A Network of Activated Cancer Signaling Pathways in a High Risk Subgroup of Acute Myeloid Leukemia Targetable By SRC-Kinase Inhibitors

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2746-2746
Author(s):  
Claudia Chiriches ◽  
Dilawar Khan ◽  
Nathalie Guillen ◽  
Michal Rokicki ◽  
Carol Guy ◽  
...  
Keyword(s):  
Stem Cells ◽  
High Risk ◽  
Signaling Pathways ◽  
Src Kinase ◽  
Class Ii ◽  
Driver Mutation ◽  
Class I ◽  
Driver Mutations ◽  
Central Node ◽  
Kinase Family

Abstract Resistance to therapy including potent and selective targeted agents remains the major clinical challenge in AML. In fact, allogeneic stem cell transplantation remains the best curative treatment option for AML patients with high-risk features. This unmet clinical need may be addressed by novel approaches based on targeting networks of activated cancer signaling pathways (NACSPs) instead of only individual pathways. Signaling pathway activation in AML is not only related to class I mutations such as FLT3-ITD, but also to class II driver mutations. This is best exemplified by AML subtypes harboring non-random chromosomal aberrations, encoding driver mutations able to induce and maintain leukemia. Despite the contribution of class I mutations to a poor prognosis, targeting these lesions does not decisively contribute to the cure of AML in the great majority of the cases. AML with the translocation t(6;9)(p23;q34) encoding the related DEK/NUP214 fusion protein is a high risk group of AML patients characterized by young age and presence of FLT3-ITD in ~ 75% of the cases, which responds only transiently to FLT3-ITD inhibitors. Using this AML subtype as a model for high risk disease, we investigated the NACSPs activated by a class II driver mutation. In these patients FLT3-ITD represents the only established recurrent genetic aberration at diagnosis in addition to the t(6;9). We have shown that the driver mutation DEK/NUP214 transforms very immature hematopoietic stem cells with the contribution of activated STAT5, present also in FLT3-ITD-negative patients. Furthermore it has been reported that AKT/mTOR is activated in DEK/NUP214-positive cells. Here we investigated whether these signaling pathways are components of a leukemogenic NACSP and are therapeutically/clinically significant. We used different inhibitors to target either selectively or to determine a inhibition pattern of a.) PI3K/AKT/mTOR signaling (BKM120, BEZ2315, RAD001, Torin1 and AZD1208) at different levels; b.) receptor tyrosine kinases (RTK- ruxolitinib, sorafenib); and c.) members of the SRC kinase family (dasatinib, ponatinib, PF114, PP2). We employed four different models of t(6;9)-positive AML: U937 cells stably transfected with DEK/NUP214, t(6;9)-positive FKH1 cells, syngeneic DEK/NUP214-driven murine AML cells and primary t(6,9)-positive AML cells. Here we show that i.) STAT5 and AKT/mTOR activation was genetically determined by the t(6;9)-DEK/NUP214; ii.) STAT5 and AKT/mTOR activation were independent of JAK2 and PI3K activation, respectively; iii) selective inhibition of the AKT/mTOR cascades strongly increased STAT5 activation; iv.) both signaling pathways form a NACP, with activated members of the SRC kinase family (SKF - LYN and SRC) as a central node; v.) the NACSP was effectively targeted by inhibitors of the SRC-kinase activity (SKI) such as dasatinib, ponatinib, PF114 and the selective SKF inhibitor PP2, resulting in cell growth arrest and induction of apoptosis in t(6;9)-positive leukemic cells; vi.) SKI not only inhibited SKF/STAT5 but also the AKT/mTOR cascade; vii.) this NACP was independent of the activation of RTKs such as PDGFR, KDR, c-KIT, FLT3 a.o, as part of the target profile of many SKIs used above, as shown by the lack of activity of inhibitors such as ruxolitinib, sorafenib or ibrutinib; viii.) addition of AKT/mTOR inhibitors strongly increased effects of low dose dasatinib or ponatinib in primary t(6;9) leukaemic cells. Our findings implicate the t(6;9)-DEK/NUP214 oncogene as a central inductor of an NACSP including SFK, AKT/mTOR and STAT5 which is independent of the recurrent FLT3-ITD signaling in these patients. This NACSP is able to maintain the leukemia in the presence of effective inhibition of FLT3-ITD signaling by Sorafenib, AC220 and other inhibitors in clinical use. Furthermore, the increase of STAT5 activation upon AKT/mTOR inhibition, suggests that the use of such a treatment would not lead to the eradication of the disease, because of the role of activated STAT5 in the maintenance of leukemic stem cells. On the other hand, SKIs target the entire NACSP. In fact, hitting the central node of the NACSP abolished the activation of both STAT5 and AKT/mTOR. Taken together these data establish SKIs as a valid therapeutic concept not only in t(6;9)-positive AMLs but also in all other AML subtypes characterised by the same NACSP. Disclosures Ottmann: Pfizer: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Fusion Pharma: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Ariad: Consultancy, Honoraria.

