Heterodimeric Cytokine Receptors Research Articles

Modular chimeric cytokine receptors with leucine zippers enhance the antitumour activity of CAR T cells via JAK/STAT signalling.

The limited availability of cytokines in solid tumours hinders maintenance of the antitumour activity of chimeric antigen receptor (CAR) T cells. Cytokine receptor signalling pathways in CAR T cells can be activated by transgenic expression or injection of cytokines in the tumour, or by engineering the activation of cognate cytokine receptors. However, these strategies are constrained by toxicity arising from the activation of bystander cells, by the suboptimal biodistribution of the cytokines and by downregulation of the cognate receptor. Here we show that replacement of the extracellular domains of heterodimeric cytokine receptors in T cells with two leucine zipper motifs provides optimal Janus kinase/signal transducer and activator of transcription signalling. Such chimeric cytokine receptors, which can be generated for common γ-chain receptors, interleukin-10 and -12 receptors, enabled T cells to survive cytokine starvation without induction of autonomous cell growth, and augmented the effector function of CAR T cells in vitro in the setting of chronic antigen exposure and in human tumour xenografts in mice. As a modular design, leucine zippers can be used to generate constitutively active cytokine receptors in effector immune cells.

Distinct Assemblies of Heterodimeric Cytokine Receptors Govern Stemness Programs in Leukemia.

Stemness is a hallmark of many cancers and is largely responsible for disease emergence, progression, and relapse. Our finding that clinically significant stemness programs in AML are directly regulated by different stoichiometries of cytokine receptors represents a hitherto unexplained mechanism underlying cell-fate decisions in cancer stem cell hierarchies. This article is highlighted in the In This Issue feature, p. 1749.

Design and Validation of Inducible TurboCARs with Tunable Induction and Combinatorial Cytokine Signaling.

Although cytokine support can enhance CAR T-cell function, coadministering cytokines or engineering CAR T cells to secrete cytokines can result in toxicities. To mitigate these safety risks, we engineered iTurboCAR T cells that coexpress a novel inducible Turbo (iTurbo) cytokine signaling domain. iTurbo domains consist of modular components that are customizable to a variety of activating inputs, as well as cytokine signaling outputs multiplexable for combinatorial signaling outcomes. Unlike most canonical cytokine receptors that are heterodimeric, iTurbo domains leverage a compact, homodimeric design that minimizes viral vector cargo. Using an iTurbo domain activated by the clinically validated dimerizer, AP1903, homodimeric iTurbo domains instigated signaling that mimicked the endogenous heterodimeric cytokine receptor. Different iTurbo domains programmed iTurboCAR T cells toward divergent phenotypes and resulted in improved antitumor efficacy. iTurbo domains, therefore, offer the flexibility for user-programmable signaling outputs, permitting control over cellular phenotype and function while minimizing viral cargo footprint.

Distinct Assemblies of Heterodimeric Cytokine Receptors Govern Stemness Programs in Leukemia

Leukemia stem cells (LSC) possess distinct self-renewal and arrested differentiation properties that are responsible for disease emergence, therapy failure and recurrence in acute myeloid leukemia (AML). Pleiotropic cytokines like interleukin (IL)-3 dimerize their receptors to signal multiple functional activities of AML cells, however, the mechanistic link between cytokine receptor dimerization and LSC fate regulation is unknown. Here, we show that the Class I heterodimeric IL3Rα/βc receptors (IL-3R) assemble as hexamers and dodecamers that segregate proliferation versus differentiation signals, respectively. Receptor stoichiometry varies across the individual cells that make up the AML hierarchy, with LSCs presenting with the highest IL3Rα/βc ratio thereby driving resultant hexamer-mediated stemness programs and poor patient survival. Our study establishes a new signaling mechanism of cytokine pleiotropy and the biased instruction of cell fate that directs the AML hierarchy; a signaling mechanism that may be generalizable to other normal and transformed cellular hierarchies.

Cell-Surface Receptor-Ligand Interaction Analysis with Homogeneous Time-Resolved FRET and Metabolic Glycan Engineering: Application to Transmembrane and GPI-Anchored Receptors.

