bluuebunny/crossref_metadata_2025 huggingface dataset:
Therapy Outcome in Childhood ALL: Experience from a Tertiary Care Centre in North India.
Authors: Marwaha Ram Kumar , Kulkarni Ketan , Trehan Amita , Bansal Deepak
Published: October 2019

Survival in childhood ALL, in resource challenged nations, has not kept pace with cure rates of over 80% in developed countries. This descriptive study was designed to assess survival data and identify risk factors for adverse outcome. Data obtained from case records of 762 patients with ALL was analyzed. Information regarding the clinical-demographic profile, therapy and course of illness were recorded. Status and duration at last follow-up were utilized to generate Kaplan-Meier survival curves. The mean age at presentation was 5.7±0.23 years (M:F::3.2:1). Caretakers of 230 (30.2%) patients opted for no therapy. There were 68 and 60 deaths in induction and remission phases respectively. Besides these, 111 children either defaulted therapy (23) or were lost to follow up (88). Relapsed disease was documented in 125 cases. The 5 year overall and event-free survival was 46 and 34% respectively. The p values for survival analysis using the Log-rank test for groups formed according to the gender, age, symptom-diagnosis interval, TLC and platelet count were 0.19, 0.02, 0.08, <0.001, 0.001 respectively. Bulk disease (p=0.047), mediastinal adenopathy (p=0.045), TLC (p=0.016), platelet count (p=0.031) and administration of 2 intensification blocks (p=0.012) were found to be significant predictors of outcome by univariate and multivariate analysis. The holistic management of ALL in children requires financial resources, a strong psychosocial structure and access to quality supportive care. Almost one third of our patients opted for no therapy. The other problem areas identified were a high proportion of therapy defaulters, lost to follow up and infection related deaths during induction and remission phases. TLC >50000/microliter and platelet count <30000/microliter had an adverse impact on survival. The administration of both early and delayed intensification improved survival. The introduction of remedial measures for tapping the difficulties identified, would undoubtedly improve cure rates in children with ALL in developing nations.
Pediatric Patients with Extramedullary Leukemia Involving the Central Nervous System Have a Superior Outcome.
Authors: Johnston Donna , Alonzo Todd , Gerbing Robert , Lange Beverly , Woods William G.
Published: October 2019

Purpose: Extramedullary leukemia (EML), formally known as chloroma, is discrete collections of leukemia cells outside of the bone marrow. EML is often seen within the central nervous system (CNS) and these are often treated in a similar fashion as leukemia cells within the cerebral spinal fluid (CSF). We previously demonstrated that the presence of leukemia cells within the CSF (CNS leukemia) does not affect overall survival. We sought to determine the outcome of patients with central nervous system EML and compare these patients with those with CNS disease and non CNS EML.

Methods: Patients enrolled on Children’s Cancer Group protocols 2861, 2891, 2941 and 2961 being treated for de novo acute myeloid leukemia (AML) with intensive timing chemotherapy were classified for the presence of CNS disease as CNS1 (<5 wbc in the CSF without blasts), CNS2 (<5 wbc in the CSF with blasts), or CNS3 (≥ 5 wbc in the CSF with blasts), as well as EML in the CNS (eg orbit, brain, etc) or non-CNS EML (eg skin, lung, etc). These patient’s outcomes were then analyzed.

Results: A total of 1459 patients treated with intensive timing chemotherapy were analyzed in this study. At diagnosis, 1113 (76%) were CNS1, 143 (13%) CNS2, 154 (11%) CNS3, 48 (3%) had CNS EML, 57 (4%) had a non-CNS EML, and only 6 patients (0.4%) with CNS EML had CNS3 status. Patients with CNS EML had a significantly higher overall survival from study entry compared to patients with non-CNS EML (83% vs 38%, p<0.001), and compared to CNS3 patients (83% vs 50%, p<0.001). The patients with CNS EML also had a significantly higher event free survival compared to patients with non-CNS EML (65% vs 34%, p<0.001), and compared to CNS3 patients (65% vs 34%, p<0.001). There was no significant difference in relapse risk, bone marrow relapse, isolated CNS relapse, or EML relapse comparing patients with CNS EML and non-CNS EML. CNS EML patients had a significantly lower relapse risk compared to CNS3 patients (29% vs 49%, p=0.025). There was not a significant difference in bone marrow relapse, isolated CNS relapse, or EML relapse comparing these 2 groups of patients.

Conclusion: Patients with extramedullary leukemia involving the CNS had a significantly better survival than patients with non-CNS EML or patients with CNS leukemia at diagnosis. This should reassure clinicians caring for these often challenging patients.
A Protease-Resistant PML-RARα Has Increased Leukemogenic Potential in a Murine Model of Acute Promyelocytic Leukemia (APL).
Authors: Uy Geoffrey L. , Welch John S. , Lane Andrew A. , Ley Timothy J.
Published: October 2019

