bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeting Drug-Resistant CML and Ph+-ALL with the Spectrum Selective Protein Kinase Inhibitor XL228.
Authors: Shah Neil P. , Kasap Corynn , Paquette Ronald , Cortes Jorge , Pinilla Javier , Talpaz Moshe , Bui Lynne A. , Clary Douglas O.
Published: October 2019

The fusion protein BCR-ABL is a hallmark of chronic myelogenous leukemia (CML) and Philadelphia-positive acute lymphocytic leukemia (Ph+-ALL), and has been demonstrated as the primary driver of these diseases. Control of CML for considerable periods of time has been achieved through use of selective ABL kinase inhibitors, particularly imatinib. Once patients fail imatinib therapy, they are commonly found to harbor a mutated and activated form of BCR-ABL which is unable to bind the inhibitor. A more recent ABL inhibitor, dasatinib, can block growth of cells harboring most of the imatinib-resistant mutations, but T315I and F317L mutations are often seen in patients relapsing after dasatinib therapy. Thus once a patient develops CML harboring these mutations, there are few therapeutic options available. XL228 is a potent multi-targeted protein kinase inhibitor with activity against IGF1R, src, and Abl. It displays low nanomolar biochemical activity against wild type Abl kinase (Ki = 5 nM), as well as the T315I form of Abl resistant to imatinib and dasatinib (Ki = 1.4 nM). XL228 also inhibits Aurora A with an IC50 of approximately 3 nM, demonstrating a more balanced inhibition profile compared to other dual Abl /Aurora inhibitors. CML and ALL cell lines were evaluated for sensitivity to XL228, and in each case the IC50 for inhibition of proliferation was less than 100 nM. XL228 inhibits phosphorylation of BCR-ABL and its substrate STAT5 in K562 cells in vitro with IC50s of 33 and 43 nM, respectively. Single-dose pharmacodynamics studies demonstrate a potent effect of XL228 on BCR-ABL signaling in K562 xenograft tumors. Phosphorylation of BCR-ABL was decreased by 50% at XL228 plasma concentrations of 3.5 μM; a similar decrease in phospho-STAT5 occurred at 0.8 μM plasma concentration. XL228 showed clear superiority to MK-0457, imatinib, and dasatinib in downregulating BCR-ABL phosphorylation in BaF3 cells expressing the T315I form of BCR-ABL in vitro (406 nM, 6912 nM, >10,000 nM, >10,000 nM, respectively), and in xenograft experiments in vivo. These results indicate that XL228 potently inhibits wild type and T315I forms of BCR-ABL, and provide a rationale for the clinical development of this agent for the treatment of patients with drug-resistant disease. A phase I dose escalation study of XL228 in subjects with CML or Ph+-ALL who have failed prior imatinib and dasatinib therapy has been initiated, focusing on safety/tolerability, pharmacodynamics, and pharmacokinetics. Pharmacodynamic assessments include a flow cytometry-based phospho-CrkL assay, quantitative PCR for BCR-ABL, and plasma markers of XL228 activity. An update on our clinical experience with XL228 in subjects resistant or intolerant to imatinib and dasatinib will be presented.
Translocation t(4;14) Is Not an Adverse Prognostic Factor in Patients with Multiple Myeloma Undergoing Allogeneic Stem Cell Transplantation.
Authors: Hansen Timon , Schilling Georgia , Shimoni Avichai , Simon-Perez Jose-Antonio , Bethge Wolfgang , Liebisch Peter , Schwerdtfeger Rainer , Bornhaeuser Martin , Otterstetter Svenja , Ayuk Francis , Bokemeyer Carsten , Zander Axel R. , San-Miguel Jesus , Nagler Arnon , Kroeger Nicolaus
Published: October 2019

We analyzed the prognostic impact of the most frequent genetic abnormalities detected by fluorescence-in situ-hybridization combined with immunofluorescent cytoplasm immunoglobulin staining (cIg-FISH) in 101 patients with multiple myeloma, who underwent allogeneic stem cell transplantation after reduced melphalan/fludarabine-based conditioning from related (n=34) and unrelated (n=67) donors. The abnormalities were del(13q14) [62%], t(11;14)(q13;q32) [11%], t(4;14)(p16.3;q32) [16%], CMYC-gains (8q24) [17%], del(17p13.1) [16%], t(14;16)(q32;q23) [4%], whereas none of the patients had t(6;14)(p25;q32). Translocation t(4;14), CMYC-gains and del 17p were frequently associated with del(13q14): 64%, 80% and 92%, respectively. The complete remission (CR) rate was 45% for all patients. Patients with del(17p13) achieved fewer complete remission than others (7% vs. 56%; p=0.001), while no difference was seen for t(4;14) and other abnormalities. Univariate analysis revealed higher relapse rates for age > 50 years (p=0.002), del(13q14) (p=0.006) and del(17p13) (p=0.003). Patients with translocation t (4;14) had a similar four year event-free survival than others (50 vs 45%). In a multivariate analysis, only del(13q14) [HR: 2.34, p=0.03] and del(17p13) [HR: 2.24; p=0.04] influenced the risk of relapse, while for event-free survival, only age [HR 2.8; p=0.01] and del(17p13) [HR: 2.05; p=0.03] retained the prognostic value. These data seem to indicate that some adverse cytogenetic risk factors such as t(4;14) can be overcome by allogeneic stem cell transplantation, probably due to the graft versus myeloma effect. Del(17p13.1) is a significant factor for a lower chance of complete remissions and shorter event free survival following allogeneic stem cell transplantation. The presented data will have implications for risk-adapted strategies in the future.
Unlike Inactivating PTEN Mutations, Pharmacological Inhibition of PTEN Does Not Increase the Sensitivity of Multiple Myeloma Cells to Inhibition of the PI3K/Akt/mTOR Pathway.
Authors: Wang Xiaojing , Zhang Shuhong , Peng Xiaodong , Garlich Joe , Chen Li , Liang Jing , Farag Sherif Shafik
Published: October 2019

