Recent Advances in Molecular Diagnostics and Targeted Therapy of Myeloproliferative Neoplasms

Somatic mutations in JAK2, calreticulin, and MPL genes drive myeloproliferative neoplasms (MPN), and recent technological advances have revealed a heterogeneous genomic landscape with additional mutations in MPN. These mainly affect genes involved in epigenetic regulation and splicing and are of diagnostic and prognostic value, predicting the risk of progression and informing decisions on therapeutic management. Thus, genetic testing has become an integral part of the current state-of-the-art laboratory work-up for MPN patients and has been implemented in current guidelines for disease classification, tools for prognostic risk assessment, and recommendations for therapy. The finding that JAK2, CALR, and MPL driver mutations activate JAK2 signaling has provided a rational basis for the development of targeted JAK2 inhibitor therapies and has fueled their translation into clinical practice. However, the disease-modifying potential of JAK2 inhibitors remains limited and is further impeded by loss of therapeutic responses in a substantial proportion of patients over time. Therefore, the investigation of additional molecular vulnerabilities involved in MPN pathogenesis is imperative to advance the development of new therapeutic options. Combination of novel compounds with JAK2 inhibitors are of specific interest to enhance therapeutic efficacy of molecularly targeted treatment approaches. Here, we summarize the current insights into the genetic basis of MPN, its use as a diagnostic and prognostic tool in clinical settings, and the most recent advances in targeted therapies for MPN.

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Current Concepts of Pathogenesis and Treatment of Philadelphia Chromosome-Negative Myeloproliferative Neoplasms

Hämostaseologie ◽

10.1055/a-1447-6667 ◽

2021 ◽

Vol 41 (03) ◽

pp. 197-205

Author(s):

Franziska C. Zeeh ◽

Sara C. Meyer

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell ◽

Mutational Analysis ◽

Myeloproliferative Neoplasms ◽

Philadelphia Chromosome ◽

Standard Of Care ◽

Driver Mutations ◽

Hematopoietic Stem ◽

Therapeutic Decisions ◽

Jak2 Inhibitors

AbstractPhiladelphia chromosome-negative myeloproliferative neoplasms are hematopoietic stem cell disorders characterized by dysregulated proliferation of mature myeloid blood cells. They can present as polycythemia vera, essential thrombocythemia, or myelofibrosis and are characterized by constitutive activation of JAK2 signaling. They share a propensity for thrombo-hemorrhagic complications and the risk of progression to acute myeloid leukemia. Attention has also been drawn to JAK2 mutant clonal hematopoiesis of indeterminate potential as a possible precursor state of MPN. Insight into the pathogenesis as well as options for the treatment of MPN has increased in the last years thanks to modern sequencing technologies and functional studies. Mutational analysis provides information on the oncogenic driver mutations in JAK2, CALR, or MPL in the majority of MPN patients. In addition, molecular markers enable more detailed prognostication and provide guidance for therapeutic decisions. While JAK2 inhibitors represent a standard of care for MF and resistant/refractory PV, allogeneic hematopoietic stem cell transplantation remains the only therapy with a curative potential in MPN so far but is reserved to a subset of patients. Thus, novel concepts for therapy are an important need, particularly in MF. Novel JAK2 inhibitors, combination therapy approaches with ruxolitinib, as well as therapeutic approaches addressing new molecular targets are in development. Current standards and recent advantages are discussed in this review.

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Weight Loss, Splenomegaly, and Hypocholesterolemia in Myeloproliferative Neoplasms: Patterns and Relevance from the Pre JAK2 Inhibitor Era.

Blood ◽

10.1182/blood.v114.22.3918.3918 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3918-3918 ◽

Cited By ~ 7

Author(s):

Ruben A. Mesa ◽

Susan Schwager ◽

Jocelin Huang ◽

Animesh D. Pardanani ◽

Kebede Hussein ◽

...