Previous work from our group has demonstrated that the azurophil granule protease, neutrophil elastase (NE) can cleave PML-RARα (PR), the fusion protein that initiates acute promyelocytic leukemia; we identified valines 420 and 432 within the PML domain as the initial sites of NE cleavage. Furthermore, neutrophil elastase deficiency reduces the penetrance of leukemia in a knock-in model of APL in which the human PR cDNA is knocked into the 5′ untranslated region of the murine cathepsin G gene (Lane and Ley, Cell 2003) In this study, we demonstrate that mutation of residues V420 and V432 to arginine (2VR) confers resistance to proteolytic cleavage by NE. V420 and V432 are also cleaved using marrow extracts derived from NE deficient mice, indicating that other marrow-derived proteases can cleave PR at these two valines; the 2VR mutation confers resistance to these proteases as well. To determine the role that cleavage of PR plays in leukemogenesis, we generated protease-resistant PR mice by inserting the PR(2VR) allele into the murine cathepsin G locus via homologous recombination. Analysis of marrow from 6–8 week old preleukemic mice indicates that myeloid progenitors from PR(WT) and PR(2VR) mice both have increased replating efficiency in methocellulose cultures with a trend towards increased efficiency with PR(2VR)mice (WT=0%, PR(WT)=0.45%, PR(2VR)=1.65% at 4th replating, p=NS). Both PR(WT) and PR(2VR) mice develop a phenotypically identical, fatal myeloid leukemia characterized by splenic infiltration with blasts that co-express CD117 (c-kit) and Gr-1, and that are morphologically promyelocytes. A tumor watch revealed that PR(2VR) mice (n=89) demonstrated a shortened latency of leukemia development (274 days) compared to PR(WT) mice (n=41, 473 days, p=0.0003). These results indicate that resistance to proteolysis confers an increased leukemogenic potential to the PR fusion protein, which may be due to increased levels of full length PR in the myeloid progenitors of these mice. Based on these observations, we suggest NE-mediated cleavage of PR is not required for its activity. Therefore, the mechanism by which NE deficiency affects APL penetrance may be indirect.
Synergistic Cytarabine:Daunorubicin Ratios Delivered by CPX-351 to Human Leukemia Xenografts Is Associated with Liposome-Mediated Bone Marrow Drug Accumulation, Intracellular Delivery of Encapsulated Agents to Leukemia Cells, and Increased Efficacy.
Authors: Mayer Lawrence , Lim Wahseng , Santos Nancy Dos , Xie Sherwin , Hopkins Andrea , Huang Ruby , Wilkins Lindsey , Kelly Sarah , Bermudes David , Harasym Troy , Tardi Paul
Published: October 2019

CPX-351, a liposomal formulation of cytarabine:daunorubicin co-encapsulated at a synergistic 5:1 molar drug ratio, has been previously shown to be highly active in various preclinical leukemia models. In the present investigations, we utilized a bone marrow engrafted human lymphocytic leukemia xenograft model in order to elucidate the pharmacodynamic basis for its superior efficacy compared to un-encapsulated cytarabine:daunorubicin combinations. In normal bone marrow, the un-encapsulated cytarabine:daunorubicin saline-based cocktail and CPX-351 provided comparable cell suppression after intravenous treatment at maximum tolerated dose. In contrast, when these formulations were administered to tumor bearing mice, CPX-351 completely ablated leukemia cells from the bone marrow for multiple weeks whereas the saline-based drug cocktail induced only transient leukemia suppression. Examination of drug levels in the bone marrow compartment revealed that cytarabine:daunorubicin concentrations were significantly elevated for CPX-351. More striking was the observation that whereas bone marrow drug concentrations over time appeared comparable after each dose of the saline-based drug cocktail, the first dose of CPX-351 promoted elevated bone marrow drug accumulation for subsequent doses. Under these conditions, the cytarabine:daunorubicin ratio in bone marrow was maintained near the administered 5:1 molar ratio for CPX- 351 while the drug ratio changed more than 100-fold within 4 hours after administration of the saline-based drug cocktail. Confocal fluorescence microscopy of leukemia cells exposed to CPX-351 in vitro revealed that CPX-351 liposomes were taken up into cytoplasmic vacuoles and subsequently released their drug contents intracellularly. Taken together, these results indicate that the improved efficacy observed following CPX-351 administration in vivo is related to liposome-mediated prolonged and direct exposure of leukemia cells to synergistic drug ratios.
Flavopiridol Down-Regulates Genes Involved in Cell Cycle Regulation and Tumor Progression in Adults with Refractory or Poor-Risk Acute Leukemia.
Authors: Resar Linda , Hillion Joelle , Alino Katrina , Rudek Michelle , Karp Judith
Published: October 2019

Acute leukemia in adults continues to be a formidable clinical challenge that demands further investigation to identify more rational therapies. To optimize anti-leukemia therapy, we are investigating the prototypical cyclin dependent kinase (cdk) inhibitor, flavopiridol, in refractory or poor-risk disease. Flavopiridol is a cytotoxic molecule that is thought to induce cell cycle arrest by blocking cyclin-dependent kinase (cdk) function, thereby interfering with RNA Polymerase II activity and globally down-regulating gene expression. In the setting of pan-cdk inhibition, E2F1 is released and appears to drive apoptosis in transformed cells. Consistent with these proposed mechanisms of action, a previous study from our group showed that flavopiridol induces apoptosis in vitro in leukemic blasts from patients with refractory leukemia. Administration of flavopiridol was associated with a decrease in one or more of the following proteins in the leukemic blasts: RNA Polymerase II, STAT3, cyclin D1, Bcl-2, and Mcl-1. Serum VEGF levels also decreased in most patients. We are now investigating mRNA levels of the genes encoding these proteins by quantitative, RT-PCR in leukemic blasts from adult patients with refractory or poor-risk leukemia before and after flavopiridol therapy. We have treated 26 patients with flavopiridol at an escalating, hybrid dose followed by ara-c and mitxantrone. Adequate RNA from leukemic blasts before and after flavopiridol administration was available from 8 of 11 patients studied thus far. All cases (8/8) exhibit a marked decrease in mRNA for VEGF following flavopiridol. mRNA levels for other putative flavopiridol target genes is also decreased in a subset of leukemic blast samples after therapy, as follows: E2F1 (6/8), STAT3 (6/8), Mcl-1 (6/8), RNA Polymerase subunit 2a (3/3), and cyclin D1 (2/3). In contrast, bcl-2 mRNA levels increased after flavopiridol in most cases (7/8), which could represent a compensatory mechanism of leukemic blasts to avoid apoptotic cell death. Our preliminary studies indicate that flavopiridol is cytotoxic in poor-risk and refractory acute leukemia. Studies are underway to determine if down-regulation of any putative target genes correlates with pharmacologic data or clinical responses.
CYP2C19*2 and *3 Polymorphisms Are Associated with High Post- Treatment Platelet Reactivity in Korean Patients with Acute Coronary Syndrome Undergoing Percutanous Coronary Intervention.
Authors: Kim In-Suk , Jeong Young-Hoon , Lee Gyeong-Won
Published: October 2019

