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Aberrant megakaryopoiesis is a hallmark of the myeloproliferative neoplasms (MPNs), a group of clonal hematological malignancies originating from hematopoietic stem cells, leading to an increase in mature blood cells in the peripheral blood. Sialylated derivatives of the glycan structure β4-N-acetyllactosamine (Galβ1,4GlcNAc or type-2 LacNAc, hereafter referred to as LacNAc) regulate platelet life span, hepatic thrombopoietin (TPO) production, and thrombopoiesis. We found increased TPO plasma levels in MPNs with high allele burden of the mutated clones. Remarkably, platelets isolated from MPNs had a significant increase in LacNAc expression that correlated with the high allele burden regardless of the underlying identified mutation. Megakaryocytes derived in vitro from these patients showed an increased expression of the B4GALT1 gene encoding β-1,4-galactosyltransferase 1 (β4GalT1). Consistently, megakaryocytes from MPN showed increased LacNAc expression relative to healthy controls, which was counteracted by the treatment with a Janus kinase 1/2 inhibitor. Altered expression of B4GALT1 in mutant megakaryocytes can lead to the production of platelets with aberrant galactosylation, which in turn promote hepatic TPO synthesis regardless of platelet mass. Our findings provide a new paradigm for understanding aberrant megakaryopoiesis in MPNs and identify β4GalT1 as a potential actionable target for therapy.

Conditioning-induced damage of the intestinal tract plays a critical role during the onset of acute graft-versus-host disease (GVHD). Therapeutic interference with these early events of GVHD is difficult, and currently used immunosuppressive drugs mainly target donor T cells. However, not donor T cells but neutrophils reach the sites of tissue injury first, and therefore could be a potential target for GVHD prevention. A detailed analysis of neutrophil fate during acute GVHD and the effect on T cells is difficult because of the short lifespan of this cell type. By using a novel photoconverter reporter system, we show that neutrophils that had been photoconverted in the ileum postconditioning later migrated to mesenteric lymph nodes (mLN). This neutrophil migration was dependent on the intestinal microflora. In the mLN, neutrophils colocalized with T cells and presented antigen on major histocompatibility complex (MHC)-II, thereby affecting T cell expansion. Pharmacological JAK1/JAK2 inhibition reduced neutrophil influx into the mLN and MHC-II expression, thereby interfering with an early event in acute GVHD pathogenesis. In agreement with this finding, neutrophil depletion reduced acute GVHD. We conclude that neutrophils are attracted to the ileum, where the intestinal barrier is disrupted, and then migrate to the mLN, where they participate in alloantigen presentation. JAK1/JAK2-inhibition can interfere with this process, which provides a potential therapeutic strategy to prevent early events of tissue damage-related innate immune cell activation and, ultimately, GVHD.

Oral ruxolitinib has been successfully used for the treatment of acute and chronic graft-versus-host disease (cGvHD) and topical ruxolitinib has demonstrated efficacy in clinical trials for vitiligo, atopic dermatitis, and psoriasis. Background: There are no FDA-approved topical treatments for cutaneous cGvHD. Topical corticosteroids are the mainstay of skin-directed therapy for the inflammatory-phase cutaneous cGvHD but are associated with significant side effects such as skin thinning, bruising, striae, infections, and acne, and may incompletely treat cutaneous cGvHD, prompting use of systemic therapies. Methods. We conducted a prospective, randomized, double-blind, vehicle- and comparator-controlled, phase 2, proof-of-concept trial evaluating the efficacy and safety of topical ruxolitinib 1.5% cream in patients 12 years of age or older with cutaneous nonsclerotic (lichen-planus like, poikilodermatous) and superficially sclerotic (lichen sclerosus, morphea-like) cGvHD with ≥2% of body surface area (BSA) affected at a single, academic transplantation center in the United States. Patients were only eligible to enroll if systemic therapy, when applicable, was stable for ≥ 4 weeks and concurrent topical therapy (including phototherapy) was not used. Patients were randomly assigned (1:1) to receive topical ruxolitinib 1.5% cream left side of face/body or right side of face/body with placebo vehicle cream to contralateral side of face/body twice daily to for 28 days, followed by an optional open label extension to both sides of 28 days for interested patients. The primary end point was efficacy as measured by BSA of the GvHD rash on the side of face/body treated with topical ruxolitinib cream vs contralateral side treated with vehicle at Day 28. Secondary endpoints were Physician's Global Assessment of clinical condition (PGA) and Composite Assessment of Index Lesion Severity (CAILS) of the ruxolitinib-treated side vs vehicle-treated side at Day 14 and 28. Interim analyses were performed once 10 patients were evaluable of the planned 24 patients. Results. Between 6/28/19 and 5/14/21, a total of 13 patients (mean age 52.6 years [SD 20.0]; 7 [54%] female and 6 [46%] male) underwent randomization; 11 patients completed Day 14 assessments, 12 patients Day 28 assessments, and 10 patients Day 56 assessments. Patients had a history of acute leukemia (N=8 [62%]), non-Hodgkin lymphoma (N=3 [23%]), myeloproliferative neoplasm (N=1 [8%]), or aplastic anemia (N=1 [8%]). Median time from transplant to enrollment was 665 days (IQR 433-1355), and from cGvHD onset to enrollment 283 days (IQR 115-867). Chronic GvHD was NIH mild (8%), moderate (23%), or severe (62%), predominantly classic (85% vs. overlap 15%), with 46% having 4 or more involved organs. Most patients were enrolled for treatment of cutaneous nonsclerotic cGvHD (N=10, 77%) with lichen planus-like (N=8), papulosquamous (N=1), and maculopapular rash/erythema features (N=1). Three patients were enrolled for treatment of lichen sclerosus-like cGvHD. Patients were heavily pretreated with 4 (31%) having 3 or more prior lines of systemic therapy for cGvHD. Most patients had failed at least 2 topical therapies, with 77% previously failed topical steroids, 24% topical calcineurin inhibitors, and 24% phototherapy. There was a trend in reduced BSA of cGvHD on the treatment side compared to the vehicle side from Day 1 (13.4 on treatment/vehicle) to Day 14 (10.9 vs 13.8; p=0.06) and continuing to Day 28 (7.7 vs 11.0; p=0.15), respectively. PGA (Day 1: 5 treatment/vehicle) of treatment side was significantly improved starting at Day 14 (3.3 treatment vs 4.4 vehicle; p=0.024), with continued improvement at Day 28 (2.5 vs 4.0; p= 0.026). CAILS (Day 1: 15.6 treatment vs 15.5 vehicle; p=0.83) of treatment side was also significantly improved starting at Day 14 (9.0 vs 13.3; p=0.02). There were no serious adverse events (SAEs) reported. One patient had a grade 1 headache which was attributed possibly to therapy. Three patients had treatment-emergent AEs (all grade 1) that were unlikely (N=1) or unrelated (N=2) to study therapy. Conclusions. Topical ruxolitinib 1.5% cream was effective in treating cutaneous nonsclerotic and superficially sclerotic GvHD as determined by PGA and CAILS. These data suggest that ruxolitinib cream might be a safe and effective treatment option for patients with cutaneous nonsclerotic and superficially sclerotic chronic GvHD. Figure 1 Figure 1. Disclosures Markova: Alira Health Ventures: Consultancy; Incyte Corporation: Research Funding; Blueprint Medicines: Consultancy; UpToDate: Patents & Royalties: Royalties for chapter on dermatologic adverse events to targeted therapies ; Amryt Pharma: Research Funding. Perales: MorphoSys: Honoraria; Omeros: Honoraria; Cidara: Honoraria; Incyte: Honoraria, Other; Equilium: Honoraria; Medigene: Honoraria; Kite/Gilead: Honoraria, Other; Karyopharm: Honoraria; Sellas Life Sciences: Honoraria; Novartis: Honoraria, Other; Nektar Therapeutics: Honoraria, Other; Servier: Honoraria; Miltenyi Biotec: Honoraria, Other; NexImmune: Honoraria; Celgene: Honoraria; Merck: Honoraria; Takeda: Honoraria; Bristol-Myers Squibb: Honoraria. Prockop: Memorial Sloan Kettering Cancer Center: Other: S Prockop receives support for the conduct of sponsored clinical trials through MSK from Atara Biotherapeutics, Jasper and AlloVir. , Patents & Royalties: S Prockop is a co-inventor on intellectual property (IP) licensed to Atara. S Prockop has waived rights to this IP to MSK and has no personal financial interests in Atara. MSK has financial interests in Atara and IP interests relevant to this abstract. ; MSK: Other: Inventor; Neovii: Consultancy; ADMA Biologics: Consultancy; Jasper: Other: support for the conduct of sponsored trials; AlloVir: Other: support for the conduct of sponsored trials; Atara Biotherapeutics: Other: support for the conduct of sponsored trials and Inventor. Ponce: Kadmon pharmaceuticals: Consultancy, Honoraria; Ceramedix: Consultancy, Honoraria; CareDx: Consultancy, Honoraria; Seres Therapeutics: Consultancy, Research Funding; Generon Pharmaceuticals: Consultancy; Takeda Pharmaceuticals: Research Funding.