scholarly journals Targeted Expression of the Class II Phosphoinositide 3-Kinase in Drosophila melanogaster Reveals Lipid Kinase-Dependent Effects on Patterning and Interactions with Receptor Signaling Pathways

2004 ◽  
Vol 24 (2) ◽  
pp. 796-808 ◽  
Author(s):  
Lindsay K. MacDougall ◽  
Mary Elizabeth Gagou ◽  
Sally J. Leevers ◽  
Ernst Hafen ◽  
Michael D. Waterfield
Keyword(s):  
Drosophila Melanogaster ◽  
Signaling Pathways ◽  
Egf Receptor ◽  
Adaptor Protein ◽  
Class Ii ◽  
Notch Receptor ◽  
Mitogen Activated Protein Kinase ◽  
Class I ◽  
Wing Venation ◽  
Receptor Signaling

ABSTRACT Phosphoinositide 3-kinases (PI3Ks) can be divided into three distinct classes (I, II, and III) on the basis of their domain structures and the lipid signals that they generate. Functions have been assigned to the class I and class III enzymes but have not been established for the class II PI3Ks. We have obtained the first evidence for a biological function for a class II PI3K by expressing this enzyme during Drosophila melanogaster development and by using deficiencies that remove the endogenous gene. Wild-type and catalytically inactive PI3K_68D transgenes have opposite effects on the number of sensory bristles and on wing venation phenotypes induced by modified epidermal growth factor (EGF) receptor signaling. These results indicate that the endogenous PI3K_68D may act antagonistically to the EGF receptor-stimulated Ras-mitogen-activated protein kinase pathway and downstream of, or parallel to, the Notch receptor. A class II polyproline motif in PI3K_68D can bind the Drk adaptor protein in vitro, primarily via the N-terminal SH3 domain of Drk. Drk may thus be important for the localization of PI3K_68D, allowing it to modify signaling pathways downstream of cell surface receptors. The phenotypes obtained are markedly distinct from those generated by expression of the Drosophila class I PI3K, which affects growth but not pattern formation.

scholarly journals Safety Standard for Special Class Damslopes Based on Reliability Analysis

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Lingzhi Huang ◽  
Zheng Si ◽  
Xiaoqi Du ◽  
Lifeng Wen ◽  
Bin Li
Keyword(s):  
High Risk ◽  
Reliability Analysis ◽  
Special Class ◽  
Slope Failure ◽  
Safety Margin ◽  
Class Ii ◽  
Class I ◽  
Safety Factors ◽  
Safety Standard ◽  
Risk Levels

The risk of slope failure is determined by the degree of damage caused by the slope slide. For the special-high slope of some high-risk water conservancy and hydropower projects, the standard should be appropriately raised. Thus, the safety standard for these slopes is explored on the basis of reliability analysis. The slopes with high risk of failure are divided into special class I and special class II slopes depending on the risk levels and acceptable risk standards. The concept of reliability theory-based relative ratio of the safety margin is utilized to establish the relationship between annual failure probability and safety factor, thereby obtaining the reasonable safety factors for different slopes. Results show that the values of safety factors for special class I and special class II are 1.40 and 1.35, respectively. These results can provide a reference for exploring the safety standards of dams with a height of more than 200 m.

scholarly journals Profile of Myeloproliferative Neoplasms in Kuwait: Population-Based Study

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5373-5373
Author(s):  
Mazyad Jamal Almazyad ◽  
Aisha S Alwehaib ◽  
Salem Alshemmari
Keyword(s):  
High Risk ◽  
Similar Pattern ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Population Based ◽  
Driver Mutation ◽  
Driver Mutations ◽  
Positive Finding ◽  
Population Based Study ◽  
Arms Pcr

