Table S1: Crystallographic data collection and refinement statistics Table S2: Inhibitory activity against other BET bromodomains Table S3: Bromodomain profiling data Fig. S1: Detailed binding interactions of compounds 1 - 5 in BRD4-1 Fig. S2: Comparison of compound 2 and JQ1 in Brd4-1 Fig. S3: Chemical structures of other compounds used in this work. Fig. S4: Statistical evaluation of kinome profiling data using the Gini coefficient Fig. S5: Original flow cytometry data of MM1.S cells Fig. S6: MM1.S cells require stimulation with IL-6 to detect phospho-STAT3 levels Fig. S7: Effect of dual BRD4-kinase inhibitors on MV-4-11 cells. Fig. S8: Time dependent alteration of c-Myc and p21Cip1 levels in MM1.S cells. Fig. S9: Effect of dual BET-kinase inhibitors on ruxolitinib sensitive MPN cell lines Fig. S10: Dose-effect analysis of UKE-1 cells treated with drug combinations Fig. S11: Cell line sensitivity by tissue type

Table S4: Kinome profiling data (Microsoft Excel sheet) Table S5: Cell line screening data (Microsoft Excel sheet)

Aberrant JAK2 tyrosine kinase signaling drives the development of Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. However, JAK2 kinase inhibitors have failed to significantly reduce allele burden in MPN patients, underscoring the need for improved therapeutic strategies. Members of the PIM family of serine/threonine kinases promote cellular proliferation by regulating a variety of cellular processes, including protein synthesis and the balance of signaling that regulates apoptosis. Overexpression of PIM family members is oncogenic, exemplified by their ability to induce lymphomas in collaboration with c-Myc. Thus, PIM kinases are potential therapeutic targets for several malignancies such as solid tumors and blood cancers. We and others have shown that PIM inhibitors augment the efficacy of JAK2 inhibitors by using in vitro models of MPNs. Here we report that the recently developed pan-PIM inhibitor INCB053914 augments the efficacy of the US Food and Drug Administration-approved JAK1/2 inhibitor ruxolitinib in both in vitro and in vivo MPN models. INCB053914 synergizes with ruxolitinib to inhibit cell growth in JAK2-driven MPN models and induce apoptosis. Significantly, low nanomolar INCB053914 enhances the efficacy of ruxolitinib to inhibit the neoplastic growth of primary MPN patient cells, and INCB053914 antagonizes ruxolitinib persistent myeloproliferation in vivo. These findings support the notion that INCB053914, which is currently in clinical trials in patients with advanced hematologic malignancies, in combination with ruxolitinib may be effective in MPN patients, and they support the clinical testing of this combination in MPN patients.

The Proviral Integration site of Moloney murine leukemia virus (PIM) serine/threonine protein kinases are overexpressed in many hematologic and solid tumor malignancies and play central roles in intracellular signaling networks important in tumorigenesis, including the Janus kinase-signal transducer and activator of transcription (JAK/STAT) and phosphatidylinositol 3-kinase (PI3K)/AKT pathways. The three PIM kinase isozymes (PIM1, PIM2, and PIM3) share similar downstream substrates with other key oncogenic kinases and have differing but mutually compensatory functions across tumors. This supports the therapeutic potential of pan-PIM kinase inhibitors, especially in combination with other anticancer agents chosen based on their role in overlapping signaling networks. Reported here is a preclinical characterization of INCB053914, a novel, potent, and selective adenosine triphosphate-competitive pan-PIM kinase inhibitor. In vitro, INCB053914 inhibited proliferation and the phosphorylation of downstream substrates in cell lines from multiple hematologic malignancies. Effects were confirmed in primary bone marrow blasts from patients with acute myeloid leukemia treated ex vivo and in blood samples from patients receiving INCB053914 in an ongoing phase 1 dose-escalation study. In vivo, single-agent INCB053914 inhibited Bcl-2-associated death promoter protein phosphorylation and dose-dependently inhibited tumor growth in acute myeloid leukemia and multiple myeloma xenografts. Additive or synergistic inhibition of tumor growth was observed when INCB053914 was combined with selective PI3Kδ inhibition, selective JAK1 or JAK1/2 inhibition, or cytarabine. Based on these data, pan-PIM kinase inhibitors, including INCB053914, may have therapeutic utility in hematologic malignancies when combined with other inhibitors of oncogenic kinases or standard chemotherapeutics.

