bluuebunny/crossref_metadata_2025 huggingface dataset:
IDH Mutations, 2-Oxoglutarate-dependent Dioxygenases, and Leukemia
Authors: Kaelin William G.
Published: October 2019

Somatic isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations have been discovered in a variety of neoplasms, including glioblastomas and acute myelogenous leukemias. Cancer-associated IDH mutants produce large quantities (low mM) of the R enantiomer of 2-hydroxyglutarate (R-2HG). R-2HG resembles 2-oxoglutarate, which is a Krebs Cycle intermediate and an essential cofactor for a variety of enzymes including the ~70 2-oxoglutarate-dependent dioxygenases. Included among these enzymes are the EglN prolyl hydroxylases that earmark the hypoxia-inducible factor (HIF) for destruction under normoxic conditions, the JmjC histone demethylases, and the ten-eleven translocation (TET) enzymes that hydroxylate methylcytosine residues. In general, both R-2HG and the alternative enantiomer, S-2HG, inhibit 2-oxoglutarate-dependent dioxygenases, with the (S) enantiomer typically being more potent, at least as measured in vitro. We recently discovered that R-2HG, in contrast to S-2HG, potentiates, rather than inhibits, EglN activity; we linked this, and subsequent downregulation of HIF, to the ability of mutant IDH1 to transform human astrocytes. Moreover, we found that R-2HG, but not S-2HG, is sufficient to promote leukemic transformation and that its effects are reversible. In this setting, inactivation of EglN, as occurs with S-HG, appears to be antithetical to transformation. R-2HG promotes leukemic transformation by inhibiting targets such as TET2, while sparing EglN activity. Recent clinical data show that inhibiting R-2HG production can induce complete remissions in some patients with IDH mutant leukemias. We are currently trying to identify the relevant downstream targets of TET2 in IDH mutant leukemias and are also exploring whether loss of EglN activity has antileukemic effects in mouse models in vivo. Finally, we are exploring whether any other 2-oxoglutarate-dependent dioxygenases, such as the RBP2/JARID1A/KDM5A, might be viable drug targets in leukemia.

Disclosures

Kaelin: Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; FibroGen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Lilly: Membership on an entity's Board of Directors or advisory committees; Nextech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Peloton: Consultancy, Membership on an entity's Board of Directors or advisory committees; TRACON: Consultancy.
The Tissue Macrophage Lineage
Authors: Merad Miriam
Published: October 2019

Despite accumulating evidence suggesting local self-maintenance of tissue macrophages in the steady state, the dogma remains that tissue macrophages derive from monocytes. In this presentation, I will discuss parabiosis, fate mapping, and adoptive transfer approaches that we developed in the laboratory to assess the contribution of monocytes to tissue resident macrophages in the steady state and after cell turnover. Using these approaches we found that monocytes contribute minimally to tissue resident macrophages in the steady state. Similarly, we found that after depletion of lung macrophages, the majority of repopulation occurs by local proliferation in a Csf-1 and Csf-2 dependent manner. This repopulation from a non-circulating population was stochastic and observed in both infection- and inflammatory-induced cytotoxicity of local macrophages. We also found that after bone marrow transplantation, host tissue macrophages retain the capacity to expand when the development of donor tissue macrophages is compromised. These results are consistent with recent results obtained in humans showing that macrophages can form in the absence of circulating monocytes. Collectively, these results indicate that tissue resident macrophages and circulating monocytes should be classified as independent mononuclear phagocyte lineages.

Disclosures

No relevant conflicts of interest to declare.
Effects of the Btk Inhibitor Ibrutinib on Monocyte Responses to Antibodies
Authors: Ren Li , Campbell Amanda , Gautam Shalini , Fang Huiqing , Fatehchand Kavin , Stiff Andrew , Mehta Payal , Mo Xiaokui , Fang Xuexun , Byrd John C. , Carson William , Butchar Jonathan P. , Tridandapani Susheela
Published: October 2019

The novel and irreversible tyrosine kinase inhibitor, Ibrutinib, has shown significant activities across a variety of B-cell tumors such as Chronic Lymphocytic Leukemia (CLL) and B-cell Non-Hodgkin lymphoma, either alone or in combination with antibody therapy. Fcγ receptors (FcγR) on effector immune cells such as macrophages play an important role in tumor-specific antibody-mediated immune responses. Here we tested the effect of Ibrutinib on FcγR-mediated activities in monocytes.

Our results indicated that Ibrutinib does not affect monocyte FcγR-mediated phagocytosis even at concentrations higher than those achieved physiologically, but suppresses FcγR-mediated cytokine production. This phenomenon was also seen in macrophages from Xid mice, in which there is a decrease in FcγR-mediated cytokine production but not phagocytosis.

One major signaling event downstream of Btk that is blocked by Ibrutinib is calcium flux. Thus, we next tested whether phagocytosis was independent of calcium flux in order to help explain the lack of effect that Ibrutinib had on monocyte phagocytic ability. Results showed that intracellular calcium flux is required for FcγR-mediated cytokine production but not for phagocytosis. In order to verify this finding, we then measured the activation of the GTPase Rac, which is responsible for actin polymerization needed for phagocytosis. Results showed that Ibrutinib did not inhibit Rac activation, nor did inhibition of calcium flux by BAPTA-AM.

We next asked whether the effect of Ibrutinib on cytokine production could be reversed by IFNγ priming since NK cells produce significant levels of IFNγ in response to antibody therapy. Treatment of monocytes with IFNγ prior to Ibrutinib treatment resulted in a lack of inhibition of cytokine production, suggesting that IFNγ signaling can overcome Ibrutinib effects. We are currently in the process of examining the mechanisms by which IFNγ can protect from Ibrutinib effects on monocytes.

These results suggest that combing Ibrutinib with monoclonal antibodies can enhance B-CLL killing while not affecting macrophage effector function.