Introduction Myeloproliferative neoplasms (MPNs) are a group of hematopoietic disorders of stem-cell origin, characterized by mutations that disrupt hematopoietic signal-transduction pathways. The Middle East lacks an MPN registry representative of the disease in our area. Here we report on the epidemiology of these neoplasms in our area, including phenotype, clinical features and relevant outcomes. Methods This population-based study reports various demographic characteristics and clinical attributes of all suspected and confirmed MPN patients from all over Kuwait referred to the research hematology lab at Kuwait University & cytogenetic lab in Kuwait Cancer Control Centre (KCCC) during the period from 2007 to 2018. Molecular determination of the patients' driver mutation status currently relies on ARMS-PCR. Confirming a diagnosis follows the WHO criteria, and its refinements, for the diagnosis of MPNs. Data entry and analysis was performed using SPSS (v.22) software. Results Most patients are ≥ 40 years old (79.8%), with a median age of 55 years. Gender distribution is almost equal, with ethnic categorization as Kuwaiti and Non-Kuwaiti showing a similar pattern. ET is the most common diagnosis (40.1%), followed by PRV (32.3%). JAK2 V617F mutation is reported positive in 89.7% of cases, followed by CALR in 8.0% of MPNs. The incidence of MPNs ranged from 0.5 to 2.1 per 100,000 in 2007 through 2018. The lowest rate was recorded in 2007 (0.511) and the highest was observed in 2011 and 2016 (2.417 and 2.101, respectively). The increase in 2011 is likely due to the introduction of a more sensitive technique using ARMS-PCR for the diagnosis of MPNs, whereas the increase in 2017 may be explained by the publication of WHO 2016 modified criteria. Moreover, throughout the years, the distribution of MPNs in different age groups showed similar pattern, with the highest incidence in patients aged ≥ 60. Driver mutations can fit with a general increase in incidence from 2007-2017, which may be attributed to increased awareness among treating physicians asking suspected cases to screen for MPNs using molecular techniques.One hundred and twenty-four (18.5%) cases were documented to have a prior history of thrombosis, with roughly equal distribution between arterial and venous sites. A large proportion (89.5%) of the thrombotic events occurred in those who are ≥ 40 years old, with most events being associated with ET (34.7%) and PRV (33.1%). Almost one-third of cases of thrombosis were associated with undetermined MPN diagnosis. Participating patients were categorized as either low or high risk for thrombotic events, with the latter being defined as age ≥ 55 years and the presence of a previous thrombotic event. The results demonstrate that a total of 46 cases were defined as high risk, most of them being associated with ET (20 cases) and PRV (19 cases). A statistically significant association was reported between gender and site of occurrence of thrombotic events, with males having more arterial thromboses, and females were documented to have more venous thromboses. Conclusion JAK2 V617F driver mutation is the most common positive finding in the participating patients. Roughly one-fifth of the participants encountered thrombotic events, and the site of thrombosis is associated with gender, demonstrating statistical significance. These results should warrant a more thorough evaluation of MPNs in Kuwait to provide a better understanding of its epidemiology. This can be achieved through optimized documentation of patients' data, and testing for additional novel driver mutations and transformation; as well as encourage physicians in primary care centers to refer suspected cases for molecular diagnosis. Disclosures No relevant conflicts of interest to declare.

scholarly journals Subtractive Interaction Proteomics Reveal a Network of Signaling Pathways Activated By an Oncogenic Transcription Factor in High Risk AML

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3917-3917
Author(s):  
Claudia Chiriches ◽  
Nathalie Guillen ◽  
Michal Rokicki ◽  
Carol Guy ◽  
Afsar Mian ◽  
...  
Keyword(s):  
High Risk ◽  
Signaling Pathways ◽  
Research Funding ◽  
Statistical Significance ◽  
Driver Mutations ◽  
Molecular Targeting ◽  
Genomic Alterations ◽  
Cancer Signaling ◽  
Depth Analysis ◽  
Interaction Proteomics