Classic Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) are driven by aberrant activity of the JAK2 tyrosine kinase. While JAK2 inhibitors rarely induce molecular remission, they provide quality of life improvements for patients and thus will be a mainstay for MPN treatment for the foreseeable future. This near ubiquitous upfront ineffectiveness of JAK2 inhibitors must be overcome if they are going to contribute to a significant disease-altering therapy for MPNs. The insulin-like growth factor-1 receptor (IGF1R) can contribute to resistance to kinase inhibitor cancer therapeutics and a recent study demonstrated a requirement of IGF1R in JAK2-V617F-induced MPN in mice. Furthermore, IGF is known to play a role in neoplastic erythroid colony formation of hematopoietic progenitors from MPN patients. Thus, there is evidence for a potential role for IGF1R signaling in the context of JAK2 inhibition, particularly in the setting of JAK2 inhibitor resistance/persistence, a concept that remains underexplored.

Abstract Synergistic action of kinase and BET bromodomain inhibitors in cell killing has been reported for a variety of cancers. Using the chemical scaffold of the JAK2 inhibitor TG101348, we developed and characterized single agents which potently and simultaneously inhibit BRD4 and a specific set of oncogenic tyrosine kinases including JAK2, FLT3, RET, and ROS1. Lead compounds showed on-target inhibition in several blood cancer cell lines and were highly efficacious at inhibiting the growth of hematopoietic progenitor cells from patients with myeloproliferative neoplasm. Screening across 931 cancer cell lines revealed differential growth inhibitory potential with highest activity against bone and blood cancers and greatly enhanced activity over the single BET inhibitor JQ1. Gene drug sensitivity analyses and drug combination studies indicate synergism of BRD4 and kinase inhibition as a plausible reason for the superior potency in cell killing. Combined, our findings indicate promising potential of these agents as novel chemical probes and cancer therapeutics. Mol Cancer Ther; 16(6); 1054–67. ©2017 AACR.

The optimism of anti-JAK2 therapy for the treatment of MPNs is largely based on the ability of JAK inhibitors to improve MPN patient constitutional symptoms. Unfortunately, such inhibitors are generally unable to induce remission or even allele burden, suggesting anti-JAK2 based therapies need refinement and optimization. Combination therapies continue to be widely investigated, however, such approaches could have significant complications when translating to patients. These include the difficulty determining proper dosing of each drug in combination in patients, the cost of treatment, and issues with combining drugs developed by different pharmaceutical companies. Recently, we and others have identified anti-BET bromodomain inhibitory properties of various kinase inhibitors, including the JAK2 inhibitors TG101209 and TG101308. Bromodomains are protein-protein interaction motifs that bind to acetylated lysine and, for example, can play roles in regulating gene expression by binding acetylated histones. These dual kinase-BET inhibitors bind to the acetylated lysine-binding pocket of BET bromodomains and thus inhibit the function of such domains. Importantly, cancer cells appear to be particularly sensitive to BET inhibitors compared to normal cells. Combining a BET inhibitor and a JAK2 inhibitor has been shown to be more effective against MPN cells than JAK2 inhibition alone. Our recent identification of dual kinase-BET inhibitors allows for the rational design of drugs with polypharmacology to inhibit more than one class of targets. To this end, we have optimized small molecules for dual anti-JAK2 and anti-BET activity. MA2-014 is a chemical derivative of TG101209 that exhibits similar anti-JAK2 activity, but about ten-fold improved anti-BET activity than TG101209. In fact, the activity of MA2-014 to target BET domains is similar to the prototypical BET inhibitor JQ1. For example, the IC50s of MA2-014 and JQ1 against the second bromodomain of the BET family member BRD4 are each about 20 nM. MA2-014 retains comparable biochemical activity against JAK2 as TG101209 and ruxolitinib (IC50s of low single digit nM, ~0.5 to 3 nM). In MPN cells, however, the ability of MA2-014 to inhibit JAK2-V617F signaling in MPN cells, as measured by P-STAT5, is about ten-fold improved over TG101209, and is comparable to ruxolitinib. Likewise, the ability of MA2-014 to inhibit the expression of c-Myc, which is widely used as a biomarker for BET inhibition, is about ten-fold better than TG101209 in MPN cells. The IC50 for MA2-014 for growth of Uke1 MPN cells is about 200 nM, compared to 500 nM for TG101209. Taken together, these data suggest that MA2-014 is a dual JAK2-BET inhibitor that exhibits superior BET inhibitory activity with similar if not better cellular JAK2 inhibitory activity than TG101209. MA2-014 efficiently inhibited the erythropoietin independent erythroid colony formation of myeloid progenitors from MPN patients, which is a hallmark of these cells and is widely used to test MPN therapeutics. The IC50 of MA2-014 in this assay is 50 nM, essentially identical to ruxolitinib, the only FDA approved JAK2 inhibitor for MPNs. The IC50 of TG101209 to inhibit this colony formation of primary MPN cells is 200 nM, four times greater than MA2-014. Interestingly, JAK2-V617F-driven MPN model cells that are resistant to ruxolitinib retained sensitivity to MA2-014. The IC50s of ruxolitinib and MA2-014 against the growth of BaF3-JAK2-V617F cells are about 100 nM. However, the same cells that are resistant to ruxolitinib (IC50 >2000 nM) remain sensitive to MA2-014 (IC50 of 240 nM). Finally, in long-term culture assays, we have determined that JAK2-V617F driven MPN Uke1 cells are not able to become resistant to MA2-014 as readily as they do to TG101209 or ruxolitinib. These data suggest MA2-014 may be more resilient to drug resistance, the major hurdle in the clinical effectiveness of JAK2 inhibitors. Collectively, our work demonstrates that rationally-designed polypharmacology may be a novel approach to develop effective therapeutics for cancer, especially diseases that are driven by aberrant kinase signaling and are also sensitive to BET inhibition, as exemplified by MPNs. The use of such an optimized polypharmacologic therapeutic may provide the benefits of combination therapy with fewer complications associated with clinical development. Disclosures No relevant conflicts of interest to declare.