PTEN-deficient multiple myeloma (MM) and other tumor cell lines have been shown to be remarkably sensitive to inhibitors of the PI3K/Akt/mTOR pathway (Shi Y, et al. Cancer Res 2002; Simth Y, et al. Proc Am Assoc Cancer Res 2002; Neshat MS, et al. Proc. Natl. Acad. Sci. USA 2001.). Inactivating mutations of PTEN, however, are very rare in human MM cells. We investigated whether pharmacological inhibition of wild type PTEN might also sensitize MM cells to inhibitors of PI3K and mTOR, and therefore provide the rationale for novel therapeutically useful combinations. We tested the effect of the PTEN antagonist, SF1740 (Semafore Inc, Indianapolis, IN), in combination with the mTOR inhibitor, rapamycin, or the PI3K inhibitor, SF1101, (also known as LY294002, from Semafore Inc, Indianapolis, IN) in U266, MM1R, MM1S myeloma cells. Using the Malachite Green assay, the IC50 for PTEN inhibition by SF1740 was 0.4 μM, and maximal inhibition was achieved at a concentration of 1μM. We treated MM cells with rapamycin (1, 5, 10 nM) or SF1101 (10, 25, 50 μM) alone or in combination with SF1740 for 24, 48, 72 and 96 hours. Cellular toxicity was assessed using the MTT assay, apoptosis by annexin V/PI staining, and the effect on intracellular signaling was monitored by Western blotting. SF1740 (1 μM) alone increased cell growth, and failed to sensitize cells to the inhibitory effects of rapamycin (1–10 nM) at all time points. There were no significant differences in cell survival between the combination of the two drugs and rapamycin alone when treated from 48–96hr. Cytotoxicity was unaffected by the sequence of drug treatment; concurrent or 24-hour pre-treatment with SF1740. Similar results were obtained using the combination of SF1740 (1 μM) and SF1101 used at concentrations of 10, 25, and 50 μM, with no difference in MM cell inhibition and survival between the SF1101 and the combination with SF1740. We conclude that in MM cells, pharmacological inhibition of wild type PTEN does not appear to replicate the effect of inactivating mutations of PTEN in increasing cell sensitivity to inihibitors of the PI3K/Akt/mTOR pathway. The results do not support the clinical use of the PTEN inhibitor SF1740 in combination with either PI3K or mTOR inhibitors. Xiaojing Wang and Shuhong Zhang contributed equally to this work.
Halofuginone, an Old Anticoccidosis Drug, Induces Apoptosisin Multiple Myeloma Cells, Associated with Down Regulation of MCL1.
Authors: Leiba Merav , Tai Yu-Tzu , Hideshima Teru , Jakubikova Jana , Hiroshi Ikeda , Li Xian-Feng , Burger Peter , Richardson Paul , Munshi Nikhil C. , Nagler Arnon , Anderson Kenneth C.
Published: October 2019

Halofuginone, a synthetic derivative of quinazolinone alkaloid, has recently been shown to have an anti-cancer effect in various solid and hematological malignancies. It is an orally available drug with a safe toxicity profile in clinical trials of inflammatory disease and has anti-angiogenic, anti-metastatic and anti-proliferative effects in preclinical studies. It blocks TGFb signaling by targeting Smad 2/3, inhibits NFkB activity, as well as downregulates extra cellular matrix (ECM) formation via the inhibition of collagen type I formation and matrix metalloproteinase 2(MMP2). Since ECM has an important role in the bone marrow microenvironment in multiple myeloma (MM) pathogenesis, we here examined whether halofuginone induces cytotoxicity against various MM cell lines, including those sensitive and resistant to conventional and novel chemotherapies. Halofuginone (12.5–400 nM) induced cytotoxicity in a dose-dependent fashion in a variety of MM cell lines (n=20), regardless of the sensitivity to conventional chemotherapy (i.e., dexamethasone, melphalan, doxorubicin) and novel therapies (i.e., lenalidomide, bortezomib) with IC50 of 50–200 nM. In contrast, halofuginone did not induce cytotoxicity against CD40-activated peripheral blood mononuclear cells from normal donors, even at high doses (500–1000nM). Importantly, neither IL6 nor IGF1, two key growth and survival factors in MM, overcame the growth inhibitory effect of halofuginone. We further examined molecular mechanisms whereby halofuginone triggers growth inhibition in MM cells. It induced both apoptosis and necrosis in a time- (2–48h) and dose (50–200 nM) dependent manner in MM1R and OPM1 MM cells, assessed by annexin V/ PI staining. Significant dose- dependent activation of caspase 3 and 8 was demonstrated after 25–200 nM of halofuginone treatment of MM1R, MM1S, and OPM1 cells. Mitochondrial membrane potential was also reduced after 24 hours of halofuginone treatment (50, 200 nM) of MM1R and OPMI cells, as evident by the accumulation of JC1 monomers. In addition, western blot analysis showed that halofuginone induces cleavage of apoptotic proteins caspase 3/8 and PARP, and downregulates anti-apoptotic protein MCL1. Taken together, these data suggest that halofuginone has significant anti-MM activities and is a potential novel therapy in MM.
Enhanced Interaction Between Hsp90 and Raptor Regulates mTOR Signaling upon T Cell Activation
Authors: Delgoffe Greg M , Kole Thomas P. , Cotter Robert J , Powell Jonathan
Published: October 2019