Keyword(s):

Weight Loss ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Prognostic Score ◽

Univariate Analysis ◽

The Body ◽

Costal Margin ◽

Additional Time ◽

Jak2 Inhibitor ◽

Jak2 Inhibitors

Abstract Abstract 3918 Poster Board III-854 BACK GROUND We have previously demonstrated that the myeloproliferative neoplasms (MPNs) of primary myelofibrosis (PMF), polycythemia vera (PV), and essential thrombocythemia (ET) can lead to weight loss, splenomegaly and constitutional symptoms (Cancer 2007;109:68–76). Additionally we have demonstrated that hypocholesterolemia in MPN patients is associated with decreased survival (Blood 2007;110:a2548). Given that current JAK2 inhibitor trials are demonstrating the ability to reverse MPN associated splenomegaly (Haematologica 2009;94(Suppl 2)439 a1088) and cachexia (Blood 2008;112(11):a1760) we sought to determine the baseline natural history for these variables in patients treated prior to the JAK2 inhibitor era. METHODS We analyzed the Mayo MPN database for patients (not treated with JAK2 inhibitors) with information on disease prognosis, presentation, therapies, height and weight at diagnosis, and outcomes. Additionally, when available, we analyzed additional weights during the clinical course, the body mass index (BMI- (weight/(height*height)), spleen size, and peripheral blood studies including lipids. Results: Patients 783 patients with MPNs (followed for a median of 51 months (range 1-871 months); 60% having expired) were identified for the analysis (PV=158, ET=255, PMF=370) with 541 (69%) having a weight at the time of diagnosis, the remainder had a weight obtained a median of 7.8 months after diagnosis. Additionally, 508 patients (65%) had a weight value available from 1–3 additional time points during the course of their disease. Corresponding measurements of splenomegaly, or absence thereof, were noted in 766 cases (98%). Lipid panels (obtained within 18 months of diagnosis) were available in 264 patients. Results by MPN disease type are listed in the Table. Impact on prognosis Univariate analysis of variables discussed which negatively impacted survival included the subtype of MPN (not surprisingly worse for PMF p<0.001), weight loss of greater than 10% during the course of follow-up (P<0.001), or development of splenomegaly of >10 cm below the left costal margin (p=0.004) whereas hypocholesterolemia was significant only for the subset of PMF patients (P=0.03). The IWG-MRT International Prognostic Score (IPSS - Cervantes et. al. Blood 2009) was the only variable prognostically relevant in multivariate analysis (P<0.001). Conclusions Progressive splenomegaly, weight loss, and hypocholesterolemia are common across all MPNs but are most prognostically detrimental in PMF. Ongoing and future trials of JAK2 inhibitors will answer whether reversal of these latter hypercatabolic and proliferative manifestations of disease will improve outcomes for MPN patients. Disclosures: No relevant conflicts of interest to declare.

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Combination Treatment with JAK2 and PI3K Inhibitors in Myeloproliferative Neoplasms

Blood ◽

10.1182/blood.v120.21.180.180 ◽

2012 ◽

Vol 120 (21) ◽

pp. 180-180

Author(s):

Meng Ling Choong ◽

Christian Pecquet ◽

Shi Jing Tai ◽

Jacklyn WY Yong ◽

Vishal Pendharkar ◽

...

Keyword(s):

Bone Marrow ◽

Combination Treatment ◽

Kinase Inhibitors ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Pi3k Inhibitor ◽