Disclosures

Byrd: Acerta Pharma BV: Research Funding.
Circadian Clock Protein CRY Controls B-Cell Intrinsic Tolerance
Authors: Cao Qi , Zhao Xuan , Gery Sigal , Chen Zhengshan , Deng Ruishu , Sun Haibo , Lin De-chen , Zhao Zhiqiang , Said Jonathan W , Li Quan-zhen , Muschen Markus , Evans Ronald M. , Koeffler H. Phillip
Published: October 2019

The circadian system regulates numerous physiological processes including adaptive immune system. Here we show that mice deficient for the circadian genes Cry1 and Cry2, (Cry double knockout [DKO]) display an autoimmune phenotype including higher serum IgG concentration than wild type (WT) mice, presence of serum anti-nuclear antibodies, precipitation of IgG, IgM and complement 3 (C3) in glomeruli, and massive infiltrations of leukocytes into the lung and kidney. A large panel of autoantigens demonstrated that the sera of the Cry DKO mice but not the WT mice, had autoantibodies covering most of the specificities reported to be present in patients with SLE, rheumatoid arthritis (RA), multiple sclerosis (MS), Sjögren's syndrome and other autoimmune disorders. Taken together, lost of the CRY circadian protein leds to severe autoimmunity. Furthermore, flow cytometry analysis of lymphoid organs showed lower pre-B cell numbers and higher mature recirculating B cells in the bone marrow as well as increased number of B2 B cells in the peritoneal cavity of Cry DKO mice.

The BCR-proximal signaling pathway plays a critical role in peripheral B cell tolerance and activation. Activation of splenic B cells from the Cry DKO mice elicited markedly enhanced and prolonged tyrosine phosphorylation of cellular proteins compared to WT mice, suggesting that a very active BCR signaling pathway may contribute to impaired B cell tolerance in the Cry DKO mice. In summary, our results suggest that B cell development, as well as the intrinsic checkpoints of immune tolerance, are under direct circadian control.

Disclosures

No relevant conflicts of interest to declare.
Tumorantigen-Specific CD40B Cells: Combining Enhanced Antigen-Presentation and Antibody-Secretion for Tumor Targeting
Authors: Wennhold Kerstin , Thelen Martin , gracia Marquez Maria , Scheid Christof , Shimabukuro-Vornhagen Alexander , von Bergwelt-Baildon Michael
Published: October 2019

Efficient antigen presentation is a prerequisite for the development of a T-cell-mediated immune response. Dendritic cells (DCs) are the most prominent professional antigen-presenting cells (APCs). However, they have several disadvantages as cellular adjuvant in cancer immunotherapy. Therefore, an alternative approach was developed, in which polyclonal B cells can serve as potent APCs by treatment with the CD40 ligand. We demonstrated that CD40-activation dramatically improves antigen presentation by B cells, efficiently inducing naive and memory CD4+ and CD8+ T-cell responses. Moreover, these CD40-activated (CD40) B cells home to secondary lymphoid organs. However, antigen presentation by antigen-specific B cells is more effective compared to polyclonal B cells. Therefore, we use tumorantigen-specific B cells to improve the antigen-presenting function of CD40B cells. Purified human and murine tumorantigen-specific B cells highly upregulate activation markers upon CD40-stimulation, which results in an enhanced CD4+ and CD8+ T cell response in vitro and in vivo. This response is significantly lower in polyclonal CD40B cells and comparable to the stimulation induced by mature dendritic cells. Moreover, antigen-specific B cells could be stimulated in vitro to differentiate into antibody-secreting plasma cells. Treatment of E.G7 lymphoma-bearing mice with a combination of antigen-specific CD40B cells and plasma cells results in inhibition of tumor growth and prolonged survival. Moreover, antigen-specific B cells home to the tumor site and the spleen, where they encounter T cells. These results provide new insights into the role of activated antigen-specific B cells as APCs and their use for cancer immunotherapy.

Disclosures

Scheid: Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.
The Second CGHC Motif of Protein Disulfide Isomerase Mediates Thrombosis
Authors: Zhou Junsong , Wu Yi , Wang Lu , Rauova Lubica , Hayes Vincent M , Poncz Mortimer , Essex David W
Published: October 2019

Protein disulfide isomerase (PDI) catalyzes thiol-disulfide exchange reactions that are critical for protein function. PDI has two distinct CGHC redox-active sites, but nothing is known about which site is important in physiologic reactions including thrombosis. To study the role of PDI and its active sites in thrombosis we used two genetically-modified models, Mx1-Cre/PDIfl/fl mice with blood cells and vessel wall cells lacking PDI, and transgenic PDI(ss-oo) mice with PDI lacking the second CGHC active site. Using laser-induced cremaster arteriole injury fibrin deposition was decreased in both mouse models. Both mouse models showed attenuation of platelet accumulation using laser arterial injury and FeCl3-induced mesenteric artery injury. The defects in fibrin deposition and platelet accumulation were rescued by infusion of recombinant PDI(oo-ss) containing only a functional second active site, confirming the second active site of PDI was critical for both fibrin formation and platelet accumulation. In vitro studies showed that platelet aggregation depended on the second CGHC motif of PDI, and that binding of PDI protein to platelets depended on the presence of the αIIbβ3 integrin. Studies on platelet secretion revealed that P-selectin expression and ATP release were dependent on a functional second active site of PDI. Treatment of the platelets with eptifibatide did not affect P-selectin expression. Together these findings suggest the second active of PDI catalyzes a reaction in αIIbβ3 supporting platelet aggregation and a reaction in a non-αIIbβ3 substrate supporting secretion. In vivo studies on coagulation showed that PDI infusion rescued fibrin formation in eptifibatide-treated mice in which platelet accumulation was completely blocked, suggesting that PDI directly contributes to activation of coagulation. In summary, our results indicate that the second CGHC active site of PDI has a role in platelet function and coagulation. Targeting this site may have a dual inhibitory effect on thrombosis by decreasing both platelet and fibrin accumulation.