Background: Cytochrome P450 (CYP) 2C19 is an enzyme showing genetic polymorphism that may cause marked interindividual and interethnic variation in the metabolism and disposition of its substrates. CYP2C19*2 and CYP2C19*3 are the most common of CYP2C19 polymorphisms, and show phenotypic poor metabolism. Recent data have shown that the CYP2C19*2 loss-of-function allele is associated with a marked decrease in platelet response to clopidogrel in healthy controls and Caucasian patients with acute coronary syndrome. However, It is unknown whether CYP2C19 *3, which is frequently noted in Asian, is also associated with platelet response to clopidogrel. Therefore, this study was conducted to analyze the effect of CYP2C19 *2 and *3 polymorphisms on high post-treatment platelet reactivity (HPPR) on clopidogrel in Korean patients with acute coronary syndrome, as a representative of Asian populations.

Methods: The study included 136 consecutive patients undergoing percutanous coronary intervention (PCI). Adenosine diphosphate (ADP)-induced platelet aggregation by light transmittance aggregometry (LTA) and the VerifyNow P2Y12 assay (Ultegra rapid platelet function assay; Accumetrics Inc., USA) were assessed after a loading dose and after the maintenance dose of clopidogrel before discharge. CYP2C19 genotype was analyzed by Snapshot method.

Results: The genotypic distributions of CYP2C19*1/*1, *1/*2, *1/*3, *2/*2, and *2/*3 were 57 (41.9%), 47 (34.6%), 12 (8.8%), 14 (10.3%), 6 (4.4%), and 6(4.4%), respectively. The frequencies of CYP2C19 mutant alleles in Korean were higher than Caucasians. The CYP2C19*2 and CYP2C19*3 polymorphisms were significantly associated with persistent higher platelet aggregation by LTA, and lower inhibition of platelet reactivity by VerifyNow P2Y12 assay after clopidogrel than CYP2C19*1 genotype. The CYP2C19*2 and *3 alleles were more frequent in clopidogrel hyporesponsiveness, defined by persistent HPPR (5 uM ADP-induced platelet aggregation >50%; p = 0.01).

Conclusions: This study suggests that the CYPC19*2 and *3 alleles influence clopidogrel hyporesponsiveness after clopidogrel in Asian patients with acute coronary syndromes undergoing PCI. These findings can have a significant impact on the future design of pharmacognetic antiaggregant strategies for acute coronary syndrome on antiplatelet treatment.
Runx1 Interactions in Stem Cell Biology.
Authors: Speck Nancy A. , Chen Michael , Yokomizo Tomomasa , Zeigler Brandon , Dzierzak Elaine
Published: October 2019

The study of developmental hematopoiesis has provided important insights into the molecules that establish and sustain this process throughout adult life. At the base of the hematopoietic hierarchy is the hematopoietic stem cell (HSC), which emerges in the mouse conceptus starting at 10.5 days post coitus (≥ 34 somite pair stages).1 HSCs have been found in several distinct sites: the yolk sac, umbilical and vitelline arteries, the dorsal aorta in the aorta/gonad/mesonephros (AGM) region, fetal liver, and, more recently, the placenta.1–4 HSCs emerge from these sites (yolk sac, umbilical and vitelline arteries, and AGM region) through the formation of intra-aortic hematopoietic clusters that develop from endothelium.5–7 Studies in mouse, zebrafish, chick, and frog embryos established that Runx1 (AML1) is the earliest specific marker of all definitive hematopoietic sites in the conceptus. Runx1 is expressed in endothelial and mesenchymal cells and in intraaortic hematopoietic clusters, and marks all committed HPs and HSCs in both the embryo and the adult.6,8–10 It has been proposed that Runx1 functions during the transition from a “hemogenic endothelium” to intra-aortic clusters and HSCs.8 Here, we show that deletion of Runx1 in vascular endothelial cadherin (VEC) positive cells blocks the emergence of intra-aortic hematopoietic clusters, HPs, and HSCs. Greater than 95% of adult bone marrow cells are marked when VEC-Cre is used to delete a Rosa26 reporter allele, demonstrating that almost all blood cells have transited through a VEC+ intermediate at some point in their life. On the other hand, Runx1 deletion with Vav-Cre, which occurs in fetal liver HPs and HSCs, does not block hematopoiesis. Collectively, these data demonstrate that Runx1 is absolutely required in endothelial cells for hematopoietic cluster, HP, and HSC formation, but after HSCs are born from endothelium, Runx1 is no longer required to maintain them.
Age-Related Changes in Tissue-Specific Stem Cells.
Authors: Wagers Amy J. , Cerletti Massimiliano , Mayack Shane R. , Kim Francis S. , Shadrach Jennifer L.
Published: October 2019