Spleen Weight ◽

Pi3k Inhibitors ◽

Jak2 Inhibitor ◽

Jak2 Inhibitors

Abstract Abstract 180 Background and Aims. The main pathogenic molecular events associated with myeloproliferative neoplasms (Polycythemia Vera, Essential Thrombocytosis, and Primary Myelofibrosis) are mutations in Janus kinase 2 (JAK2) or in the thrombopoietin receptor that arise in the hematopoietic stem/progenitor cells. Both type of mutations lead to constitutive activation of the JAK2 signaling pathways. The approved JAK2 inhibitor (Ruxolitinib) is not expected to be selective for the mutant JAK2/receptor signaling or to completely suppress the multiple signaling pathways activated by the aberrant JAK2 signaling. We postulate that myeloproliferative neoplasms can be treated more effectively if we target the constitutive JAK2 signaling by a JAK2 inhibitor together with another kinase inhibitor targeting a specific pathway that is co-activated by the aberrant JAK2 signaling. This should increase targeting specificity, reduce JAK2 inhibitor dosages, and minimize potential side effects of these drugs. To this end, we constructed cell line models of myeloproliferative neoplasms and tested the models using a JAK2 inhibitor in combination with a panel of kinase inhibitors to identify combination pairs that give the best synergism. The synergistic pair was further confirmed in mouse models of myeloproliferative neoplasms. Methods. Mouse Ba/F3 cells were engineered to express either JAK2 WT, or JAK2 V617F, or TpoR W515L, or TpoR JAK2 WT, or TpoR JAK2 V617F, or Bcr-Abl. The effect of two JAK2 inhibitors (Ruxolitinib and TG101348) in combination with a panel of 15 various kinase inhibitors (one JNK, one B-Raf, one ROCK-1, one TIE-2, one PI3K, two CDK, two MAPK, three p38, and three mTOR inhibitors). An 8×8 constant ratio Latin square design were used for testing inhibition of cell proliferation/survival in these cell line models. Calculations were carried out using the Chou-Talalay method to determine which drug-pair demonstrated synergism in inhibiting cell growth. Further eight PI3K inhibitors were acquired and tested when we found strong synergism between the JAK2 inhibitors and the PI3K inhibitor ZSTK474 in the first panel. The engineered Ba/F3 cells were also inoculated into female BALB/c nude mice to generate the JAK2 mutant mouse model. These mice were treated intravenously with Ruxolitinib and the PI3K inhibitor GDC0941. Blood profile and physical parameters of the mice were measured for 14 days post treatment. Bone marrow cells from mice reconstituted with bone marrow from JAK2 V617F knock-in mice were plated for colony formation in the presence or absence of Ruxolitinib and the PI3K inhibitor GDC0941. Primary Epo-independent colonies from CD34+ cells of one PV patient were assessed in two independent experiments in the presence or absence of combination drugs. Results. Out of 15 kinase inhibitors tested, three PI3K inhibitors (ZSTK474, GDC0941 and BEZ235), synergized with JAK2 inhibitors (Ruxolitinib and TG101348) in inhibiting cell growth. The combination index was less than 0.5 in all 8×8 dose combination ratios. The JAK2-PI3K inhibitors combination was specific for JAK2 signaling as growth of Ba/F3 cells expressing Bcr-Abl (at equivalent STAT5 activation levels) was unaffected by this combination treatment. Balb/c mice inoculated with Ba/F3 cells expressing TpoR JAK2 V617F were found to have increased spleen weight due to proliferation of autonomous cells. Our combination treatment using Ruxolitinib and GDC0941 could drastically reduce spleen weight compared to treatment with either compound alone. Endogenous erythroid colony forming unit (CFU-E) and burst forming unit (BFU-E) formation from JAK2 V617F knock-in bone marrow cells was reduced significantly by the combined use of Ruxolitinib and GDC0941 compared to individual drugs. Similarly, Epo-independent BFU-E colony formation from peripheral CD34+ cells of one JAK2 V617F-positive PV patient was reduced significantly by the drug combination. Conclusions. Our findings of strong synergy between the JAK2 inhibitors and PI3K inhibitors suggested that we may be able to administer these drugs at lower concentrations than when the drugs are used individually. It provides a framework for combination trials using compounds in these two classes in patients with myeloproliferative neoplasms. Disclosures: No relevant conflicts of interest to declare.