Since its approval in 2011, the Janus kinase 1/2 (JAK1/2) inhibitor ruxolitinib has evolved to become the centerpiece of therapy for myelofibrosis (MF), and its use in patients with hydroxyurea resistant or intolerant polycythemia vera (PV) is steadily increasing. Several other JAK2 inhibitors have entered clinical testing, but none have been approved and many have been discontinued. Importantly, the activity of these agents is not restricted to patients with JAK2 V617F or exon 12 mutations. Although JAK2 inhibitors provide substantial clinical benefit, their disease-modifying activity is limited, and rational combinations with other targeted agents are needed, particularly in MF, in which survival is short. Many such combinations are being explored, as are other novel agents, some of which could successfully be combined with JAK2 inhibitors in the future. In addition, new JAK2 inhibitors with the potential for less myelosuppression continue to be investigated. Given the proven safety and efficacy of ruxolitinib, it is likely that ruxolitinib-based combinations will be a major way forward in drug development for MF. If approved, less myelosuppressive JAK2 inhibitors such as pacritinib or NS-018 could prove to be very useful additions to the therapeutic armamentarium in MF. In PV, inhibitors of histone deacetylases and human double minute 2 have activity, but their role, if any, in the future treatment algorithm is uncertain, given the availability of ruxolitinib and renewed interest in interferons. Ruxolitinib is in late-phase clinical trials in essential thrombocythemia, in which it could fill an important void for patients with troublesome symptoms.

Protein acetylation is an important contributor to cancer initiation. Histone deacetylase 6 (HDAC6) controls JAK2 translation and protein stability and has been implicated in JAK2-driven diseases best exemplified by myeloproliferative neoplasms (MPNs). By using novel classes of highly selective HDAC inhibitors and genetically deficient mouse models, we discovered that HDAC11 rather than HDAC6 is necessary for the proliferation and survival of oncogenic JAK2-driven MPN cells and patient samples. Notably, HDAC11 is variably expressed in primitive stem cells and is expressed largely upon lineage commitment. Although Hdac11is dispensable for normal homeostatic hematopoietic stem and progenitor cell differentiation based on chimeric bone marrow reconstitution, Hdac11 deficiency significantly reduced the abnormal megakaryocyte population, improved splenic architecture, reduced fibrosis, and increased survival in the MPLW515L-MPN mouse model during primary and secondary transplantation. Therefore, inhibitors of HDAC11 are an attractive therapy for treating patients with MPN. Although JAK2 inhibitor therapy provides substantial clinical benefit in MPN patients, the identification of alternative therapeutic targets is needed to reverse MPN pathogenesis and control malignant hematopoiesis. This study establishes HDAC11 as a unique type of target molecule that has therapeutic potential in MPN.

Activation-induced cytidine deaminase (AID) initiates somatic hypermutation and class switch recombination of the immunoglobulin genes. As a trade-off for its physiological function, AID also contributes to tumor development through its mutagenic activity. In chronic lymphocytic leukemia (CLL), AID is overexpressed in the proliferative fractions (PFs) of the malignant B lymphocytes, and its anomalous expression has been associated with a clinical poor outcome. Recent preclinical data suggested that ibrutinib and idelalisib, 2 clinically approved kinase inhibitors, increase AID expression and genomic instability in normal and neoplastic B cells. These results raise concerns about a potential mutagenic risk in patients receiving long-term therapy. To corroborate these findings in the clinical setting, we analyzed AID expression and PFs in a CLL cohort before and during ibrutinib treatment. We found that ibrutinib decreases the CLL PFs and, interestingly, also reduces AID expression, which correlates with dampened AKT and Janus Kinase 1 signaling. Moreover, although ibrutinib increases AID expression in a CLL cell line, it is unable to do so in primary CLL samples. Our results uncover a differential response to ibrutinib between cell lines and the CLL clone and imply that ibrutinib could differ from idelalisib in their potential to induce AID in treated patients. Possible reasons for the discrepancy between preclinical and clinical findings, and their effect on treatment safety, are discussed. Fil: Morande, Pablo Elías. Instituto Pasteur de Montevideo; Uruguay. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Sivina, Mariela. University of Texas Health Science Center at Houston. University of Texas Md Anderson Cancer Center; Estados Unidos Fil: Uriepero, Angimar. Instituto Pasteur de Montevideo; Uruguay Fil: Seija, Noé. Instituto Pasteur de Montevideo; Uruguay Fil: Berca, Catalina. Instituto Pasteur de Montevideo; Uruguay Fil: Fresia, Pablo. Instituto Pasteur de Montevideo; Uruguay Fil: Landoni, Ana Inés. Ministerio de Salud.Hospital Maciel. Administración de los Servicios de Salud del Estado; Uruguay Fil: Di Noia, Javier M.. University of Montreal; Canadá. Cliniques de Montreal. Institut de Recherches. Division of Immunity and Viral Infections; Canadá Fil: Burger, Jan A.. University of Texas Health Science Center at Houston. University of Texas Md Anderson Cancer Center; Estados Unidos Fil: Oppezzo, Pablo. Instituto Pasteur de Montevideo; Uruguay

The regulation of toll-like receptor (TLR) signaling in a tumor microenvironment is poorly understood despite its importance in cancer biology. To address this problem, TLR7-responses of chronic lymphocytic leukemia (CLL) cells were studied in the presence and absence of a human stromal cell-line derived from a leukemic spleen. CLL cells alone produced high levels of tumor necrosis factor (TNF)-α and proliferated in response to TLR7-agonists. A signal transducer and activator of transcription 3 -activating stromal factor, identified as interleukin (IL)-6, was found to upregulate microRNA (miR)-17 and miR-19a, target TLR7 and TNFA messenger RNA, and induce a state of tolerance to TLR7-agonists in CLL cells. Overexpression of the miR-17-92 cluster tolerized CLL cells directly and miR-17 and miR-19a antagomiRs restored TLR7-signaling. Inhibition of IL-6 signaling with antibodies or small-molecule Janus kinase inhibitors reversed tolerization and increased TLR7-stimulated CLL cell numbers in vitro and in NOD-SCIDγc (null) mice. These results suggest IL-6 can act as tumor suppressor in CLL by inhibiting TLR-signaling.