Aging of multicellular organisms typically involves progressive decline in the body’s ability to maintain homeostatic cell replacement and to regenerate tissues and organs after injury. In both the blood and the skeletal muscle, aging significantly impairs regenerative activity and can dysregulate normal homeostatic production of mature cells. These age-acquired defects in tissue function profoundly impact the health of older individuals, as evidenced by the high incidence of age-related muscle deterioration (sarcopenia), bone marrow failure, immune dysfunction, and blood cancers in the elderly. How aging causes deterioration of tissue function is poorly understood, but several lines of evidence suggest that loss or functional impairment of tissue-specific stem cells directly contributes to age-dependent failures in tissue repair. Interestingly, the effects of aging on tissue stem cell function appear to arise at least in part from alterations in the aged tissue environment, which can inhibit stem cell activity in older animals and may be regulated by factors that circulate naturally in the bloodstream. By making use of sensitive in vivo and in vitro approaches, including direct cell isolation by FACS, we are investigating the extrinsic factors and interactions that control stem cell function in aged animals. Our current studies have pointed us toward a discrete set of metabolic regulators and inflammatory cytokines, which may alter the signals that stem cells receive from their environment in aged animals. The knowledge we gain from these ongoing studies will help to define novel strategies to delay or reverse the onset of age-related disease, extending the healthful life of aging individuals.
Challenges and Opportunities for Effective NOTCH1 Targeting in T-ALL.
Authors: Ferrando Adolfo A.
Published: October 2019

The NOTCH pathway is an evolutionarily conserved mechanism responsible for the direct transduction of developmental signals at the cell surface into changes in gene expression in the nucleus. In the hematopoietic system, the NOTCH1 signaling pathway is required for the commitment of multipotent hematopoietic progenitors to the T cell lineage and, later on, to support cell growth, proliferation, and survival at multiple stages of thymocyte development. These physiological functions are disrupted by activating mutations in the NOTCH1 gene in over 50% of T-cell acute lymphoblastic leukemias (T-ALL). NOTCH1 mutations result in ligand-independent activation or increased protein stability, leading to deregulated cell proliferation and survival in T cell progenitors and T cell transformation. Importantly, small molecule inhibitors of the γ-secretase complex (GSIs) effectively abrogate NOTCH1 signaling, making NOTCH1 a promising therapeutic target for the treatment of T-ALL. However, the clinical development of GSIs has been hampered by our incomplete understanding of the effector pathways controlled by NOTCH1, the lack of clinical responses to GSI therapy, and the development of gastrointestinal toxicity secondary to inhibition of NOTCH signaling in the gut. Recent progress in the identification of the transcriptional regulatory networks that control T cell transformation downstream of NOTCH1 has shown a close relationship between oncogenic NOTCH1 signaling and the transcriptional control of cell growth and metabolism. Thus, NOTCH1 directly controls multiple genes involved in anabolic pathways and further promotes cell growth via direct transcriptional upregulation of MYC and indirect upregulation of the PI3K-AKT signaling pathway via HES1-mediated downregulation of PTEN. Moreover, oncogenic NOTCH1 signaling promotes activation of the NFKB pathway and interacts with the function of p53. Although the specific mechanisms mediating some of these interactions have not been elucidated yet, these results suggest that combinations of GSIs with chemotherapy or drugs targeting the PI3K-AKT and NFKB pathways may have a synergistic effect in the treatment of T-ALL. Moreover, inhibitory antibodies capable of selectively blocking the activation of NOTCH1 and inhibitors of the nuclear transcriptional complex mediating the activation of NOTCH1 target genes offer alternative approaches for the inhibition of oncogenic NOTCH1 signaling. Each of these strategies offers the promise to induce potent antileukemic effects and/or to ameliorate the gastrointestinal toxicity associated with systemic inhibition of NOTCH signaling and may ultimately lead to the development of rationally-designed and highly effective anti-NOTCH1 therapies for T-ALL.
Notch and Leukemia: Lessons from Mouse Models.
Authors: Pear Warren S.
Published: October 2019