Full T cell activation requires antigen recognition (Signal 1) in the context of costimulation (Signal 2). Our group and others have determined that the mammalian Target of Rapamycin (mTOR) plays an important role in integrating costimulatory signals. Specifically, Th1 T cell activation in the absence of mTOR activation leads to tolerance in the form of T cell anergy. mTOR is an evolutionarily-conserved serine/threonine kinase which has been shown to interpret environmental cues in mammalian cells. mTOR is activated by an array of diverse inputs including insulin, amino acids, growth factors and CD28. mTOR signals through two signaling complexes: TORC1 and TORC2. A critical component of TORC1 signaling is the Regulatory Associated Protein of TOR (raptor). One of the central questions in understanding mTOR function is determining how diverse upstream signals can lead to diverse downstream functional consequences. To address this issue in T cells we undertook a proteomic approach to identify novel binding proteins for raptor. Jurkat T cells were either incubated in serum-free media or hyper-activated. Raptor was immunoprecipitated (IP) from the two lysates, separated by SDS-PAGE and silver stained. Next, protein bands that were differentially bound to raptor in the lysates from stimulated versus unstimulated cells were identified. One band located near 90 kDa was excised and using mass spectrometry subsequently determined to be Hsp90, a chaperone protein necessary for the correct folding of many protein “clients”. These findings were next confirmed in primary T cells. Upon activation with anti-CD3 and anti-CD28, IP of raptor led to co-IP of Hsp90 and IP of Hsp90 led to the concomitant precipitation of raptor. To further determine the role of the Hsp90-raptor interaction in T cells we used 17-AAG, an Hsp90 inhibitor. Primary T cells were stimulated in the presence of rapamycin or 17-AAG. Incubation with 17-AAG but not rapamycin led to a decrease in raptor protein levels consistent with the concept that raptor is an Hsp90 client. Functionally, this led to a decrease in TORC1 activation as measured by phosphorylation of S6K-1. More importantly, although17-AAG did not inhibit IL-2 production upon initial stimulation, primary T cells stimulated with anti-CD3 and anti-CD28 in the presence of 17-AAG failed to produce IL-2 upon rechallenge 5 days later; they were anergic. Overall, our findings demonstrate a novel activation-induced interaction between Hsp90 and raptor in T cells. This interaction can regulate the decision between TCR-induced activation and tolerance. Hsp90 inhibitors are currently being evaluated as anti-neoplastic agents, and while such agents do not acutely inhibit T cell function, they may induce anergy in activated Th1 cells. Thus, Hsp90 inhibitors might be incorporated into novel immunosuppressive regimens to treat and prevent GVHD and transplant rejection through the promotion of T cell tolerance.
Platelets Regulate Tumor-Induced Angiogenesis
Authors: Chang Yali , Zhang Jing , Lin Huifeng , Italiano Joseph , Handin Robert I.
Published: October 2019

The hypothesis that tumors require a vascular network to grow beyond a critical size is now widely accepted. There is also circumstantial evidence implicating the blood platelet in the process of angiogenesis. We have previously shown that the platelet sequesters pro- and anti-angiogenic proteins in discrete subsets of alpha granules and differentially releases them in vitro in response to PAR1 and PAR4 activating peptides. We have extended these studies by injecting two murine -- Lewis Lung Carcinoma (LLC), B16 F10 melanoma and a human renal cell carcinoma (RCC 786-O) into the perivitelline space of wild type and Fli1-GFP transgenic zebrafish embryos 48 hours post fertilization (hpf). One day after injecting 2000 tumor cells suspended in Matrigel, a network of new blood vessels had grown up toward the site of tumor injection. By 3 to 4 days post-injection (dpi) the vascular network had entered and enveloped the tumor mass. We then blocked the production of zebrafish thrombocytes, the fish equivalent of mammalian platelets, by injecting an anti-sense morpholino directed against c-mpl, the thrombopoietin receptor, into single cell embryos followed, at 48 hpf, by the injection of tumor cells. We have previously shown that this c-mpl morpholino inhibits thrombocyte production for 5–7 days. The initial wave of angiogenesis was normal in all of the c-mpl morphants but there was almost complete inhibition of subsequent angiogenesis and blood vessel extension into the LLC, B16 melanoma and RCC 1080 tumors. In contrast, control fish that had received a scrambled morpholino and had a normal number of circulating thrombocytes, had normal angiogenesis within the tumor mass. We then transplanted LLC and B16 F10 cells into CD41-GFP embryos that have fluorescent thrombocytes in their circulation. Three dpi we observed the arrest and adhesion of GFP+ thrombocytes to vessels within the tumor mass. We also noted that thrombocytes were present in tumor vessels before blood flow had been established. We conclude that platelets/thrombocytes play an important role in TIA and may serve as a vehicle for the delivery of angiogenic factors to human and murine tumors. Thrombocytes appear to be particularly important for the penetration of new vessels into the tumor mass. These studies raise the interesting possibility that inhibiting platelet production or granule secretion might help to inhibit TIA.
Targeting STAT3 Signaling to Augment the Immunogenicity of B-Cell Lymphomas
Authors: Wang Hongwei , Cheng F. , Wright K. , Tao J. , Smith M. , Pinilla J. , Dessureault S. , Sebti S. , Sotomayor E.M.
Published: October 2019