Because both phosphatidylinositol 3-kinase δ (PI3Kδ) and Janus kinase (JAK)-signal transducer and activator of transcription pathways contribute to tumor cell proliferation and survival in B-cell malignancies, their simultaneous inhibition may provide synergistic treatment efficacy. This phase 1 dose-escalation/expansion study assessed the safety, efficacy, pharmacokinetics, and pharmacodynamics of INCB040093, a selective PI3Kδ inhibitor, as monotherapy or combined with itacitinib (formerly INCB039110), a selective JAK1 inhibitor, in adult patients with relapsed or refractory (R/R) B-cell lymphomas. Final results are reported. Overall, 114 patients were treated (monotherapy, n = 49; combination therapy, n = 72 [7 patients crossed over from monotherapy to combination]). INCB040093 100 mg twice daily (monotherapy) and INCB040093 100 mg twice daily + itacitinib 300 mg once daily (combination) were the recommended phase 2 doses. One dose-limiting toxicity (gastrointestinal bleed secondary to gastric diffuse large B-cell lymphoma [DLBCL] regression) occurred with monotherapy. The most common serious adverse events with monotherapy were pneumonia (n = 5) and pyrexia (n = 4), and with combination Pneumocystis jiroveci pneumonia (n = 5), pneumonia (unrelated to P jiroveci; n = 5), and pyrexia (n = 4). Grade 3 or higher transaminase elevations were less common with combination. INCB040093 was active across the B-cell lymphomas; 63% of patients (5/8) with follicular lymphoma responded to monotherapy. Adding itacitinib provided promising activity in select subtypes, with responses of 67% (14/21) in classic Hodgkin lymphoma (vs 29% [5/17] with monotherapy) and 31% (4/13) in nongerminal center B-cell-like DLBCL. INCB040093 with/without itacitinib was tolerated and active in this study, and is a promising treatment strategy for patients with select R/R B-cell lymphomas. This trial was registered at www.clinicaltrials.gov as #NCT01905813.

Graft-versus-host-disease (GVHD) is a severe complication of allogeneic hematopoietic cell transplantation (allo-HCT) characterized by the production of high levels of proinflammatory cytokines. Activated Janus kinases (JAKs) are required for T-effector cell responses in different inflammatory diseases, and their blockade could potently reduce acute GVHD. We observed that inhibition of JAK1/2 signaling resulted in reduced proliferation of effector T cells and suppression of proinflammatory cytokine production in response to alloantigen in mice. In vivo JAK 1/2 inhibition improved survival of mice developing acute GVHD and reduced histopathological GVHD grading, serum levels of proinflammatory cytokines, and expansion of alloreactive luc-transgenic T cells. Mechanistically, we could show that ruxolitinib impaired differentiation of CD4(+) T cells into IFN-γ- and IL17A-producing cells, and that both T-cell phenotypes are linked to GVHD. Conversely, ruxolitinib treatment in allo-HCT recipients increased FoxP3(+) regulatory T cells, which are linked to immunologic tolerance. Based on these results, we treated 6 patients with steroid-refractory GVHD with ruxolitinib. All patients responded with respect to clinical GVHD symptoms and serum levels of proinflammatory cytokines. In summary, ruxolitinib represents a novel targeted approach in GVHD by suppression of proinflammatory signaling that mediates tissue damage and by promotion of tolerogenic Treg cells.

The JAK1/JAK2 inhibitor ruxolitinib produced significant reductions in splenomegaly and symptomatic burden and improved survival in patients with myelofibrosis (MF), irrespective of their JAK2 mutation status, in 2 phase III studies against placebo (COMFORT-I) and best available therapy (COMFORT-II). We performed a comprehensive mutation analysis to evaluate the impact of 14 MF-associated mutations on clinical outcomes in 166 patients included in COMFORT-II. We found that responses in splenomegaly and symptoms, as well as the risk of developing ruxolitinib-associated anemia and thrombocytopenia, occurred at similar frequencies across different mutation profiles. Ruxolitinib improved survival independent of mutation profile and reduced the risk of death in patients harboring a set of prognostically detrimental mutations (ASXL1, EZH2, SRSF2, IDH1/2) with an hazard ratio of 0.57 (95% confidence interval: 0.30-1.08) vs best available therapy. These data indicate that clinical efficacy and survival improvement may occur across different molecular subsets of patients with MF treated with ruxolitinib.

Inhibition of Janus-kinase 1/2 (JAK1/2) is a mainstay to treat myeloproliferative neoplasms (MPN). Sporadic observations reported the co-incidence of B-cell non-Hodgkin lymphomas during treatment of MPN with JAK1/2 inhibitors. We assessed 626 patients with MPN, including 69 with myelofibrosis receiving JAK1/2 inhibitors for lymphoma development. B-cell lymphomas evolved in 4 (5.8%) of 69 patients receiving JAK1/2 inhibition compared with 2 (0.36%) of 557 with conventional treatment (16-fold increased risk). A similar 15-fold increase was observed in an independent cohort of 929 patients with MPN. Considering primary myelofibrosis only (N = 216), 3 lymphomas were observed in 31 inhibitor-treated patients (9.7%) vs 1 (0.54%) of 185 control patients. Lymphomas were of aggressive B-cell type, extranodal, or leukemic with high MYC expression in the absence of JAK2V617F or other MPN-associated mutations. Median time from initiation of inhibitor therapy to lymphoma diagnosis was 25 months. Clonal immunoglobulin gene rearrangements were already detected in the bone marrow during myelofibrosis in 16.3% of patients. Lymphomas occurring during JAK1/2 inhibitor treatment were preceded by a preexisting B-cell clone in all 3 patients tested. Sequencing verified clonal identity in 2 patients. The effects of JAK1/2 inhibition were mirrored in Stat1 (−/−) mice: 16 of 24 mice developed a spontaneous myeloid hyperplasia with the concomitant presence of aberrant B cells. Transplantations of bone marrow from diseased mice unmasked the outgrowth of a malignant B-cell clone evolving into aggressive B-cell leukemia-lymphoma. We conclude that JAK/STAT1 pathway inhibition in myelofibrosis is associated with an elevated frequency of aggressive B-cell lymphomas. Detection of a preexisting B-cell clone may identify individuals at risk.

In this issue of Blood , Phillips et al demonstrate an exciting approach to improve efficacy and decrease the toxicity of phosphatidylinositol 3-kinase (PI3K) inhibitors. 1 In the past decade, we have learned of the pivotal role PI3K plays in the growth and survival of many cancers, including B-cell malignancies. PI3Ks are also important in the microenvironment, where they have effects on recruitment and differentiation of cells that support the survival of the malignant B cells. There are 4 isoforms: α, β, δ, and ϒ. The expression of these isoforms varies by tissue. Preclinical models suggested that specifically inhibiting the δ isoform might provide significant efficacy in B-cell malignancies while decreasing potential toxicities of pan inhibitors because its expression is restricted to leukocytes. However, autoimmune problems, particularly colitis, were also seen in these preclinical models. 2 There are now 2 PI3K inhibitors that have received regulatory approval for the treatment of B-cell malignancies in the United States. The first was idelalisib, a PI3K δ isoform-specific inhibitor. The clinical results of idelalisib are very much in line with what one would have predicted from the preclinical studies, substantial efficacy in lymphoma and chronic lymphocytic leukemia, but also with the colitis, as seen in the mouse models. Liver toxicity has also been observed, which appears to be immune mediated. 3

Establishing the diagnosis of focal brain lesions in patients with unexplained neurologic symptoms represents a challenge. The goal of this study is to provide evidence supporting functional roles for CXC chemokine ligand (CXCL)13 and interleukin (IL)-10 in central nervous system (CNS) lymphomas and to evaluate the utility of each as prognostic and diagnostic biomarkers. We demonstrate for the first time that elevated CXCL13 concentration in cerebrospinal fluid (CSF) is prognostic and that CXCL13 and CXCL12 mediate chemotaxis of lymphoma cells isolated from CNS lymphoma lesions. Expression of the activated form of Janus kinase 1 supported a role for IL-10 in prosurvival signaling. We determined the concentration of CXCL13 and IL-10 in CSF of CNS lymphoma patients and control cohorts including inflammatory and degenerative neurologic disease in a multicenter study involving 220 patients. Bivariate elevated CXCL13 plus IL-10 was 99.3% specific for primary and secondary CNS lymphoma, with sensitivity significantly greater than reference standard CSF tests. These results identify CXCL13 and IL-10 as potentially important biomarkers of CNS lymphoma that merit further evaluation and support incorporation of CXCL13 and IL-10 into diagnostic algorithms for the workup of focal brain lesions in which lymphoma is a consideration.