Classical myeloproliferative neoplasms (MPNs) are hematopoietic stem cell disorders that exhibit excess mature myeloid cells, bone marrow fibrosis, and risk of leukemic transformation. Aberrant JAK2 signaling plays an etiological role in MPN formation. Because neoplastic cells in patients are largely insensitive to current anti-JAK2 therapies, effective therapies remain needed. Members of the PIM family of serine/threonine kinases are induced by JAK/STAT signaling, regulate hematopoietic stem cell growth, protect hematopoietic cells from apoptosis, and exhibit hematopoietic cell transforming properties. We hypothesized that PIM kinases may offer a therapeutic target for MPNs. We treated JAK2-V617F-dependent MPN model cells as well as primary MPN patient cells with the PIM kinase inhibitors SGI-1776 and AZD1208 and the JAK2 inhibitor ruxolitinib. While MPN model cells were rather insensitive to PIM inhibitors, combination of PIM inhibitors with ruxolitinib led to a synergistic effect on MPN cell growth due to enhanced apoptosis. Importantly, PIM inhibitor mono-therapy inhibited, and AZD1208/ruxolitinib combination therapy synergistically suppressed, colony formation of primary MPN cells. Enhanced apoptosis by combination therapy was associated with activation of BAD, inhibition of downstream components of the mTOR pathway, including p70S6K and S6 protein, and activation of 4EBP1. Importantly, PIM inhibitors re-sensitized ruxolitinib-resistant MPN cells to ruxolitinib by inducing apoptosis. Finally, exogenous expression of PIM1 induced ruxolitinib resistance in MPN model cells. These data indicate that PIMs may play a role in MPNs and that combining PIM and JAK2 kinase inhibitors may offer a more efficacious therapeutic approach for MPNs over JAK2 inhibitor mono-therapy.