Abstract Acute myeloid leukemias (AML) are characterized by recurrent genomic alterations, often in transcriptional regulators, which form the basis on which current prognostication and therapeutic intervention is overlaid. Three subtypes of AML carrying specific translocations, namely t(15;17), t(11;17) and t(6;9), are notable for being associated with a smaller number of co-existing driver mutations than e.g. AML with normal karyotype. This strongly suggests that the function of their aberrant gene products, PML/RAR and DEK/CAN, respectively, may subsume the functions of other driver mutations. Thus we hypothesized that these functions, while as yet elusive, not necessarily require sequential acquisition of secondary genomic alterations. We elected to study AML with the t(6;9), defined as a distinct entity by the WHO classification, because of its particular biological and high risk clinical features and unmet clinical needs. Most t(6;9)-AML patients are young, with a median age of 23-40 years, complete remission rates do not exceed 50% and median survival after diagnosis is only about 1 year. We used a novel "subtractive interaction proteomics" (SIP) approach to understand the mechanisms by which the t(6;9)-DEK/CAN nuclear oncogene induces this highly resistant leukemic phenotype. Based on Tandem Affinity Precipitation (TAP) for the enrichment of proteins complexes associated with SILAC-technology followed by LC-MS/MS we developed SIP as a comparison between the interactome of an oncogene and those of its functionally inactive mutants in order to obtain eventually only relevant interaction partners (exclusive binders) in the same genetic background. This is achieved by the subtraction of binders that are common to four functionally inactive mutants classifying them as not relevant. Bioinformatic network analysis of the 9 exclusive binders of DEK/CAN revealed by SIP (RAB1A, RAB6A, S100A7, PCBD1, Clusterin, RPS14 and 19, IDH3A, SerpinB3) using BioGrid, IntAct and String together with Ingenuity© Pathway Analysis (IPA), indicated a functional relationship with ABL1-, AKT/mTOR-, MYC- and SRC family kinases-dependent signaling. Interestingly, we found all these signaling pathways strongly activated in an autonomous manner in four DEK/CAN-positive leukemia models, DEK/CAN expressing U937 cells, t(6;9)-positive FKH-1 cells, primary syngeneic murine DEK/CAN-driven leukemias, and t(6;9)-positive patient samples. Bioinformatic analysis of the phopshoproteomic profile of FKH1 cells upon molecular targeting of single pathways (imatinib for ABL1, PP2 for SFKs, dasatinib for ABL1/SFK and Torin1 or NVP-BEZ-235 for mTOR/AKT) revealed that these signaling pathways were organized in clusters creating a network with nodes that are credible candidates for combinatorial therapeutic interventions. On the other hand inhibition of individual outputs had the potential to activate interconnected pathways in a detrimental manner with consequential clinical impact e.g. the activation of STAT5 by the inhibition of mTOR/AKT in these cells. Treatment of mice injected with primary syngeneic DEK/CAN-induced leukemic cells with dasatinib (10mg/kg) and NVP-BEZ-235 (45mg/kg) alone and in combination for 14 days led to a strong reduction of leukemia burden in all cohorts (each cohort n=7). In fact, as compared to untreated controls (146.6 +/- 36mg), mice treated with NVP-BEZ 235 alone and in combination (61.7 +/-4.7mg and 65.3+/- 4.6mg, respectively) showed a statistically significant reduction of spleen size whereas those treated with dasatinib alone (77.5 8 +/- 5.4mg) did not reach statistical significance. Taken together the here presented results reveal specific interdependencies between a nuclear oncogene and kinase driven cancer signaling pathways providing a foundation for the design of therapeutic strategies to better address the complexity of cancer signaling. In addition, it provides evidence for the need of a more in depth analysis of indirect effects of molecular targeting strategies in a preclinical setting not only in AML but in all cancer types. Disclosures Ottmann: Novartis: Consultancy; Pfizer: Consultancy; Fusion Pharma: Consultancy, Research Funding; Amgen: Consultancy; Celgene: Consultancy, Research Funding; Takeda: Consultancy; Incyte: Consultancy, Research Funding.

The First Demonstration of Sequential Acquisition of Class I and Class II Gene Mutations in Formation of Human Acute Myeloid Leukemia Stem Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 859-859
Author(s):  
Takahiro Shima ◽  
Yoshikane Kikushige ◽  
Toshihiro Miyamoto ◽  
Koichi Akashi
Keyword(s):  
Stem Cells ◽  
Class Ii ◽  
Myelogenous Leukemia ◽  
Class I ◽  
Leukemic Stem Cells ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Kit Mutation ◽  
Normal Differentiation