One recently identified subtype of pediatric B-precursor acute lymphoblastic leukemia (ALL) has been termed BCR-ABL1–like or Ph-like because of similarity of the gene expression profile to BCR-ABL1 positive ALL suggesting the presence of lesions activating tyrosine kinases, frequent alteration of IKZF1, and poor outcome. Prior studies demonstrated that approximately half of these patients had genomic lesions leading to CRLF2 overexpression, with half of such cases harboring somatic mutations in the Janus kinases JAK1 and JAK2. To determine whether mutations in other tyrosine kinases might also occur in ALL, we sequenced the tyrosine kinome and downstream signaling genes in 45 high-risk pediatric ALL cases with either a Ph-like gene expression profile or other alterations suggestive of activated kinase signaling. Aside from JAK mutations and 1 FLT3 mutation, no somatic mutations were found in any other tyrosine kinases, suggesting that alternative mechanisms are responsible for activated kinase signaling in high-risk ALL.

In this issue of Blood, Das and colleagues report their results on the use of the Janus kinase 1/2 (JAK1/2) inhibitor ruxolitinib in murine models of hemophagocytic lymphohistiocytosis (HLH), and the HLH-sibling macrophage activation syndrome (MAS).

The bone marrow (BM) provides a protective microenvironment to support the survival of leukemic cells and influence their response to therapeutic agents. In acute myeloid leukemia (AML), the high rate of relapse may in part be a result of the inability of current treatment to effectively overcome the protective influence of the BM niche. To better understand the effect of the BM microenvironment on drug responses in AML, we conducted a comprehensive evaluation of 304 inhibitors, including approved and investigational agents, comparing ex vivo responses of primary AML cells in BM stroma-derived and standard culture conditions. In the stroma-based conditions, the AML patient cells exhibited significantly reduced sensitivity to 12% of the tested compounds, including topoisomerase II, B-cell chronic lymphocytic leukemia/lymphoma 2 (BCL2), and many tyrosine kinase inhibitors (TKIs). The loss of TKI sensitivity was most pronounced in patient samples harboring FLT3 or PDGFRB alterations. In contrast, the stroma-derived conditions enhanced sensitivity to Janus kinase (JAK) inhibitors. Increased cell viability and resistance to specific drug classes in the BM stroma-derived conditions was a result of activation of alternative signaling pathways mediated by factors secreted by BM stromal cells and involved a switch from BCL2 to BCLXL-dependent cell survival. Moreover, the JAK1/2 inhibitor ruxolitinib restored sensitivity to the BCL2 inhibitor venetoclax in AML patient cells ex vivo in different model systems and in vivo in an AML xenograft mouse model. These findings highlight the potential of JAK inhibitors to counteract stroma-induced resistance to BCL2 inhibitors in AML.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome, characterized by severe hyperinflammation and immunopathological manifestations in several tissues. These features result from organ infiltration by overactivated CD8 T-cells and macrophages, which produce high levels of pro-inflammatory cytokines, such as IFN-γ, TNF-α, IL-6, and IL-18. Recently, several Janus kinase 1/2 (JAK1/2) inhibitors, such as ruxolitinib, have been developed as immunosuppressive agents. They have proven beneficial effects in the treatment of myeloproliferative disorders and inflammatory conditions. To determine whether pharmacological inhibition of the JAK1/2 not only prevents the onset of HLH immunopathology but also is effective against existing HLH, cytotoxicity-impaired Prf1(-/-) and Rab27a(-/-) mice with full-blown HLH syndrome were treated with a clinically relevant dose of ruxolitinib. In vivo, ruxolitinib treatment suppressed signal transducer and activator of transcription 1 activation and led to recovery from HLH manifestations in both murine models. In the Prf1(-/-) mice, these beneficial effects were evidenced by a greater survival rate, and in both murine models, they were evidenced by the correction of blood cytopenia and a rapid decrease in serum IL-6 and TNF-α levels. During ruxolitinib treatment, liver tissue damage receded concomitantly with a decrease in the number of infiltrating inflammatory macrophages and an increase in the number of alternatively activated macrophages. In Rab27a(-/-) mice, central nervous system involvement was significantly reduced by ruxolitinib therapy. Our findings demonstrate that clinically relevant doses of the JAK1/2 inhibitor ruxolitinib suppresses the harmful consequences of macrophage overactivation characterizing HLH in 2 murine models. The results could be readily translated into the clinic for the treatment of primary, and perhaps even secondary, forms of HLH.

The discovery of JAK2617F mutation paved the way for the development of small molecule inhibitors of JAK1/2 resulting in first approved JAK1/2 inhibitor, ruxolitinib, for the treatment of patients with myelofibrosis (MF). Although JAK1/2 inhibitor therapy is effective in decreasing the burden of symptoms associated with splenomegaly and MF-related constitutional symptoms, it is neither curative nor effective in reducing the risk of leukemic transformation. Presently, allogeneic hematopoietic cell transplantation (HCT) is the only curative therapy for MF. A significant risk of regimen-related toxicities, graft failure, and GVHD are major barriers to the success of HCT in MF. Because of significant HCT-associated morbidity and mortality, divergent opinions regarding its appropriate role in this clinical situation have emerged. In this review, the risk-benefit ratios of modern drug therapy compared with HCT in MF patients are analyzed. A risk-adapted approach individualized to each patient's biologic characteristics and comorbidities is described, which is currently warranted in determining optimal treatment strategies for patients with MF. Inclusion of JAK1/2 inhibitor therapy in future transplant conditioning regimens may provide an opportunity to overcome some of these barriers, resulting in greater success with HCT for MF patients.

Ruxolitinib is JAK1/JAK2 inhibitor with established clinical benefit in myelofibrosis (MF). We analyzed long-term outcomes of 107 patients with intermediate-2 or high-risk MF receiving ruxolitinib at MD Anderson Cancer Center (MDACC) on phase 1/2 trial. After a median of 32 months of follow-up, 58 patients (54%) were still receiving ruxolitinib, with overall survival (OS) of 69%. The splenomegaly and symptom reductions achieved with ruxolitinib were sustained with long-term therapy. Therapy was well tolerated; discontinuation rates at 1, 2, and 3 years were 24%, 36%, and 46%, respectively. OS of 107 MDACC patients was significantly better (P = .005) than that of 310 matched (based on trial enrollment criteria) historical control patients, primarily because of highly significant difference in OS in the high-risk subgroup (P = .006). Furthermore, among MDACC patients, those with high-risk MF experienced the same OS as those with intermediate-2 risk. Patients with ≥ 50% reduction in splenomegaly had significantly prolonged survival versus those with < 25% reduction (P < .0001). Comparison of discontinuation rates and reasons for stopping the therapy to those reported for other 51 patients in the phase 1/2 trial, and 155 ruxolitinib-treated patients in phase 3 COMFORT-I study, suggest that continued therapy with ruxolitinib at optimal doses contributes to the benefits seen, including OS benefit.

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

Although several cytokines have been demonstrated to be critical regulators of development of multiple blood cell lineages, it remains disputed to what degree they act through instructive or permissive mechanisms. Signaling through the FMS-like tyrosine kinase 3 (FLT3) receptor and the hematopoietin IL-7 receptor alpha (IL-7Ralpha) has been demonstrated to be of critical importance for sustained thymopoiesis. Signaling triggered by IL-7 and thymic stromal lymphopoietin (TSLP) is dependent on IL-7Ralpha, and both ligands have been implicated in T-cell development. However, we demonstrate that, whereas thymopoiesis is abolished in adult mice doubly deficient in IL-7 and FLT3 ligand (FLT3L), TSLP does not play a key role in IL-7-independent or FLT3L-independent T lymphopoiesis. Furthermore, whereas previous studies implicated that the role of other cytokine tyrosine kinase receptors in T lymphopoiesis might not involve permissive actions, we demonstrate that ectopic expression of BCL2 is sufficient not only to partially correct the T-cell phenotype of Flt3l(-/-) mice but also to rescue the virtually complete loss of all discernable stages of early T lymphopoiesis in Flt3l(-/-)Il7r(-/-) mice. These findings implicate a permissive role of cytokine receptors of the hematopoietin and tyrosine kinase families in early T lymphopoiesis.

