Rethinking JAK2 inhibition: towards novel strategies of more specific and versatile Janus kinase inhibition.

This corrects the article DOI: 10.1038/leu.2017.43.

Rethinking JAK2 inhibition: towards novel strategies of more specific and versatile Janus kinase inhibition.

Janus kinases (JAKs) are required for cytokine receptor signaling. Since the discovery of the highly prevalent JAK2 V617F mutation in myeloproliferative neoplasms (MPNs), JAK2 became a prime target for inhibition. Only one approved JAK2 inhibitor exists, with positive, but not curative effects in MPNs, and promising effects in autoimmune diseases and cancer. On the basis of recent advances in the structural features regulating both normal and mutant JAKs, as well as in small-molecule targeting, we review the current state of JAK2 inhibitor development and present novel avenues of selecting JAK2 inhibitors, with broad and narrow specificities and extend these approaches to other JAKs.

Persistent STAT5 activation in myeloid neoplasms recruits p53 into gene regulation.

STAT (Signal Transducer and Activator of Transcription) transcription factors are constitutively activated in most hematopoietic cancers. We previously identified a target gene, LPP/miR-28 (LIM domain containing preferred translocation partner in lipoma), induced by constitutive activation of STAT5, but not by transient cytokine-activated STAT5. miR-28 exerts negative effects on thrombopoietin receptor signaling and platelet formation. Here, we demonstrate that, in transformed hematopoietic cells, STAT5 and p53 must be synergistically bound to chromatin for induction of LPP/miR-28 transcription. Genome-wide association studies show that both STAT5 and p53 are co-localized on the chromatin at 463 genomic positions in proximal promoters. Chromatin binding of p53 is dependent on persistent STAT5 activation at these proximal promoters. The transcriptional activity of selected promoters bound by STAT5 and p53 was significantly changed upon STAT5 or p53 inhibition. Abnormal expression of several STAT5-p53 target genes (LEP, ATP5J, GTF2A2, VEGFC, NPY1R and NPY5R) is frequently detected in platelets of myeloproliferative neoplasm (MPN) patients, but not in platelets from healthy controls. In conclusion, persistently active STAT5 can recruit normal p53, like in the case of MPN cells, but also p53 mutants, such as p53 M133K in human erythroleukemia cells, leading to pathologic gene expression that differs from canonical STAT5 or p53 transcriptional programs.

JAK/STAT signaling in hematological malignancies.

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway is central to signaling by cytokine receptors, a superfamily of more than 30 transmembrane proteins that recognize specific cytokines, and is critical in blood formation and immune response. Many of those receptors transmit anti-apoptotic, proliferative and differentiation signals, and their expression and functions are critical for the formation of blood lineages. Several cancers, including blood malignancies, have been associated with constitutive activation of members of the STAT family, which normally require JAK-mediated tyrosine phosphorylation for transcriptional activation. More recently, human myeloproliferative neoplasms were discovered to be associated with a unique acquired somatic mutation in JAK2 (JAK2 V617F), rare exon 12 JAK2 mutations, or thrombopoietin receptor mutations that constitutively activate wild-type JAK2. Prompted by these observations, many studies have explored the possibility that JAKs, cytokine receptors, or other components of the JAK/STAT pathway are mutated or upregulated in several hematological malignancies. This has been observed in certain pediatric acute lymphoblastic leukemias and adult T-cell lymphoblastic leukemias, and overexpression of JAK2 seems to be important in Hodgkin lymphoma. Here we discuss the nature and respective contribution of mutations dysregulating the JAK/STAT pathway in hematological malignancies and present examples in which such mutations drive the disease, contribute to the phenotype, or provide a survival and proliferative advantage. JAK inhibitors are making their way into the therapeutic arsenal (for example, in myelofibrosis), and we discuss the possibility that other hematological diseases might benefit from treatment with these inhibitors in combination with other agents.

Effects of erythropoietin receptors and erythropoiesis-stimulating agents on disease progression in cancer.