Ligand-binding assays are the linchpin of drug discovery and medicinal chemistry. Cell-surface receptors and their ligands have traditionally been characterized by radioligand-binding assays, which have low temporal and spatial resolution and entail safety risks. Here, we report a powerful alternative (GlycoFRET), where terbium-labeled fluorescent reporters are irreversibly attached to receptors by metabolic glycan engineering. For the first time, we show time-resolved fluorescence resonance energy transfer between receptor glycans and fluorescently labeled ligands. We describe GlycoFRET for a GPI-anchored receptor, a G-protein-coupled receptor, and a heterodimeric cytokine receptor in living cells with excellent sensitivity and high signal-to-background ratios. In contrast to previously described methods, GlycoFRET does not require genetic engineering or antibodies to label receptors. Given that all cell-surface receptors are glycosylated, we expect that GlycoFRET can be generalized with applications in chemical biology and biotechnology, such as target engagement, receptor pharmacology, and high-throughput screening.

Interferon-γ is a master checkpoint regulator of cytokine-induced differentiation

Cytokines are protein mediators that are known to be involved in many biological processes, including cell growth, survival, inflammation, and development. To study their regulation, we generated a library of 209 different cytokines. This was used in a combinatorial format to study the effects of cytokines on each other, with particular reference to the control of differentiation. This study showed that IFN-γ is a master checkpoint regulator for many cytokines. It operates via an autocrine mechanism to elevate STAT1 and induce internalization of gp130, a common component of many heterodimeric cytokine receptors. This targeting of a receptor subunit that is common to all members of an otherwise diverse family solves the problem of how a master regulator can control so many diverse receptors. When one adds an autocrine mechanism, fine control at the level of individual cells is achieved.

Role of the Histone Deacetylase Inhibitor Givinostat (ITF2357) in Treatment of CRLF2 Rearranged Acute Lymphoblastic Leukemia

B Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) represents 35% of all cancers in pediatric age group. The cure rate for this disease approaches 90% with current treatment regimens, however only a third of patients with relapse are cured. Therefore, there is an urgent need to focus on subgroups of patients with hallmarks of bad prognosis that could benefit from novel therapeutic approaches. Alterations of Cytokine Receptor-like Factor 2 (CRLF2), a negative prognostic factor in pediatric BCP-ALL, have been identified in up to 10% of patients. However these patients represent half of the high risk Ph-like ALL and of Down Syndrome-associated BCP-ALL. Rearrangements of CRLF2 result in the overexpression of this component of the heterodimeric cytokine receptor for thymic stromal lymphopoietin (TSLP) and is associated with activating mutations of the JAK-STAT pathway. Together these cause hyperactivation of JAK/STAT and PI3K/mTOR signaling. Inhibition of CRLF2/JAK2 signaling has the potential to become a therapeutic targeted intervention for this subgroup of poor prognostic patients.Previous studies have shown that the HDAC inhibitor Givinostat/ITF2357 has potent anti-tumor activity against hematological malignancies, particularly JAK2V617F mutated myeloproliferative neoplasms (MPN) such as polycythemia vera, for which it has already a clinic application and established safety profile. We therefore studied the in vitro and in vivo efficacy of Givinostat in cases with CRLF2 rearrangements. Here we demonstrated that Givinostat inhibited proliferation and induced apoptosis of BCP-ALL CRLF2-rearranged MHH-CALL4 and MUTZ5 cell lines positive for exon 16 JAK2 mutations. Of note, the observed IC50 values for MHH-CALL4 were lower than those for the SET2 cell line positive control bearing JAK2V617F mutation, both for proliferation (IC50: 0.08±0.05µM vs. 0.14±0.03µM) and apoptosis (IC50: 0.17±0.03µM vs. 0.22±0.04µM). We next investigated the effect of Givinostat on blasts from CRLF2 rearranged BCP-ALL patient samples. For this purpose we developed xenograft models of human CRLF2 rearranged ALL to expand cells from patients and to recapitulate human leukemia in recipient mice. ALL blasts isolated from xenografts were co-cultured on OP9 stroma to perform ex vivo assays. Consistent with our findings in cell lines, Givinostat (0.2µM) reduced the % of live cells (Annexin V/Sytox negative) in all xenografts treated with the drug. In particular, after 72 hours, Givinostat was able to kill up to >90% of blast cells in all xenografts in contrast with the vehicle-treated samples which showed 25-60% of blasts still alive after treatment. The induction of cell death in Givinostat treated primografts was confirmed on primary samples from diagnosis using CyTOF which allowed us to observe that CD10+/CRLF2+ blasts were preferentially killed by the drug whereas CD45 high expressing cells (normal residue) remained unaffected by the treatment. Moreover, at low doses (0.2 µM), Givinostat downregulated genes of the JAK/STAT pathway (STAT5A, JAK2, IL7Rα, CRLF2, BCL2L1 and cMYC) and inhibited the basal and ligand induced signaling, reducing the phoshporylation of STAT5 in all tested primografts (mean fold decrease of pSTAT5: 2.4+0.6). Most importantly, to understand if the transcriptional downregulation of CRLF2 resulted in a functional effect, the downmodulation of CRLF2 protein was observed by flow cytometry (mean fold decrease 3.55+1.38). In vivo, Givinostat significantly reduced engraftment of human blasts in xenograft models of CRLF2 positive BCP-ALL (ranging from 1.9 to 34 fold decrease in bone marrow). Furthermore, Givinostat augmented the effect of chemotherapy in inhibiting proliferation and inducing apoptosis in CRLF2 rearranged cell lines and in primografts, in vitro. After 72 hours, the combined treatment reached 4.6-8.8 fold lower % of remaining viable blasts than chemotherapy alone (6.3-35.3% viable cells in chemotherapy-treated samples vs 1.4-4.3% of combination), 2.5-8.5 fold lower than Givinostat alone (4.3-36.4% vs 1.4-4.3%) and 2.4-13 fold lower than Methyl-prednisolone (5.2-39.1 vs 1-16.3%).In conclusion, Givinostat may represent a novel and effective tool, in combination with current chemotherapy, to treat this difficult to handle subset of ALL and these data strongly argue for the translation of Givinostat in combination with conventional therapy into human trials. DisclosuresDavis:Fluidigm, Inc: Honoraria. Nolan:Fluidigm, Inc: Equity Ownership.

Distinct Acute Lymphoblastic Leukemia (ALL)-associated Janus Kinase 3 (JAK3) Mutants Exhibit Different Cytokine-Receptor Requirements and JAK Inhibitor Specificities

JAK1 and JAK3 are recurrently mutated in acute lymphoblastic leukemia. These tyrosine kinases associate with heterodimeric cytokine receptors such as IL-7 receptor or IL-9 receptor, in which JAK1 is appended to the specific chain, and JAK3 is appended to the common gamma chain. Here, we studied the role of these receptor complexes in mediating the oncogenic activity of JAK3 mutants. Although JAK3(V674A) and the majority of other JAK3 mutants needed to bind to a functional cytokine receptor complex to constitutively activate STAT5, JAK3(L857P) was unexpectedly found to not depend on such receptor complexes for its activity, which was induced without receptor or JAK1 co-expression. Introducing a mutation in the FERM domain that abolished JAK-receptor interaction did not affect JAK3(L857P) activity, whereas it inhibited the other receptor-dependent mutants. The same cytokine receptor independence as for JAK3(L857P) was observed for homologous Leu(857) mutations of JAK1 and JAK2 and for JAK3(L875H). This different cytokine receptor requirement correlated with different functional properties in vivo and with distinct sensitivity to JAK inhibitors. Transduction of murine hematopoietic cells with JAK3(V674A) led homogenously to lymphoblastic leukemias in BALB/c mice. In contrast, transduction with JAK3(L857P) induced various types of lymphoid and myeloid leukemias. Moreover, ruxolitinib, which preferentially blocks JAK1 and JAK2, abolished the proliferation of cells transformed by the receptor-dependent JAK3(V674A), yet proved much less potent on cells expressing JAK3(L857P). These particular cells were, in contrast, more sensitive to JAK3-specific inhibitors. Altogether, our results showed that different JAK3 mutations induce constitutive activation through distinct mechanisms, pointing to specific therapeutic perspectives.