The development of the dual Janus kinase 1/2 (JAK1/2) inhibitor ruxolitinib for the treatment of myeloproliferative neoplasms (MPNs) has led to studies of ruxolitinib in other clinical contexts, including JAK-mutated acute lymphoblastic leukemia (ALL). However, the limited ability of JAK inhibition to induce molecular or clinicopathological responses in MPNs suggests a need for development of better therapies for JAK kinase-dependent malignancies. Here, we demonstrate that heat shock protein 90 (HSP90) inhibition using a purine-scaffold HSP90 inhibitor in early clinical development is an effective therapeutic approach in JAK-dependent ALL and can overcome persistence to JAK-inhibitor therapy in ALL cells.

Robust CD8+ T-cell activation is vital for the recovery from many viral infections and is orchestrated via the integration of signals delivered through surface molecules, including the T-cell antigen receptors (TcRs) and cytokine receptors. Little is known about how virus-specific T cells interpret sequential or combined stimulation through these receptors, which must undoubtedly occur in vivo during antiviral immune responses. When measured in real time, peptide antigen and the cytokines, interleukin 12 (IL-12) and IL-18, independently regulate the on/off kinetics of protective (interferon γ, tumor necrosis factor α) and immunomodulatory (IL-2, CD40L) cytokine production by activated T cells and memory T cells. The remarkable differences in effector functions elicited by innate or adaptive signals (IL-12/ IL-18 or peptide, respectively) illustrate the complex and stringent regulation of cytokine expression by CD8+ T cells. Together, these results indicate how antiviral T cells incorporate multiple signals from their local microenvironment and tailor their cytokine responses accordingly.

SHP-1, an src homology 2 (SH2) domain containing protein tyrosine phosphatase, functions as a negative regulator of signaling downstream of cytokine receptors, receptor tyrosine kinases and receptor complexes of the immune system. Dephosphorylation of receptors and/or receptor-associated kinases has been described as the mechanism for the function of SHP-1. Here we demonstrate a novel mechanism by which SHP-1 down-regulates the Janus kinase–2 (Jak2)/signal transducer and activator of transcription-5 (Stat5) pathway downstream of the prolactin receptor (PRLR) and the erythropoietin receptor (EPOR) in a catalytic activity–independent manner. Structural/functional analysis of SHP-1 defined the C-terminal tyrosine residues (Y278, Y303, Y538, Y566) within growth factor receptor–bound protein 2 (Grb-2) binding motif to be responsible for delivering the inhibitory effects. Our results further indicate that these tyrosine residues, via recruitment of the adaptor protein Grb-2, are required for targeting the inhibitory protein suppressor of cytokine signaling–1 (SOCS-1) to Jak2 kinase. Finally, loss of SOCS-1 expression in SOCS-1–/– mouse embryonic fibroblast (MEF) cells led to attenuation in SHP-1 function to down-regulate PRL-induced Stat5 activation. All together, our results indicate that SHP-1 inhibits PRLR and EPOR signaling by recruitment and targeting of SOCS-1 to Jak2, highlighting a new mechanism of SHP-1 regulation of cytokine-receptor signaling.

Human cytomegalovirus (HCMV) has developed multiple strategies to escape immune recognition. Here, we demonstrate that HCMV down-regulates HLA-DR expression in infected interferon γ (IFN-γ)–stimulated fibroblasts at 1 day after infection. Decreased HLA-DR expression was not observed on cells infected with an HCMV strain lacking the pp65 gene (RVAD65), but was observed on cells transfected with the pp65 gene. HLA-DR expression accumulated in vacuoles near the nucleus in HCMV-infected, but not in uninfected or RVAD65-infected cells. In addition, the HLA-DR α-chain, but not the β-chain or HLA-DM, was degraded in HCMV-infected but not in RVAD65-infected cells. Thus, the HCMV protein pp65 mediates decreased expression of HLA-DR, by mediating an accumulation of HLA class II molecules in lysosomes that results in degradation of the HLA-DR α-chain.

In the current issue of Blood , Degryse and coauthors[1][1] report the transforming potential of a series of JAK3 mutations identified in primary T-cell acute lymphoblastic leukemia (T-ALL) samples and pave the way toward multitargeted JAK1 and JAK3 therapy in T-ALL. ![Figure][2] IL7R

Janus kinases (JAKs) are non-receptor tyrosine kinases that are generally found bound to cytokine receptors. JAKs have well established roles in activating signal transducers and activators of transcription (STATs) in response to cytokine stimulation in the cytoplasm. However, the epigenetic regulation of gene transcription by nuclear JAK2 through phosphorylation of tyrosine 41 on the histone protein H3 has recently been discovered. Our very recent work demonstrated that this histone targeting mechanism applies to another member of the JAK family, JAK1, which is activated by the autocrine cytokines IL6 and IL10 in activated B-cell-like diffuse large B cell lymphoma (ABC DLBCL). Genome-wide mapping of the H3Y41p histone mark revealed 2956 potential JAK1 target genes, 91% of which do not have a STAT motif in their promoter region. Here we investigated the JAK1 nuclear transfer mechanism in ABC DLBCL. Using cell fractionation and immunofluorescence assays, we detected a nuclear pool of JAK1 in both HEK 293T cells and ABC DLBCL cell lines. In searching for a putative nuclear localization signal (NLS) of JAK1, we identified a basic amino acid enriched sequence (KRKK…KHKK, starting from amino acid 342) that was reminiscent of a classical bipartite NLS. Immunofluorescence with GFP-tagged JAK1 truncations and full length JAK1 with mutations in putative NLS revealed that the first group of amino acids (KRKK) was the NLS that was responsible for the nuclear transport of JAK1. Our results, however, indicate that the active transport of JAK1 into the nucleus is independent of its activation status, since nucleocytoplasmic distribution did not change either in HEK293T cells when constitutively activated JAK1 was expressed or in ABC DLBCL cells with IL-6 stimulation or in GCB DLBCL cells that lack cytokine signaling. To identify potential importins that recognize the NLS of JAK1 and subsequently transport JAK1 into the nucleus, we used immunoprecipitation and GST pull down assays. We found that importin a4, a5 and a7 (encoded by KPNA3, KPNA1, and KPNA6 respectively) physically interacted with the NLS of JAK1 and these interactions were abolished when the 4 amino acids in the NLS were mutated to alanine, suggesting these alpha importins recognize the NLS of JAK1 (KRKK) and mediate the nuclear transport of JAK1. To test whether the NLS-mediated nuclear transfer that allows JAK1 to function in the nucleus is important for the fitness of ABC DLBCL cells, we performed JAK1 knockdown and rescue experiments in TMD8 and OCI-Ly10 cells, in which endogenous JAK1 was knocked down by an shRNA and an shRNA-resistant cDNA of either wild type or NLS-defective JAK1 was constantly expressed. The results revealed that knockdown of JAK1 led to reduced viable cells in the culture, suggesting that JAK1 expression is required for cancer cell survival. As expected, overexpression of wild type JAK1 completely reversed the toxicity due to knockdown of endogenous JAK1. However, overexpression of the NLS-defective JAK1 did not rescue the toxic effects of JAK1 shRNA. Thus, the findings suggest that nuclear transport of JAK1 is an essential process to maintain the survival of ABC DLBCL, and targeting the JAK1 NLS might be a potential targeted therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

