bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Epigenetic Readers in Pathogenesis and Therapy of Acute Leukemias
Authors: Huntly Brian J. P.
Published: October 2019

Acute myeloid leukemia (AML) is an aggressive hematological malignancy with a dismal survival rate of less than 30 percent and is thus a disease where novel therapeutics are urgently required. AML is highly heterogeneous at both the molecular and clinical level. However, recurrent themes of altered transcriptional and epigenetic control are evident from sequencing and mechanistic studies. Recently, critical epigenetic regulators have surfaced as mediators of leukemogenesis and potential therapeutic targets in AML and other hematological malignancies. We and others have recently demonstrated that the Bromodomain and Extra Terminal (BET) protein family of transcriptional adapter proteins are required for transformation by MLL-fusion proteins, where they facilitate the localization of aberrant transcriptional complexes to chromatin at genes critical for leukemogenesis. Furthermore, we and others have demonstrated that small molecule inhibitors, such as I-BET151 and JQ1, of the protein-protein interactions between these proteins and acetylated lysine residues in histone tails are effective in pre-clinical models of MLL leukemias. We have recently extended studies of BET inhibition to other forms of AML using I-BET151 and found a range of sensitivities across a panel of AML cell lines and patient samples with common recurrent mutations. These cell lines also demonstrate a common cellular response to MLL-fusion leukemias, with induction of apoptosis and G1/0 arrest, suggesting that similar transcriptional programs may be targeted. In further investigating this, we identified a common transcriptional program downregulated in sensitive leukemia subtypes. In addition, using diagnostic gene expression profiles, we also demonstrate that this program can classify AML patients, suggesting that the program may provide predictive biomarkers and potentially identify those patients most likely to respond to BET inhibitions. Focusing on the most common mutation in AML, mutations of the nucleophosmin gene (NPM1c mutations), we present evidence to suggest that wild-type NPM1 has an inhibitory influence on BRD4, which is relieved upon NPM1c mutation and cytosplasmic dislocation. NPM1c mutation allows upregulation of the core transcriptional program facilitating leukemia development, and this program is abrogated by I-BET therapy. Finally, we demonstrate the efficacy of I-BET151 in human cell lines, a unique murine model, and in primary patient samples of NPM1c AML. Taken together, we have used BET inhibitors as a laboratory tool to dissect out mechanisms of aberrant transcriptional control in AML. Furthermore our pre-clinical evaluation supports clinical trials of BET inhibitors across multiple AML subtypes and identifies potential predictive biomarkers of sensitivity and response to inform these studies.

Disclosures:

No relevant conflicts of interest to declare.
Mechanisms of Neutrophil Release from the Bone Marrow
Authors: Link Daniel
Published: October 2019

Neutrophils are an essential component of the innate immune response and a major contributor to inflammation. Consequently, neutrophil homeostasis in the blood is tightly regulated. This is achieved by balancing neutrophil production and release from the bone marrow with clearance of senescent neutrophils from the circulation. Here, we highlight recent studies elucidating the signals that regulate neutrophil homeostasis. Constitutive chemokine expression by bone marrow stromal cells plays a key role in regulating neutrophil egress. Specifically, CXCL12 from CXCL12-abundant reticular (CAR) cells and possibly endothelial cells serves to retain neutrophils in the bone marrow. Conversely, CXCL1/2 expression from endothelial cells may promote neutrophil egress. Consistent with these observations, gain-of-function mutations of CXCR4 (the major receptor for CXCL12) or loss-of-function mutations of CXCR2 (the major receptor for CXCL1/2) are associated with myelokathexis in humans. G-CSF promotes neutrophil release from the bone marrow, in large part, by decreasing CXCL12 expression in bone marrow stromal cells, while increasing CXCL2 expression in bone marrow endothelial cells. CXCL12 production from bone marrow stromal cells may be a target of a feedback loop involving the clearance of senescent neutrophils. Specifically, macrophage engulfment of senescent neutrophils in the bone marrow attenuates CXCL12 expression, thereby facilitating neutrophil egress. Recent data suggest that signals from gut microbiota may play a significant role in regulating neutrophil homeostasis. In particular, mice raised under germ-free conditions display marked neutropenia. Gut microbiota appear to regulate neutrophil homeostasis, at least in part through toll-like receptor signaling. These data suggest new pathways that might be targeted therapeutically to modulate neutrophil number in the blood.