Disclosures

No relevant conflicts of interest to declare.
Increased Levels of Histone in Human Plasma in Septic Patients with DIC
Authors: Kawasugi Kazuo , Yamamoto Tadashi , Shirafuji Naoki , Yamada Shingo , Takashi Ito , Maruyama Ikuro
Published: October 2019

Sepsis is a life-threatening condition that is characterized by a whole-body inflammatory state (called a systemic inflammatory response syndrome, SIRS). Histones are essential for packing DNA into the cell nucleus and also play role in regulating gene expression but the new study (Jun Xu et al, Nature Medicine 15. 1318, 2009) have shown histones released into blood stream at the onset of sepsis can have devastating effects (endothelial damage and organ failure etc). However, plasma levels of histone are unclear in septic patients with DIC. Therefore, We developed a sandwich ELISA to quantify serum or plasma histone H3 levels and measured plasma levels of histone H3 in septic patients with DIC (n=30).

DIC was diagnosed on the Japanese Ministry of Health and Welfare (JMHW) DIC diagnostic criteria. The thrombin antithrombin complexes (TAT) levels were higher in septic patients with DIC as reported by others. The working range of this ELISA was 3-1000 ng/ml. In healthy volunteers, plasma levels of histone (H3) were not detectable. We detected high levels of extracellular hisotone (H3) in the plasma of all septic patients with DIC by sandwich ELISA. Mean plasma levels of hisotone (H3) were 70.5 ± 63.2 (12.9-336.5) ng/ml in septic patients with DIC. However, plasma levels of extracellular histone (H3) were negative in acute promyelocytic leukemia (APL)-induced DIC (n=5). Also, Plasma levels of histone were 10 ng/ml or less by sandwich ELISA in septic patients without DIC (n=8).

These results suggest that assess of extracellular hisotones in the plasma may be helpful in the making the diagnosis in septic patients with DIC. Also, it appears that extracellular hisotones in the plasma may contribute to develop DIC in septic patients.

Disclosures

No relevant conflicts of interest to declare.
Hematopoietic Stem Cells Develop in the Absence of Endothelial Cadherin 5 Expression
Authors: Anderson Heidi , Patch Taylor , Reddy Pavan , Hagedorn Elliott , Tamplin Owen J. , Bauer Daniel E. , Paw Barry H. , Vestweber Dietmar , Zon Leonard I. , Orkin Stuart H. , Daley George Q. , Shah Dhvanit I.
Published: October 2019

Rare endothelial cells in the aorta-gonad-mesonephros (AGM) transition into hematopoietic stem cells (HSCs) during embryonic development. Lineage tracing experiments indicate that HSCs emerge from Cadherin 5 (Cdh5, VE-cadherin)+ endothelial precursors, and isolated populations of Cdh5+ cells from mouse embryos and embryonic stem (ES) cells can be differentiated into hematopoietic cells. Cdh5 has also been widely implicated as a marker of AGM-derived hemogenic endothelial cells. Since Cdh5-/- mice embryos die before the first HSCs emerge, it is unknown if Cdh5 has a direct role in HSC emergence. Our previous genetic screen yielded malbec (mlbbw306), a zebrafish mutant for cdh5, with normal embryonic and definitive blood. Utilizing time-lapse imaging, parabiotic surgical pairing of zebrafish embryos, and blastula transplantation assays, we show that HSCs emerge, migrate, engraft, and differentiate in the absence of cdh5 expression. By tracing Cdh5-/- GFP+/+ cells inchimeric mice, we demonstrated that Cdh5-/- GFP+/+ HSCs emerging from E10.5 and E11.5 AGM or derived from E13.5 fetal liver not only differentiate into hematopoietic colonies but also engraft and reconstitute multi-lineage adult blood. These data establish that Cdh5, a marker of hemogenic endothelium in the AGM, is dispensable for the transition of hemogenic endothelium to HSCs.

Disclosures

Bauer: Biogen: Research Funding; Editas Medicine: Consultancy. Zon:FATE Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Scholar Rock: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder. Orkin:Editas Medicine: Membership on an entity's Board of Directors or advisory committees; Biogen: Research Funding; Pfizer: Research Funding; Sangamo Biosciences: Consultancy.
Thrombopoietin (TPO) Induces Hematopoietic Differentiation of Mouse ES Cells Via HIF-1 Alpha-Dependent Activation of a BMP4 Autoregulatory Loop
Authors: Zahabi Azadeh , Morishima Tatsuya , Pramono Andri , Lan Dan , Kanz Lothar , Welte Karl , Skokowa Julia
Published: October 2019

Understanding the molecular mechanisms underlying hematopoietic differentiation of embryonic stem (ES) cells may help to ascertain the optimal conditions for the production of hematopoietic cells as a source for transplantation or experimental use. Previously, we found that patients with congenital amegakaryocytic thrombocytopenia (CAMT), who develop pancytopenia early after birth, harbor mutations within the thrombopoietin (TPO) receptor, c-mpl. This knowledge, together with observations in vitro and in animal models in vivo, suggests that TPO/c-mpl signaling promotes early hematopoiesis. However, the downstream mechanisms underlying TPO signaling are not fully elucidated. Here, we describe for the first time a direct connection between the TPO and bone morphogenetic protein 4 (BMP4) signaling pathways in the hematopoietic fate decision of ES cells. BMP4 is a classical morphogen and a well-known inducer of early hematopoietic differentiation of ES cells. Treatment of ES cells with TPO induced the autocrine production of BMP4 by ES cells with concomitant upregulation of the BMP receptor, BMPR1A, phosphorylation of Smad1, 5, and 8 and activation of the specific target genes, Id1, 2, and 3, and Msx1 and 2. This was mediated by TPO-dependent binding of the HIF-1α transcription factor to the BMP4 gene promoter, resulting in further activation of the BMP4-autoregulatory loop. Treatment of ES cells with the BMP antagonist noggin substantially reduced TPO-dependent hematopoietic differentiation of ES cell. Taken together, our findings contribute to the understanding the mechanisms of hematopoietic differentiaiton of ES cells and might help to establish new methods for the efficient production of hematopoietic stem cells in vitro.