STAT3 signaling has emerged as a negative regulator of inflammatory responses in immune cells. In bone-marrow derived antigen-presenting cells (APCs), genetic or pharmacologic disruption of STAT3 led to inflammatory cells that effectively prime antigen-specific T-cell responses and restore the responsiveness of tolerized T-cells. In contrast, enhanced Stat3 activity in APCs resulted in increased production of the immunosuppressive cytokine IL-10 and induction of T-cell tolerance1. B-cell lymphomas being tumors derived from B-lymphocytes display intrinsic antigen-presenting capabilities. Augmentation of this APC function has been shown to result in effective anti-lymphoma immunity2. In this study we determined whether targeting Stat3 signaling might influence the intrinsic APC function of malignant B-cells and the responsiveness –or not- of antigen-specific CD4+ T-cells. First, we specifically block STAT3 signaling in A20 lymphoma B-cells by using a dominant negative variant of STAT3, Stat3b. Inhibition of STAT3 resulted in tumor cells capable not only of fully priming naïve antigen-specific CD4+T-cells but also able of restoring the responsiveness of tolerant T-cells from lymphoma bearing mice. Conversely, transfection of A20 B-cells with Stat3c, a constitutively activated mutant form of STAT3, led to T-cell unresponsiveness. Of note, manipulation of STAT3 in B cell tumors was associated with changes in the mRNA expression and protein levels of IL-10. Second, we evaluated the effects of two novel Stat3 inhibitors, CPA-7 (a platinum-containing compound that disrupts STAT3 DNA binding activity) and S3I-201 (inhibitor of Stat3:Stat3 complex formation and Stat3 DNA binding and transcriptional activities) in a murine model of Mantle Cell Lymphoma (MCL). In vitro treatment of FC-muMCL1 cells - derived from a tumor elicited in Em-Cyclin D1 transgenic mice- with increasing concentrations of either CPA-7 or S3I-201 resulted in an enhanced presentation of OVA-peptide to naïve CD4+ T-cells specific for a MHC class II restricted epitope of ovalbumin (OT-II cells). Indeed, these T-cells produce higher levels of IL-2 and IFN-gamma compared to anti-OVA T cells that encountered cognate antigen in untreated FC-muMCL1 cells. More importantly, MCL cells treated with CPA-7 restored the responsiveness of tolerized anti-OVA CD4+ T-cells. Finally, in vivo treatment of MCL-bearing mice with CPA-7 (5 mg/kg/iv given on days +21, +24 and +27 after tumor challenge) resulted in significant inhibition of p-Stat3 in malignant B-cells and augmentation of their APC function. Taken together, STAT3 signaling is involved in the regulation of the antigen-presenting capabilities of B-cell lymphomas and as such represents a novel molecular target to augment the immunogenicity of these tumors.
NKX3.1 Is a Direct TAL1 Target Gene That Mediates the TAL1 Dependent Proliferation of Human T-Cell Acute Lymphoblastic Leukemia (T-ALL)
Authors: Kusy Sophie , Goardon Nicolas , Armstrong Florence , Pflumio Francoise , Romeo paul-Henri
Published: October 2019

The TAL1/SCL gene encodes a bHLH (basic Helix-Loop-helix) protein that acts as a master gene in hematopoiesis. The TAL1/SCL gene is also the most frequently activated gene in human T-ALL but the oncogenic transcriptional programs, downstream of TAL1 in human T-ALL, are not well characterized. Using RNA interference to knockdown TAL1 expression, we show that TAL1 regulates both cell proliferation and death of human T-ALL cells. To determine the TAL1 target genes in human T-ALL, we combine TAL1 knockdown and gene expression profiling and show that TAL1 activates and repress a common subset of genes in cell lines. This subset includes known TAL1 target genes but also the NKX3.1 gene that is a homeobox gene, specifically expressed in the prostate epithelium during prostate development and in adulthood. NKX3.1 gene inactivation is one of the earliest events that occur in prostate cancer initiation, defining NKX3.1 as a major tumor suppressor gene of this cancer. TAL1 expression is associated with NKX3.1 activation in human T-ALL cell lines and NKX3.1 is expressed in TAL1 expressing human T-ALL blasts. TAL1 and GATA-3 are specifically bound in vivo to the [−870/−570] region of the human NKX3.1 gene promoter, and ex vivo, TAL1 can either directly binds an E-box [position −738] or be recruited by GATA-3 on a GATA binding site [position −697]. Finally, functional analyses of the NKX3.1 promoter indicate that these binding sites mediate the transcriptional activity of this promoter in T-cell lines. Sequences analysis of the human and mouse NKX3.1 promoters show that the regulatory sequences involved in the TAL1 activation of the human NKX3.1 gene are not conserved in the mouse gene, indicating why the NKX3.1 gene is not expressed in mouse models of TAL1 mediated leukemogenesis. NKX3.1 knockdown shows that NKX3.1 is necessary for the proliferation of TAL1 expressing T-ALL cell lines and NKX3.1 overexpression can complement the proliferation defects associated with TAL1 knockdown in T-ALL cell lines. Microarray analyses show that TAL1 and NKX3.1 regulate a common subset of genes in T-ALL that includes numerous genes encoding proteins known to be involved in T-cell proliferation and/or signaling. Finally, using a new culture system that enables proliferation of primary human leukemic cells, we show that the NKX3.1 gene is specifically activated in human TAL1 expressing T-ALL together with the defined potential TAL1 and/or NKX3.1 target genes. These results characterize NKX3.1 as the first gene directly activated by TAL1 and involved in the TAL1 dependent proliferation of human T-cell Acute Lymphoblastic Leukemia.
Inhibition of HIV-1 by DpT-Based Iron Chelators
Authors: Debebe Zufan , Kumar Krishna , Ammosova Tatiana , Niu Xiaomei , Richardson Des R , Gordeuk Victor R. , Nekhai Sergei
Published: October 2019

HIV-1 transcription is activated by HIV-1 Tat protein, which recruits transcriptional co-activators to the HIV-1 promoter. Elongation of HIV-1 transcription is mediated by the interaction of Tat with host cell cycle-dependent kinase 9 (CDK9)/cyclin T1, which phosphorylates the C-terminal domain of RNA polymerase II. Tat itself is phosphorylated by host cell cycle-dependent kinase 2 (CDK2) [1] and inhibition of CDK2 by tridentate iron chelators such as 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone, (311) or ICL670 (deferasirox) inhibits HIV-1 transcription [2]. In addition to the inhibition of CDK2, 311 and ICL670 also prevent association of CDK9 with cyclin T1 [2], which could lead to inhibition of CDK9 activity and also to inhibition of HIV-1 transcription. Recently, a group of novel di-2-pyridylketone thiosemicarbazone (DpT) based tridentate iron chelators were shown to exhibit marked antiproliferative activity in vivo [3]. Here we screened DpT-based and also 2-benzoylpyridine thiosemicarbazone (BpT)-based tridentate iron chelators and identified three chelators, Dp44mT, Bp4eT and Bp4aT, that inhibited HIV-1 transcription but were not cytotoxic as determined by propidium iodide uptake, LDH release and calcein-AM uptake. The inhibition of HIV-1 transcription was observed in CEM HIV-1 LTR-GFP cells infected with Adeno-Tat and in 293T cells transiently transfected with HIV-1-LTR LacZ and Tat-expressing vectors with IC50s in the mid-nanomolar range. These new iron chelators also inhibited HIV-1 replication in CEM and THP-1 cells at 10 mM concentration. Analysis of the molecular mechanism of HIV-1 inhibition revealed that the DpT- and BpT-based iron chelators inhibited the activities of both CDK9/cyclin T1 and CDK2. The CDK9/cyclin T1 complex was disrupted in the cells treated with iron chelators, suggesting a possible mechanism for the inhibition of CDK9. In conclusion, our findings provide further evidence that iron chelators may inhibit HIV-1 transcription by deregulating CDK2 and CDK9. The projected therapeutic index of the selected DpT-based iron chelators was over 103 suggesting their potential usefulness as future anti-retroviral therapeutics.
Impact of Autologous Dendritic Cell Immunotherapy (Arcelis™) on B Cell Subsets in HIV-1-Infected Subjects Receiving Antiretroviral Therapy
Authors: Boulassel Mohamed-Rachid , Yassine-Diab Bader , Healey Don , Nicolette Charles , Sékaly Rafick-Pierre , Routy Jean Pierre
Published: October 2019