It has been shown that interferons (IFNs) exert their signals through receptor-associated Janus kinases (JAKs) and signal transducers and activators of transcription (STATs). However, molecular mechanism of regulation of IFN signaling has not been fully understood. We have reported novel cytokine-inducible SH2 protein (CIS) and JAK binding protein (JAB) family genes that can potentially modulate cytokine signaling. Here we report that JAB is strongly induced by IFN-γ but not by IFN-β in mouse myeloid leukemia M1 cells and NIH-3T3 fibroblasts. NIH-3T3 cells ectopically expressing JAB but not CIS3 lost responsiveness to the antiviral effect of IFN-β and IFN-γ. M1 leukemic cells stably expressing JAB were also resistant to IFN-γ and IFN-β–induced growth arrest. In both NIH-3T3 and M1 transformants expressing JAB, IFN-γ did not induce tyrosine phosphorylation and DNA binding activity of STAT1. Moreover, IFN-γ–induced activation of JAK1 and JAK2 and IFN-β–induced JAK1 and Tyk2 activation were inhibited in NIH-3T3 JAB transformants. These results suggest that JAB inhibits IFN signaling by blocking JAK activity. We also found that IFN-resistant clones derived from LoVo cells and Daudi cells expressed high levels of JAB without stimulation. In IFN-resistant Daudi cells, IFN-induced STAT1 and JAK phosphorylation was partially reduced. Therefore, overexpression of JAB could be, at least in part, a mechanism of IFN resistance. © 1998 by The American Society of Hematology.

Cytokines and growth factors induce activation of the family of signal transducers and activators of transcription (Stats) that directly activate gene expression. Recently, constitutively activated Stat1, Stat3, and Stat5 were identified in nuclear extracts of acute myeloid leukemia (AML) patients, suggesting involvement of constitutive Stat activity in the events of leukemogenesis. In the present study, blasts of nine AML cases were investigated for the constitutive binding activity of the recently identified transcription factor LIL-Stat (LPS- and IL-1-inducible Stat). Band-shift assays were performed using the LPS-and IL-1-responsive element (LILRE) oligonucleotide, a gamma interferon activation site-like site that is present in the human IL-1β promoter. Constitutive LIL-Stat binding activity was observed in three leukemic cell lines and in seven out of nine AML cases. Transient transfection studies with a reporter plasmid containing three sequential LIL-Stat binding sites showed distinct transcriptional activity of LIL-Stat only in those AML blasts that constitutively expressed LIL-Stat. In CD34+ cells LIL-Stat also constitutively bound to its consensus sequence. However, when these cells were cultured in the presence of macrophage-colony stimulating factor (M-CSF) and stem cell factor (SCF) for differentiation along the monocytic lineage, the LIL-Stat binding activity disappeared totally. In agreement with these findings neither mature monocytes nor granulocytes showed constitutive or inducible LIL-Stat binding activity. We conclude that the LIL-Stat transcription factor is constitutively activated in undifferentiated and leukemic hematopoietic cells, but not in mature cells. This may suggest a role for this transcription factor in the process of differentiation. © 1998 by The American Society of Hematology.

CYTOKINE RECEPTORS are cell-surface glycoproteins that bind specifically to cytokines and transduce their signals. These receptors enable cells to communicate with the extracellular environment by responding to signals generated in the vicinity or in other parts of the organism. Thus, the initial

RECEPTORS FOR MANY cytokines and growth factors are triggered by the binding of their cognate ligands to assemble into an active multi-subunit complex that frequently comprises a homodimer of the ligand-binding subunit. This results in the activation of intracellular protein tyrosine kinase

Janus kinases (Jak) play essential roles in cytokine and growth factor signaling. Conventional gene targeting of Jak2, creating a null allele, leads to a block in definitive erythropoiesis as a result of failing signal transduction at the homomeric erythropoietin receptor (EpoR) and at the heteromeric interferon γ receptor (IFNGR). To investigate the in vivo relevance of the activation loop of Jak2, a Jak2-YY1007/1008FF knockin mutation was introduced into the germline of mice. The phenotype of the Jak2(FF/FF) mouse line reveals that tyrosine residues 1007/1008 are absolutely essential for kinase function and signal transduction at the homomeric EpoR. Detailed studies using the Jak2 activation loop mutant uncover an essential scaffolding function of Jak2 within the IFNGR receptor complex and reveal that Jak1 can mediate a semi-redundant function for IFNGR signal transduction. These studies are highly important for the molecular understanding of cytokine and growth factor signaling and provide new insights for future strategies in the design of pharmacological blockers of Jak2.

Colony-stimulating factor 1 (CSF-1) causes the activation of STAT1 and STAT3 transcription factors in bone marrow macrophages (BMM), in the macrophage cell line BAC1.2F5, and in fibroblasts that express the wild-type receptor for CSF-1. Fibroblasts expressing a mutant receptor in which the tyrosine 809 is replaced with phenylalanine do not activate STAT proteins in response to CSF-1. The activation of the STAT proteins in BMM is accompanied by tyrosine phosphorylation of Tyk2. In fibroblasts, the activation of the STAT proteins is accompanied by tyrosine phosphorylation of Tyk2 and JAK1. We propose that these JAK kinases are subjected to very rapid phosphorylation in response to CSF-1, followed by rapid dephosphorylation. Furthermore, we propose that kinases other than JAK kinase may be involved in the phosphorylation of the STAT proteins in response to CSF-1.

In the present study, we have used a human erythroleukemia cell line, TF-1, that proliferates in response to granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3), and interleukin-5 (IL-5) to investigate the role of receptors for these cytokines in signal transduction mechanisms involved in proliferative responses. The receptors for GM-CSF, IL-3, and IL-5 each possess a cytokine specific alpha subunit, but all three share a common beta chain. Using an immunoblotting system designed to detect phosphotyrosine containing proteins and a permeabilized cell system to detect rapid changes in phosphate turnover on proteins, we show that while GM-CSF and IL-3 use tyrosine phosphorylation to mediate mitogenic signal transduction, IL-5 uses tyrosine dephosphorylation in its signaling pathway. The use of different signaling pathways by these cytokines can be confirmed in a biologic system whereby the proliferation induced in culture by GM-CSF and IL-3 is inhibited by tyrosine kinase inhibitors, but that induced by IL-5 is enhanced. Conversely, GM-CSF- and IL-3-induced proliferation is stimulated by a tyrosine phosphatase inhibitor, yet IL-5-induced proliferation is inhibited. Inhibitors of protein kinase C inhibit IL-3- and GM-CSF-, but not IL-5-induced proliferation. We suggest that, because all these cytokines share the identical beta chain of their receptors, the cytokine specific alpha chain mediates the linkage of each receptor to the individual biochemical signal transduction pathways responsible for the different biologic activities of these cytokines.

CRLF2 rearrangements, JAK1/2 point mutations, and JAK2 fusion genes have been identified in Philadelphia chromosome (Ph)–like acute lymphoblastic leukemia (ALL), a recently described subtype of pediatric high-risk B-precursor ALL (B-ALL) which exhibits a gene expression profile similar to Ph-positive ALL and has a poor prognosis. Hyperactive JAK/STAT and PI3K/mammalian target of rapamycin (mTOR) signaling is common in this high-risk subset. We, therefore, investigated the efficacy of the JAK inhibitor ruxolitinib and the mTOR inhibitor rapamycin in xenograft models of 8 pediatric B-ALL cases with and without CRLF2 and JAK genomic lesions. Ruxolitinib treatment yielded significantly lower peripheral blast counts compared with vehicle (P < .05) in 6 of 8 human leukemia xenografts and lower splenic blast counts (P < .05) in 8 of 8 samples. Enhanced responses to ruxolitinib were observed in samples harboring JAK-activating lesions and higher levels of STAT5 phosphorylation. Rapamycin controlled leukemia burden in all 8 B-ALL samples. Survival analysis of 2 representative B-ALL xenografts demonstrated prolonged survival with rapamycin treatment compared with vehicle (P < .01). These data demonstrate preclinical in vivo efficacy of ruxolitinib and rapamycin in this high-risk B-ALL subtype, for which novel treatments are urgently needed, and highlight the therapeutic potential of targeted kinase inhibition in Ph-like ALL.