Mammalian Notch1 was first identified by Dr. Jeffrey Sklar (Yale University) and coworkers as a chromosomal translocation partner in a rare subset of human T-ALLs carrying a t(7;9) translocation. Notch moved from a bit player to center stage when Drs. Jon Aster (Brigham & Women’s Hospital), A. Thomas Look (Dana-Farber Cancer Institute), and colleagues identified Notch1 activating mutations in the majority of human T-ALLs. These mutations occur in the Notch extracellular domain in the heterodimerization domain, allowing for ligand-independent signaling (HD mutations), or in the C-terminal PEST domain (PEST mutations), which increases Notch protein stability. In retrospect, the frequent association of Notch mutations with human T-ALL may have been predicted given that Notch is a potent T cell oncogene in mice. Notch-induced T-ALL shares many features with human T-ALL, suggesting that oncogenic Notch signaling may have similar functions in T cell tumorigenesis in mice and people. Notch functions as a T cell oncogene in both retroviral and transgenic models, where it is introduced as the primary oncogenic event, as well as in models where it is likely acquired as a collaborating event. Notch1 PEST mutations are frequent in the latter. Acquired Notch mutations in both humans and mouse T-ALL have only been identified in Notch1, whereas ectopic expression of the intracellular domain of Notch1, 2, and 3 all give rise to T cell neoplasms in mice. These differences may be related to variation in signal strength between these different Notch family members. Although intracellular Notch1 (Notch1-IC) is a potent inducer of T-ALL in retroviral and transgenic mouse models, this form of Notch is rarely present in human T-ALL. We recently assayed more commonly mutated Notch1 alleles in the retroviral model and found that HD, PEST, and HD + PEST mutations were weak inducers of T-ALL. When assayed on a background that predisposes to T-ALL, such as oncogenic ras, these weakly leukemogenic alleles shortened disease latency and gave rise to cell lines that were Notch-dependent. These data suggest that the common Notch mutations found in human T-ALL are weak tumor initiators but still give rise to tumors that are “addicted” to Notch. This strengthens the rationale for inhibiting Notch in T-ALL. The mouse also appears to be a useful model for testing Notch-targeted therapeutics, as the murine tumors are frequently susceptible to Notch inhibition, and mice exhibit some of the same toxicities as people when exposed to continuous Notch inhibition. A challenge for the field is to identify a safe therapeutic index for Notch inhibition. As a transcriptional regulator, an area of active investigation is identifying oncogenic Notch1 transcriptional targets. Several targets have been identified in both mouse and human, such as c-myc and regulators of T cell identify. Ongoing studies will likely reveal additional transcriptional targets, which is important for understanding the function of Notch in the pathogenesis of T-ALL.
The JunB Transcriptional Network: from Hematopoietic Stem Cells to Myeloid Function.
Authors: Passegué Emmanuelle
Published: October 2019

Hematopoietic development is organized hierarchically, starting with a rare population of hematopoietic stem cells (HSCs) that gives rise to a series of committed myeloid progenitors and mature cells with particular functional and immunophenotypic properties. HSCs are operationally defined by their ability to provide long-term multilineage reconstitution when transplanted into hematopoietically compromised recipient, and are the only cells within the myeloid series that self-renew throughout life. Under steady-state conditions, HSCs are largely quiescent, slowly cycling cells. However, in response to environmental stresses, HSCs can undergo dramatic expansion and contraction to ensure proper homeostatic replacement of blood cells. HSCs are found predominantly in the bone marrow (BM) associated with several recently described vascular and endosteal niches. A complex balance of cell extrinsic factors present in these niches maintains HSCs in relatively dormant states and regulate their trafficking to and from these BM microenvironments. Recent studies have highlighted the roles of specific signal transducer (Pten), polycomb group protein (Bmi1), transcription factors (JunB, Gfi-1, Hox genes), and extrinsic regulatory pathways (Notch, TGF-beta, Wnt) in controlling HSC selfrenewal, proliferation, and differentiation. Here, we will review the various experimental approaches used to identify and functionally assess HSC and myeloid progenitor activity in the mouse. We will discuss how HSCs are molecularly equipped to integrate signals from the microenvironment, and how such regulations control the balance between proliferation and early myeloid differentiation. The case of the JunB/AP-1 transcription factor will be used as an example of pathological situation. Inactivation of JunB leads to the development of a myloproliferative disease (MPD) arising from the HSC compartment. JunB normally controls HSC proliferation by regulating the expression levels of key cell cycle regulators, including Bmi1, and limits the rate at which HSCs produce early myeloid progenitors by maintaining appropriate responsiveness to both Notch and TGF-beta signaling pathways. The transforming effect of JunB loss is, therefore, due to a broad deregulation of the regulatory mechanisms controlling HSC differentiation, leading to overproduction of myeloid progenitors and MPD development in vivo. These results provide insights into fundamental issues in hematopoiesis and leukemogenesis.
Mild Pro-Inflammatory State and Anemia in Older Persons.
Authors: Longo Dan L. , Ferrucci Luigi , Ershler William
Published: October 2019

Aging is characterized by a progressive dysregulation of homeostatic equilibrium. Inflammation is perhaps the most studied aspect of such homeostatic dysregulation, particularly in the context of anemia. Older persons and even “extremely healthy” older individuals are often affected by a mild pro-inflammatory state marked by high levels of pro-inflammatory markers, such as IL-6 and C-reactive protein (CRP). Patients with overt inflammatory diseases tend to develop anemia, usually of mild severity. Four mechanisms by which inflammation may affect anemia have been hypothesized:

inhibition of proliferation and differentiation of erythroid precursors by inflammatory cytokines and/or downregulation of the EPO biological response; blunted EPO production; upregulation of hepcidin synthesis and consequent impairment of intestinal absorption and iron recycling; and reduced erythrocyte survival, not fully compensated by increased erythropoiesis.

