 mg twice a day, those with platelets between 100 to 200 × 10 9 /L start at 15 mg twice a day, and those with platelets between 50 to 99 × 10 9 /L start at 5 mg twice a day. Ruxolitinib is not recommended for patients with lower platelet values. Ruxolitinib dosing should not be adjusted in the first 4 weeks of therapy and, thereafter, not more often than every 2 weeks.


The most common nonhematologic adverse events reported in patients using ruxolitinib, include diarrhea and peripheral edema, but these were minor. Of particular concern are the potential infectious complications associated with ruxolitinib because this drug is immunosuppressive. In COMFORT I, herpes zoster occurred in 10.3% of subjects randomized to ruxolitinib and 13.5% of subjects who crossed over to ruxolitinib treatment. In COMFORT II, the rates of urinary tract infection were 24.6%, pneumonia, 13.1%; herpes zoster reactivation, 11.5%; sepsis, 7.9%; and tuberculosis 1%. In addition, there was 1 report of progressive multifocal leukoencephalopathy and a case of cytomegalovirus retinitis.


Based on the rarity of nonhematologic events, cytopenias are undoubtedly the most common reason for treatment discontinuation and limit which patients can initiate this drug. A recent study demonstrating that Jak2 is not required for terminal megakaryopoiesis and platelet production in mice suggests that ruxolitinib-related thrombocytopenia may occur as a result of JAK-STAT inhibition upstream in the hematopoietic hierarchy, namely in stem and/or progenitor cells, rather than in megakaryocytes and platelets.


Another significant concern with ruxolitinib use is the potential for rapid rebound of symptoms and splenomegaly on discontinuation. This is of particular concern in patients who are approaching transplantation as optimal timing or necessity of discontinuation before transplant remains unknown. However, a trial is planned to address the feasibility of maintaining subjects on ruxolitinib during the conditioning and early pretransplant period to address this issue.


Although ruxolitinib represents a major treatment advance for patients with MF, this drug is associated with a high discontinuation rate mainly due to loss of efficacy and cytopenias. In addition, ruxolitinib does not seem to alter the disease biology of MF and leads to small changes in JAK2V617F allele burden and in bone marrow fibrosis. A recent analysis of patients on ruxolitinib revealed that, of the 236 JAK2V617F mutant subjects in the COMFORT studies, only 20 subjects achieved partial and 6 subjects achieved complete molecular remissions, with a median time to respond of 22.2 and 27.5 months, respectively. Moreover, ruxolitinib does not eliminate risk of progression to acute leukemia. Long-term follow-up from the COMFORT II study shows similar rates of progression to acute leukemia in ruxolitinib-treated subjects compared with those who received BAT.


It should be noted that ruxolitinib is efficacious for MF patients regardless of their MPN phenotypic driver mutational status because activated JAK-STAT signaling is a common feature of MPN and ruxolitinib is not a JAK2V617F mutant-specific inhibitor. However, JAK-STAT inhibition in normal hematopoietic cells also explains some of the on-target toxicity of ruxolitinib; for example, anemia.


There has been a lot of speculation about why ruxolitinib is associated with a survival advantage despite its lack of clonal selectivity for JAK2V617F-mutant or CALR -mutant cells and its limited disease-modifying activity. Ruxolitinib is associated with a reduction in circulating cytokines that may be responsible for the reduction in symptoms and spleen size. This in turn leads to improvement in functional status, which may then lead to improved outcomes. It is unclear if ruxolitinib has additional effects on halting disease progression through other as yet undefined mechanisms. Notably, the use of historical control cohorts for survival analysis comparisons has not helped in trying to clarify this issue. The advantages and disadvantages of ruxolitinib in the treatment of MF are summarized in Fig. 2 and ongoing studies are summarized in Table 1 .




Fig. 2


Summary of the pros and cons of JAK2 inhibitor treatment in MPN patients.


Table 1

Ongoing studies using ruxolitinib for myelofibrosis




























































































































Drug Drug Class Phase Trial Primary Endpoint
Studies with ruxolitinib
Ruxolitinib JAK1/2 inhibitor IB NCT01317875 Determine dose for MF patients with platelets <100×10 9 /L
II NCT01795677 Before allogeneic transplant safety or efficacy
III NCT02598297 Early MF with high-risk mutations
I-II NCT02806375 With post-transplant cyclophosphamide in subjects with MF
II–III NCT02962388 ET-ruxolitinib vs BAT failure-free time
III NCT02962388 ET and PV-ruxolitinib vs BAT efficacy
Combination studies
Ruxolitinib + INCB050465 PI3K-delta inhibitor II NCT02718300 Safety or efficacy
Ruxolitinib + TGR-1202 PI3 kinase inhibitor I NCT02493530 DLT, safety
Ruxolitinib + Idelalisib PI3K delta kinase inhibitor IB NCT02436135 DLT, safety
Ruxolitinib + Vismodegib Hedgehog inhibitor I-II NCT02593760 Efficacy
Ruxolitinib + PIM447 PIM inhibitor IB NCT02370706 DLT, safety
Ruxolitinib + Glasdegib Hedgehog pathway inhibitor II NCT02226172 Safety, efficacy
Ruxolitinib and Decitabine Hypomethylating agent I-II NCT02076191 MTD, safety efficacy
Ruxolitinib and Azacitidine Hypomethylating agent II NCT01787487 Efficacy
Ruxolitinib + Pracinostat HDAC inhibitor II NCT03069326 Efficacy, tolerability
Ruxolitinib + Peg-Interferon Alpha-2a Interferon I-II NCT02742324 Safety, efficacy
Ruxolitinib + Sotatercept Soluble activin receptor type wA IgG-FC fusion protein II NCT01712308 Anemia response
Thalidomide Immunomodulatory agent II NCT03069326 Efficacy
Pomalidomide Immunomodulatory agent IB-II NCT01644110 Efficacy
Idelalisib P13Kδ inhibitor I NCT02436135 Safety

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Sep 14, 2017 | Posted by in HEMATOLOGY | Comments Off on The Development and Use of Janus Kinase 2 Inhibitors for the Treatment of Myeloproliferative Neoplasms

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