Erythropoiesis-stimulating agents (ESAs) increase red blood cell (RBC) production in bone marrow by activating the erythropoietin receptor (EpoR) on erythrocytic-progenitor cells. Erythropoiesis-stimulating agents are approved in the United States and Europe for treating anaemia in cancer patients receiving chemotherapy based on randomised, placebo-controlled trials showing that ESAs reduce RBC transfusions. Erythropoiesis-stimulating agent-safety issues include thromboembolic events and concerns regarding whether ESAs increase disease progression and/or mortality in cancer patients. Several trials have reported an association between ESA use and increased disease progression and/or mortality, whereas other trials in the same tumour types have not provided similar findings. This review thoroughly examines available evidence regarding whether ESAs affect disease progression. Both clinical-trial data on ESAs and disease progression, and preclinical data on how ESAs could affect tumour growth are summarised. Preclinical topics include (i) whether tumour cells express EpoR and could be directly stimulated to grow by ESA exposure and (ii) whether endothelial cells express EpoR and could be stimulated by ESA exposure to undergo angiogenesis and indirectly promote tumour growth. Although assessment and definition of disease progression vary across studies, the current clinical data suggest that ESAs may have little effect on disease progression in chemotherapy patients, and preclinical data indicate a direct or indirect effect of ESAs on tumour growth is not strongly supported.

Aberrant signal transduction pathways in myeloproliferative neoplasms.

The BCR-ABL-negative myeloproliferative neoplasms (MPNs), polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), entered the spotlight in 2005 when the unique somatic acquired JAK2 V617F mutation was described in >95% of PV and in 50% of ET and PMF patients. For the very rare PV patients who do not harbor the JAK2 V617F mutation, exon 12 JAK2 mutants were discovered also to result in activated forms of JAK2. A minority of ET and PMF patients harbor mutations that constitutively activate the thrombopoietin receptor (TpoR). In bone marrow reconstitution models based on retroviral transduction, the phenotype induced by JAK2 V617F is less severe and different from the rapid fatal myelofibrosis induced by TpoR W515L. The reasons for these differences are unknown. Exactly by which mechanism(s) one acquired somatic mutation, JAK2 V617F, can promote three different diseases remains a mystery, although gene dosage and host genetic variation might have important functions. We review the recent progress made in deciphering signaling anomalies in PV, ET and PMF, with an emphasis on the relationship between JAK2 V617F and cytokine receptor signaling and on cross-talk with several other signaling pathways.

Clinical implications of JAK2 mutations in myeloproliferative disorders.

The V617F mutation of JAK2 is the key molecular event in 90% of polycythaemia vera (PV), 50% of essential thrombocythaemia (ET) and 50% of primary myelofibrosis (PMF). JAK2 exon 12 and MPLW515 mutations are less frequent. Because JAK2 V617F is specific for myeloid neoplasms, and because it can be detected in peripheral blood granulocytes, it offers a powerful tool that facilitates the diagnosis of these BCR-ABL negative myeloproLiferative disorders. These discoveries provide the rationale for a revision of the current WHO diagnostic criteria for PV, ET and PMF and could ultimately lead to the development of a specific targeted therapy.

JAK kinases overexpression promotes in vitro cell transformation.

Constitutive activation of the JAK-STAT pathway is frequent in cancer and contributes to oncogenesis. Here, we took advantage of the Ba/F3 cell line, a murine proB cell line dependent on IL-3 for growth, to analyse mechanisms of constitutive STAT activation in vitro. Cytokine-independent and tumorigenic Ba/F3 cell lines were derived from a two-step selection process. Cells transfected with a defective IL-9 receptor acquire IL-9 responsiveness during a first step of selection, and progress after a second selection step to autonomously growing tumorigenic cells. Microarray analysis pointed to JAK1 overexpression as a key genetic event in this transformation. Overexpression of JAK1 not only increased the sensitivity to IL-9 but also allowed a second selection step toward cytokine-independent growth with constitutive STAT activation. This progression was dependent on a functional FERM and kinase JAK1 domain. Similar results were observed after JAK2, JAK3 and TYK2 overexpression. All autonomous cell lines showed an activation of STAT5, ERK1-2 and AKT but only TYK2-overexpressing cell lines showed a constitutive activation of STAT3. Thus, JAK overexpression can be considered as one of the oncogenic events leading to the constitutive activation of the JAK-STAT pathway.