Requirement of Lipid Raft Integrity for Signaling by JAK2-V617F

AbstractAbstract 4191JAK2 is a cytoplasmic tyrosine kinase that plays an important role in signaling following activation of various cytokine receptors. JAK2 activation promotes growth, survival, and differentiation of various cell types. Mutation of JAK2 is seen in numerous hematopoietic diseases, most notably in myeloproliferative neoplasms (MPNs). JAK2-V617F is a frequent mutation found in the classical MPNs: polycythemia vera, essential thrombocythemia, and primary myelofibrosis. The single amino acid change of valine to phenylalanine occurs in the pseudokinase domain of JAK2, relieving auto-inhibition of the kinase domain and allowing constitutive kinase activity. Numerous mouse models have demonstrated that JAK2-V617F can induce MPN-like disorders in mice. Thus, this point mutation, as well as other less common JAK2 mutations, is believed to play an important etiologic role in the development of MPNs in humans. The development and use of JAK2 inhibitors in clinical trials has shown promising results, again demonstrating the important role JAK2 plays in MPNs. While the JAK2-V617F mutation, as well as other JAK2 mutations, decreases auto-inhibition of JAK2 kinase activity, it is clear that mutated JAK2 still requires the expression of cytokine receptors to induce activation of transforming signals in hematopoietic cells. Normally, JAK2 binds to homodimeric and heterodimeric cytokine receptors through specific receptor motifs and is activated by various structural changes induced by cytokine stimulation. Following activation it utilizes receptor tyrosines as substrates for phosphorylation, leading to recruitment of downstream signaling molecules, such as STAT5, among others. JAK2 then activates STAT5 via phosphorylation and STAT5 then translocates to the nucleus to regulate transcription of target genes. JAK2-V617F does not require ligand for activation, but still requires the scaffolding function of cytokine receptors to facilitate its full activation and activation of downstream signaling via phosphorylation. Lipid rafts are microdomains of the plasma membrane that are enriched in cholesterol and sphingolipids. They have gained appreciation in signal transduction as sites of localization of signaling mediators, including membrane-bound receptors. Congregation of signaling proteins in lipid rafts within the plasma membrane promotes complex formation and signaling cascade activation. We have recently demonstrated that JAK2 is present in lipid rafts during erythropoietin signaling and that lipid raft integrity is required for erythropoietin-mediated signal transduction (Blood 2009, 114: 292). In our current study, we demonstrate that constitutive JAK-STAT signaling driven by JAK2-V617F is sensitive to lipid raft disruption. Human erythroleukemia (HEL) cells express constitutive activation of JAK-STAT signaling due to the presence of JAK2-V617F. Treatment of these cells with methyl-beta-cyclodextrin to disrupt lipid rafts abolished JAK2, STAT5, and STAT3 activation. Similar results are obtained in other cell lines harboring JAK2-V617F and that exhibit JAK-STAT activation that is dependent on this activated form of JAK2. We also demonstrate that JAK2-V617F co-localizes with lipid rafts, as shown by immunofluorescence, and that this co-localization is abolished by lipid raft disruption. This suggests the loss of JAK2-V617F-mediated JAK-STAT activation we observe following lipid raft disruption may be due to an inhibition of properly localized protein complex formation in the plasma membrane that is necessary for JAK2-V617F signaling. Lipid rafts may provide a site for an accumulation of JAK2-V617F-containing signaling complexes and may be necessary for the cellular signals initiated by JAK2-V617F. Our data show JAK2-V617F-driven JAK-STAT pathway activation is vulnerable to lipid raft disrupting agents and suggest lipid raft integrity as a potential therapeutic target for JAK2-V617F positive neoplasms. Targeting lipid rafts in combination with JAK2 kinase inhibitors may allow for more effective kinase inhibition at lower doses, potentially decreasing undesirable side effects associated with kinase inhibitor treatment. Disclosures:No relevant conflicts of interest to declare.

