Structure-Function Correlation of G6, a Novel Small Molecule Inhibitor of Jak2: INDISPENSABILITY OF THE STILBENOID CORE*

Somatic mutations in the Jak2 protein, such as V617F, cause aberrant Jak/STAT signaling and can lead to the development of myeloproliferative neoplasms. This discovery has led to the search for small molecule inhibitors that target Jak2. Using structure-based virtual screening, our group recently identified a novel small molecule inhibitor of Jak2 named G6. Here, we identified a structure-function correlation of this compound. Specifically, five derivative compounds of G6 having structural similarity to the original lead compound were obtained and analyzed for their ability to (i) inhibit Jak2-V617F-mediated cell growth, (ii) inhibit the levels of phospho-Jak2, phospho-STAT3, and phospho-STAT5; (iii) induce apoptosis in human erythroleukemia cells; and (iv) suppress pathologic cell growth of Jak2-V617F-expressing human bone marrow cells ex vivo. Additionally, we computationally examined the interactions of these compounds with the ATP-binding pocket of the Jak2 kinase domain. We found that the stilbenoid core-containing derivatives of G6 significantly inhibited Jak2-V617F-mediated cell proliferation in a time- and dose-dependent manner. They also inhibited phosphorylation of Jak2, STAT3, and STAT5 proteins within cells, resulting in higher levels of apoptosis via the intrinsic apoptotic pathway. Finally, the stilbenoid derivatives inhibited the pathologic growth of Jak2-V617F-expressing human bone marrow cells ex vivo. Collectively, our data demonstrate that G6 has a stilbenoid core that is indispensable for maintaining its Jak2 inhibitory potential.     

Cell death induced by the Jak2 inhibitor, G6, correlates with cleavage of vimentin filaments

Hyperkinetic Jak2 tyrosine kinase signaling has been implicated in several human diseases including leukemia, lymphoma, myeloma, and the myeloproliferative neoplasms. Using structure-based virtual screening, we previously identified a novel Jak2 inhibitor named G6. We showed that G6 specifically inhibits Jak2 kinase activity and suppresses Jak2-mediated cellular proliferation. To elucidate the molecular and biochemical mechanisms by which G6 inhibits Jak2-mediated cellular proliferation, we treated Jak2-V617F expressing human erythroleukemia (HEL) cells for 12 h with either vehicle control or 25 μM of the drug and compared protein expression profiles using two-dimensional gel electrophoresis. One differentially expressed protein identified by electrospray mass spectroscopy was the intermediate filament protein, vimentin. It was present in DMSO treated cells but absent in G6 treated cells. HEL cells treated with G6 showed both time- and dose-dependent cleavage of vimentin as well as a marked reorganization of vimentin intermediate filaments within intact cells. In a mouse model of Jak2-V617F mediated human erythroleukemia, G6 also decreased the levels of vimentin protein, in vivo. The G6-induced cleavage of vimentin was found to be Jak2-dependent and calpain-mediated. Furthermore, we found that intracellular calcium mobilization is essential and sufficient for the cleavage of vimentin. Finally, we show that the cleavage of vimentin intermediate filaments, per se, is sufficient to reduce HEL cell viability. Collectively, these results suggest that G6-induced inhibition of Jak2-mediated pathogenic cell growth is concomitant with the disruption of intracellular vimentin filaments. As such, this work describes a novel pathway for the targeting of Jak2-mediated pathological cell growth.     