JAK2, the JAK2 V617F mutant and cytokine receptors.

Recently, a unique recurrent somatic mutation was identified as a major molecular event in polycythemia vera, essential thrombocythemia and idiopathic myelofibrosis. Expression of this mutant in cytokine-dependent hematopoietic cell lines induces autonomous growth. This effect is enhanced by overexpression of cytokine receptors, and can be inhibited by co-expression at higher levels of the wild type JAK2, which may compete for a limited pool of receptors. In JAK2-deficient cells, we showed that JAK2 V617F can transmit signals from ligand-activated TpoR or EpoR. Furthermore, the mutant JAK2 can be demonstrated to stimulate traffic of the EpoR. Thus, JAK2 V617F mutant must be able to interact via its intact FERM-SH2 domains with the cytosolic domains of cytokine receptors. A synergy between JAK2 V617F and insulin-like growth factor 1 receptor (IGF1R) can be detected in cytokine-dependent cell proliferation. Once cells are rendered autonomous by expression of JAK2 V617F, IGF1 acquires the ability to activate the JAK-STAT pathway. Thus, expression of JAK2 V617F may explain the described hypersensitivity of PV erythroid progenitors to IGF1. The V617 is conserved in two other mammalian JAKs, JAK1 and Tyk2. The homologous mutants JAK1 V658F and Tyk2 V678F are also active in proliferation and transcriptional assays. Such mutants may be found in human cancers or autoimmune diseases. In contrast, the JAK3 M592F does not lead to activation of JAK3. Current hypotheses on how JAK2 V617F contributes to three myeloproliferative diseases, and which other events may favor one disease versus another, are discussed.

The erythropoietin receptor cytosolic juxtamembrane domain contains an essential, precisely oriented, hydrophobic motif.

We report that the erythropoietin receptor cytosolic juxtamembrane region is conformationally rigid and contains a hydrophobic motif, composed of residues L253, I257, and W258, that is crucial for Janus kinase 2 (JAK2) activation and receptor signaling. Alanine insertion mutagenesis shows that the orientation of this motif and not its distance from the membrane bilayer is critical. Intragenic complementation studies suggest that L253 is contained within an alpha helix functionally continuous to the transmembrane alpha helix. The alpha-helical orientation of L53 is required not for JAK2 activation but for activated JAK2 to induce phosphorylation of the erythropoietin receptor. This motif is highly conserved among cytokine receptors and couples ligand-induced conformational changes in the receptor to intracellular activation of JAK2.

The N-terminal domain of Janus kinase 2 is required for Golgi processing and cell surface expression of erythropoietin receptor.

We show that Janus kinase 2 (JAK2), and more specifically just its intact N-terminal domain, binds to the erythropoietin receptor (EpoR) in the endoplasmic reticulum and promotes its cell surface expression. This interaction is specific as JAK1 has no effect. Residues 32 to 58 of the JAK2 JH7 domain are required for EpoR surface expression. Alanine scanning mutagenesis of the EpoR membrane proximal region reveals two modes of EpoR-JAK2 interaction. A continuous block of EpoR residues is required for functional, ligand-independent binding to JAK2 and cell surface receptor expression, whereas four specific residues are essential in switching on prebound JAK2 after ligand binding. Thus, in addition to its kinase activity required for cytokine receptor signaling, JAK is also an essential subunit required for surface expression of cytokine receptors.

Stem cell generation and choice of fate: role of cytokines and cellular microenvironment.