Activation of JAK2-V617F by Components of Heterodimeric Cytokine Receptors

The JAK2-V617F mutation is an important etiologic factor for the development of myeloproliferative neoplasms. The mechanism by which this mutated tyrosine kinase initiates deregulated signals in cells is not completely understood. It is believed that JAK2-V617F requires interactions with homodimeric cytokine receptors to elicit its transforming signal. In this study, we demonstrate that components of heterodimeric cytokine receptors can also activate JAK2-V617F. Expression of IL27Ra, a heterodimeric receptor component, enhanced the activation of JAK2-V617F and subsequent downstream signaling to activation of STAT5 and ERK. In addition, expression of components of the interleukin-3 receptor, IL3Ra and the common beta chain, activated JAK2-V617F as well as STAT5 and ERK. Importantly, expression of IL27Ra functionally replaced the requirement of a homodimeric cytokine receptor to promote the activation and transforming activity of JAK2-V617F in BaF3 cells. Tyrosine phosphorylation of IL27Ra was not required to induce activation of JAK2-V617F or STAT5, or to enhance the transforming activity of JAK2-V617F. Expression of IL3Ra or the common beta chain in BaF3 cells also enhanced the ability of JAK2-V617F to transform these hematopoietic cells. However, the heterodimeric receptor component IL12RB1 did not enhance the activation or transforming signals of JAK2-V617F in BaF3 cells. IL27Ra also activated the K539L and R683G JAK2 mutants. Together our data demonstrate that in addition to homodimeric receptors, some heterodimeric receptor components can support the activation and transforming signals of JAK2-V617F and other JAK2 mutants. Therefore, heterodimeric receptors may play unappreciated roles in JAK2 activation in the development of hematopoietic diseases including myeloproliferative neoplasms.

Activation of JAK2-V617F by Heterodimeric Receptor Components.

Abstract 2955Poster Board II-931Mutational activation of Janus Kinase 2 (JAK2) is an important etiologic factor for the development of myeloproliferative neoplasms (MPNs). JAK2 is mutated in nearly all patients with polycythemia vera and about half of patients with essential thrombocythemia and primary myelofibrosis. The most prevalent mutation of JAK2 is valine 617 mutated to phenylalanine (V617F). The normal function of JAK2 is to interact with and become activated by cytokine receptors following their interaction with ligand. Interestingly, while JAK2-V617F is considered a constitutively activated kinase, there is evidence that it still requires interaction with a cytokine receptor to elicit its transforming signal. When expressed at less than or near endogenous JAK2 levels in hematopoietic cells, JAK2-V617F requires co-expression of a homodimeric receptor in order to become activated, transduce downstream signals, and induce transformation. When expressed at high levels in hematopoietic cells, co-expression of a homodimeric cytokine receptor is not needed. However, a functional cytokine receptor interacting domain is still required, suggesting that even when expressed at high levels JAK2-V617F requires interaction with a receptor. Also, a functional cytokine receptor interacting domain in JAK2-V617F is required for it to induce MPN-like disease in mouse models. The ability of cytokine receptor expression to activate JAK2-V617F has focused on homodimeric receptors. In this study we demonstrate that single components of heterodimeric receptors can also activate JAK2-V617F. Expression of interleukin-27 receptor alpha (IL27Ra), the ligand-binding component of the IL-27 receptor, enhances phosphorylation of JAK2-V617F on tyrosines 1007 and 1008. This activation of JAK2 also leads to tyrosine phosphorylation of signal transducers and activators of transcription-5 (STAT5), a main downstream effector of JAK2 activation. We obtain similar results when we utilize interleukin-12 receptor beta 1 (IL12RB1), a receptor belonging to the same family as IL27Ra. To extend these studies to other heterodimeric cytokine receptor components, we utilized the components of the interleukin-3 receptor, interleukin-3 receptor alpha (IL3Ra) and the beta common chain, which is also utilized in the receptors for interleukin-5 and granulocyte macrophage colony stimulating factor. Expression of each of these receptor subunits activates JAK2-V617F as well as STAT5. Importantly, we demonstrate that expression of IL27Ra can functionally replace the expression of a homodimeric cytokine receptor to support the activation of JAK2-V617F in BaF3 cells as well as promote the transforming activity of JAK2-V617F in these cytokine dependent hematopoietic cells. Interestingly, while IL12RB1 expression activates JAK2-V617F in 293T cells, it is not capable of enhancing the transforming signals of JAK2-V617F in BaF3 cells. Expression of IL3Ra or the beta common chain in BaF3 cells also enhances the ability of JAK2-V617F to transform these cells to cytokine independence. However, this enhancement is not immediate as it only becomes evident at later time points. Together our data demonstrate that in addition to homodimeric receptors, some heterodimeric receptor components may contribute to JAK2-V617F activation. It should also be considered that such receptors may play a role in JAK2-V617F-negative MPNs, perhaps through altered expression or activating receptor mutations, analogous to mutated thrombopoietin receptor proteins that play a role in the development of disease in a fraction of these MPN patients. Disclosures:No relevant conflicts of interest to declare.