Bromodomain (BRD)-containing proteins are essential for the recognition of acetylated lysine residues of histones during transcriptional activation. The BRD-containing proteins have emerged as promising drug targets for a number of diseases, including many cancers, that are characterized by changes in the epigenetic cell signature. Recent reports have shown that targeting BRD4 with small molecules may represent a new way to treat prostate and breast cancer, acute myeloid leukemia and melanoma. We have identified many type 1 and type 2 kinase inhibitors which also inhibit BRD4 by robotic co-crystallization screening of kinase inhibitor libraries against BRD4. In each case the co-crystal structure unambiguously revealed the inhibitor bound to the acetyl lysine site of BRD4-1. The identified BRD4 ligands were subjected to differential scanning fluorimetry (DSF) and AlphaScreen assay to assess their binding and inhibitory potentials against BRD4. As shown previously for other BRD-inhibitor-protein complexes, the melting temperatures of BRD4-kinase inhibitor complexes were logarithmically proportional to their IC50 values. We now report the design, synthesis, structural analysis and biological evaluation of next-generation nanomolar BET-selective and nanomolar dual-activity BET-JAK2 inhibitors, based on the initial co-crystallization screening hits. Structure activity relationships were developed using both DSF and co-crystallization of the ligands with BRD4, to assess binding potential and binding modes, respectively. We report initial evaluation of the anticancer potential of compounds possessing dual potent BRD4 and JAK2 inhibitory properties. In addition to myeloma cell lines, this includes the evaluation of dual BRD4-JAK2 inhibitor compounds against JAK2-driven myeloproliferative neoplasm cell lines and primary cells from patients. Citation Format: Steven Gunawan, Ayaz Muhammad, Stuart W. J. Ember, Jin-Yi Zhu, Rebecca A. Jacobsen, Norbert Berndt, Que T. Lambert, Gary W. Reuther, Harshani R. Lawrence, Ernst Schonbrunn, Nicholas J. Lawrence. Targeting the acetyl-lysine binding site of BRD4 with dual nanomolar BET-JAK2 inhibitors: A new anticancer therapeutic strategy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3643. doi:10.1158/1538-7445.AM2015-3643

Myeloproliferative neoplasms (MPNs) are a group of hematopoietic stem cell disorders characterized by the abnormal production of various myeloid cells. Aberrant JAK2 signaling (e.g. induced by JAK2-V617F) plays an etiological role in MPN formation. While JAK2 inhibitors improve patient symptoms, they do not induce cell death as neoplastic cells appear to be rather insensitive to JAK2 inhibition and effectively rapidly become resistant to treatment. Therefore, the development of additional therapeutic approaches for MPNs is needed. Pim kinases are serine/threonine kinases that protect hematopoietic cells from apoptosis and also play a role in regulating hematopoietic stem cell growth. In mouse models, elevated Pim expression contributes to the development of lymphoma. Pims are constitutively active and thus regulated by protein expression, which is controlled by Pim gene expression and Pim protein stability. Pim1 gene expression is normally induced by JAK2/STAT5 signaling in response to extracellular growth factor stimulation, as Pim1 is a direct transcriptional target of STAT5. The deregulated JAK2 signaling in MPNs also induces Pim expression. STAT5 is required for MPN disease in mouse models, suggesting genes transcriptionally regulated by STAT5 are required for MPN disease formation. Together with the anti-apoptotic signaling and transforming properties of Pims, this suggests Pims may play a role in MPNs. We hypothesized that Pim kinases may offer a therapeutic target for MPNs and that Pim kinase inhibitors in combination with JAK inhibitors may cause neoplastic cytotoxicity, improving on current JAK2-inhibitor mono-therapy for MPNs. JAK2-V617F-dependentMPN model cells (HEL, SET2, Uke1, and BaF3/JAK2-V617F, including cells that are resistant to the JAK2 inhibitor ruxolitinib) as well as MPN patient cells, were treated with Pim kinase inhibitors, SGI-1776 and AZD1208, and the JAK2 inhibitor, ruxolitinib. The effects on cell growth, cell cycle, viability, and cell signaling were studied. High concentration SGI-1776 (10 μM) inhibited cell growth and viability of MPN model cells while lower doses (1 and 3 μM) had little effect on the growth and viability of these cells. Combination of 3 μM SGI-1776 with low dose ruxolitinib significantly enhanced growth inhibition and cell death of HEL and SET2 cells. Similar results were obtained with the much more effective and selective Pim inhibitor, AZD1208. We show that ruxolitinib inhibits Pim expression in MPN cells, and Pim expression is restored in ruxolitinib-resistant cells. Importantly, low dose SGI-1776 or AZD1208 (100 nM) re-sensitized ruxolitinib-resistant MPN cells to ruxolitinib treatment. Significantly, as single agents, both SGI-1776 and AZD1208 inhibited erythropoietin-independent erythroid colony formation of primary cells from MPN patients, but not erythroid colonies of normal controls. The combination of AZD1208 and ruxolitinib exhibited enhanced inhibition of colony formation of primary cells from MPN patients compared to treatment with either drug alone. These data indicate that Pim kinase inhibitors in combination with a JAK2 inhibitor may offer a more efficacious therapeutic approach over JAK2 inhibitor mono-therapy for MPNs. Disclosures No relevant conflicts of interest to declare.