Disclosures

No relevant conflicts of interest to declare.
Interferon Alpha Mediated Activation of the Bone Marrow Stem Cell Vascular Niche
Authors: Prendergast Áine M. , Kuck Andrea , Von Essen Mieke , Essers Marieke A. G.
Published: October 2019

Endothelial cells (ECs) significantly influence the response of an organism to inflammation and infection. In the bone marrow, these cells form a major part of the bone marrow vascular niche, which regulates stem cell function and influences stem cell fate. The primary response to infection involves synthesis of immune-modulatory cytokines, such as interferon alpha (IFNα). We, and others, have shown that in contrast to the anti-proliferative effect of IFNα on hematopoietic stem cells (HSCs) in vitro, in vivo, IFNα induces cell cycle entry of quiescent HSCs (Essers et al. 2009). Given the contrasting outcome of in vitro and in vivo exposure of HSCs to IFNα, it is probable that niche cells and molecular maintenance signals from the niche are required for IFNα-induced activation of quiescent HSCs. Here, we now show that although interferon signaling itself in niche cells is not required for HSC activation, niche components do respond to IFNα stimulation. Of these, bone marrow ECs are rapidly and indirectly stimulated following IFNα treatment in vivo. The vascular system, lined by ECs, is a central primary responder following inflammatory insult with a multifaceted role, including transport of immune cells and promotion of a rapid return to homeostasis. We have found that IFNα stimulation in vivo results in an increased bone marrow vascularity, visualized by fluorescence imaging of cryo-sectioned murine femurs immunostained with the vascular basement membrane protein, laminin. IFNα-mediated activation of ECs involves the expression of key inflammatory and endothelial-stimulatory markers, including VE-cadherin and ESAM, and also an increased vascular leakiness in the bone marrow, demonstrated by the Evans blue assay. In accordance with this finding of vascular activation, we confirmed that VEGF, which is an established regulator of vascular dynamics, is rapidly up regulated in the bone marrow supernatant of treated mice. Furthermore, we can also demonstrate a key role for VEGF in our observed IFNα-mediated stimulation of ECs by abrogation of this activation using co-treatment with Avastin (bevacizumab) in vivo. We are now investigating the feedback from this activation of ECs on the hematopoietic system itself. In summary, these data indicate a rapid and indirect stimulation of the bone marrow vascular niche in an inflammatory setting. In addition, they support a previously undescribed cellular instruction from an activated hematopoietic system to a niche component.

Disclosures

No relevant conflicts of interest to declare.
5-Fluorouracil Treatment Leads to Activation of Stem Cell Niche By Reconstructing Mesenchymal Stromal Cells and Exert a Distinct Microenvironmental Impact on Normal and Leukemic Cells
Authors: Jeong Seon-Yeong , Kim Jin-A , Oh Il-Hoan
Published: October 2019

Reactivation of endogenous hematopoietic stem cells (HSCs) are initiated by stimulation of bone marrow niche triggered by various injury signals. Here, we show that treatment with 5-fluorouracil (5-FU) leads to reconstruction of bone marrow (BM) microenvironment to establish an activated niche stimulating hematopoietic stem cells (HSCs). First, we show that pre-treatment with 5-FU leads to engraftment of donor cells in non-irradiated recipient mice without affecting the homing efficiency of HSCs into BM. The HSC activation effects were reproduced in-vitro by co-culturing hematopoietic cells with CD45-Ter119- stromal cells derived from 5-FU treated BM, but not by co-culture with CD45+ cells or stromal cells obtained from enzymatic digestion of bone from the same mice. Examination of BM mesenchymal cells after 5-FU treatment revealed a rapid emergence of high-proliferating mesenchymal progenitors exhibiting large size colony (CFU-F) and higher self-renewal of colonogenic cells 3-5 days after 5-FU treatment, which was concomitantly associated with regeneration of CD34+Lin-Sca-1+c-kit+ (LSK) cells in the same BM. The cellular changes in mesenchymal stroma was associated with rapid emergence of characteristic mesenchymal cell populations (PDGFR-a+/Leptin receptor+/SSEA-3+: PLS) with 650-folds increase of the PLS cells in BM in 3 days after 5-FU treatment. However, the increase of these PLS mesenchymal cells were not associated with increase in mitotic activity of mesenchymal cells (<5% BrdU+ cells), indicating phenotypic conversion of subpopulation in BM. Moreover, cellular changes in mesenchymal niche were associated with rapid increase of mesenchymal cells expressing cross-talk molecules such as CXCL-12 (20-folds), Jagged-1 (13-folds) and DLL-1 (15-folds). Furthermore, in-vivo administration of chemicals blocking CXCL-12 and notch signaling during the recovery from the 5-FU treatment led to the significant loss of LSK-SLAM cells in the regenerated BM. Interestingly, the BM niche activated by 5-FU exerted a distinct effect on normal and leukemic cells in a manner that it provide higher support on the primitive state of normal HSCs than for MN-1 induced leukemia cells. Thus, leukemic mice engrafted with MN-1 cells exhibited a decrease in primitive leukemic cell (Lin-c-kit+) and higher survival by 5-FU treatment than those treated by radiation. Taken together, our study reveals the cellular reconstruction of mesenchymal niche in BM during stimulus-induced niche activation and provides an insight on the selective niche targeting as a novel therapeutic strategies for hematological diseases.