Activated Jak2 with the V617F point mutation promotes G1/S phase transition

Hematopoietic stem cells in myeloproliferative diseases mostly retain the potential to differentiate but are characterized by hyper-responsiveness to growth factors, as well as partial factor-independent growth. The V617F activating point mutation in Jak2 has recently been associated with myeloproliferative disorders. Using various cell line models, mechanisms that contribute to Jak2V617-mediated signaling were investigated. Treatment of the Jak2V617F mutant-expressing erythroid leukemia cell line HEL with a small molecule Jak2 inhibitor was associated with a dose-dependent G(1) cell cycle arrest. This inhibition correlated with decreased expression of cyclin D2 and increased expression of the cell cycle inhibitor p27(Kip). Inhibition of Jak2V617F with a Jak2-targeted small interfering RNA approach resulted in a similar phenotype. Mechanisms leading to altered p27(Kip) and cyclin D2 likely involve inhibition of STAT5, a major target of Jak2 in hematopoietic cells, because a constitutively active form of STAT5 reduced p27(Kip) and increased cyclin D2 expression. Jak2V617F and constitutively active STAT5 also induced high levels of reactive oxygen species, which are sufficient to promote G(1)/S phase transition. In contrast, treatment of HEL cells with the antioxidant N-acetylcysteine decreased cell growth or expression of cyclin D2 and increased expression of p27(Kip). Similar results were obtained in BaF3 cells transfected with Jak2V617F, but these cells required coexpression of the erythropoietin receptor for optimal signaling. These results suggest that regulation of cyclin D2 and p27(Kip) in combination with redox-dependent processes promotes G(1)/S phase transition downstream of Jak2V617F/STAT5 and therefore hint at potential novel targets for drug development that may aid traditional therapy.     

DNA damage stress and inhibition of Jak2-V617F cause its degradation and synergistically induce apoptosis through activation of GSK3β

The cytoplasmic tyrosine kinase Jak2 plays a crucial role in cytokine receptor signaling in hematopoietic cells. The activated Jak2-V617F mutant is present in most cases of BCR/ABL-negative myeloproliferative neoplasms and constitutively activates downstream signals from homodimeric cytokine receptors, such as the erythropoietin receptor (EpoR). Here we examine the effects of DNA damage stress on Jak2 or Jak2-V617F and on induction of apoptosis in hematopoietic cells. Etoposide or doxorubicin dose-dependently decreased the expression level of Jak2 in UT7 or 32D cells expressing EpoR in the absence of Epo and that of exogenously expressed Jak2-V617F in UT7 cells when cotreated with the Jak2 inhibitor JakI-1 or AG490. Studies with pharmacological inhibitors and genetic manipulations further showed that downregulation of the PI3K/Akt pathway leading to the activation of GSK3β may be involved in downregulation of Jak2 or Jak2-V617F as well as in synergistic induction of Bax activation and apoptosis. The downregulation of Jak2 was inhibited by the proteasome inhibitor MG132 or by expression of both of loss-of-function mutants of c-Cbl and Cbl-b, E3 ubiquitin ligases which facilitated ubiquitination of Jak2-V617F when co-expressed in 293T cells. The pan-caspase inhibitor Boc-d-fmk also inhibited the Jak2 downregulation as well as appearance of a 100-kDa fragment that contained the N-terminal portion of Jak2 in response to DNA damage. Together, these data suggest that DNA damage stress with simultaneous inhibition of the kinase activity causes degradation of Jak2 or Jak2-V617F by caspase cleavage and proteasomal degradation through GSK3β activation, which is closely involved in synergistic induction of apoptosis in hematopoietic cells.     

Proliferation and Survival Signaling from Both Jak2-V617F and Lyn Involving GSK3 and mTOR/p70S6K/4EBP1 in PVTL-1 Cell Line Newly Established from Acute Myeloid Leukemia Transformed from Polycythemia Vera