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Mouse Models of Myeloproliferative Neoplasms and Their Use In Preclinical Drug Testing

Blood ◽

10.1182/blood.v116.21.sci-35.sci-35 ◽

2010 ◽

Vol 116 (21) ◽

pp. SCI-35-SCI-35

Author(s):

Richard A. Van Etten

Keyword(s):

Board Of Directors ◽

Targeted Therapies ◽

Myeloproliferative Neoplasms ◽

Normal Population ◽

Hematopoietic Cells ◽

Advisory Committees ◽

Early Therapy ◽

Jak2 Inhibitor ◽

Advantages And Disadvantages ◽

Jak2 Inhibitors

Abstract Abstract SCI-35 The myeloproliferative neoplasms (MPNs) and related conditions, many of which are characterized by dysregulated tyrosine kinase (TK) signalling, can be modelled in mice by expressing the relevant mutant signalling molecules (for example, BCR-ABL1 or mutant JAK2) in mouse hematopoietic cells. There are two strategies to accomplish this: retroviral- or lentiviral gene transfer into hematopoietic cells followed by transplantation, and expression via a chromosomal transgene. Each method has advantages and disadvantages for modeling MPNs and for pre-clinical evaluation of molecularly targeted therapies. For BCR-ABL1, such preclinical studies have proven useful in predicting clinical responses to TK inhibitors in patients (Hu et al., Nat. Genet. 2004;36:453). The retroviral strategy has been used to model MPNs induced by JAK2V617F, JAK2 exon 12 mutants, and MPL W515L/K mutants (Lacout et al., Blood 2006;108:1652; Zaleskas et al., PLoS ONE 2006;1:e18; Pikman et al., PLoS Med. 2006;3:e270). For JAK2V617F, the models recapitulate predominantly the erythroid phenotype of PV, with polycythemia, splenomegaly, endogenous erythroid colonies (EEC), and progression to myelofibrosis (MF). Treatment with small molecule JAK2 inhibitors reverses polycythemia and splenomegaly, but the effects on EEC frequency, JAK2V617F allele burden, and MF are in general less profound, possibly reflecting a lack of discrimination between endogenous and mutant JAK2 by these drugs in vivo. MPL W515L induces more fulminant MPN and MF in mice, and while early therapy with a JAK2 inhibitor can prolong survival and decrease MF (Koppikar et al., Blood 2010;115:2919), it is less clear whether established MF responds to treatment. With the transgenic approach, a TK can be expressed at more physiologic levels. As JAK2V617F must associate with EpoR or MPL for signaling activity, competition between endogenous and mutant JAK2 might influence disease phenotype. Several transgenic models of JAK2V617F MPN have been published recently (Tiedt et al., Blood 2008;111:3931; Shide et al., Leukemia 2008;22:87), including “knock-in” models where JAK2V617F is conditionally expressed from the endogenous promoter (Akada et al., Blood 2010; 15:3589; Marty et al., Blood 2010;Epub May 14; Li et al., Blood 2010;Epub May 20). These models lend some experimental support for the concept that expression of JAK2V617F at levels similar to or higher than endogenous JAK2 causes erythrocytosis whereas lower expression favors thrombocytosis, but several variables, including origin of JAK2 and mouse strain, may confound the picture. The response of these transgenic mice to treatment with JAK2 inhibitors may differ from MPN patients in that they lack a normal population of HSC in their marrow and spleen, and are wholly dependent on JAK2V617F–associated hematopoiesis for blood cell production. Overall, these models should prove useful for guiding clinical trials of targeted therapies in the Ph− MPNs. Disclosures: Van Etten: AstraZeneca Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Cephalon Oncology: Membership on an entity's Board of Directors or advisory committees. Off Label Use: INCB18424 (JAK2 inhibitor) TG101348 (JAK2 inhibitor) AZD1480 (JAK2 inhibitor).