Disclosures:

No relevant conflicts of interest to declare.
Characteristics and Prognostic Significance of CRLF2 High Expression and the Co-Existence with JAK1 Mutations and Ikaros Deletion in Adult Acute Lymphoblastic Leukemia
Authors: Ge Zheng , Liu Juan , Zhang Run , Guo Xing , Xu Jing-Yan , Li Min , Qiao Chun , Li Jian-Yong
Published: October 2019

Objective Cytokine receptor-like factor 2 (CRLF2) play an important role in differentiation and proliferation of lymphoid precursor cells through activation of JAK signaling pathway. Increased CRLF2 expression associates with mutations in JAK2, a combination that transforms hematopoietic cells, suggesting that mutants in JAK family members and CRLF2 may cooperate to contribute to acute lymphoblastic leukemia (ALL) pathogenesis. Moreover, the Ikaros deletion is also associated with the development of T-/B-cell ALL with poor outcome and relapse of high-risk leukemia. The aim of this study was to determine the clinical characterization and prognostic values of CRLF2 high expression and its concomitant expression with JAK1 mutations and Ikaros deletion in adult ALL patients.

Methods Quantitative PCR (qPCR) was performed to detect the expression of CRLF2 in 133 newly diagnosed adult patients with ALL. Genomic DNA was amplified to detect the mutations of the exon 13, 14, 16, 18 and 19 of JAK1, and IKZF1 exons 4 through 7 deletions (△4–7) by direct sequencing or sequencing after cloning. The CD34, CD13, CD33 and other markers were detected on the leukemia cells from bone marrow of the patients by flow cytometry, and the correlations of the CRLF2 high expression with the clinical features, survival, and with co-expression of JAK1 mutations and Ikaros deletion were statistically analyzed with Pearson's chi-square test or Fisher's exact test and Kaplan–Meier curves analysis.

Results CRLF2 high expression was detected in 22.8% of newly diagnosed adult ALL. The patients with CRLF2 high expression has significantly higher percentage of CD34, CD13 or CD33 positive than those with low expression（91.3% vs 62%, P=0.008; 76.2% vs 46.3%, P=0.016; 80.0% vs 37.9%, P=0.001), higher frequency of splenomegaly（60.0% vs 32.0%, P=0.040) in the adult ALL and shorter overall survival and event-free survival（9.5 months vs 16 months, P=0.029; 3 months vs 9 months, P=0.030）in the Philadelphia chromosome negative ALL. Moreover, the 4 JAK1 point mutations with amino acid changes were detected in the patients, which had significant CRLF2 high expression compared to that without mutation（75% vs 21.3%, P=0.037). The co-existence of CRLF2 high expression and IKZF1 exons 4-7 deletion (isoform Ik6) was found in 4 of 10 patients.

Conclusion CRLF2 high expression predicts poor survival, and significantly co-exists with JAK1 mutation and Ikaros deletion in adult ALL patients. Our result also suggested that CRLF2, JAK1 and IKZF1 could be integrated in future prognostic model of adult ALL as possible markers for high-risk leukemia.

Disclosures

No relevant conflicts of interest to declare.
Antibodies from Donor B Cells Are Not Required for Initiating but Required for Persisting Scleroderma in Chronic Gvhd
Authors: Jin Hua , Ni Xiong , Deng Ruishu , Young James , Johnston Heather F , Liu Qifa , Zeng Defu
Published: October 2019