The gain of function mutation JAK2-V617F is very frequently found in myeloproliferative neoplasms (MPNs) and is strongly implicated in pathogenesis of these and other hematological malignancies. Here we report establishment of a new leukemia cell line, PVTL-1, homozygous for JAK2-V617F from a 73-year-old female patient with acute myeloid leukemia (AML) transformed from MPN. PVTL-1 is positive for CD7, CD13, CD33, CD34, CD117, HLA-DR, and MPO, and has complex karyotypic abnormalities, 44,XX,-5q,-7,-8,add(11)(p11.2),add(11)(q23),−16,+21,−22,+mar1. Sequence analysis of JAK2 revealed only the mutated allele coding for Jak2-V617F. Proliferation of PVTL-1 was inhibited and apoptosis was induced by the pan-Jak inhibitor Jak inhibitor-1 (JakI-1) or dasatinib, which inhibits the Src family kinases as well as BCR/ABL. Consistently, the Src family kinase Lyn was constitutively activated with phosphorylation of Y396 in the activation loop, which was inhibited by dasatinib but not by JakI-1. Further analyses with JakI-1 and dasatinib indicated that Jak2-V617F phosphorylated STAT5 and SHP2 while Lyn phosphorylated SHP1, SHP2, Gab-2, c-Cbl, and CrkL to induce the SHP2/Gab2 and c-Cbl/CrkL complex formation. In addition, JakI-1 and dasatinib inactivated the mTOR/p70S6K/4EBP1 pathway and reduced the inhibitory phosphorylation of GSK3 in PVTL-1 cells, which correlated with their effects on proliferation and survival of these cells. Furthermore, inhibition of GSK3 by its inhibitor SB216763 mitigated apoptosis induced by dasatinib but not by JakI-1. Together, these data suggest that apoptosis may be suppressed in PVTL-1 cells through inactivation of GSK3 by Lyn as well as Jak2-V617F and additionally through activation of STAT5 by Jak2-V617F. It is also speculated that activation of the mTOR/p70S6K/4EBP1 pathway may mediate proliferation signaling from Jak2-V617F and Lyn. PVTL-1 cells may provide a valuable model system to elucidate the molecular mechanisms involved in evolution of Jak2-V617F-expressing MPN to AML and to develop novel therapies against this intractable condition.     

Inhibition of the PI3K/Akt/GSK3 Pathway Downstream of BCR/ABL,Jak2-V617F,or FLT3-ITD Downregulates DNA Damage-Induced Chk1 Activation as Well as G2/M Arrest and Prominently Enhances Induction of Apoptosis

Constitutively-activated tyrosine kinase mutants, such as BCR/ABL, FLT3-ITD, and Jak2-V617F, play important roles in pathogenesis of hematopoietic malignancies and in acquisition of therapy resistance. We previously found that hematopoietic cytokines enhance activation of the checkpoint kinase Chk1 in DNA-damaged hematopoietic cells by inactivating GSK3 through the PI3K/Akt signaling pathway to inhibit apoptosis. Here we examine the possibility that the kinase mutants may also protect DNA-damaged cells by enhancing Chk1 activation. In cells expressing BCR/ABL, FLT3-ITD, or Jak2-V617F, etoposide induced a sustained activation of Chk1, thus leading to the G2/M arrest of cells. Inhibition of these kinases by their inhibitors, imatinib, sorafenib, or JakI-1, significantly abbreviated Chk1 activation, and drastically enhanced apoptosis induced by etoposide. The PI3K inhibitor GD-0941 or the Akt inhibitor MK-2206 showed similar effects with imatinib on etoposide-treated BCR/ABL-expressing cells, including those expressing the imatinib-resistant T315I mutant, while expression of the constitutively activated Akt1-myr mutant conferred resistance to the combined treatment of etoposide and imatinib. GSK3 inhibitors, including LiCl and SB216763, restored the sustained Chk1 activation and mitigated apoptosis in cells treated with etoposide and the inhibitors for aberrant kinases, PI3K, or Akt. These observations raise a possilibity that the aberrant kinases BCR/ABL, FLT3-ITD, and Jak2-V617F may prevent apoptosis induced by DNA-damaging chemotherapeutics, at least partly through enhancement of the Chk1-mediated G2/M checkpoint activation, by inactivating GSK3 through the PI3K/Akt signaling pathway. These results shed light on the molecular mechanisms for chemoresistance of hematological malignancies and provide a rationale for the combined treatment with chemotherapy and the tyrosine kinase or PI3K/Akt pathway inhibitors against these diseases.     

Erlotinib effectively inhibits JAK2V617F activity and polycythemia vera cell growth

JAK2(V617F), a mutant of tyrosine kinase JAK2, is found in most patients with polycythemia vera (PV) and a substantial proportion of patients with idiopathic myelofibrosis or essential thrombocythemia. The JAK2 mutant displays a much increased kinase activity and generates a PV-like phenotype in mouse bone marrow transplant models. This study shows that the anti-cancer drug erlotinib (Tarceva) is a potent inhibitor of JAK2(V617F) activity. In vitro colony culture assays revealed that erlotinib at micro-molar concentrations effectively suppresses the growth and expansion of PV hematopoietic progenitor cells while having little effect on normal cells. Furthermore, JAK2(V617F)-positive cells from PV patients show greater susceptibility to the inhibitor than their negative counterparts. Similar inhibitory effects were found with the JAK2(V617F)-positive human erythroleukemia HEL cell line. These data suggest that erlotinib may be used for treatment of JAK2(V617F)-positive PV and other myeloproliferative disorders.     