Disclosures

No relevant conflicts of interest to declare.
A Human Bone Marrow Failure Syndrome Caused By a Homozygous Mutation in MYSM1
Authors: Bahrami Ehsan , Racek Tomas , Witzel Maximilian , Puchalka Jacek , Greif-Kohistani Naschla , Kotlarz Daniel , Orth Michael , Lauber Kirsten , Walz Christoph , Herz Waleed Al , Horny Hans-Peter , Schmidt Heinrich , Albert Michael H. , Klein Christoph
Published: October 2019

Inherited bone marrow failure syndromes comprise a heterogeneous group of genetic disorders characterized by dysfunction of hematopoietic stem or progenitor cells. We present a consanguineous pedigree with two siblings with early-onset, progressive bone marrow failure (neutropenia, anemia, thrombocytopenia) associated with trigonocephaply, hyperplastic gingiva, cataract, accessory mammilla, dental changes and mild neurocognitive developmental delay. Pancytopenia at birth as well as non-compaction cardiomyopathy was observed in one of the patients.

Bone marrow histology showed marked hypocellularity with decrease of all hematopoietic cell lines but without cellular atypia, monocytosis, blast excess or fibrosis. Fanconi anemia was excluded by mitomycin C induced chromosomal breakage studies.

Immunophenotyping of peripheral blood cells showed a reduction of mature B cells, reduced switched memory B cells, whereas the level of activated B-cells and plasma blasts were increased. T cell proliferation upon PHA and specific antigens was normal.

Using whole exome sequencing in patients and both parents we could identify a single potentially disease causing homozygous stop codon mutation (NM_001085487: c.1168G>T: p.E390*) in MYSM1 (Myb-Like, SWIRM And MPN Domain-Containing Protein 1). A family with two affected patients carrying the same mutation has been reported previously (Alsultan A et al, Blood 122:3844, 2013). MYSM1 is a Histone H2A deubiquitinase that has previously been implicated in controlling hematopoietic stem cells, progenitor B cells, and NK cells in mice (Nijnik A et al, Blood 119:1370, 2012).

MYSM1 protein was absent in patients' EBV-transformed B cell lines (EBV-LCLs). MYSM1-deficient EBV-LCLs had elevated γ-H2AX levels, indicative of increased genomic instability. Upon exposure to UV light, we observed a sustained expression of p53 and phospho-p53 as well as p38 MAPK and phospho-p38 MAPK in patients' fibroblasts in comparison to fibroblasts from healthy individuals.

In view of the progressive pancytopenia, both siblings underwent allogeneic hematopoietic stem cell transplantation from 10/10 HLA-matched family donors after reduced intensity conditioning with alemtuzumab, fludarabine and treosulfan at 2 and 4 years of age, respectively. The procedure was well tolerated, and both siblings exhibit full donor hematopoietic reconstitution at 21 and 26 months after transplant.

In summary, we here identify a novel human bone marrow failure syndrome caused by MYSM1-deficiency that can be cured by allogeneic hematopoietic stem cell transplantation.

Disclosures

No relevant conflicts of interest to declare.
Allogeneic Hematopoietic Stem Cell Transplant for GATA2 Deficiency
Authors: Hickstein Dennis D. , Shah Nirali N , Freeman Alexandra F , Zerbe Christa , Holland Steven M. , Parta Mark
Published: October 2019

Background: Mutations in the zinc finger transcription factor GATA2 are responsible for: MonoMAC, monocytopenia with nontuberculous mycobacterial (NTM) infections; DCML, dendritic cell, monocyte and lymphoid cell deficiency; Emberger's syndrome with lymphedema and monosomy 7; and familial myelodysplastic syndrome (MDS)/acute myelogenous leukemia (AML). Allogeneic hematopoietic stem cell transplant (HSCT) represents the only definitive therapy for GATA2 deficiency.

Methods: Eleven patients with GATA2 deficiency received a myeloablative-conditioning regimen (2 matched related donors or MRD, 4 matched unrelated donors or URD, and 5 haploidentical related donors. MRD and URD received busulfan 3.2 mg/kg/day and fludarabine 40 mg/m2/day on days -6, -5, -4, and -3. Haploidentical related donors received cyclophosphamide 14.5 mg/kg on day's -6 and -5, fludarabine 30 mg/m2/day on day's -6 to -2, busulfan 3.2 mg/kg/day on day's -4 and -3, and 200 cGy TBI on day -1. MRD and URD recipients received tacrolimus and short course methotrexate post-transplant, while haploidentical related donor recipients received cyclophosphamide 50 mg/kg/day on days + 3 and +4 followed by tacrolimus and mycophenolate mofetil as post-transplant immunosuppression for graft-versus-host disease.

Results: Ten of the 11 (91%) of patients are alive and disease-free at a mean follow-up of 12 months (range 1 mo to 24 mo). One URD recipient died from persistent acute myelogenous leukemia. Four patients developed graft-versus-host disease, one case Grade 4. All 10 patients who survived had complete reconstitution of the monocyte, NK, and B-lymphocyte compartments, the three cell compartments that were severely deficient pre-transplant. All 10 patients had reversal of the infection susceptibility phenotype. In particular, there were no recurrences of NTM infections. Importantly, all 10 patients had correction of the cytogenetic abnormalities present pre-transplant (5 patients with trisomy 8 and 1 patient with monosomy 7).