Abstract Abstract 859 Hematopoietic stem cells (HSCs) should be the main target for accumulation of mutational events, which eventually leads to formation of leukemic stem cells. These leukemogenic mutations have been classified at least into class I (providing the proliferative and survival advantage) and class II (impairing the differentiation activity) gene abnormalities. It has been proposed that acquisition of both class I and class II mutation are essential for the development of leukemia. Although several experimental animal studies suggest this model, there is no direct evidence that class I and class II mutations collaborate to contribute to development of human leukemias. Here we demonstrate that the acquisition of 8;21 translocation, which encodes the AML1-ETO (a class II chimeric fusion gene), and of mutational c-Kit (a class I mutation) is sequentially occurred in human acute myelogenous leukemia (AML). It has been shown that in t(8;21) AML patients treated with chemotherapy, a small amount of AML1-ETO mRNA was never disappeared even in patients maintaining remission for more than 10 years. We have demonstrated that this AML1-ETO mRNA in “cured” patients is derived from t(8;21)+ HSCs that consisted only a few percent of HSCs in remission (Miyamoto et al., PNAS 2000; 97: 7521–7526). The t(8;21)+ HSCs possessed normal differentiation at least into myeloerythroid cells and B cells. These data strongly suggest that acquisition of the AML1-ETO fusion is not sufficient for development of t(8;21) AML, and that t(8;21)+ HSCs are preleukemic HSCs. We hypothesized that acquisition of additional class I mutation might transform the AML1-ETO+ preleukemic HSCs into AML stem cells. We therefore searched for class I mutations in t(8;21) AML samples, and found that in 13 out of 33 t(8;21) AML patients, AML cells have c-Kit mutations (but not other class I such as FLT3-ITD and N-Ras mutations) at diagnosis. We then tested whether the AML1-ETO+ preleukemic HSCs in remission marrow have the c-Kit mutation. Six out of these 13 t(8;21) AML patients with c-Kit mutation maintaining long-term remission were enrolled in this study. To confirm the coexistence of AML1-ETO and c-Kit mutation in single leukemic stem cells, CD34+CD38− AML cells were purified from the bone marrow of patients at diagnosis, and tested for the presence of AML1-ETO and c-Kit mutation by single cell PCR. In all of 910 single CD34+CD38− AML cells, both AML1-ETO and c-Kit mutations were detected. Then, CD34+CD38− HSCs in remission were tested for the presence of AML1-ETO and c-Kit mutation. In 1728 single CD34+CD38− HSCs of remission marrow, 0.9% (16 cells) of these cells expressed AML1-ETO. Surprisingly, none of these AML1-ETO+ preleukemic HSCs possessed c-Kit mutation, indicating that AML1-ETO+ clones in long-term remission are independent from the original t(8;21) AML clones in terms of the presence of c-Kit mutation. We then performed colony-forming assays to evaluate the differentiation potential of these AML1-ETO+ preleukemic HSCs. HSCs of remission marrow-derived colonies were picked up, and tested for the presence of AML1-ETO and c-Kit mutation. In 7187 colonies formed in the culture of remission marrow, 1.2% (89 colonies) of these colonies were positive for AML1-ETO, and all of these colonies were negative for c-Kit mutation. These data collectively suggest that the acquisition of c-Kit mutation is the second step for formation of t(8;21) AML stem cells: Normal HSCs acquire t(8;21) and express resultant AML1-ETO (Class II) but it is not sufficient for full transformation into AML stem cells. These preleukemic HSCs possess normal differentiation activity, but additional c-Kit mutation (Class I) might be critical in transforming into AML stem cells. This is the first clear-cut evidence that HSCs transform into AML stem cells by stepwise acquisition of Class I and Class II mutations. Disclosures: No relevant conflicts of interest to declare.

scholarly journals MP32: The Pulmonary Embolism Severity Index (PESI) score and disposition decisions in Calgary emergency departments

CJEM ◽  
2017 ◽  
Vol 19 (S1) ◽  
pp. S76
Author(s):  
S. Brunet ◽  
D. Wang ◽  
E. Lang
Keyword(s):  
Pulmonary Embolism ◽  
High Risk ◽  
Mortality Rate ◽  
Class Ii ◽  
Low Risk ◽  
Class I ◽  
Class Iii ◽  
Class V ◽  
Risk Class ◽  
Class Iv