A shift in the salt bridge interaction of residues D620 and E621 mediates the constitutive activation of Jak2-H538Q/K539L

Jak2 mutations in the exon 14 and exon 12 regions that cause constitutive activation have been associated with myeloproliferative neoplasms. We have previously shown that a pi stacking interaction between F617 and F595 is important for the constitutive activation of Jak2-V617F (Gnanasambandan et al., Biochemistry 49:9972-9984, 2010). Here, using a combination of molecular dynamics (MD) simulations and in vitro mutagenesis, we studied the molecular mechanism for the constitutive activation of the Jak2 exon 12 mutation, H538Q/K539L. The activation levels of Jak2-H538Q/K539L were found to be similar to that of Jak2-V617F, and Jak2-H538Q/K539L/V617F. Data from MD simulations indicated a shift in the salt bridge interactions of D620 and E621 with K539 in Jak2-WT to R541 in Jak2-H538Q/K539L. When compared to Jak2-WT, K539A mutation resulted in increased activation, while K539D or K539E mutations diminished Jak2 activation by 50 %. In the context of Jak2-H538Q/K539L, R541A mutation reduced its activation by 50 %, while R541D and R541E mutations returned its activation levels to that of Jak2-WT. Collectively, these results indicate that a shift in the salt bridge interaction of D620 and E621 with K539 in Jak2-WT to R541 in Jak2-H538Q/K539L is critical for constitutive activation of this Jak2 exon 12 mutant.     

The Jak2 Small Molecule Inhibitor,G6, Reduces the Tumorigenic Potential of T98G Glioblastoma Cells In Vitro and In Vivo

Glioblastoma multiforme (GBM) is the most common and the most aggressive form of primary brain tumor. Jak2 is a non-receptor tyrosine kinase that is involved in proliferative signaling through its association with various cell surface receptors. Hyperactive Jak2 signaling has been implicated in numerous hematological disorders as well as in various solid tumors including GBM. Our lab has developed a Jak2 small molecule inhibitor known as G6. It exhibits potent efficacy in vitro and in several in vivo models of Jak2-mediated hematological disease. Here, we hypothesized that G6 would inhibit the pathogenic growth of GBM cells expressing hyperactive Jak2. To test this, we screened several GBM cell lines and found that T98G cells express readily detectable levels of active Jak2. We found that G6 treatment of these cells reduced the phosphorylation of Jak2 and STAT3, in a dose-dependent manner. In addition, G6 treatment reduced the migratory potential, invasive potential, clonogenic growth potential, and overall viability of these cells. The effect of G6 was due to its direct suppression of Jak2 function and not via off-target kinases, as these effects were recapitulated in T98G cells that received Jak2 specific shRNA. G6 also significantly increased the levels of caspase-dependent apoptosis in T98G cells, when compared to cells that were treated with vehicle control. Lastly, when T98G cells were injected into nude mice, G6 treatment significantly reduced tumor volume and this was concomitant with significantly decreased levels of phospho-Jak2 and phospho-STAT3 within the tumors themselves. Furthermore, tumors harvested from mice that received G6 had significantly less vimentin protein levels when compared to tumors from mice that received vehicle control solution. Overall, these combined in vitro and in vivo results indicate that G6 may be a viable therapeutic option against GBM exhibiting hyperactivation of Jak2.     

Octa-arginine mediated delivery of wild-type Lnk protein inhibits TPO-induced M-MOK megakaryoblastic leukemic cell growth by promoting apoptosis

Background

Lnk plays a non-redundant role by negatively regulating cytokine signaling of TPO, SCF or EPO. Retroviral expression of Lnk has been shown to suppress hematopoietic leukemic cell proliferation indicating its therapeutic value in cancer therapy. However, retroviral gene delivery carries risks of insertional mutagenesis. To circumvent this undesired consequence, we fused a cell permeable peptide octa-arginine to Lnk and evaluated the efficacy of inhibition of leukemic cell proliferation in vitro.