Molecular Mechanisms of JAK2V617F Oncogenic Activation: The JAK2- V617F Mutant Utilizes the SH2 Domain for Constitutive Kinase Activity in the Presence of Unstimulated Heterodimeric Cytokine Receptor.

The JAK2-V617F mutation has been reported in the majority of MPDs including PV, ET, and IMF. This mutation leads to the constitutive activation of the JAK2 tyrosine kinase activity and overexpression of JAK2V617F renders hematopoietic cell lines growth factor-independent. However, the molecular mechanism leading to constitutive activation of JAK2V617F is largely unclear and the requirement of homodimeric or heterodimeric cytokine receptors needs to be determined. Here we show that oncogenic JAK2-V617F requires an intact SH2 domain for constitutive kinase activity. To this end we mutated the conserved arginine 426 within the SH2 domain to a lysine. Ba/F3 cells expressing JAK2V617F grew IL-3-independent and showed constitutive activation of JAK2, STAT5, and ERK1/2. In contrast, introduction of the SH2 mutation in JAK2V617F abrogated both transformation as well as constitutive activation of downstream signaling pathways. Accordingly, reconstitution of JAK2 mutants in a JAK2-negative cell line with IL-3R co-expression revealed reduced activation of JAK2 when the SH2 domain was mutated. It has been reported that JAK2 binding to homodimeric type I cytokine receptor may facilitate JAK2V617F-mediated transformation. Interestingly, co-expression of the homodomeric EpoR with SH2 mutated JAK2V617F rescues the phenotype indicating that the SH2 domain is required for JAK2 signaling in the presence of heterodimeric but not homodimeric cytokine receptors. Membrane localization studies showed equal membrane distribution of SH2-mutated and unmutated JAK2-V617F indicating that the SH2 domain mutation does not affect subcellular distribution of JAK2. However, co-IP experiments revealed a possible role for the SH2 domain in the dimerization and transphosphorylation of JAK2. Consequently, reduced transphosphorylation was seen in IL-3R- but not in EpoR-expressing cells. In a BM transplantation model we found that an intact SH2 domain in JAK2V617F was required for the induction of a MPD-like disease. Thus, our results points to an important role of the SH2 domain for the constitutive activation of JAK2V617F in cells expressing heterodimeric cytokine receptors.