Introduction: The Pulmonary Embolism Severity Index (PESI) score predicts short-term mortality from pulmonary embolism and low-risk patients suitable for home therapy. However, it is unknown if it is a driver for disposition decisions for emergency department (ED) patients. The primary objective of this study was to define the relationship between disposition decisions and the PESI score in Calgary zone hospitals. Methods: The PESI score was calculated retrospectively for 576 patients presenting to one of four Calgary zone hospitals for pulmonary embolism over the last 2 years. The calculated PESI score allowed the mortality risk of each patient to be estimated for very low risk (Class I, 0-1.6% 30-day mortality rate), low risk (Class II, 1.7-3.5% 30-day mortality rate), intermediate risk (Class III, 3.2-7.1% 30-day mortality rate), high risk (Class IV, 4.0-11.4% 30-day mortality rate), and very high risk (Class V, 10.0- 24.5% 30- day mortality rate). The patients were grouped based on being admitted to the hospital for inpatient care, or discharged for outpatient care. Descriptive statistics were used to describe the data. Results: Of the 576 patients, 317 (55%) were discharged and 259 (45%) were admitted to the hospital for inpatient care. Among admitted patients, 20.5% were considered Class I, 29.3% were Class II, 24.3% were Class III, 17.6% were Class IV, and 8.1% were Class V. Among discharged patients, 53.9 % were Class I, 25.6% were Class II, 15.5% were Class III, 4.4% were Class IV, and 0.6% were Class V. Of the 25 very high-risk (Class V) patients, 2 (8.0%) were discharged from the ED and treated as outpatients. Of the 223 very low risk (Class I) patients, 171 (76.7%) were discharged and 52 (23.3%) were admitted to hospital. Conclusion: A significant percentage of pulmonary embolism patients admitted to Calgary Zone hospital wards are PESI low risk (29.3%) or very low risk (20.5%). Implementation of a PESI score-based disposition pathway could improve the safety, cost-effectiveness and quality of ED disposition decisions for PE.

scholarly journals Split tolerance induced by the intrathymic adoptive transfer of thymocyte stem cells.

1988 ◽  
Vol 168 (1) ◽  
pp. 143-156 ◽  
Author(s):  
R P Shimonkevitz ◽  
M J Bevan
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Lymph Nodes ◽  
Mhc Class I ◽  
Transient State ◽  
Class Ii ◽  
Class I ◽  
Donor Cells ◽  
Split Tolerance ◽  
Spleen And Lymph Nodes

The intrathymic transfer of semiallogeneic CD4/CD8 double-negative (DN) thymocyte stem cells into irradiated host mice resulted in a transient state of chimerism in adoptive host thymus, spleen, and lymph nodes. Host-derived T cells, isolated from the thymus and periphery of the chimeric mice, were found to be specifically nonresponsive to the MHC antigens of the semiallogeneic DN donor in cytotoxicity assays. This nonresponsiveness was not permanent, but persisted as long as appreciable numbers of Thy-1 alloantigen-positive progeny of the DN donor cells could be detected in the spleen and lymph nodes of adoptive host mice. FACS sorting of DN donor cells before intrathymic transfer indicated that nonresponsiveness could be induced by Thy-1+ cells and was therefore not attributable to contaminating thymic macrophages, dendritic cells, or B cells. When FACS-sorted Thy-1+ (bm5 x bm12)F1 DN cells were transferred intrathymically into C57BL/6 hosts, nonresponsiveness to DN donor MHC class I but not class II alloantigen (split tolerance) was observed. These experiments were repeated using FACS-sorted Thy-1+ DN donor cells that were semiallogeneic to the irradiated adoptive host at either MHC class I or class II locus with similar results. Limiting dilution analysis showed that host-derived CTL precursors were tolerant of DN donor MHC class I alloantigen and no evidence for the involvement of suppressor T cells was found. The data indicate that murine thymocytes themselves are capable of tolerizing to MHC class I but not class II alloantigen after intrathymic transfer. The implications for intrathymic T cell differentiation and maintenance of self tolerance are discussed.

scholarly journals Myeloproliferative Neoplasm (MPN) Driver Mutations Are Enriched during Hematopoietic Stem Cell Differentiation in Patterns That Correlate with Clinical Phenotype and Treatment Response

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4317-4317 ◽  
Author(s):  
Ghaith Abu-Zeinah ◽  
Silvana Di Giandomenico ◽  
Claudia Sosner ◽  
Niamh Savage ◽  
Spencer Krichevsky ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Research Funding ◽  
Clinical Phenotype ◽  
Driver Mutation ◽  
Driver Mutations ◽  
Myeloid Differentiation ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Stem