We demonstrated the enhancement of CD8-specific responses following the administration of an immune-based therapy consisting of dendritic cells (DC) electroporated with autologous amplified HIV-1 RNA and CD40 ligand (CD40 L) RNA manufactured by the Arcelis™ process in HIV patients receiving antiretroviral therapy (ART). We conducted a sub study on circulating B cell populations to further assess changes induced by this autologous DC therapy as CD40L is a major B cell co-stimulatory factor. To this end, we assessed B cell subset changes in relation to the proliferative capacity of CD4+ and CD8+ T cells response to DC targets containing the 4 HIV-1 antigens (Gag, Vpr, Rev, Nef). The co-expression of CD19, CD38, IgD, CD10, CD23, CD27, CD5, and CD138 were analyzed by multi-parametric flow cytometry to assess circulating B cell subsets such as naïve resting B-cells (Bm1), activated naïve B cells (Bm2), GC founder cells (Bm2’), centroblasts and centrocytes (Bm3 and Bm4), early memory B cells (eBm5), memory B cells (Bm5), IgD memory cells, plasma cells, and B-1 cells. Changes in B cells subsets were analyzed before and after the four intradermal injections of this immunotherapeutic product containing 1.2 × 107 DC. Ten ART treated subjects with undetectable viral load (< 50 copies/ml), median CD4+ count of 440 cells/μl (range: 316–1102), and with a CD4+ nadir > 200 cells/μl were studied. Throughout the study, no significant changes in CD4+ cell count, CD4/CD8 ratio, and no viral blips were noticed. The percentage of total B cells, Bm1, Bm2, Bm2′, eBm5, IgD memory, plasma cells, and B-1 cell subsets did not significantly change. However, a decrease in the percentage of Bm3 and Bm4 cells was found (0.36 [0.06–0.86] versus 0.11 [0.04–0.36]; P=0.05). Conversely, an important increase in the Bm5 cell subset was evidenced (10.4 [1.6–24.2] versus 18.1 [5.1–27.5]; P=0.005) suggesting a proliferation of B memory cells induced by DC immunization. In addition, the multifunctional and polyvalent CD8+ T cell proliferative responses to the 4 HIV genes used in this immunotherapy were noticed in 8 out of 9 subjects available for analysis and characterized by an effector memory phenotype. No CD4+ T cell immune responses were detected, consistent with the endogenous HLA class I loading of the antigens. Collectively, these results indicate that this immunotherapy induces an increase in the B memory cell population in the absence of inducing any clinically apparent autoimmunity along with strong HIV specific multifunctional CD8+ T cell specific immune responses.
Immunomodulatory Derivatives of Thalidomide (IMiD)-Induced Neutropenia Is Associated with PU.1 Downregulation and Myeloid Maturation Arrest
Authors: Pal Rekha , Roodman G. David , Mapara Markus , Mocsinski Lynn , List Alan F , Lentzsch Suzanne
Published: October 2019

Previous clinical trials have shown that immunomodulatory derivatives of thalidomde (IMiDs) including lenalidomide or pomalidomide induce high response rates in patients with relapsed or refractory multiple myeloma. However, the most dose limiting adverse effect of IMiDs is severe neutropenia. This is in contrast to our findings that IMiDs promote a shift in hematopoietic progenitor lineage commitment toward myeloid precursors with a subsequent maturation stop.

In colony forming assays using human CD34+ hematopoietic precursors treated with IMiDs, we observed a highly significant increase in myeloid progenitor formation at the expense of erythroid colonies. Treatment of hematopoietic progenitors with IMiDs showed an upregulation of CD33, a marker for immature myeloid cells. Further, in vitro treatment with IMiDs resulted in downregulation of the transcription factor PU.1. PU.1 gene knock-down models show increased granulopoiesis with impaired neutrophil maturation, resulting in an accumulation of promyelocytes. In accordance with that, Cathepsin G as a promyelocytic marker was highly upregulated under treatment with IMiDs measured by high-density oligonucleotide microarray, quantitative RT-PCR, western blot and ELISA, confirming the maturation arrest of neutrophil granulocytes. To evaluate medullary changes in granulopoiesis in human subjects treated with lenalidomide, we evaluated sequential bone marrows from 6 patients comparing bone marrow features prior to treatment, during treatment, and at the time of grade 4 neutropenia. Marrows at the time of neutropenia showed an accumulation of myeloid precursors (myelocytes, metamyelocytes) supporting impaired myeloid maturation. In 5 out of 6 patients the myeloid:erythroid (M:E) ratio increased with an M:E median of 1.95 at pretreatment and 9.2 at the time of the neutrophil nadir. At the same time bone marrow cellularity was unchanged.