A Component of a Heterodimeric Receptor Can Functionally Replace a Homodimeric Receptor in Supporting the Activation and Transforming Activity of JAK2-V617F

We have recently identified IL27Ra as a gene from an AML patient that transforms hematopoietic cells to a state of cytokine-independent growth. Importantly, we have found that the gene product of IL27Ra, intereukin-27 receptor (IL27R), is expressed on the cell surface of the leukemic cells of AML patients. IL27R, also know as TCCR and WSX-1, is a component if a heterodimeric type I cytokine receptor that functions as the ligand-binding subunit of the receptor for IL-27. Normally, IL27R functions with the gp130 co-receptor to induce signal transduction in response to IL-27. However, we have demonstrated that IL27R can induce the transformation of hematopoietic cells in a ligand- and gp130-independent manner. IL27R, which contains a JAK-binding Box 1 motif, requires JAK family kinase activity to transform cells. In a potential mechanism of IL27R-mediated transformation, IL27R may function as a homodimer to activate JAK family members. In an effort to determine if IL27R can function as a homodimer, we tested to see if IL27R could activate the V617F mutation of JAK2 commonly found in various myeloproliferative neoplasms (MPNs). It is believed that JAK2-V617F utilizes a homodimeric cytokine receptor complex as a scaffold in order to multimerize and become fully activated via transphosphorylation. Expression of IL27R activated the kinase activity of JAK2-V617F, suggesting that IL27R may form homodimers in cells. In addition, co-expression of IL27R and JAK2-V617F led to transformation of BaF3 cells to cytokine-independence, whereas IL27R expressed with wildtype JAK2 or JAK2-V617F expressed alone did not induce rapid cytokine-independent growth of these cells. This is similar data to that obtained with co-expression of JAK2-V617F with homodimeric receptors (e.g. EpoR, GCSFR), demonstrating that a component of a heterodimeric cytokine receptor can functionally replace a homodimeric receptor in mediating JAK2-V617F activation. Chemical crosslinking showed that IL27R could dimerize suggesting that IL27R may form a scaffold for JAK2-V617F-mediated transformation. In addition to providing a scaffold for binding and activation of JAK2-V617F, it has been proposed that homodimeric receptors also provide an important substrate of the JAK2 tyrosine kinase activity to induce cell signaling and transformation. Tyrosine phosphorylation of the scaffolding receptor presumably recruits signaling molecules (e.g. STATs) to the receptor where they can be activated by JAK2 phosphorylation. We have mutated the two tyrosine residues in the cytoplasmic domain of IL27R and used these receptor mutants to determine if tyrosine phosphorylation of IL27R is required to induce the transforming signal of JAK2-V617F. Upon initial cytokine removal, there is a slight lag in the growth of JAK2-V617F cells expressing an IL27R receptor that lacks both intracellular tyrosines, suggesting these tyrosines may play a role in signaling. However, this lag is transient and cytokine-independent growth becomes similar to cells expressing wildtype IL27R and JAK2-V617F. This suggests tyrosine phosphorylation of the receptor is not required to induce the transforming signal of JAK2-V617F. Our studies demonstrate for the first time that single chain components of heterodimeric receptors can functionally replace homodimeric receptors in mediating transformation of hematopoietic cells by JAK2-V617F. Our work also suggests that JAK2-V617F does not need to phosphorylate its receptor scaffold to mediate its transforming signal. The requirement for other endogenous cytokine receptors as substrates for activated JAK2-V617F receptors remains a possibility and is currently under investigation. Together, our studies demonstrate that IL27R, and perhaps other single chain components of heterodimeric receptors, may play unappreciated roles in myeloid diseases such as AML and MPNs.

Role of SH2 Domain in JAK2-V617F Mediated Transformation.