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The role of JAK2 inhibitors in MPNs 7 years after approval

Blood ◽

10.1182/blood-2018-01-791491 ◽

2018 ◽

Vol 131 (22) ◽

pp. 2426-2435 ◽

Cited By ~ 28

Author(s):

Francesco Passamonti ◽

Margherita Maffioli

Keyword(s):

Myeloproliferative Neoplasms ◽

Symptom Relief ◽

Primary Myelofibrosis ◽

Driver Mutations ◽

Volume Response ◽

Symptom Response ◽

Definition Of ◽

Jak2 Inhibitors

Abstract Myeloproliferative neoplasms (MPNs) include essential thrombocythemia, polycythemia vera (PV), and primary myelofibrosis (MF). Phenotype-driver mutations of JAK2, CALR, and MPL genes are present in MPNs and can be variably combined with additional mutations. Driver mutations entail a constitutive activation of the JAK2/STAT pathway, the key signaling cascade in MPNs. Among JAK2 inhibitors (JAKis), ruxolitinib (RUX) has been approved for the treatment of intermediate and high-risk MF and for PV inadequately controlled by or intolerant of hydroxyurea. Other JAKis, such as fedratinib and pacritinib, proved to be useful in MF. The primary end points in MF trials were spleen volume response (SVR) and symptom response, whereas in PV trials they were hematocrit control with or without spleen response. In advanced MF, RUX achieved a long lasting SVR of >35% in ∼60% of patients, establishing a new benchmark for MF treatment. RUX efficacy in early MF is also remarkable and toxicity is mild. In PV, RUX achieved hematocrit control in ∼60% of cases and SVR in 40%. Symptom relief was evident in both conditions. In the long-term, however, many MF patients lose their SVR. Indeed, the definition of RUX failure and the design of new trials in this setting are unmet needs. Decrease of hemoglobin/platelet levels and increased infection rates are the most common side effects of RUX, and nonmelanoma skin tumors need to be monitored while on treatment. In conclusion, the introduction of JAKis raises the bar of treatment goals in MF and PV.

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From Janus kinase 2 to calreticulin: the clinically relevant genomic landscape of myeloproliferative neoplasms

Blood ◽

10.1182/blood-2014-03-530865 ◽

2014 ◽

Vol 123 (24) ◽

pp. 3714-3719 ◽

Cited By ~ 116

Author(s):

Mario Cazzola ◽

Robert Kralovics

Keyword(s):

Essential Thrombocythemia ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Janus Kinase ◽

Driver Mutations ◽

Mutation Status ◽

Genomic Landscape ◽

Exon 10 ◽

Stat Pathway

Abstract Our understanding of the genetic basis of myeloproliferative neoplasms began in 2005, when the JAK2 (V617F) mutation was identified in polycythemia vera, essential thrombocythemia, and primary myelofibrosis. JAK2 exon 12 and MPL exon 10 mutations were then detected in subsets of patients, and subclonal driver mutations in other genes were found to be associated with disease progression. Recently, somatic mutations in the gene CALR, encoding calreticulin, have been found in most patients with essential thrombocythemia or primary myelofibrosis with nonmutated JAK2 and MPL. The JAK-STAT pathway appears to be activated in all myeloproliferative neoplasms, regardless of founding driver mutations. These latter, however, have different effects on clinical course and outcomes. Thus, evaluation of JAK2, MPL, and CALR mutation status is important not only for diagnosis but also for prognostication. These genetic data should now also be considered in designing clinical trials.

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JAK2 Inhibitor Therapy in Chronic Myeloproliferative Neoplasms Ph(-): Hematologic, Clinical and Molecular Responses

Blood ◽

10.1182/blood.v120.21.5059.5059 ◽

2012 ◽

Vol 120 (21) ◽

pp. 5059-5059

Author(s):

Ana Esther Kerguelen Fuentes ◽

Dolores Hernández-Maraver ◽

Miguel Angel ◽

Canales Albendea ◽

Ana Rodriguez de la Rua

Keyword(s):

Clinical Response ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Molecular Response ◽