Conclusions: Myeloablative HSCT in GATA2 deficiency results in uniform engraftment and reversal of the hematologic, cytogenetic, and clinical manifestations of GATA2 deficiency. There was a low regimen-related toxicity, even in this cohort of patients with considerable co-morbidities. We anticipate that with HSCT earlier in the clinical course, before significant organ damage or clonal evolution of MDS to AML or CMML occurs, the outcome of allogeneic HSCT in patients with GATA2 deficiency will continue to improve. Haploidentical related donor transplant appears to be particularly well suited for this disease, especially when the disease presents as a hypocellular myelodysplastic syndrome.

Disclosures

No relevant conflicts of interest to declare.
Microrna-9 Promotes Proliferation of Leukemia Cells in Adult CD34 Positive Acute Myeloid Leukemia with Normal Karyotype By Down-Regulation of Hes1
Authors: Tian Chen , Zheng Guoguang , You M. James , Zhang Yizhuo
Published: October 2019

Acute myeloid leukemia (AML) is a group of heterogeneous hematopoietic malignancies sustained by a small population of leukemic stem cells (LSCs) that can resist treatment and act as barriers to cure. Previously, we observed that Hes1 and p21 expression was down-regulated in AML cell lines compared to that of normal bone marrow mononuclear cells. However, the activation status of Hes1-p21 pathway and its regulation in LSCs as well as normal hematopoietic stem cells (HSCs) in AML has not been elucidated. In this study, the Hes1-p21 pathway in LSCs and leukemic progenitors (LPs) was studied in adult CD34+ AML with normal karyotype and no genetic mutations and the upstream miRNA regulators were screened. Our results showed that the level of either Hes1 or p21 was lower in LSCs or LPs than that of HSCs whereas the level of miR-9 was higher in LSCs or LPs than HSCs. An inverse correlation was observed in the expression of Hes1 and miR-9. Furthermore, we validated miR-9 as one of the regulators of Hes1 by reporter gene analysis. Knockdown of miR-9 by lentivirus infection suppressed the proliferation of AML cells by the induction of G0 arrest and apoptosis in vitro. Moreover, knockdown of miR-9 resulted in decreased circulating leukemic cell counts in peripheral blood and bone marrow, attenuated splenomegaly, and prolonged survival in a xenotransplant mouse model. Our results indicate that the miR-9-Hes1-p21 pathway plays an important role in supporting AML cell growth and survival, and that miR-9 has a potential to be a therapeutic target for suppressing AML.

Disclosures

No relevant conflicts of interest to declare.
ROS-Mediated Jab1-Thioredoxin Interaction in Acute Monocytic Leukemia
Authors: Zhou Fuling
Published: October 2019

SUMMARY

Increased production of reactive oxygen species (ROS) or an inefficient antioxidant system leads to oxidative stress, which influences hematopoietic cell function in acute myeloid leukemia (AML); however, the mechanisms of this stress remain poorly understood. Investigations were carried out on matched samples from the same 58 patients with AML-M5 (33 males and 25 females) who experienced a relapse after treatment. They had a median age of 43.90 ± 17.39 years (range, 14-77 years) at diagnosis and 44.86 ± 16.28 years at relapse. The mean white blood cell (WBC) counts were 16.58 x 109/L at diagnosis and 24.41 x 109/L at relapse (P < 0.05). Here, our studies showed that abnormally high ROS levels and low antioxidant enzyme capacity were characteristic of AML-M5 at both diagnosis and relapse. It is worth mentioning Jab1 and Trx protein levels were higher in all leukemia cell lines tested (THP-1, U937, and MOLM-13 cells), as well as in leukemic cells derived from AML-M5 patients, than in CD34+ cells from healthy donors (Figure 1A-D). A chi-square test revealed that patients with high Jab1 levels were less sensitive to Ara-c-based chemotherapy (Figure 1E). Consistently, high gene expression levels of thioredoxin (Trx) and Jab1 were associated with poor survival rates in AML-M5 patients(Figure 1F-H). Moreover, stimulating AML-M5 cells with a low concentration of hydrogen peroxide increased Jab1 and Trx expression. Additionally, depletion of Jab1 inhibited cellular proliferation and invasion, which was accompanied by a decrease in Trx expression.

Mechanistically, we demonstrate that Jab1 controls a major antioxidative cellular molecule Trx by interacting with and enhancing its transcriptional levels through AP-1 binding sites. To assess whether Jab1 and Trx interact we performed co-immunoprecipitation assays and demonstrated that Jab1 and Trx proteins interacted specifically with each other using endogenous proteins or ectopically expressed tagged-proteins, in U937 and THP-1 cells (Figure 2A and B). Furthermore, our results demonstrate that Jab1 knockdown remarkably reduced Trx levels (Figure 2C). Inversely, Jab1 overexpression induced higher endogenous Trx levels (Figure 2D). However, neither Trx silencing nor Trx overexpression affected Jab1 levels. Thus, our findings indicate that Jab1 is an upstream regulator and positively controls Trx expression in AML-M5 cells. To further delineate whether Jab1 regulates transcriptionally Trx, we performed a luciferase reporter assays with the human Trx promoter. Our data demonstrated that transactivation of Trx expression is driven by Jab1 through the AP-1 binding site (Figure 2E-H). In conclusion, these results elucidate a novel Jab1 and Trx axis, which is a critical cellular process in the pathobiology of AML-M5 and could promotes AML-M5 relapse.

Disclosures

No relevant conflicts of interest to declare.
Wild-Type Kras Inhibits NrasQ61R/+-Induced Leukemias
Authors: Wen Zhi , Zhang Jing
Published: October 2019

Ras proteins belong to the super family of small GTPases. Although RAS mutations are oncogenic, it remains highly controversial whether wild-type (WT) RAS facilitates or inhibits tumorigenesis in the presence of oncogenic RAS. Previous studies suggest that this might depend on cell type, specific RAS isoforms, and/or stage of tumorigenesis (e.g. tumor initiation versus tumor progression). Recently, we found that deletion of WT Kras in oncogenic Kras mice promotes myeloidproliferative neoplasm (MPN) in a cell-autonomous manner. This is through promoting activation of all Ras isoforms and thus enhancing cytokine signaling in vivo. However, it remains unknown whether WT Kras plays a similar role in oncogenic Nras-induced leukemogenesis.