Methodology/Principal Findings

In this study, proliferation assays, flow cytometry, Western Blot analyses were performed on wild-type (WT), mutant Lnk R8 or BSA treated M-MOK cells. We found that delivered WT, but not mutant Lnk R8 blocked TPO-induced M-MOK megakaryoblastic leukemic cell proliferation. In contrast, WT Lnk R8 showed no growth inhibitive effect on non-hematopoietic HELA or COS-7 cell. Moreover, we demonstrated that TPO-induced M-MOK cell growth inhibition by WT Lnk R8 was dose-dependent. Penetrated WT Lnk R8 induced cell cycle arrest and apoptosis. Immunoprecipitation and Western blots data indicated WT Lnk R8 interacted with endogeneous Jak2 and downregulated Jak-Stat and MAPK phosphorylation level in M-MOK cells after TPO stimulation. Treatment with specific inhibitors (TG101348 and PD98059) indicated Jak-Stat and MAPK pathways were crucial for TPO-induced proliferation of M-MOK cells. Further analyses using TF-1 and HEL leukemic cell-lines showed that WT Lnk R8 inhibited Jak2-dependent cell proliferation. Using cord blood-derived CD34+ stem cells, we found that delivered WT Lnk R8 blocked TPO-induced megakaryopoiesis in vitro.

Conclusions/Significance

Intracellular delivery of WT Lnk R8 fusion protein efficiently inhibited TPO-induced M-MOK leukemic cell growth by promoting apoptosis. WT Lnk R8 protein delivery may provide a safer and more practical approach to inhibit leukemic cell growth worthy of further development.     

STAT1 is involved in signal transduction in the EPO induced HEL cells

Erythropoietin(EPO) is the major regulator of mamalian erythropoisis,which stimulates the growth and differentiation of hematopoietic cells through interaction with its receptor(EPO-R),Here we use HEL cells (a human erythro-leukemia cell line) as a model to elucidate the pathway of signal transduction in the EPO-induced HEL cells.Our data show that the EPOR (EPO receptor) on the surface of HEL cells interacts with the Janus tyrosine protein kinase(Jak2) to transduce intracellular signals through phosphorylation of cytoplasmic proteins in EPO-treated HEL cells.Both STAT1 and STAT5 in this cell line are tyrosine-phosphorylated and translocated to nucleus following the dinding of EPO to HEL cells.Furthermore,the dinding of both STAT1 and STAT5 proteins to specific DNA elements(SIE and PIE elements) is revealed in an EPO-dependent manner,Our data demonstrate that the pathway of signal transduction following the binding of EPO to HEL cells is similar to immature eryhroid cell from the spleen of mice infected with anemia strain of Friend virus.     

Jak2 FERM domain interaction with the erythropoietin receptor regulates Jak2 kinase activity

Janus kinases are essential for signal transduction by a variety of cytokine receptors and when inappropriately activated can cause hematopoietic disorders and oncogenesis. Consequently, it can be predicted that the interaction of the kinases with receptors and the events required for activation are highly controlled. In a screen to identify phosphorylation events regulating Jak2 activity in EpoR signaling, we identified a mutant (Jak2-Y613E) which has the property of being constitutively activated, as well as an inactivating mutation (Y766E). Although no evidence was obtained to indicate that either site is phosphorylated in signaling, the consequences of the Y613E mutation are similar to those observed with recently described activating mutations in Jak2 (Jak2-V617F and Jak2-L611S). However, unlike the V617F or L611S mutant, the Y613E mutant requires the presence of the receptor but not Epo stimulation for activation and downstream signaling. The properties of the Jak2-Y613E mutant suggest that under normal conditions, Jak2 that is not associated with a receptor is locked into an inactive state and receptor binding through the FERM domain relieves steric constraints, allowing the potential to be activated with receptor engagement.     