Abstract Introduction. Philadelphia negative (Ph-neg) Myeloproliferative Neoplasms (MPNs) differ in clinical phenotype and outcomes despite harboring identical driver mutations. The biology behind phenotypic heterogeneity has been attributed to mutational burden, co-occurring mutations and mutation order, but remains uncertain. It is known that MPN mutations in JAK2, CALR, and MPL result in augmentation of cytokine signaling at different stages of hematopoietic differentiation towards myeloid lineages. Consistent with this, the JAK2V617F driver mutation is enriched in certain myeloid lineages compared to the hematopoietic stem and progenitor cell (HSPC) compartment (Anand et al. Blood 2011). We therefore hypothesized that the lineage-specific patterns of clonal enrichment that arise during hematopoietic differentiation from stem cells to mature progeny would account for phenotypic heterogeneity in MPNs. Methods. Peripheral blood and/or bone marrow specimens were prospectively collected from 87 MPN patients (pts) providing informed consent between July 2017 and July 2018. Data on age, gender, diagnosis, date of diagnosis, symptoms, spleen size, blood counts, bone marrow findings, mutation profile, and treatment was collected for all pts. Each specimen was deconvoluted into 11 well-defined and strictly validated hematopoietic populations using a combination of density gradient separation (Ficoll), immunomagnetic selection (CD34) and fluorescence-activated cell sorting (FACS). Final purification of specimens was performed by multiparameter FACS to isolate CD15+/CD16+ polymorphonucleated cells (PMNs), CD14+/CD11b+ monocytes, CD3+ T cells, CD19+ B cells, CD71+/CD36+ erythroblasts and 6 well-defined HSPC populations (Manz et al. PNAS. 2002, Majeti et al. Cell Stem Cell 2007). Functional and morphologic characteristics were validated for all populations. DNA was extracted from the sorted populations and the variant allelic frequency (VAF) of the driver mutation in JAK2, CALR, or MPL was quantified by droplet digital PCR. Individual patterns of mutation enrichment were represented by the VAF plotted on a hematopoietic hierarchy (Fig 1). A composite VAF hierarchy was established for each of the WHO defined MPN subtypes of Polycythemia Vera (PV), Essential Thrombocythemia (ET), and Primary Myelofibrosis (PMF) prior to treatment, during course of treatment, and in secondary myelofibrosis. Clustering of individual patterns in relation to composite trees was performed using principal component analysis (PCA), which allowed clinical validation of clustering patterns. Results. A total of 135 samples were collected from 87 pts. Demographic and clinical features of the cohort are shown in Table 1. The composite pattern of MPN mutation enrichment differed in PV, ET and PMF (Fig 1). Pts with untreated PV or ET harbored a small proportion of mutated stem cells. The VAF in PV patients implied clonal dominance in mature myeloid progeny while there was minimal enrichment of the driver mutation in pts with JAK2 ET. In comparison, pts with PMF had a significantly higher VAF in the HSC compartment, but had minimal enrichment of the driver mutation during myeloid differentiation. Using PCA, individual patterns from a sample of 34 JAK2V617F MPN pts were clustered in reference to the pre-established composite patterns (heatmap in Fig. 2). This was done in order to validate consistency of patterns with clinical diagnoses (see example in Fig 2). Finally, we found that interferon treated PV pts had a unique decline in VAF with myeloid differentiation (Fig 3) but persistence of the mutant allele within the stem cell compartment: a finding that potentially explains the persistence of bone marrow abnormalities in pts achieving molecular response determined by whole blood (WB) VAF. Conclusion. The pattern of clonal enrichment of an MPN stem cell during hematopoiesis is unique to individual pts and varies significantly among the 3 major WHO subtypes of PV, ET, and PMF. This pattern also differs in relation to the disease stage, and is informative of treatment effects. WB VAF does not predict the proportion of immature HSPC harboring driver mutation. For these reasons, evaluating the clonal heterogeneity of MPN driver mutations, particularly those in HSPCs, may provide a useful surrogate measure to qualify response to novel agents in pre-clinical and clinical studies in MPNs. This is currently being tested. Disclosures Ritchie: NS Pharma: Research Funding; Incyte: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Research Funding; ARIAD Pharmaceuticals: Speakers Bureau; Astellas Pharma: Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding, Speakers Bureau; Celgene: Consultancy, Other: Travel, Accommodations, Expenses, Speakers Bureau.