T cell Acute Lymphoblastic Leukemia (T-ALL) is an aggressive hematologic malignancy comprising 15% of pediatric and 25% of adult cases of ALL. With current treatment options, T-ALL survival rates have reached 50-60% in adults and 85% in children. Despite great strides in the treatment of this subset of ALL, T-ALL still shows resistance to first-line therapies in over 50% of adults and 25% of children, and relapse is often chemorefractory. Mutations in the Janus Activating Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathways and overexpression of the B cell lymphoma-2 (Bcl-2) protein are highly linked to the progression of T-ALL, and to the resistance of a number of available treatments for lymphoid malignancies. JAK/STAT is one of the main cytokine signaling pathways involved in hematopoietic cell growth and development. Indeed, the JAK/STAT pathway is often constitutively activated by T-ALL. The Bcl-2 pathway also plays an important role in cell survival. Overexpression of Bcl-2, an anti-apoptotic member of the Bcl-2 family, promotes cell survival by binding and neutralizing pro-apoptotic members. Inhibition of key proteins in both of these pathways has been greatly explored individually, but little is known about their combined effects on T-ALL. It is hypothesized that T-ALL manipulates both of these pathways as a means of escaping individual inhibition of either JAK/STAT or Bcl-2. Our hypothesis is that inhibiting both the JAK/STAT and Bcl-2 pathways with two small molecule inhibitors; Ruxolitinib (JAK 1/2 inhibitor) and Venetoclax (BH3 mimetic targeting Bcl-2), will inhibit T-ALL growth and survival. Proliferation of T-ALL was assessed by MTT assay and viability was measured by trypan blue and flow cytometry at 24, 48 and 72-hour time points post-treatment. Single-drug dose responses were conducted for both inhibitors. Six doses of both Ruxolitinib and Venetoclax were tested from a range of 0.156uM-5uM for Ruxolitinib and 1.56nM-50nM of Venetoclax. A response was seen for the three highest doses of both inhibitors (1.25uM, 2.5uM, and 5uM for Ruxolitinib and 12.5nM, 25nM, 50nM for Venetoclax). However, a synergistic effect was only achieved when combining 1.25uM Ruxolitinib with 25nM Venetoclax or 2.5uM Ruxolitinib with 12.5nM or 25nM Venetoclax. The combination dose of 1.25uM Ruxolitinib and 25nM Venetoclax demonstrated the greatest combined synergistic effect (CI Disclosures No relevant conflicts of interest to declare.

Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein that stimulates proliferation and differentiation of progenitor cells of neutrophils by signaling through its receptor (G-CSFR). Although the G- CSFR belongs to the cytokine receptor superfamily, which lacks an intracellular kinase domain, G-CSF-induced tyrosine phosphorylation of cellular proteins is critical for its biologic activities. We report here that JAK1 and JAK2 tyrosine kinases are tyrosine phosphorylated in response to G-CSF induction. We also demonstrate that the DNA-binding protein STAT3 (also called the acute-phase response factor [APRF], activated by interleukin-6) is an early target of G-CSF-induced tyrosine phosphorylation. G-CSF induces two DNA-binding complexes; the major complex contains tyrosine phosphorylated STAT3 protein and the minor complex appears to be a heterodimer of the STAT1 (previously p91, a component of DNA-binding complexes activated by interferons) and STAT3 proteins. Antiphosphotyrosine antibody interferes with the DNA binding activity of activated STAT3, indicating that tyrosine phosphorylation of STAT3 is important for the DNA binding activity. These results identify a signal transduction pathway activated in response to G-CSF and provide a mechanism for the rapid modulation of gene expression by G-CSF.

We have recently reported inactivation of the tyrosine phosphatase PTPN2 (also known as TC-PTP) through deletion of the entire gene locus in ∼ 6% of T-cell acute lymphoblastic leukemia (T-ALL) cases. T-ALL is an aggressive disease of the thymocytes characterized by the stepwise accumulation of chromosomal abnormalities and gene mutations. In the present study, we confirmed the strong association of the PTPN2 deletion with TLX1 and NUP214-ABL1 expression. In addition, we found cooperation between PTPN2 deletion and activating JAK1 gene mutations. Activating mutations in JAK1 kinase occur in ∼ 10% of human T-ALL cases, and aberrant kinase activity has been shown to confer proliferation and survival advantages. Our results reveal that some JAK1 mutation–positive T-ALLs harbor deletions of the tyrosine phosphatase PTPN2, a known negative regulator of the JAK/STAT pathway. We provide evidence that down-regulation of Ptpn2 sensitizes lymphoid cells to JAK1-mediated transformation and reduces their sensitivity to JAK inhibition.

JAK-STAT signaling is involved in the regulation of cell survival, proliferation, and differentiation. JAK tyrosine kinases can be transiently activated by cytokines or growth factors in normal cells, whereas they become constitutively activated as a result of mutations that affect their function in tumors. Specifically, the JAK2V617F mutation is present in the majority of patients with myeloproliferative disorders (MPDs) and is implicated in the pathogenesis of these diseases. In the present study, we report that the kinase CK2 is a novel interaction partner of JAKs and is essential for JAK-STAT activation. We demonstrate that cytokine-induced activation of JAKs and STATs and the expression of suppressor of cytokine signaling 3 (SOCS-3), a downstream target, are inhibited by CK2 small interfering RNAs or pharmacologic inhibitors. Endogenous CK2 is associated with JAK2 and JAK1 and phosphorylates JAK2 in vitro. To extend these findings, we demonstrate that CK2 interacts with JAK2V617F and that CK2 inhibitors suppress JAK2V617F autophosphorylation and downstream signaling in HEL92.1.7 cells (HEL) and primary cells from polycythemia vera (PV) patients. Furthermore, CK2 inhibitors potently induce apoptosis of HEL cells and PV cells. Our data provide evidence for novel cross-talk between CK2 and JAK-STAT signaling, with implications for therapeutic intervention in JAK2V617F-positive MPDs.

Although significant strides are being made in the molecular classification of mature T-cell lymphomas and leukemias (MTCLL),2 the genetic steps that lead to the neoplastic transformation of mature T cells and drive the emergence, progression, and clinical outcome of these malignancies remain unsolved. Some glimpse of the initial genomic and signaling events supporting the uncontrolled, clonal outgrowth of postthymic T-cell subsets in vivo is now being gained through the window of newly described murine models of MTCLL. Some of these models highlight the role of T-cell receptor (TCR)-activated pathways such as TCL1/AKT and SYK/ITK (reviewed by Warner et al3), while others reveal the importance of epigenetic mechanisms in T-cell transformation, as shown by interleukin-15 transgenic mice.4 The relative importance of genetic vs epigenetic events in the neoplastic transformation of human mature T-cells is an open question with significant clinical implications, and a very preliminary look at ongoing next-generation sequencing studies, including the one from Kiel et al, suggests that the rate of somatic mutations and the frequency of epigenetic aberrations may differ significantly across the spectrum of human MTCLL. As Kiel et al demonstrate, mapping the genetic landscape of MTCLL is already providing new opportunities for understanding the common pathogenesis and targeting the specific molecular drivers that are at play in these cancers. Among the key mediators of intracellular signaling in response to regulatory cytokines during blood cell development and the immune responses, the members of the JAK/STAT pathway have long been known to play a central role.5 Not surprisingly, somatic mutations of JAK/STAT genes and constitutive activation of JAK and STAT proteins have been observed in myeloid and lymphoid neoplasms6 and are now surfacing as recurrent genetic hits in different types of MTCLL.7-9 However, our knowledge of the specific role that the JAK/STAT family members play in T-cell oncogenesis remains limited. In this respect, the study of Kiel et al significantly expands our understanding of the impact of this key regulatory pathway in MTCLL by revealing that STAT5B, in addition to the previously described JAK1 and JAK3,7,8 is the most common target of somatic mutations in T-PLL. T-PLL is a rare and highly chemoresistant leukemic neoplasm of mature CD3+, TCRαβ, CD4+ T cells, affecting older adults (median age, 61 years).10 Prior to the introduction of alemtuzumab (Campath-1H), complete responses (CRs) were rare and median survival was only 7 months. With alemtuzumab, most patients achieve a CR, but in the absence of an allogeneic hematopoietic stem cell transplant, relapses are the rule and median survival is still