In summary we show that IMiDs downregulate PU.1, a key transcription factor involved in granulocyte differentiation. The loss of PU.1 results in medullary accumulation of immature myeloid precursors, with corresponding neutropenia in the peripheral blood. Further high amounts of azurophilic granules of promyelocytes subsequently increase Cathepsin G and neutrophil elastase 2 resulting in enhanced platelet aggregation and a possible higher risk for venous thromboembolism. Our findings show that IMiDs do not induce neutropenia by marrow suppression but rather by transient block of maturation, which might be overcome by application of G-CSF.
Recombinant Human HPA-1A Antibodies for Treatment of Fetomaternal Alloimmune Thrombocytopenia (FMAIT): Proof of Principle in An in Vivo Murine Model and Human Volunteer Studies
Authors: Ghevaert Cedric , Wilcox David A. , Fang Juan , Armour Kathryn , Clark Mike R , Ouwehand Willem H. , Williamson Lorna M
Published: October 2019

Fetomaternal alloimmune thrombocytopenia (FMAIT) is caused by maternal generation of antibodies specific for paternal platelet antigens and can lead to fetal intracranial hemorrhage. A single nucleotide polymorphism (SNP) in the gene encoding integrin β3 causes a clinically important maternal-paternal antigenic difference; Leu33 generates the human platelet antigen 1a (HPA-1a), whereas Pro33 generates HPA-1b. As a potential treatment to prevent fetal intracranial hemorrhage in HPA-1a alloimmunized pregnancies, we generated an antibody that blocks the binding of maternal HPA-1a–specific antibodies to fetal HPA-1a1b platelets by combining a high-affinity human HPA-1a–specific scFv (B2) with an IgG1 constant region modified to minimize Fcγ receptor–dependent platelet destruction (G1Δnab). B2G1Δnab saturated HPA-1a+ platelets at micromolar concentration and substantially inhibited binding of clinical HPA-1a–specific sera to HPA-1a+ platelets. The response of monocytes to B2G1Δnab-sensitized platelets was substantially less (<15%) than their response to unmodified B2G1, as measured by chemiluminescence. In addition, B2G1Δnab inhibited chemiluminescence induced by B2G1 and HPA-1a–specific sera. We set out to prove the principle of our strategy in vivo using a murine model. Beta3-deficient mice (WT) were transplanted with littermate bone marrow transduced with a lentiviral vector containing the human ITGB3 encoding either Leucine-33 or Proline-33. B2G1 and polyclonal Ig preparations from clinical HPA-1a–specific sera reduced circulating HPA-1a+ platelets, concomitant with transient thrombocytopenia whilst WT mice and HPA-1b+ mice remained unaffected. Given the differences between murine and human Fcγ receptors, F(ab)2 B2G1 was used as a non-destructive proof of principle blocking antibody and prevented the in vivo platelet destruction seen with B2G1 and polyclonal HPA-1a–specific antibodies. These results provide rationale for human clinical studies, which are now underway. Eight HPA-1a1b heterozygotes volunteers have been recruited and from each of them we generate 2 pools of autologous platelets. Each pool is sensitized with either B2G1 or B2G1Δnab and radiolabeled with either Cr-51 or In-111. After re-injection into the volunteer, platelet survival is followed by the means of the radiolabel in order to show the differential effect on platelet destruction of the G1Δnab constant region when compared to the reference IgG1 destructive antibody. Full data of the volunteer studies will be presented in support of further clinical trials.
Identification of Mir-145 and Mir-146a as Micrornas Involved in the Pathogenesis of 5q- Syndrome
Authors: Starczynowski Daniel T. , Kuchenbauer Florian , Argiropoulos Bob , Sung Sandy , Morin Ryan , Muranyi Andrew L , Hirst Martin , Hogge Donna E. , Marra Marco , Wells Richard A. , Lam Wan , Humphries R. Keith , Karsan Aly
Published: October 2019

Deletion of the long arm of chromosome 5 is the most common cytogenetic alteration in myelodysplastic syndromes and is classified as a distinct subtype of the disease (5q- syndrome). Patients with 5q- syndrome present with macrocytic anemia, variable neutropenia, normal or elevated platelet counts, and megakaryocyte dysplasia. Over time these patients progress to marrow failure or acute myeloid leukemia. The commonly deleted region in 5q- syndrome contains numerous genes that are implicated in hematopoiesis, however no single gene loss recapitulates all features of 5q- syndrome in vivo. We examined the expression of microRNAs (miR) within the deleted region of chromosome 5q and found reduced expression of miR-145 and miR-146a in patients with del (5q). Knockdown of miR-145 or miR-146a in normal mouse hematopoietic cells resulted in increased megakaryopoiesis. We identified TIRAP and TRAF6 as critical mRNA targets of miR-145 and miR-146a, respectively. Expression of TIRAP alone resulted in autoactivation of TRAF6, implicating activation of a common pathway with loss of either miR-145 or miR-146a. Transplantation of lethally-irradiated mice with marrow cells expressing retrovirally-transduced TRAF6 resulted in neutropenia, anemia, thrombocytosis, and hypolobulated megakaryocytes. Long-term follow up of chimeric mice over 12 months revealed progression in approximately half the mice to marrow failure or acute leukemia. Further experiments revealed that recapitulation of 5q- syndrome-like features were mediated by paracrine effects induced by IL-6, as well as by cell autonomous TRAF6 effects. These findings suggest that several of the features of 5q- syndrome may be secondary to loss of microRNAs within the deleted region of chromosome 5q.
A Stapled BIM BH3 Helix Overcomes the MCL-1-Mediated Apoptotic Blockade of Refractory Hematologic Cancers
Authors: Labelle James L. , Lawrence Chelsea E. , Bird Gregory H. , Walensky Loren D.
Published: October 2019