Spleen Size ◽

Molecular Responses ◽

Allele Burden ◽

Jak2 Inhibitor ◽

Chronic Myeloproliferative Neoplasms ◽

Jak2 Inhibitors

Abstract Abstract 5059 JAK2 inhibitors are known to improve symptoms, to control myeloproliferation and to reduce splenomegaly in patients diagnosed with chronic myeloproliferative neoplasms (MPNs)Ph(-). However their ability to decrease the allele burden and achieve molecular responses is controversial. Objective: To evaluate hematologic, clinical and molecular responses according to the criteria of the European LeukemiaNet and European Myelofibrosis Network in 13 patients treated with JAK2 inhibitors. Material and Methods: We performed a prospective study in the Haematology Service of the Hospital La Pazbetween 1987 and 2012 in 13 patients diagnosed with NMP Ph (-) and treated with of JAK2 inhibitors: 5 secondary mylofibrosis (SFM)to homozygous polycythemia vera JAK (+), 4 SFM to essential thrombocythemias JAK (-), 2 primary myelofibrosis (one JAK (-) and one heterozygous JAK (+)) and 2 homozygous PV JAK (+) resistant to hydrea. The RT-PCR was performed at 6 or 12 months after the first determination of the allelic burden. Median follow-up was 3 months (1 – 15). A) Hematologic Response (HR): 3/5 SFM to PV(1)/TE JAK(-)(2) reached HR at 3 months of initiation of JAK2 inhibitor to 20mg/day. Molecular and clinical response were not evaluated. B) Clinical Response: Three patients had a reduction in the spleen size. Only one patient in the SFM group had a reduction in the spleen size (18 cm before the drug was commenced to 13. 7 cm) and the allele burden decrease from 55% to 23% after 5 months of therapy with JAK2 inhibitor at 25mg/12h (increase of 5mg/12h after 15 days of initiation of medication). 2/3 MFS to TE JAK(-) had a reduction from 15, 3 cm before the drug was commenced to 9 cm after 3 months of therapy with JAK2 inhibitor at 20 mg/12h. 3/3 MFP JAK(-) had a 6cm reduction in spleen size. Twenty cm splenomegaly was documented before starting JAK2 inhibitor to 15 mg/day. C) Molecular Response: 2/5 SFM to PV decreased the previous allele burden value. One patient decreased by 25% the previous allele burden value (99. 28%) at 6 months of JAK2 inhibitor. Second patient decreased by 13% the previous allele burden value (55%) at 6 months of starting JAK2 inhibitor to 25 mg/day. In 1/2 PV, the previous allele burden value (93. 17%) decreased by 11. 4% at 6 months of starting JAK2 inhibitor at 100mg/24h. D) Lack of response and disease progression: One patient with SMF secondary to JAK 2 (-) ET had dose reductions from 20 mg twice a day secondary to grade IV thrombocytopenia and renal toxicity. Patient finally developed acute leukemia. Conclusions: Our study confirms that JAK2 inhibitors reduce splenomegaly in MPNs JAK(-)and JAK(+). Prospective studies with an adequate sample size are necessary to demonstrate whether splenomegaly and symptom reductions achieved with inhibition of JAK2 could be associated to decrease the allele burden and achieve molecular responses in MPNs JAK(+). Disclosures: No relevant conflicts of interest to declare.

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Recent advances in understanding myelofibrosis and essential thrombocythemia

F1000Research ◽

10.12688/f1000research.8081.1 ◽

2016 ◽

Vol 5 ◽

pp. 700 ◽

Cited By ~ 21

Author(s):

William Vainchenker ◽

Stefan N. Constantinescu ◽

Isabelle Plo

Keyword(s):