To address this issue, we conditionally down-regulated WT Kras expression in a novel leukemia model induced by endogenous NrasQ61R/+. Detailed characterization of NrasQ61R allele reveals that NrasQ61R/+ displays an intermediate leukemogenic activity between NrasG12D/+ and NrasG12D/G12D. NrasQ61R/+ mice developed MPN (100%) and acute T-cell lymphoblastic leukemia/lymphoma (T-ALL) (~10%) at the moribund stage with a median survival of 197 days. Therefore, NrasQ61R/+ mice provide an excellent experimental system to test genetic lesions promoting or inhibiting oncogenic Nras-induced leukemogenesis. We observed that 3-weeks after activation of NrasQ61R/+ and deletion of WT Kras, the double mutant mice displayed more severe MPN phenotypes than NrasQ61R/+ mice, as demonstrated by more prominent hepatosplenomegaly and further expanded myeloid compartment in bone marrow and spleen. Consistent with these MPN phenotypes, NrasQ61R/+; Kras-/- myeloid progenitor and precursor cells showed prolonged and hyperactivated GM-CSF signaling compared to NrasQ61R/+ cells. In addition, NrasQ61R/+; Kras-/- mice displayed further expanded common lymphoid progenitor compartment and a less differentiated phenotype in thymic T-cells than NrasQ61R/+ mice. In NrasQ61R/+; Kras-/- mice, early thymic T cell progenitors demonstrated enhanced cell proliferation and stronger ERK1/2 signaling compared to NrasQ61R/+ cells. Consequently, NrasQ61R/+; Kras-/- mice survived significantly shorter than NrasQ61R/+ mice (median survival: 97 days; P < 0.001) and 100% of them developed both MPN and T-ALL at the moribund stage. Preliminary results from bone marrow transplantation assay indicate that loss of WT Kras expression promoted NrasQ61R/+-induced MPN and T-ALL in a cell-autonomous manner. Taken together, our results suggest that loss of WT Kras promotes leukemogenesis in NrasQ61R/+ mice. We are currently studying on the underlying mechanisms.

Disclosures

No relevant conflicts of interest to declare.
Towards a Targeted Therapy:Phosphoproteomics Reveals Signaling Pathways That Are Normalized in AML Cells Following Treatment with Anti-CD44 Antibodies
Authors: Jalal El-Din Heba M. , Merzaban Jasmeen S.
Published: October 2019

Acute myeloid leukemia (AML) is a clonal malignant disease characterized by a blockage in the differentiation of myeloid cells resulting in the accumulation of highly proliferating immature blast cells. With the success of All Trans Retinoic acid (ATRA) in acute promyelocytic leukemia (AML3), differentiation therapy has become a very attractive treatment option. Ligation of CD44 (a cell surface antigen) with anti-CD44 monoclonal antibodies (mAbs) is reported to reverse the blockage of differentiation and suppress the proliferation of blasts derived from most AML subtypes. However, the molecular mechanisms underlying this apparent 'normalization' (reversal) of AML cells induced by CD44 have not been fully elucidated. To expand our understanding of the cellular regulation and circuitry involved, we aimed to apply a quantitative phosphoproteomic approach using Stable Isotope Labeling with Amino acids in Cell culture (SILAC) to monitor dynamic changes of phosphorylation states in HL60 cells following treatment with CD44 mAbs. Phosphoproteomic analysis identified differentially phosphorylated proteins among CD44 mAb treated and control HL60 cells that are involved in a number of major signaling pathways as determined by the Ingenuity Pathway analysis (IPA) platform. Among others, Rho signaling emerged as a major pathway significantly changed by CD44 mAb treatment. Rho GTPases are well-recognized regulators of the actin cytoskeleton but have also been implicated in diverse cellular events such as cell polarity, microtubule dynamics, membrane trafficking, transcriptional regulation, cell growth control and development. An interesting Rho family member, PAK-2 was identified in our search. PAK-2 is a ubiquitously expressed serine/threonine protein kinase, which is a direct target for small GTPases and has been identified as a switch between cell survival and cell death signaling depending on its mode of activation. Western-blot analysis of cell lysates of CD44 mAb treated and control HL60 cells confirmed that the phosphorylation of Pak-2 was altered as early as 5 minutes following treatment. Further validation and characterization of the activation mode, phosphorylation dynamics and protein-protein interactions of PAK-2 are essential in understanding its role in AML.

Disclosures

No relevant conflicts of interest to declare.
Effect of Melphalan 140 Mg/m2 Versus 200 Mg/m2 on Toxicities and Outcomes in Multiple Myeloma Patients Undergoing Single Autologous Stem Cell Transplantation
Authors: Katragadda Lakshmikanth , McCullough Lindsay , Dai Yunfeng , Hsu Jack W , Hiemenz John W , May W. Stratford , Cogle Christopher R. , Norkin Maxim , Brown Randy A , Wingard John R. , Chang Myron , Moreb Jan S
Published: October 2019

Introduction: Although melphalan at a dose of 140 mg/m2 (MEL-140) is known to be an effective preparative regimen for autologous hematopoietic stem cell transplantation (ASCT) in multiple myeloma (MM) patients, there are very few studies comparing it to the most commonly used dose of 200 mg/m2 ( MEL-200).

Methods: We retrospectively reviewed the records of all myeloma patients who underwent an ASCT between 2001 and 2010 at our institution. We then identified patients who received melphalan as their preparative regimen at doses of 140 mg/m2 or 200 mg/m2. Patients who received any other drug as conditioning regimen or had more than one ASCT or had documented amyloidosis were excluded. Data were collected for variables known to possibly affect prognosis of MM patients. We assessed effect of melphalan dose on toxicities and outcomes.