MCL-1 is an anti-apoptotic member of the BCL-2 family of proteins and has emerged as a major resistance factor in hematologic cancer. Pharmacologic targeting of anti-apoptotic proteins is an effective strategy for reactivating apoptosis in cancers that evade cell death by neutralizing pro-apoptotic BCL-2 family proteins. Anti-apoptotic proteins contain a hydrophobic groove of differing specificity that binds and sequesters the critical BH3 the BH3-only members such as BID and BIM that transmit signals of cellular stress to that oligomerize upon activation to permeabilize the outer mitochondrial membrane. Small molecules such as ABT-737 that selectively inhibit the anti-apoptotic proteins BCL-2 and BCL-XL induce apoptosis of tumors that are especially dependent upon this subset of survival proteins. However, the compounds are rendered ineffective by cellular expression of alternate anti-apoptotic proteins such as MCL-1, which lie outside their range of binding specificity. We previously developed Stabilized Alpha Helices of BCL-2 domains (SAHBs) that are structurally stable, protease-resistant, and cell-permeable compounds capable of targeting BCL-2 family proteins in situ with high affinity. By inserting a hydrocarbon staple into the BH3 domain of pro-apoptotic BIM, we have generated a broad spectrum BCL-2 family targeting agent that binds all anti-apoptotic proteins with nanomolar affinity and also directly triggers the activation of pro-apoptotic BAX. Here we report that BIM SAHB, but not a point mutant control, induces dosedependent apoptosis of ABT-737-resistant acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, diffuse large B-cell lymphoma, and multiple myeloma cell lines. Importantly, we demonstrate by co-immunoprecipitation that BIM SAHB broadly targets BCL-2 family proteins in these MCL-1-expressing hematologic cancers. Thus, by exploiting the native binding spectrum of BIM BH3 for both anti- and pro-apoptotic BCL-2 family members, we demonstrate the therapeutic potential of BIM SAHB in overcoming MCL-1-mediated resistance in hematologic cancers.
p38MAPK Inhibits Neutrophil Development through Phosphorylation of C/EBPα on Serine 21
Authors: Buitenhuis Miranda , Geest Christian R. , Laarhoven Annemieke G. , Bruin Marrie C.A. , Bierings Marc B. , Vellenga Edo , Coffer Paul J.
Published: October 2019

Many extracellular stimuli regulate growth, survival and differentiation responses through activation of the dual specificity kinase MAPK kinase 3 (MKK3) and its downstream effector p38 Mitogen-Activated Protein Kinase (MAPK). Using CD34+ hematopoietic progenitor cells, here we describe a novel role for MKK3-p38MAPK in the regulation of myelopoiesis. Inhibition of p38MAPK utilising the pharmacological inhibitor SB203580, enhanced neutrophil development ex-vivo, but conversely reduced eosinophil differentiation. In contrast, constitutive activation of MKK3 dramatically inhibited neutrophil differentiation. Transplantation of β2-microglobulin−/− NOD/SCID mice with CD34+ cells ectopically expressing constitutively active MKK3 resulted in reduced neutrophil differentiation in vivo, whereas eosinophil development was enhanced. Inhibitory phosphorylation of C/EBPα on serine 21 was induced upon activation of p38MAPK. Moreover, ectopic expression of a non-phosphorylatable C/EBPα mutant was sufficient to abrogate MKK3 induced inhibition of neutrophil development. Furthermore, treatment of CD34+ progenitors from patients with severe congenital neutropenia with SB203580 restored neutrophil development. These results establish a novel role for MKK3-p38MAPK in the regulation of lineage choices during myelopoiesis through modulation of C/EBPα activity. This signaling module may thus provide an important therapeutic target in the treatment of bone marrow failure.
Autosomal Recessive Hereditary Thrombocytosis: Functional Analysis of a Novel C-Mpl Mutation Resulting in An Unsual Mode of Constitutive Receptor Activation
Authors: Stockklausner Clemens , Gehring Niels , Echner Nicole , Mulla Naima Ali Al , Kulozik Andreas E.
Published: October 2019

Thrombopoiesis is tightly regulated by the interaction between thrombopoietin (TPO) and its receptor c-Mpl. Ligand binding results in dimerization of the receptor, internalization of the complex, and activation of downstream signaling. This activation pathway also leads to the clearance of TPO from the plasma thus establishing a negative feedback loop. Gain of function mutations of TPO and c-Mpl are exceedingly rare but provide interesting models for the biological understanding of the molecular interactions that are required for a functional TPO pathway. Here, we describe a consanguineous Qatari family with hereditary thrombocytosis. Genotype analysis revealed a novel c-Mpl P106L mutation that was found homozygously in 3 affected patients with platelet counts between 700–800/nl. The TPO gene sequence was normal. Notably, platelet counts in heterozygous relatives were within the normal range. Surprisingly, TPO plasma levels were elevated to 10 to 15-fold in homozygotes but within the normal range in heterozygotes. A simple gain of function mechanism of the c-Mpl mutation could thus be excluded. Therefore, we next functionally analyzed and compared the normal and the P106L mutated c-Mpl receptor. An analysis of HeLa and BaF3 cells that were transiently or stably, respectively, transfected with c-Mpl expression vectors showed that the P106L mutation leads to abnormal subcellular receptor distribution. Whereas normal c-Mpl was detected in the Golgi-apparatus and in the plasma membrane, c-Mpl P106L was predominantly found within the ER. Furthermore, we showed that c-Mpl P106L cannot be processed to the normal, tunicamycin sensitive, strongly glycosylated form. Functional analysis of the TPO/c-Mpl signaling pathway in stably transfected BaF3 cells demonstrated an elevated phosphorylation of Stat5 with P106L when compared to normal c-Mpl. Moreover, cells transfected with the mutant but not with the normal receptor gene were proliferating independently of extracellular growth factors such as IL3. These data indicate that c-Mpl P106L activates downstream signaling and thus stimulates cell division and platelet production in a ligand-independent fashion. The recessive mode of inheritance suggests that the presence of normal receptor molecules prevents this abnormal function and promotes the maintenance of the negative feedback loop regulating TPO plasma levels by an unknown mechanism. The raised TPO plasma levels in homozygotes and the lack of detectable mutated c-Mpl in the plasma membrane indicate that the mutated receptor does not bind and internalize its normal ligand. In conclusion, we hypothesize that the novel c-Mpl P106L receptor mutation results in spontaneous dimerization and ligand independent activation even before the receptor reaches the Golgi apparatus and is fully glycosylated and transported to the plasma membrane.
Nation-Wide Survey of Infant Leukemia in Japan: A Report from the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG).
Authors: Miyamura Takako , Koh Katsuyoshi , Tomizawa Daisuke , Sugita Kanji , Kato Koji , Sato Takashi , Takahashi Yoshihiro , Ogawa Atsushi , Hirayama Masahiro , Kikuchi Akira , Oda Megumi , Hasegawa Daiichiro , Koike Kazutoshi , Saikawa Yutaka , Hatanaka Michiki , Horibe Keizo , Ishii Eiichi
Published: October 2019