Frequent Loss of HLA Alleles From Hematopoietic Stem Cells in Patients with Hepatitis-Associated Aplastic Anemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2407-2407
Author(s):  
Takamasa Katagiri ◽  
Aiko Sato-Otsubo ◽  
Koichi Kashiwase ◽  
Satoko Morishima ◽  
Yusuke Sato ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
High Risk ◽  
Time Lag ◽  
Class I ◽  
Allelic Loss ◽  
Hematopoietic Stem ◽  
Blood Samples ◽  
Risk Alleles

Abstract Abstract 2407 Background: Hepatitis-associated aplastic anemia (HAA), a subset of acquired AA which accounts for 5% of all AA, is thought to have an immune-mediated pathogenesis. Although the attack by CD8+ T cells may be responsible for both hepatitis and bone marrow failure, the exact mechanisms underlying the development of HAA, as well as the reason for the time lag from its onset to that of hepatitis remain unclear. We recently found HLA-class I allelic loss from leukocytes due to copy-number neutral LOH in chromosome 6p (6pLOH) in 15% of AA patients, and identified four class I alleles (HLA-A*02:01, A*02:06, A*31:01, and B*40:02) that are overrepresented in AA, based on the finding that the missing HLA haplotype of 6pLOH(+) patients frequently contained one of the four alleles. Such immunologically-selected hematopoiesis caused by HLA allelic loss may be more common in patients with HAA than in non-HAA patients because HAA presents with homogenous pathophysiology, and a kinetic analysis of the HLA-missing leukocytes may help to understand the immune pathophysiology of HAA. Objectives/Methods: To gain insight into the immune pathophysiology of HAA, 17 patients with HAA (11 males and 6 females) aged 1 to 63 (median 23 years) whose 10 HLA allelic data were available were analyzed for this study. Blood samples from seven of the 17 patients were examined for the presence of 6pLOH and leukocytes lacking HLA-A antigens using Affymetrix® 500K SNP arrays and flow cytometry (FCM) using allele-specific monoclonal antibodies. For three HAA patients whose blood samples before therapy were available, peripheral blood was serially examined for the presence of HLA-A missing leukocytes. The frequency of high risk HLA alleles in HAA patients were compared to those of non-HAA patients and of 6,629 registries from the Japan Marrow Donor Program who had received an unrelated allogeneic bone marrow transplant. Results: 6pLOH was detected in 4 (57%) of the 7 HAA patients, which was more frequently than in non-HAA patients (12%, P=5.1 × 10−4). Granulocytes with unilateral HLA-A expression were detectable in all four patients at a frequency of 23% to 99%. The loss of HLA-A expression in the 6pLOH(+) cases was found in multiple lineages of leukocytes, including granulocytes, monocytes, B cells, and to a lesser extent, in T cells and NK cells. The missing HLA haplotypes of the 4 patients included at least one of the four high risk alleles. The frequency of patients possessing one or more of the high risk alleles was 77% in HAA patients, 64% in non-HAA patients, and 52% in the control population (P=0.03 for HAA vs. control; P=0.25 for HAA vs. non-HAA; P=2.0 × 10−8 for AA vs. control). In one patient who developed severe hepatitis and AA simultaneously, HLA-A missing leukocytes were not detectable before ATG, but all granulocytes that appeared 16 days after ATG therapy lacked an HLA-A antigen. On the other hand, in two patients who developed AA 1 month and 2 months after the onset of hepatitis, HLA-missing leukocytes were detectable before therapy at a frequency of 31% and 37%. The percentages remained unchanged in both a patient who responded to ATG (Figure) and another non-responding patient. Conclusions: HLA allelic loss from leukocytes and the high risk class I alleles were more strongly associated with HAA than idiopathic AA, suggesting that cytotoxic lymphocyte (CTL) attack against hematopoietic stem cells (HSCs) is involved in the pathogenesis of HAA. A 2-step mechanism may explain the time lag between hepatitis and the onset of AA; CTLs simultaneously attack hepatocytes and HSCs, but it takes several weeks or months until the secondary immune response can suppress HSCs enough to develop pancytopenia, because subsequent cytokine secretion is needed to cause BM failure. Disclosures: No relevant conflicts of interest to declare.

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