Cytokines and their receptors regulate haemopoiesis by controlling cellular growth, survival and differentiation. Thus it is not surprising that mutations of cytokine receptors contribute to the formation of haemopoietic disorders, including cancer. We recently identified transforming properties of IL27R, the ligand-binding component of the receptor for interleukin-27. Although wild-type IL27R exhibits transforming properties in haemopoietic cells, in the present study we set out to determine if the transforming activity of IL27R could be enhanced by mutation. We identified three mutations of IL27R that enhance its transforming activity. One of these mutations is a phenylalanine to cysteine mutation at residue 523 (F523C) in the transmembrane domain of the receptor. The two other mutations identified involve deletions of amino acids in the cytoplasmic juxtamembrane region of the receptor. Expression of each of these mutant IL27R proteins led to rapid cytokine-independent transformation in haemopoietic cells. Moreover, the rate of transformation induced by these mutants was significantly greater than that induced by wild-type IL27R. Expression of these IL27R mutants also induced enhanced activation of JAK (Janus kinase)/STAT (signal transducer and activator of transcription) signalling compared with wild-type. An activating deletion mutation of IL27R enhanced homodimerization of the receptor by a mechanism that may involve disulfide bonding. These transforming IL27R mutants displayed equal or greater transforming activity than bona fide haemopoietic oncogenes such as BCR-ABL (breakpoint cluster region-Abelson murine leukaemia viral oncogene homologue) and JAK2-V617F. Since IL27R is expressed on haemopoietic stem cells, lymphoid cells and myeloid cells, including acute myeloid leukaemia blast cells, mutation of this receptor has the potential to contribute to a variety of haemopoietic neoplasms.

Recent work has highlighted roles for JAK (Janus kinase) family members in haemopoietic diseases. Although sequencing efforts have uncovered transforming JAK1 mutations in acute leukaemia, they have also identified non-transforming JAK1 mutations. Thus with limited knowledge of the mechanisms of JAK1 activation by mutation, sequencing may not readily identify transforming mutations. Therefore we sought to further understand the repertoire of transforming mutations of JAK1. We identified seven randomly generated transforming JAK1 mutations, including V658L and a deletion of amino acids 629–630 in the pseudokinase domain, as well as L910P, F938S, P960S, K1026E and Y1035C within the kinase domain. These mutations led to differential signalling activation, but exhibited similar transforming abilities, in BaF3 cells. Interestingly, these properties did not always correlate with JAK1 activation-loop phosphorylation. We also identified a JAK1 mutant that did not require a functional FERM (4.1/ezrin/radixin/moesin) domain for transformation. Although we isolated a mutation of JAK1 at residue Val658, which is found mutated in acute leukaemia patients, most of the mutations we identified are within the kinase domain and have yet to be identified in patients. Interestingly, compared with cells expressing JAK1-V658F, cells expressing these mutants had higher STAT1 (signal transducer and activator of transcription 1) phosphorylation and were more sensitive to interferon-γ-mediated growth inhibition. The differential STAT1 activation and interferon-sensitivity of JAK1 mutants may contribute to the determination of which specific JAK1 mutations ultimately contribute to disease and thus are identified in patients. Our characterization of these novel mutations contributes to a better understanding of mutational activation of JAK1.

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.

Abstract 2955 Poster Board II-931 Mutational 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.

We identified the IIIb C2 epithelial cell-specific splice variant of fibroblast growth factor receptor 2 (FGFR2 IIIb C2) receptor tyrosine kinase in a screen for activated oncogenes expressed in T-47D human breast carcinoma cells. We found FGFR2 IIIb C2 expression in breast carcinoma cell lines and, additionally, expression of the mesenchymal-specific FGFR2 IIIc splice variant in invasive breast carcinomas,. FGFR2 IIIc expression was associated with loss of epithelial markers and gain of mesenchymal markers. Although FGFR2 IIIb is expressed in epithelial cells, previous studies on FGFR2 IIIb transformation have focused on NIH 3T3 fibroblasts. Therefore, we compared the transforming activities of FGFR2 IIIb C2 in RIE-1 intestinal cells and several mammary epithelial cells. FGFR2 IIIb C2 caused growth transformation of epithelial cells but morphologic transformation of only NIH 3T3 cells. FGFR2 IIIb C2-transformed NIH 3T3, but not RIE-1 cells, showed persistent activation of Ras and increased cyclin D1 protein expression. NIH 3T3 but not RIE-1 cells express keratinocyte growth factor, a ligand for FGFR2 IIIb C2. Ectopic treatment with keratinocyte growth factor caused FGFR2 IIIb C2-dependent morphologic transformation of RIE-1 cells, as well as cyclin D1 up-regulation, indicating that both ligand-independent and stromal cell-derived, ligand-dependent mechanisms contribute to RIE-1 cell transformation. Our results, support cell context distinct mechanisms of FGFR2 IIIb C2 transformation.