Infant leukemia consists of wide spectrum of disease phenotype which includes acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) and mixed lineage leukemia, and has unique biological and clinical features. However, there are few studies which have focused on its diversity from the cross-sectional view of this specific age group. We here conducted a nation-wide survey to analyze the disease distribution, characteristics, clinical courses, and outcomes of these patients in Japan. Questionnaires were sent to 170 institutions participating in the JPLSG which covers more than 95% of the hospitals in Japan, and total of 225 cases with infant leukemia and related disorders aged less than 24 months at onset who were diagnosed between 2004 and 2007 were registered. Median age at onset was 12 months, ranged from 0 to 22 months. No gender differences were observed (male, 118 cases; female, 107 cases). Thirty-nine were diagnosed as ALL with positive MLL gene rearrangements (MLL-R ALL), 74 as ALL with germline MLL (MLL-G ALL), 83 as AML, 9 as mixed lineage leukemia, and 20 as other disorders. In MLL-R ALL group, the frequency of t(4;11) was high in patients of <12 months of age (18/29 cases), whereas hyperdiploidy was detected in 20% of cases of MLL-G ALL group (12/61 cases). In AML, positive MLL gene rearrangements were detected in only 15% (13/83 cases), while t(1;22)(p13q13) or OTT-MAL was detected in 4 cases. Regardless of disease groups, early infant group of <6 months of age showed poor prognosis compared with other age groups. Particularly, all cases of ALL diagnosed at <4 weeks after birth harbored MLL gene rearrangements and either died or relapsed, indicating that more appropriate strategy is needed to improve the outcome for this specific group. Patients with MLL-G ALL less than 12 months old showed a favorable prognosis compared to those with MLL-R ALL. In AML, majority of cases were myelomonocytic/monocytic leukemia (M4/M5) or megakaryoblastic leukemia (M7), and showed favorable outcome. In conclusion, infant leukemia is still a challenging disease harboring diverse features, therefore, it is necessary to keep on-going the prospective trials, to register and follow-up these cases systematically and to standardize its treatment including supportive cares, especially for those with younger age onset.
A Randomized Trial Investigating an Exercise Program to Prevent Osteoporosis and Motor Problems during Treatment for Childhood Acute Lymphoblastic Leukemia.
Authors: te Winkel Mariel L. , Hartman Annelies , van Beek Robert D. , de Muinck Keizer-Schrama Sabine M.P.F. , Kemper Han C.G. , Hop Wim C.J. , van den Heuvel-Eibrink Marry M. , Pieters Rob
Published: October 2019

Reduction in bone mineral density (BMD), fractures, altered body composition, deteriorated motor performance and impaired passive ankle dorsiflexion are side-effects of chemotherapy during childhood acute lymphoblastic leukemia (ALL). Because only scarce information is available on the value of regular exercise to prevent these side-effects, we performed a randomized trial to investigate the effects of intervention with an exercise program. At diagnosis 51 ALL patients (median age: 5.4 years) were randomized into a group receiving intervention (including twice-daily high-intensity weight-bearing activities throughout the two-year during treatment period), or a control group receiving standard care. BMD of total body (BMDTB) and lumbar spine (BMDLS) and body composition parameters were measured using dual energy X-ray absorptiometry (DEXA). We measured motor performance with the Bayley Scales of Infant Development and the Movement-ABC, and passive ankle dorsiflexion with a goniometer. The investigators were blinded to the randomization. Repeated measurements analysis (ANOVA) was used. BMD decreased equally in the intervention and control group during treatment (delta-BMDTB: −0.75 SDS vs −0.96 SDS, p=0.65 and delta-BMDLS: −0.15 SDS vs −0.04 SDS, p=0.83), and increased equally during the year after treatment (delta-BMDTB: 0.42 SDS vs 0.35 SDS, p=0.70 and delta-BMDLS: 0.10 SDS vs 0.14 SDS, p=0.84). Body-fat increased during treatment in both groups (delta-fat: 1.04 SDS vs 1.56 SDS, p=0.25). In the intervention group a more rapid decline of body-fat was observed during the year after completion of therapy than in the control group (delta-fat: −1.08 SDS vs −0.49 SDS, p=0.01). Lean body mass (LBM) of both groups decreased equally during treatment (delta-LBM: −0.61 SDS vs −0.12 SDS, p=0.16) and increased equally the year after stop of treatment (delta-LBM: 0.29 SDS vs 0.22 SDS, p=0.66). Both groups showed similar changes in passive ankle dorsiflexion mobility (−5.2º vs −4.6º, p=0.76) and motor performance (0.37 SDS vs 0.68 SDS, p=0.44) during treatment. Adherence to the exercise program varied considerably: 11% of the patients performed exercises daily, 37% more than once a week, 16% once weekly and 36% less than once a week. The exercise program was not more beneficial in preventing reduction in BMD, motor performance and passive ankle dorsiflexion than standard care, most likely due to unsatisfactory compliance. However, the faster recovery after stop of treatment of the excess of body-fat in the intervention group than in the control group may be due to the educational effect of the intervention program.