A point mutation in JAK2 (V617F) has been described recently in patients with myeloproliferative diseases like polycythemia vera (PV), essential thrombocythemia (ET) and chronic idiopathic myelofibrosis (IMF). This V617F point mutation in JAK2 has been shown to activate several downstream pathways including STAT5 and ERK. This mutation also renders haematopoietic progenitors cytokine-independent. The role of the V617F mutation in oncogenesis is not fully understood. In this study we aim to dissect the role of the SH2 domain in JAK2-V617F mediated transformation.Stable Ba/F3 cell lines expressing JAK2-wild type (wt), JAK2-V617F, JAK2-R439K (SH2 domain mutation) and JAK2-V617F/R439K mutants were generated. Cell proliferation assays showed that JAK2-V617F transforms Ba/F3 cells and renders them IL3 independent, while wild type JAK2 and JAK2-R439K could not. Surprisingly, JAK2-V617F/R439K was not able to induce a transformed phenotype in Ba/F3 cells. Imunoblotting revealed strong activation of JAK2, STAT5 and ERK in cells expressing JAK2-V617F, whereas no such activation could be found in JAK2-wt, JAK2-R439K and in JAK2-V617F/R439K expressing cells. Thus the SH2 domain in JAK2-V617F seems to play a crucial role in the transformation of Ba/F3 cells containing a heterodimeric (IL-3) cytokine receptor.It has been demonstrated that JAK2-V617F induces cellular transformation more efficiently in cells expressing a homodimeric cytokine receptor such as the erythropoetin receptor. We therefore established Ba/F3 cells overexpressing EpoR together with JAK2-wt, JAK2-V617F, JAK2-R439K and JAK2-V617F/R439K. In contrast to parental Ba/F3 cells, EpoR expressing Ba/F3 cells could be transformed by both JAK2-V617F as well as JAK2-V617F/R439K. Both the single and double mutant Ba/F3 cells showed strong activation of STAT5 and ERK. This suggests that an intact SH2 domain is not required for homodimeric cytokine receptor expressing cells.These results show that transformation by JAK2-V617F requires an intact SH2 domain only in cells expressing a heterodimeric cytokine receptor. In contrast, cells containing a homodimeric cytokine receptor are able to induce transformation in the presence of JAK2-V617F with an additional SH2 mutation. Further progress in understanding the role of the SH2 domain in JAK2-V617F mediated transformation may help in delineating downstream signalling with therapeutic implications.

A peptide mimetic of human interferon (IFN)-beta.

Type I interferons (IFNs) are cytokines that are used clinically as antiviral and antitumour agents. The interaction of IFNs with their heterodimeric type I IFN receptor comprised of IFNAR1 and IFNAR2 is a first step to inducing biological actions. Here, we describe the successful mimicry of IFN-beta by a peptide isolated by phage-display screening using a neutralizing anti-IFN-beta monoclonal antibody. The 15-mer peptide, designated SYR6, was shown to compete with IFN-beta for binding to type I IFN receptor in a concentration-dependent manner, and was shown to elicit antiviral activity on cultured cells. This antiviral activity was not eliminated in the presence of neutralizing monoclonal antibodies to IFN-alpha, -beta and -gamma, and a low concentration of soluble type I IFN receptor, suggesting that it was not due to IFN contamination or the induction of endogenous IFNs by SYR6. This peptide might be a potent agonist to provide a mechanism of activating heterodimeric cytokine receptors.

Structure of the activation domain of the GM-CSF/IL-3/IL-5 receptor common β-chain bound to an antagonist

Heterodimeric cytokine receptors generally consist of a major cytokine-binding subunit and a signaling subunit. The latter can transduce signals by more than 1 cytokine, as exemplified by the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), and IL-6 receptor systems. However, often the signaling subunits in isolation are unable to bind cytokines, a fact that has made it more difficult to obtain structural definition of their ligand-binding sites. This report details the crystal structure of the ligand-binding domain of the GM-CSF/IL-3/IL-5 receptor β-chain (βc) signaling subunit in complex with the Fab fragment of the antagonistic monoclonal antibody, BION-1. This is the first single antagonist of all 3 known eosinophil-producing cytokines, and it is therefore capable of regulating eosinophil-related diseases such as asthma. The structure reveals a fibronectin type III domain, and the antagonist-binding site involves major contributions from the loop between the B and C strands and overlaps the cytokine-binding site. Furthermore, tyrosine421 (Tyr421), a key residue involved in receptor activation, lies in the neighboring loop between the F and G strands, although it is not immediately adjacent to the cytokine-binding residues in the B-C loop. Interestingly, functional experiments using receptors mutated across these loops demonstrate that they are cooperatively involved in full receptor activation. The experiments, however, reveal subtle differences between the B-C loop and Tyr421, which is suggestive of distinct functional roles. The elucidation of the structure of the ligand-binding domain of βc also suggests how different cytokines recognize a single receptor subunit, which may have implications for homologous receptor systems.