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.

From a patient with acute myeloid leukemia (AML), we have identified IL-27Ra (also known as TCCR and WSX1 ) as a gene whose expression can induce the transformation of hematopoietic cells. IL-27Ra (IL-27R) is a type I cytokine receptor that functions as the ligand binding component of the receptor for IL-27 and functions with the glycoprotein 130 (gp130) coreceptor to induce signal transduction in response to IL-27. We show that IL-27R is expressed on the cell surface of the leukemic cells of AML patients. 32D myeloid cells transformed by IL-27R contain elevated levels of activated forms of various signaling proteins, including JAK1, JAK2, STAT1, STAT3, STAT5, and ERK1/2. Inhibition of JAK family proteins induces cell cycle arrest and apoptosis in these cells, suggesting the transforming properties of IL-27R depend on the activity of JAK family members. IL-27R also transforms BaF3 cells to cytokine independence. Because BaF3 cells lack expression of gp130, this finding suggests that IL-27R-mediated transformation of hematopoietic cells is gp130-independent. Finally, we show that IL-27R can functionally replace a homodimeric type I cytokine receptor in the activation of JAK2-V617F, a critical JAK2 mutation in various myeloproliferative disorders (MPDs). Our data demonstrate that IL-27R possesses hematopoietic cell-transforming properties and suggest that, analogous to homodimeric type I cytokine receptors, single-chain components of heterodimeric receptors can also enhance the activation of JAK2-V617F. Therefore, such receptors may play unappreciated roles in MPDs.

Ras guanine nucleotide releasing proteins (RasGRPs) function as guanine nucleotide exchange factors for Ras proteins. Thus, RasGRPs are direct activators of Ras proteins and contribute an important role in various cell-signaling pathways that are regulated by the activation state of Ras proteins. RasGRPs are regulated by the second messengers diacylglycerol and intracellular calcium and are also known as CalDAG-GEFs or calcium and diacylglycerol-regulated guanine nucleotide exchange factors. RasGRPs couple signaling events that generate these second messengers in the cell into activation of signaling pathways that are regulated by Ras. RasGRPs, therefore, increase the repertoire of extracellular stimuli that lead to activation of Ras. Analyzing the regulation of RasGRP activity should continue to play an important role in understanding the mechanisms by which signal transduction pathways use RasGRP proteins to activate Ras proteins in cells.