Hematopoietic stem cells (HSC) have provided a model for the isolation, enrichment and transplantation of stem cells. Gene targeting studies in mice have shown that expression of the thrombopoietin receptor (TpoR) is linked to the accumulation of HSCs capable to generate long-term blood repopulation when injected into irradiated mice. The powerful increase in vivo in HSC numbers by retrovirally transduced HOX4B, a homeotic gene, along with the role of the TpoR, suggested that stem cell fate, renewal, differentiation and number can be controlled. The discovery of the precise region of the mouse embryo where HSCs originate and the isolation of supporting stromal cell lines open the possibility of identifying the precise signals required for HSC choice of fate. The completion of human genome sequencing coupled with advances in gene expression profiling using DNA microarrays will enable the identification of key genes deciding the fate of stem cells. Downstream from HSCs, multipotent hematopoietic progenitor cells appear to co-express a multiplicity of genes characteristic of different blood lineages. Genomic approaches will permit the identification of the select group of genes consolidated by the commitment of these multipotent progenitors towards one or the other of the blood lineages. Studies with neural stem cells pointed to the unexpected plastic nature of these cells. Isolation of stem cells from multiple tissues may suggest that, providing the appropriate environment/ signal, tissues could be regenerated in the laboratory and used for transplantation. A spectacular example of influence of the environment on cell fate was revealed decades ago by using mouse embryonic stem cells (ES). Injected into blastocysts, ES cells contribute to the formation of all adult tissues. Injected into adult mice, ES cells become cancer cells. After multiple passages as ascites, when injected back into the blastocyst environment, ES- derived cancer cells behaved again as ES cells. More recently, the successful cloning of mammals and reprogramming of transferred nuclei by factors in the cytoplasm of oocytes turned back the clock by showing that differentiated nuclei can be "re-booted" to generate again the stem cells for different tissues.

Activation of the erythropoietin receptor by the gp55-P viral envelope protein is determined by a single amino acid in its transmembrane domain.

The spleen focus forming virus (SFFV) gp55-P envelope glycoprotein specifically binds to and activates murine erythropoietin receptors (EpoRs) coexpressed in the same cell, triggering proliferation of erythroid progenitors and inducing erythroleukemia. Here we demonstrate specific interactions between the single transmembrane domains of the two proteins that are essential for receptor activation. The human EpoR is not activated by gp55-P but by mutation of a single amino acid, L238, in its transmembrane sequence to its murine counterpart serine, resulting in its ability to be activated. The converse mutation in the murine EpoR (S238L) abolishes activation by gp55-P. Computational searches of interactions between the membrane-spanning segments of murine EpoR and gp55-P provide a possible explanation: the face of the EpoR transmembrane domain containing S238 is predicted to interact specifically with gp55-P but not gp55-A, a variant which is much less effective in activating the murine EpoR. Mutational studies on gp55-P M390, which is predicted to interact with S238, provide additional support for this model. Mutation of M390 to isoleucine, the corresponding residue in gp55-A, abolishes activation, but the gp55-P M390L mutation is fully functional. gp55-P is thought to activate signaling by the EpoR by inducing receptor oligomerization through interactions involving specific transmembrane residues.

The anemic Friend virus gp55 envelope protein induces erythroid differentiation in fetal liver colony-forming units-erythroid.

The gp55 envelope proteins of the spleen focus-forming virus initiate erythroleukemia in adult mice. Because the gp55 from the polycythemic strain (gp55-P), but not from the anemic strain (gp55-A), activates the erythropoietin receptor (EpoR) for proliferation of hematopoietic cell lines, the mechanism by which gp55-A initiates erythroleukemia has remained a mystery. We show here that gp55-A activates the EpoR in fetal liver cells. In contrast to previous studies using bone marrow cells from phenylhydrazine-treated, anemic mice, we find that both gp55-A and gp55-P induce erythroid differentiation from colony-forming unit-erythroid (CFU-E) progenitors in fetal liver cells. The effects on CFU-Es of both gp55-A and -P are mediated by the EpoR, because no colonies are seen upon expression of either gp55 in EpoR-/- fetal liver cells. However, only gp55-P induces erythroid bursts from burst-forming unit-erythroid progenitors and only gp55-P induces Epo independence in Epo-dependent cell lines. Using chimeric gp55 P/A proteins, we extend earlier work showing that the transmembrane sequence determines the capacity of gp55 proteins to differentially activate EpoR signaling. We discuss the possibilities for different signaling capacities of gp55-A and -P in fetal liver and bone marrow-derived erythroid progenitor cells.