Essential Thrombocythemia ◽

Myeloproliferative Neoplasms ◽

Great Majority ◽

Primary Myelofibrosis ◽

Driver Mutations ◽

New Therapeutic Approaches ◽

Recent Advances ◽

Downstream Effectors ◽

Thrombopoietin Receptor

The classicBCR-ABL-negative myeloproliferative neoplasms (MPNs), a form of chronic malignant hemopathies, have been classified into polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). ET and PMF are two similar disorders in their pathogenesis, which is marked by a key role of the megakaryocyte (MK) lineage. Whereas ET is characterized by MK proliferation, PMF is also associated with aberrant MK differentiation (myelodysplasia), leading to the release of cytokines in the marrow environment, which causes the development of myelofibrosis. Thus, PMF is associated with both myeloproliferation and different levels of myelodysplastic features. MPNs are mostly driven by mutated genes called MPN drivers, which abnormally activate the cytokine receptor/JAK2 pathway and their downstream effectors. The recent discovery ofCALRmutations has closed a gap in our knowledge and has shown that this mutated endoplasmic reticulum chaperone activates the thrombopoietin receptor MPL and JAK2. These genetic studies have shown that there are two main types of MPNs: JAK2V617F-MPNs, including ET, PV, and PMF, and the MPL-/CALR-MPNs, which include only ET and PMF. These MPN driver mutations are associated with additional mutations in genes involved in epigenetics, splicing, and signaling, which can precede or follow the acquisition of MPN driver mutations. They are involved in clonal expansion or phenotypic changes or both, leading to myelofibrosis or leukemic transformation or both. Only a few patients with ET exhibit mutations in non-MPN drivers, whereas the great majority of patients with PMF harbor one or several mutations in these genes. However, the entire pathogenesis of ET and PMF may also depend on other factors, such as the patient’s constitutional genetics, the bone marrow microenvironment, the inflammatory response, and age. Recent advances allowed a better stratification of these diseases and new therapeutic approaches with the development of JAK2 inhibitors.

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CYT387, a novel JAK2 inhibitor, induces hematologic responses and normalizes inflammatory cytokines in murine myeloproliferative neoplasms

Blood ◽

10.1182/blood-2009-05-223727 ◽

2010 ◽

Vol 115 (25) ◽

pp. 5232-5240 ◽

Cited By ~ 164

Author(s):

Jeffrey W. Tyner ◽

Thomas G. Bumm ◽

Jutta Deininger ◽

Lisa Wood ◽

Karl J. Aichberger ◽

...

Keyword(s):

Cell Lines ◽

Inflammatory Cytokines ◽

Kinase Inhibitor ◽

Myeloproliferative Neoplasms ◽

Symptomatic Improvement ◽

Janus Kinase ◽

Jak2 Inhibitor ◽

Cell Counts ◽

Tyrosine Kinase 2 ◽

Jak2 Inhibitors

Abstract Activating alleles of Janus kinase 2 (JAK2) such as JAK2V617F are central to the pathogenesis of myeloproliferative neoplasms (MPN), suggesting that small molecule inhibitors targeting JAK2 may be therapeutically useful. We have identified an aminopyrimidine derivative (CYT387), which inhibits JAK1, JAK2, and tyrosine kinase 2 (TYK2) at low nanomolar concentrations, with few additional targets. Between 0.5 and 1.5μM CYT387 caused growth suppression and apoptosis in JAK2-dependent hematopoietic cell lines, while nonhematopoietic cell lines were unaffected. In a murine MPN model, CYT387 normalized white cell counts, hematocrit, spleen size, and restored physiologic levels of inflammatory cytokines. Despite the hematologic responses and reduction of the JAK2V617F allele burden, JAK2V617F cells persisted and MPN recurred upon cessation of treatment, suggesting that JAK2 inhibitors may be unable to eliminate JAK2V617F cells, consistent with preliminary results from clinical trials of JAK2 inhibitors in myelofibrosis. While the clinical benefit of JAK2 inhibitors may be substantial, not the least due to reduction of inflammatory cytokines and symptomatic improvement, our data add to increasing evidence that kinase inhibitor monotherapy of malignant disease is not curative, suggesting a need for drug combinations to optimally target the malignant cells.

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JAK2 L884P Mutation Confers Resistance To The Type II JAK2 Inhibitor NVP-BBT594 When Co-Occurring With JAK2 R683G But Not JAK2 V617F

Blood ◽

10.1182/blood.v122.21.1429.1429 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1429-1429

Author(s):

Nadja Kopp ◽

Jordy C. Van der Zwet ◽

Jacob Layer ◽

Oliver Weigert ◽

Eric Vangrevelinghe ◽

...