Results: A total of 129 eligible patients were identified, with 33 receiving MEL-140 and 96 receiving MEL-200. As was expected significantly higher percentage of patients in the MEL-140 arm were older than 65 years (P=<0.001) or had cardiac ejection fraction < 50 (P=0.0001) or had Karnofsky score < 80 (P=0.01) or had creatinine > 2 either at diagnosis (P=0.004) or the time of ASCT (P=0.001). Rest of the patient and disease characteristics including Durie-Salmon stage, myeloma subtype and disease status at ASCT were not significantly different between the 2 arms. Patients in MEL-140 needed significantly longer time to ANC engraftment (P=0.037) and also had significantly higher frequency of neutropenic fever (P=0.003). There were no significant differences in mucositis (including grade), nausea, vomiting, diarrhea, bacteremia, or length of hospital stay and frequency of repeat hospitalizations among both groups. There was no treatment related mortality in either group. At a median follow up of 74 months (range, 52-140) from ASCT, there were no significant differences in relapse free survival (RFS) (P=0.4988) and overall survival (OS) (P=0.6936) between the two groups. Five year OS for MEL-140 and MEL-200 is 71.6% and 78.9%, while RFS is 23.9% and 34%, respectively. Proportion of patients whose myeloma status improved to ≥ VGPR at 3 months post ASCT was also not different (P=0.385). Importantly, similar proportions of patients received various post ASCT maintenance therapy (P=0.605). In multivariate cox proportional hazards model only disease status of ≥VGPR at the time of ASCT significantly affected RFS (P=0.024) but did not impact OS (P=0.104).

Conclusion: MM patients who received MEL-140 had similar long term outcomes as those who received MEL-200 despite their older age, lower performance status and renal insufficiency.

Disclosures

No relevant conflicts of interest to declare.
A Comparison of Lamivudine and Entecavir for the Prophylaxis of Hepatitis B Virus Reactivation in Recipients of Allogeneic Hematopoietic Stem Cell Transplantation: A Single Institutional Experience
Authors: Shang Jin , Sun Jing , Fan Zhiping , Huang Fen , Zhang Yu , Jiang Qianli , Zhou Hongsheng , Liu Qifa
Published: October 2019

Backgroud:

To compare the efficacy of lamivudine and entecavir in the prevention of hepatitis B virus (HBV) reactivation in HBsAg-positive patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT).

Method:

A total of 216 consecutive HBV surface antigen (HBsAg) positive patients undergoing allo-HSCT at our institute between March 2008 and March 2014 were enrolled in this retrospective investigation.

Results:

Of the 216 patients, 119 received lamivudine and 97 received entecavir to prevent HBV reactivation. The cumulative rate of HBV reactivation at 24 th, 48 th, and 96 th post-transplantation were 7.0% and 0.0%, 15.0% and 1.0%, and 24.0% and 2.0% in lamivudine and entecavir groups, respectively. The cumulative rate of severe hepatitis attributable to HBV reactivation were 5.9% and 0.0% (p = 0.01) in lamivudine and entecavir groups at week 96 post-transplantation, respectively. On the 12th, 24th, and 48th weeks of anti-viral therapythe average serum HBV-DNA levels reduction in entecavir group were greater than in the lamivudine Drug-resistant mutation sites were detected in 25 (21.0%) patients in the lamivudine group and in one patient (1.0%) in the entecavir group.

Conclusion:

Our data indicates that entecavir might reduce the incidence of HBV reactivation and severe HBV reactivation hepatitis in HBsAg-positive allo-HSCT recipients compared to lamivudine.

Disclosures

No relevant conflicts of interest to declare.
Design and Development of a Novel Orally-Active, PI3K-p110Beta/Delta Inhibitor with Combined Tumor Immunotherapeutic, Growth Inhibition and Anti-Metastatic Activity
Authors: Shuttleworth Stephen Joseph
Published: October 2019

The phosphatidylinositide 3-kinases (PI3Ks) have emerged as important therapeutic targets for the treatment of hematological malignancies and solid tumors. In hematological cancer therapy, the majority of clinical activity has centered on the inhibition of the class IA isoform p110δ and the class IB enzyme p110γ, whereas for solid tumor treatment the landscape has been dominated by inhibitors of all four class I PI3Ks (p110α, β, δ and γ) or dual class I/mTOR inhibitors of limited selectivity. Our research has focused on the emerging role of p110β and p110δ in both cancer types, in which overexpression, mutation and PTEN-defectiveness have been shown to be key features in disease pathogenesis and progression. We have designed and developed a uniquely selective series of small molecules that inhibit the p110β/δ isoforms, and which display extremely high specificity in the human kinome. The design of such molecules has enabled us to interrogate the function of p110δ in tumor immunotherapy and p110β in primary tumor growth and metastasis; our lead compounds show promising oral activity in syngeneic models, in which they inhibit tumor growth and metastatic spread whilst also impacting on downstream biomarker responses. Additionally, through our ability to tune selectivity over p110α and p110γ, elimination of the mechanism-related toxicities associated with those isoforms can be realized, making selective dual p110β/δ inhibition potentially more conducive to combination therapy than for pan-PI3K inhibitors. Described herein is data for our clinical development candidate, KA2237, which is an orally-active, potent and selective p110β/δ inhibitor. This molecule displays potent in vitro growth inhibition and PD responses in a spectrum of hematological tumor lines, including in MCL and PTEN-null GCB-DLBCL, and has potential to treat BTK inhibitor-resistant malignancies of the hematopoietic system. KA2237 will enter Phase I studies in hematological cancer at the end of 2015.

Disclosures

No relevant conflicts of interest to declare.