Studies have shown that the pro-inflammatory state of aging inflammation is associated with EPO levels higher than expected in non-anemic individuals and lower than expected in anemic individuals, suggesting the existence of a pre-anemic state where EPO resistance is compensated by increased EPO levels. Only when this compensatory mechanism fails does anemia become clinically evident. Consistent with this hypothesis, individuals with high EPO and high levels of inflammatory markers are at high risk of developing anemia. The mediating role of hepcidin in the pathogenesis of anemia of inflammation is suggested by an extensive literature. By interacting with the unique iron transporter, ferroportin, hepcidin controls iron intestinal absorption and mobilization from hepatocytes and macrophages, thereby causing low serum iron and ineffective erythropoiesis. Patients affected by disease characterized by overt inflammation, such as rheumatoid arthritis, abscess, and sepsis, show increased hepcidin urinary excretion. There is some evidence that the mild-pro-inflammatory state of aging does not cause hepcidin upregulation, although it is associated with low serum iron, lower transferrin saturation, and higher ferritin concentration. Thus, the iron lowering effect of moderate inflammation may be mediated by some still-unknown mechanism other than hepcidin. Studies are needed to test the hypothesis that the mild inflammation affects erythrocyte lifespan.
Platelet Death.
Authors: Josefsson Emma C. , Schoenwaelder Simone , White Michael , Goschnick Matthew , Roberts Andrew W. , Mason Kylie D. , Jackson Shaun , Huang David C. , Kile Benjamin T.
Published: October 2019

Human platelets exhibit a circulating lifespan of ~10 days, mouse platelets ~5 days. This finite existence is circumscribed by members of the Bcl-2 family of proteins, which control the intrinsic apoptosis pathway. Pro-survival Bcl-xL is the critical regulator of platelet lifespan, functioning to keep pro-death Bak and Bax in check, thereby maintaining platelet viability. After 5–10 days in the circulation, platelets not consumed in hemostatic processes initiate a Bak and Bax-dependent cell death program and clearance from the bloodstream. Mutations in Bcl-xL reduce platelet lifespan in a dose-dependent fashion, while deletion of Bak and Bax extend it. Studies with the BH3 mimetic compound ABT-737, which inhibits pro-survival Bcl-xL, have shown that platelets induced to undergo cell death in vitro exhibit many of the hallmarks of apoptosis in nucleated cells, including mitochondrial damage, caspase activation and externalization of membrane phosphatidylserine (PS). Whether any of these features occur during physiological platelet clearance remains unclear. Certainly, mitochondrial damage can reduce the recovery of transfused platelets, but whether PS – which is known to promote the pro-coagulant activity of agonist-activated platelets – also acts as a clearance signal for dying platelets in vivo is yet to be established. Conversely, Bak and Bax may play a role in mediating PS exposure triggered by activation. Supporting the idea that there may be crosstalk between classical platelet signaling pathways and the intrinsic apoptosis pathway is recent evidence that platelet agonists can also activate caspases. Intriguingly, elements of the intrinsic pathway may also contribute to the generation of platelets by megakaryocytes. Several groups have demonstrated that megakaryocytes contain activated caspases and that their inhibition can block platelet shedding by cultured cells. Preliminary evidence we have generated suggests that Bcl-2 family proteins may be required for platelet production in vivo. Thus, it appears that there is much to be understood about the role of the intrinsic apoptosis pathway in the regulation of platelet biogenesis, function, and death.
Nitric Oxide Effects in Sickle Cell Disease.
Authors: Styles Lori
Published: October 2019

Sickle cell disease (SCD) is a complex hemoglobinopathy characterized by microvascular occlusion and hemolytic anemia. Patients suffer from a myriad of both acute and chronic problems affecting virtually every organ system. Historically, microvascular occlusion has been the focus of scientific investigations into these manifestations and the chronic hemolysis of SCD was overlooked. More recently, however, the importance of the pathophysiology of hemolysis has been appreciated and related to a subset of the clinical manifestations of SCD, including pulmonary hypertension, priapism, skin ulcers, and possibly stroke. This subphenotype of SCD has been convincingly related to impaired nitric oxide (NO) homeostasis due to hemolysis. NO has pleiotropic effects including vaso-dilatory, antioxidative, anti-adhesion, and anti-thrombotic properties, which are all potentially important in the pathophysiology of SCD. Perturbation of NO homeostasis, therefore, could profoundly impact patients with SCD. Animal and human data support a state of “NO resistance” in SCD patients. Human studies have shown that SCD patients have a decreased response to exogenous NO donors and that is likely due to the scavenging of NO by free plasma hemoglobin that results from ongoing hemolysis. “NO resistance” is further augmented by the increased levels of reactive oxygen species (ROS) known to occur in SCD patients. High levels of ROS favor additional hemolysis through increased oxidant stress on the sickle red blood cell and reduce NO bioavailability by inactivation of circulating NO. With the substantial human and animal data to support a role for “NO resistance” in the pathophysiology of SCD, investigation with NO-based therapy have begun. Several approaches to overcoming “NO resistance” can be devised including increasing the precursors to NO, decreasing hemolysis, direct NO donors, and decreasing oxidant stress. To date, studies evaluating arginine (NO precursor), inhaled NO, and sildenafil (NO donor) have been reported. Oral arginine showed no benefit in a large clinical trial, and a preliminary trial of inhaled NO had only minimal benefit. Sildenafil may be more promising and is under further study. Lastly, although impaired NO bioavailability has been related to a subset of patients with pulmonary hypertension, skin ulcers and priapism, it will be important to determine what impact NO has on other manifestations, such as vaso-occlusive pain episodes and whether NO modulation can also be used therapeutically in this setting.
Functional and Biochemical Changes in Stored Human Red Blood Cells.
Authors: McMahon Timothy J.
Published: October 2019