Keyword(s):

De Novo ◽

Myeloproliferative Neoplasms ◽

Acquired Resistance ◽

Genetic Resistance ◽

Random Mutagenesis ◽

Jak2 V617f ◽

Type I ◽

Type Ii ◽

Jak2 Inhibitor ◽

Jak2 Inhibitors

Abstract Approximately 50% of myeloproliferative neoplasms (MPNs) harbor the JAK2 V617F mutation while approximately 50% of B-cell acute lymphoblastic leukemias (B-ALLs) with CRLF2 rearrangements harbor JAK2 exon 16 mutations that primarily involve R683. Multiple enzymatic inhibitors of JAK2 are in clinical development for the treatment of patients with malignant and nonmalignant conditions that depend on constitutive JAK2 signaling. Most of these drugs are ATP-mimetics that block JAK2 signaling in the active conformation (so-called “type I JAK2 inhibitors”). Resistance to type I JAK2 inhibitors can occur through heterodimerization between activated JAK2 and either JAK1 or TYK2 (Koppikar et al. Nature 2012). In addition, E864K, Y931C, and G935R mutations in the kinase domain of JAK2 (JH1 domain) confer resistance to a panel of type I JAK2 inhibitors (including ruxolitinib, tofacitinib, TG101348, JAK inhibitor I) without drastically affecting signaling by JAK2 (Weigert et al. J Exp Med 2012). Resistance caused by these mutations is independent of whether in the context of CRLF2 with JAK2 R683G or EPOR with JAK2 V617F (Weigert et al. J Exp Med 2012). In contrast to type I inhibitors, type II JAK2 inhibitors bind to and stabilize the inactive confirmation of JAK2 and prevent the activation loop from being phosphorylated. Thus, transphosphorylation of JAK2 by JAK1 or TYK2 does not confer resistance to the type II JAK2 inhibitor NVP-BBT594 (BBT594) (Koppikar et al. Nature 2012). In this study we report the first evidence that mutation of JAK2 can also confer resistance to type II Jak2 inhibitors. BBT594 had similar potency to the type I JAK2 inhibitor NVP-BVB808 (BVB808) in murine lymphoblast BaF3 cells dependent on CRLF2 with JAK2 R683G (IC50 8.5nM vs 15.7nM) or EPOR with JAK2 V617F (IC50 29nM vs 10nM). In contrast, the Y931C mutation conferred >3-fold resistance to BVB808 in BaF3 cells expressing CRLF2 with JAK2 R683G but no significant change in sensitivity to BBT594. Thus, type II JAK2 inhibitors can overcome genetic resistance to type I JAK2 inhibitors. We performed a random mutagenesis screen of JAK2 R683G and expressed the mutagenized library in BaF3 cells that express CRLF2. Selecting in the presence of 3uM BBT594 resulted in a large number of clones, of which all screened (n>30) harbored the same JAK2 L884P mutation. Structural modeling of this mutation predicted changes in the JH1 domain that may impact the conformation of the P-loop and helix C, and thereby compromise the sub-pocket required for type II inhibitor binding. In contrast to mutations that confer resistance to type I JAK2 inhibitors, the L884P mutation only conferred resistance to BBT594 in the context of CRLF2/JAK2 R683G (IC50 504nM versus 8.5nM for R683G alone) and not EPOR/JAK2 V617F. To our knowledge, this is the first mechanism of resistance specific to JAK2 R683G. BaF3 cells expressing CRLF2 with JAK2 R683G L884P Y931C remained resistant to BBT594. Transduction of the mutagenized JAK2 R683G library into BaF3 cells expressing CRLF2 followed by selection in both BVB808 and BBT594 did not yield any resistant colonies. In conclusion, mutations that affect the binding of type I JAK2 inhibitors do not affect the potency of the type II JAK2 inhibitor BBT594. The L884P mutation confers resistance to BBT594 when co-occurring with the activating mutation R683G but not with V617F. Thus, combinations of multiple JAK2 inhibitors with distinct mechanisms may be useful in overcoming de novo and acquired resistance to JAK2 inhibitors. Disclosures: Vangrevelinghe: Novartis: Employment. Radimerski:Novartis: Employment. Weinstock:Novartis: Consultancy, Research Funding.

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