Curcumin induces apoptosis in JAK2‐mutated cells by the inhibition of JAK2/STAT and mTORC1 pathways

Myeloproliferative neoplasms are chronic myeloid cancers divided in Philadelphia positive and negative. The JAK2 V617F is the most common mutation in Philadelphia negative patients and results in a constitutive activation of the JAK/STAT pathway, conferring a proliferative advantage and apoptosis inhibition. Recent studies identified a functional crosstalk between the JAK/STAT and mTOR pathways. The identification of an effective therapy is often difficult, so the availability of new therapeutic approaches might be attractive. Previous studies showed that curcumin, the active principle of the Curcuma longa, can suppress JAK2/STAT pathways in different type of cancer and injuries. In this study, we investigated the anti‐proliferative and pro‐apoptotic effects of curcumin in JAK2 V617F‐mutated cells. HEL cell line and cells from patients JAK2 V617F mutated have been incubated with increasing concentrations of curcumin for different time. Apoptosis and proliferation were evaluated. Subsequently, JAK2/STAT and AKT/mTOR pathways were investigated at both RNA and protein levels. We found that curcumin induces apoptosis and inhibition of proliferation in HEL cells. Furthermore, we showed that curcumin inhibits JAK2/STAT and mTORC1 pathways in JAK2 V617F‐mutated cells. This inhibition suggests that curcumin could represent an alternative strategy to be explored for the treatment of patients with myeloproliferative neoplasms.     

c-Abl Activates Janus Kinase 2 in Normal Hematopoietic Cells

Jak2 is involved in cytokine growth factor-stimulated signal transduction, but the mechanism of its activation is largely unknown. Here, we investigated Jak2 activation in a normal hematopoietic cell line, 32D mouse myeloid cells. The bimolecular fluorescence complementation studies showed that c-Abl formed a stable complex with Jak2 in live cells. Co-immunoprecipitation results showed that c-Abl bound to the βc chain of IL-3/IL-5/GM-CSF receptors. The kinase activities of both c-Abl and Jak2 were stimulated by IL-3 in 32D cells. Decreasing c-Abl protein expression in 32D cells by inducible shRNA decreased Jak2 activity and resulted in the failure of Jak2 activation in response to IL-3. Treatment of IL-3 and serum-starved 32D cells with 1 μm imatinib mysylate inhibited IL-3 stimulated kinase activities of both c-Abl and Jak2. In addition, the kinase-deficient Bcr-Abl mutant (p210K1172R) was defective for activation of Jak2 in 32D cells and impaired IL-3 independent growth, which was rescued by overexpression of c-Abl (+Abl). IL-3 efficiently inhibited apoptosis of 32Dp210K/R+Abl cells induced by imatinib mysylate but not Jak2 kinase inhibitor TG101209. In summary, our findings provide evidence that the kinase function of c-Abl and its C-terminal CT4 region is crucial for its interaction with Jak2 and its activation. c-Abl kinase activity induced by IL-3 is required for IL-3-stimulated Jak2 and Jak1 activation. Our findings reveal a novel regulatory role of c-Abl in Jak2 activation induced by IL-3 cytokine growth factor in 32D hematopoietic cells.     

Mpl Traffics to the Cell Surface Through Conventional and Unconventional Routes

Myeloproliferative neoplasms (MPNs) are often characterized by JAK2 or calreticulin (CALR) mutations, indicating aberrant trafficking in pathogenesis. This study focuses on Mpl trafficking and Jak2 association using two model systems: human erythroleukemia cells (HEL; JAK2V617F) and K562 myeloid leukemia cells (JAK2WT). Consistent with a putative chaperone role for Jak2, Mpl and Jak2 associate on both intracellular and plasma membranes (shown by proximity ligation assay) and siRNA‐mediated knockdown of Jak2 led to Mpl trapping in the endoplasmic reticulum (ER). Even in Jak2 sufficient cells, Mpl accumulates in punctate structures that partially colocalize with ER‐tracker, the ER exit site marker (ERES) Sec31a, the autophagy marker LC3 and LAMP1. Mpl was fused to miniSOG, a genetically encoded tag for correlated light and electron microscopy. Results suggest that a fraction of Mpl is taken up into autophagic structures from the ER and routed to autolyososomes. Surface biotinylation shows that both immature and mature Mpl reach the cell surface; in K562 cells Mpl is also released in exosomes. Both forms rapidly internalize upon ligand addition, while recovery is primarily attributed to immature Mpl. Mpl appears to reach the plasma membrane via both conventional ER‐Golgi and autolysosome secretory pathways, as well as recycling.      

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 β 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 β 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.