Transfusion of red blood cells (RBCs) is associated with excess mortality and morbidity in some anemic patients with critical illness, and no benefit in others, but the underlying mechanisms are poorly understood. Specifically, little is known of how “storage lesions” of the RBC may contribute to adverse outcomes or lack of benefit from a transfusion. The recent discovery of an active role of the human RBC in regulating blood flow – a major determinant of its own function, O2 delivery – prompts a reexamination of storage-induced changes in this and related RBC functions, and motivates specific identification of the relevant mediators. RBCs release the vasodilators S-nitrosothiols (SNOs) and ATP upon physiological exposure to hypoxia, contributing to O2-dependent blood flow regulation. The result is hypoxic vasodilation peripherally and modulation of hypoxic vasoconstriction in the lung. We show that conventionally stored human RBCs become deficient in SNOs within three hours of acquisition and independent of exposure to storage solution or leukofiltration; both hemoglobin-bound SNO and membrane SNO are depressed. Early changes (hours) are also seen in the function of stored RBCs, namely in RBC regulation of vascular tone in vitro and in the lungs of intact mice in vivo, whereas declines in RBC deformability take place more gradually (from days to weeks). We also confirm storage-induced depletion of RBC ATP, a lesion which may contribute to the functional declines in both deformability and in RBC-dependent vasoactivity. Time courses, therefore, vary widely for several storage-induced functional and biochemical defects that may contribute to adverse clinical outcomes following RBC transfusion. Strategies that either prevent the loss of vasoactive mediators during RBC storage, or replete bioactive (S)NO after RBC storage, are reviewed and may inform the rational design of strategies to improve the risk-benefit balance associated with RBC transfusion in critically ill and other anemic patients.
Genetic Origin of Myeloma.
Authors: Chesi Marta , Bergsagel Peter Leif , Kuehl Michael
Published: October 2019

Multiple myeloma (MM) is a tumor of isotype-switched plasma cells that accumulate in the bone marrow, replacing normal plasma cells and leading to bone destruction and marrow failure. It is often preceded by a stable monoclonal gammopathy (MG) with limited (<10%) bone marrow plasmacytosis that progresses stochastically to MM at a rate of between 1–2% per year. Primary genetic events thought to be associated with disease initiation are present at similar frequencies in both MG and MM. In approximately 40% of cases, there are recurrent chromosome translocations involving the immunoglobulin heavy chain gene that have the hallmarks of being mediated by errors in switch recombination (90%) or somatic hypermutation (10%). These involve recurrent loci that fall into three categories: Cyclin D (15% CCND1 at 11q13, <1% CCND2 at 12p13, 2% CCND3 at 6p21), MAF (5% c-maf at 16q23, 2% MAFB at 20q12, <1% MAFA at 8q24.3), and MMSET (usually with FGFR3 at 4p16, 15%). Approximately one-half of cases are hyperdiploid (48–75 chromosomes) with trisomies involving chromosomes 3, 5, 7, 9, 11, 15, 19 and 21. A unifying feature of MM is dysregulated expression of a cyclin D gene, either directly by Ig gene translocation, by transactivation by MAF (CCND2), or by unknown mechanisms (MMSET – CCND2, Hyperdiploid – CCND1). Interestingly, about 2% of patients express no cyclin D, and one-half of these patients have bi-allelic deletion of RB, highlighting the critical role of the CCND/RB axis in the pathogenesis of MM. Deletion of chromosome 13 is seen in one-half of MM but is much more common in the tumors that express CCND2 (>80%) than in those that express CCND1 (~30%). Although with largely redundant functions, CCND1 has additional ability to antagonize RB not shared by CCND2, suggesting that CCND2 tumors may be more critically dependent on the absolute level of RB protein. A variety of secondary genetic events associated with disease progression occur with a similar frequency in hyperdiploid and non-hyperdiploid tumors (17p loss or p53 mutations, RAS mutations, secondary Ig translocations, MYC translocations, and inactivation of p18INK4c or RB1). In contrast, a promiscuous array of mutations that activate the NFKB pathway are less common in hyperdiploid MM, which, nonetheless, has high levels of presumably ligand-dependent NFKB activity and appears particularly dependent on the bone marrow microenvironment. Both some of the primary (MAF, MMSET) and secondary (17p deletion) genetic events are associated with a poor prognosis with melphalan-based regimens and appear to benefit from the early introduction of bortezomib.
Genomics and Systems Biology.
Authors: Golub Todd R.
Published: October 2019

Genomics holds particular potential for the elucidation of biological networks that underlie disease. For example, gene expression profiles have been used to classify human cancers, and have more recently been used to predict graft rejection following organ transplantation. Such signatures thus hold promise both as diagnostic approaches and as tools with which to dissect biological mechanism. Such systems-based approaches are also beginning to impact the drug discovery process. For example, it is now feasible to measure gene expression signatures at low cost and high throughput, thereby allowing for the screening libraries of small molecule libraries in order to identify compounds capable of perturbing a signature of interest (even if the critical drivers of that signature are not yet known). This approach, known as Gene Expression-Based High Throughput Screening (GE-HTS), has been shown to identify candidate therapeutic approaches in AML, Ewing sarcoma, and neuroblastoma, and has identified tool compounds capable of inhibiting PDGF receptor signaling. A related approach, known as the Connectivity Map (www.broad.mit.edu/cmap) attempts to use gene expression profiles as a universal language with which to connect cellular states, gene product function, and drug action. In this manner, a gene expression signature of interest is used to computationally query a database of gene expression profiles of cells systematically treated with a large number of compounds (e.g., all off-patent FDA-approved drugs), thereby identifying potential new applications for existing drugs. Such systems level approaches thus seek chemical modulators of cellular states, even when the molecular basis of such altered states is unknown.
Donor T Cell Gene Expression and GVHD.
Authors: Perreault Claude
Published: October 2019