MicroRNAs are endogenously expressed small noncoding RNAs that regulate gene expression on the posttranscriptional level. The miR-17-92 cluster (encoding miR-17, -18a, -19a/b, -20a, and miR-92a) is highly expressed in tumor cells and is up-regulated by ischemia. Whereas miR-92a was recently identified as negative regulator of angiogenesis, the specific functions of the other members of the cluster are less clear. Here we demonstrate that overexpression of miR-17, -18a, -19a, and -20a significantly inhibited 3-dimensional spheroid sprouting in vitro, whereas inhibition of miR-17, -18a, and -20a augmented endothelial cell sprout formation. Inhibition of miR-17 and miR-20a in vivo using antagomirs significantly increased the number of perfused vessels in Matrigel plugs, whereas antagomirs that specifically target miR-18a and miR-19a were less effective. However, systemic inhibition of miR-17/20 did not affect tumor angiogenesis. Further mechanistic studies showed that miR-17/20 targets several proangiogenic genes. Specifically, Janus kinase 1 was shown to be a direct target of miR-17. In summary, we show that miR-17/20 exhibit a cell-intrinsic antiangiogenic activity in endothelial cells. Inhibition of miR-17/20 specifically augmented neovascularization of Matrigel plugs but did not affect tumor angiogenesis indicating a context-dependent regulation of angiogenesis by miR-17/20 in vivo.

Gene expression profiling of 207 uniformly treated children with high-risk B-progenitor acute lymphoblastic leukemia revealed 29 of 207 cases (14%) with markedly elevated expression of CRLF2 (cytokine receptor-like factor 2). Each of the 29 cases harbored a genomic rearrangement of CRLF2: 18 of 29 (62%) had a translocation of the immunoglobulin heavy chain gene IGH@ on 14q32 to CRLF2 in the pseudoautosomal region 1 of Xp22.3/Yp11.3, whereas 10 (34%) cases had a 320-kb interstitial deletion centromeric of CRLF2, resulting in a P2RY8-CRLF2 fusion. One case had both IGH@-CRLF2 and P2RY8-CRLF2, and another had a novel CRLF2 rearrangement. Only 2 of 29 cases were Down syndrome. CRLF2 rearrangements were significantly associated with activating mutations of JAK1 or JAK2, deletion or mutation of IKZF1, and Hispanic/Latino ethnicity (Fisher exact test, P < .001 for each). Within this cohort, patients with CRLF2 rearrangements had extremely poor treatment outcomes compared with those without CRLF2 rearrangements (35.3% vs 71.3% relapse-free survival at 4 years; P < .001). Together, these observations suggest that activation of CRLF2 expression, mutation of JAK kinases, and alterations of IKZF1 cooperate to promote B-cell leukemogenesis and identify these pathways as important therapeutic targets in this disease. This trial was registered at www.clinicaltrials.gov as #NCT00005603.

Abstract 2674 Poster Board II-650 Human plasmacytoid dendritic cells (pDC) play a central role in regulating adaptive T cell responses in the course of neoplastic, viral and autoimmune disorders. In several of these diseases, elevated extracellular levels of the serine protease granzyme B (GrB) are observed. We found that human pDC can be an abundant source of GrB based on FACS analysis, ELISpot, ELISA, Sensizyme, Western immunoblotting, RT-PCR, and fluorescence microscopy. GrB is actively secreted by pDCs and reaches maximal levels up to two logs higher than those produced by classical GrB producers such as CTL or NK cells. However, pDC GrB production is not accompanied by perforin secretion. Spinning disk confocal microscopy revealed that GrB+ pDC bind to and transfer active GrB to T cells. Importantly, this GrB transfer induces a suppression of T cell proliferation in a GrB-dependent, perforin-independent manner, a process reminiscent of regulatory T cells. GrB expression in pDC is regulated on a transcriptional level by JAK1, STAT3 and STAT5. IL-3 and IL-10 enhance GrB production by pDCs while GrB production is inhibited by toll-like-receptor agonists and CD40 ligand. These findings suggest that GrB production by pDCs is involved in the complex interactions between pDC and T cells and that GrB-secreting pDC may play a regulatory role related to anti-tumor immunity, anti-viral immune responses, and autoimmune processes. Disclosures: No relevant conflicts of interest to declare.

Activin A, BMP2, and BMP4, 3 members of the transforming growth factor-β family, are involved in the regulation of hematopoiesis. Here, we explored the role of these molecules in human megakaryopoiesis using an in vitro serum-free assay. Our results highlight for the first time that, in the absence of thrombopoietin, BMP4 is able to induce CD34+ progenitor differentiation into megakaryocytes through all stages. Although we have previously shown that activin A and BMP2 are involved in erythropoietic commitment, these molecules have no effect on human megakaryopoietic engagement and differentiation. Using signaling pathway-specific inhibitors, we show that BMP4, like thrombopoietin, exerts its effects on human megakaryopoiesis through the JAK/STAT and mTor pathways. Inhibition of the BMP signaling pathway with blocking antibodies, natural soluble inhibitors (FLRG or follistatin), or soluble BMP receptors reveals that thrombopoietin uses the BMP4 pathway to induce megakaryopoiesis, whereas the inverse is not occurring. Finally, we show that thrombopoietin up-regulates the BMP4 autocrine loop in megakaryocytic progenitors by inducing their production of BMP4 and up-regulating BMP receptor expression. In summary, this work indicates that BMP4 plays an important role in the control of human megakaryopoiesis.

SOCS1 and SHP1 negatively regulate the Janus kinase/signal transducer and activator of transcription (Jak/STAT) signaling pathway. The role of promoter hypermethylation leading to epigenetic inactivation of SOCS1 and SHP1 in myeloma was investigated. The methylation-specific polymerase chain reaction (MSP) was used to define SOCS1 and SHP1 methylation in 34 diagnostic myeloma samples. For SOCS1, MSP primers 3′ to the translation start site were unreliable and gave positive results in normal controls. However, primers in the 5′ promoter region were specific, although no myeloma samples showed methylation. For SHP1, 27 of 34 (79.4%) myeloma samples showed SHP1 hypermethylation. The biologic significance of SHP1 methylation was investigated in the U266 human myeloma line. U266 contained completely methylated SHP1. Furthermore, there was constitutive STAT3 phosphorylation. Treatment with 5-azacytidine led to progressive demethylation of SHP1 on days 2 to 5, with consequent increasing reexpression of SHP1 as shown by reverse transcription-polymerase chain reaction (RT-PCR). Concomitant with increasing SHP1, a parallel down-regulation of phosphorylated STAT3 occurred, so that by day 5 phosphorylated STAT3 was barely detectable. The overall survivals of patients with and without SHP1 methylation were similar. SHP1 methylation leading to epigenetic activation of the Jak/STAT pathway might have a tentative role in the pathogenesis of myeloma, which should be further confirmed by functional studies in primary myeloma samples. (Blood. 2004;103:4630-4635)

To the editor: Ruxolitinib is the first JAK inhibitor approved as therapy for high- and intermediate-risk myelofibrosis (MF). Two phase 3 trials have shown that ruxolitinib provides significant clinical benefit to patients with MF by reducing spleen size and ameliorating debilitating disease