Proc Amer Assoc Cancer Res, Volume 47, 2006 5512 Nerve growth factor (NGF) mediates the phosphorylation and signaling through the receptor tyrosine kinase TrkA, which is expressed and active in the early hemopoietic progenitor cells, as well as in the K562 and TF1 human leukemia cell lines and AML-ETO-expressing primary human acute myelogenous leukemia (AML) cells. In primary AML cells, a 75-amino acid deletion mutant of TrkA (ΔTrkA) has also been demonstrated to be constitutively active as a pro-growth and pro-survival protein through mechanisms involving ERK1/2 and Akt activities. In previous reports, we have shown that the acute leukemia associated Bcr-Abl and mutant FLT-3 are chaperoned by hsp90 and the geldanamycin analogue hsp90 inhibitors induce misfolding, polyubiquitylation and proteasomal degradation of Bcr-Abl and mutant FLT-3. In the present studies, we determined whether TrkA is a hsp90 client protein. We also determined the effect of the novel hsp90 inhibitor DMAG (Kosan Biosciences Inc.) on TrkA levels and activity in mouse myeloid 32D cells with or without the ectopic expression of ΔTrkA (32D/ΔTrkA cells), as well as on the endogenous levels of wild-type (WT) TrkA in K562 and TF1 cells. Exposure to 0.25 or 1.0 μM DMAG attenuated the levels of TrkA in K562, TF1 and 32D, as well as ΔTrkA in 32D/ΔTrkA cells. Co-treatment with the proteasome inhibitor bortezomib (100 nM) restored DMAG mediated depletion of TrkA in K562 cells. In K562 cells, immunoprecipitation (IP) with monoclonal anti-TrkA antibody followed by immunoblot (IB) analyses with anti-hsp90, anti-p23 and anti-cdc37 antibodies showed that TrkA binds to hsp90 and its co-chaperones, p23 and cdc37. This association is inhibited by treatment with DMAG. Following suspension of K562 cells in a serum free medium containing 100 ng/ml of NGF, within 5 to 10 minutes the levels of pTrkA, pERK1/2 and pAkt were significantly upregulated. Co-treatment with 1.0 μM DMAG inhibited the induction of pTrkA and pERK1/2. Exposure of K562 cells to DMAG also depleted the levels of other hsp90 client proteins, including c-Raf, Akt and Bcr-Abl in K562 cells. This was associated with growth arrest and apoptosis in a dose-dependent manner. Additionally in the rat pheochromocytoma PC-12 cells, NGF mediated neurite formation was significantly inhibited by co-treatment with 17-DMAG. In two samples of primary CML cells, treatment with DMAG attenuated the levels of TrkA, which was also associated with loss of survival. These findings demonstrate that TrkA is an hsp90 client protein, and hsp90 inhibition by treatment with DMAG depletes WT or mutant TrkA levels and activity in acute leukemia cells. These findings suggest that hsp90 inhibitors may be effective treatment against human acute leukemia cells in which TrkA mediated signaling supports growth and survival.

Nerve growth factor (NGF) mediates the phosphorylation and signaling through the receptor tyrosine kinase TrkA, which has been shown to be expressed and active in the early hemopoietic progenitor cells, as well as in the K562 and TF1 leukemia cell lines and AML-ETO-expressing human acute leukemia cells. In AML, a 75-amino acid deletion mutant of TrkA (ΔTrkA) has also been demonstrated to be constitutively active as a pro-growth and pro-survival protein through ERK1/2 and Akt activation. We have previously reported that that the ATP bound molecular chaperone hsp90 binds the leukemia associated Bcr-Abl and FLT-3 tyrosine kinases as client proteins, maintaining them in a properly folded and active conformation, and that geldanamycin analogue hsp90 inhibitors disrupt this chaperone association, resulting in polyubiquitylation and proteasomal degradation of the client proteins. In the present studies, we investigated a) whether TrkA is a client protein of hsp90 and b) the effect of the novel and highly soluble hsp90 inhibitor DMAG (Kosan Biosciences Inc.) on TrkA levels and activity in mouse myeloid 32D cells with or without the ectopic expression of ΔTrkA (32D/ΔTrkA cells), as well as on endogenous levels of wild-type (WT) TrkA in K562 and TF1 cells. Exposure to 0.25 or 1.0 μM DMAG attenuated the levels of WT TrkA in K562, TF1 and 32D, as well as ΔTrkA in 32D/ΔTrkA cells. Co-treatment with the proteasome inhibitor bortezomib (100 nM) restored DMAG mediated depletion of WT TrkA in K562 cells, suggesting that DMAG induced the polyubiquitylation and degradation of TrkA by the 26S proteasome. In K562 cells, immunoprecipitation (IP) with monoclonal anti-TrkA antibody followed by immunoblot (IB) analyses with anti-hsp90 antibody (or IP with anti-hsp90 followed by IB with anti-TrkA antibody) showed that TrkA binds to hsp90, which is inhibited by treatment with DMAG. Following suspension of K562 cells in a serum free medium containing 100 ng/ml of NGF, the levels of pTrkA, pERK1/2 and pAkt significantly increased within 5 to 10 minutes. Co-treatment with 1.0 μM DMAG inhibited pTrkA and pERK1/2 induction, suggesting that hsp90 chaperone function may be required for TrkA activity. Exposure to DMAG also depleted the levels of the other hsp90 client proteins, including c-Raf, Akt and Bcr-Abl in K562 cells, which was associated with growth arrest and apoptosis in a dose-dependent manner. These findings demonstrate that TrkA may be an hsp90 client protein, and hsp90 inhibition by treatment with DMAG would deplete WT or mutant TrkA levels and activity in human leukemia cells. These findings suggest that hsp90 inhibitors may be effective against human acute leukemia cells that may depend on the activity of mutant or WT TrkA for growth and survival.