GVHD is initiated by donor T cell responses to host alloantigens. However, the occurrence and severity of GVHD are not determined solely by the level of histoincompatibility between donor and recipient. Two MHC-identical subjects will display over 50 minor histocompatibility antigen differences. If histoincompatibility is sufficient for triggering GVHD, the rate of GVHD in MHC-matched recipients of allogeneic hematopoietic cell transplantation (HCT) that receive no immunosuppressive agents should be 100%. Under these conditions, however, GVHD is found in only 50% and 73% of mouse and human recipients, respectively. Histoincompatibility is thus necessary but not sufficient to elicit GVHD. We tested the hypothesis that some donors may be “stronger alloresponders” than others, and consequently more likely to elicit GVHD. To this end, we studied the gene expression profiles of CD4 and CD8 T cells from 50 HCT donors using microarrays and qRT-PCR. We found that gene expression profiling before HCT was able to distinguish those donors whose cells caused GVHD from those whose cells did not. The “dangerous donor” trait (GVHD+ recipient) is under polygenic control and is shaped by the activity of genes that regulate TGF-β signaling and cell proliferation. The donor gene profile defined on day 0 shows strong correlation with that of recipient CD4 and CD8 T cells harvested one year post-AHCT. The latter correlation provides compelling evidence that a significant portion of the differential gene profiles between GVHD+ and GVHD– donors is imprinted at the hematopoietic stem cell level. Moreover, stability of the gene expression profiles over a one-year period suggests that the profiles result from inherited genetic traits as opposed to environmental factors. The gene with the best GVHD-predictive accuracy was SMAD3, a key component of the TGF-β pathway. By testing a cohort of 450 subjects using qRT-PCR, we found that amounts of SMAD3 transcripts varied over a 6-fold range. In mice and humans, SMAD3 is constitutively activated (as evidenced by phosphorylation and accumulation in the nucleus) in many leukocyte subsets. We found in mice that induction of TGF-β signaling in donor T cells is an early event following AHCT and that Smad3-deficient donors trigger more severe GVHD than wild-type littermates. These findings strongly suggest that the donor gene expression profile has a dominant influence on the occurrence of GVHD. In allogeneic HCT, the ability to discriminate strong and weak alloresponders using gene expression profiling could help select low-risk donors and permit tailoring GVHD prophylaxis regimens according to the probability of GVHD occurrence.
Developing a Therapeutic Anti - Dendritic Cell Antibody to Prevent Graft Versus Host Disease.
Authors: Hart Derek , Seldon Therese , Jones Martina , Sheng Yonghua , Palkova Anna , Cullup Hannah , Munro Trent , Sinfield Laura , Rice Alison M , Wilson John , Gray Peter , Barnard Ross , Mahler Stephen , Munster David
Published: October 2019

Abstract 3554

Poster Board III-491

Introduction

Host and donor dendritic cells (DC) stimulate alloreactive donor T lymphocytes, and initiate GVHD. We have shown that polyclonal antibody to the DC surface activation marker human CD83 (anti hCD83), which depletes activated DC, can prevent human DC and T cell induced lethal xenogeneic GVHD in SCID mice without impairing T cell mediated anti-leukaemic and anti-viral (CMV and influenza) immunity (J Exp Med 2009; 206: 387). Therefore, we made and tested a polyclonal anti mouse CD83 (RAM83) antibody in murine HSCT models and developed a human mAb against hCD83 as a potential new therapeutic immunosuppressive agent.

Methods

RAM83 specificity and function was tested by flow cytometry and allogeneic MLRs. It was administered as a single dose on day -1 of murine syngeneic and allo HSCT. Human and mouse single chain variable fragment (scFv) phage libraries were panned on recombinant human CD83 extracellular domain. After screening for specificity and affinity, the clones were reformatted to human IgG1 and expressed by transfected CHO cells. The purified mAb were tested for their ability to block a mixed leucocyte reaction (MLR).

Results

Lin-,Sca1+,Kit+ murine stem or progenitor cells were CD83-ve. RAM83 treated mice receiving syngeneic HSCT survived >35days (normal day 14 WBC). Full MHC mismatched (2× 60mg/kg cyclophosphamide + 2× 500cGy conditioned), B6 [H-2b] into BALB/c [H-2d]) recipient mice treated with a single dose of RAM83 developed GVHD later and survived longer (mean 26.9 days ±3.3) compared to controls (RAneg 12.3±1.3days, p=0.001; Nil antibody 7.7±1.5 days, p=0.0008). Daily high dose (50mg/kg) cyclosporin A delayed GVHD by a mean 18.2±3.3 days. Four phage clones that bind to human CD83 have been reformatted. Of the two tested to date, one mouse/human chimeric mAb has lost binding affinity, probably as a result of immunoglobulin variable region glycosylation. The other reformatted fully human anti-CD83 (3C12) mAb blocked a MLR, by an ADCC mechanism.

Conclusions

mAb targeting of activated DC is a promising novel approach to immunosuppression. The 3C12 clone and others will be subjected to preclinical testing in the xenogeneic SCID mouse model. The human mAb, which prevents GVHD without impairing the desired graft versus leukaemia effect will be developed for phase 1 clinical trials in clinical alloHSCT.

Disclosures:

Hart: CRC-BT: Consultancy.