We recently reported that in a chronic graft versus host disease (GVHD) model of DBA/2 donor to MHC-matched BALB/c recipient, donor CD4+ T and B cell interaction resulted in not only hyperglobulinemia and glomerulonephritis but also scleroderma (J. Immunol. 2012). It is well known that glomerulonephritis is caused by immune complex deposition. However, the role of antibodies from donor B cells in the pathogenesis of scleroderma remains unclear. To address this question, we generated DBA/2 mice whose B cells have APC function but cannot secrete antibodies by backcrossing IgHµg1 mice from Dr. Rajewsky’s lab (JEM 2007). We observed that, while transplanting T-cell-depleted bone marrow (TCD-BM) and spleen cells from littermate control mice induced proteinuria and scleroderma, transplanting BM and spleen cells from IgHµg1 DBA/2 mice induced no proteinuria, but the recipients developed scleroderma ~35 days after HCT. Interestingly, the scleroderma gradually recovered ~55 days after HCT. 40 days after HCT, scleroderma recipients transplanted with WT spleen cells (Rec-WT) or recipients transplanted with IgHµg1 spleen cells (Rec-IgHµg1) both had high percentage (~12%) of IFN-g+ or IL-17+ CD4+ T cells in the peripheral lymph node (PLN) and skin tissues, as compared to that (~3%) of GVHD-free recipients given TCD-BM alone (Rec-TCD). While Rec-WT had severe reduction of CD4+CD8+ thymocytes, the Rec-IgHµg1 had no reduction of the thymocytes, as compared to that of Rec-TCD. By day 60 after HCT, the Rec-WT with ongoing scleroderma still had ~10% IFN-g+ or IL-17+ CD4+ T cells in the PLN and skin tissues; in contrast, although the Rec-IgHµg1 with reversal of scleroderma still had >10% IFN-g+or IL-17+ CD4+ T cells in the PLN, those cells in the skin had reduced to <2%. This reduction was associated with DC upregulation of B7H1 and T cell upregulation of PD-1. These results suggest that antibodies from B cells are required for maintaining inflammatory status of tissue DCs and persistence of scleroderma in chronic GVHD. (This work was supported by NIH R01 AI066008).

Disclosures

No relevant conflicts of interest to declare.
Targeted Next Generation Sequencing of the Five Hemochromatosis Genes in Italian Patients with Iron Overload and Non-Diagnostic First Level Genetic Test: A Pilot Study
Authors: Badar Sadaf , Busti Fabiana , Zamperin Giampiero , Ferrarini Alberto , Bozzini Paolo , Manna Daniele , Campostrini Natascia , De Matteis Giovanna , Delledonne Massimo , Girelli Domenico
Published: October 2019

Background and Aim: Molecular diagnosis of HFE-related hereditary hemochromatosis (HH) is typically made by searching for the C282Y and H63D mutations (first level genetic test). However, in the Mediterranean area up to one third of patients with HH phenotype do not have the “diagnostic” genotypes (C282Y homozygosity, or C282Y/H63D compound heterozygosity). This pilot study was designed to develop a “second level” next generation sequencing (NGS)-based test for rapid and simultaneous analysis of the five HH genes (HFE, HFE2, HAMP, TFR2 and SLC40A1).

Methodology: we studied 61 patients with relevant biochemical signs of iron overload (IO) and non-diagnostic first level genetic test suggesting a possible “non-HFE” HH. The five HH genes were captured by Halo-Plex™ technology, and then sequenced using aNGS platform (Illumina HiSeq 1000). Sequenced reads were aligned against human reference HG19 and analyzed by GoldenHelix™ software to annotate all the variants possibly involved in the disease.

Results: In IO patients a large number of new non-synonymous variants (according to bioinformatics tools based on publicly available databases including the 1000-genomes project) were found. Many of them were relatively frequent and detected also in controls, thus being considered likely “non-pathogenic”, unless clearly enriched in patients. On the other hand, some rare variants (i.e. limited to a single or very few individuals), particularly in SCL40A1, TFR2, and HFE, were found exclusively in patients, and could be considered “potentially pathogenic”.

Conclusions: The combination of the Halo-Plex™ approach with NGS platform and GoldenHelix™ algorithm appears a suitable approach for a better molecular characterization of patients with unexplained HH phenotype, and could represent a good option for second level genetic testing in referral centers. However, establishing the clinical relevance of NGS-detected “novel” genetic variants in a prevalently autosomal recessive disorder like HH remains a difficult task, requiring further functional studies and national/international collaborative efforts.

Disclosures

No relevant conflicts of interest to declare.
Clinical Impact of CD8+ Cells in Hodgkin Lymphoma Lymphadenopaties. Contribution of Flow Cytometry
Authors: Alonso Sara , Vidriales Maria Belen , Caballero Maria Dolores , Blanco Oscar , Galende J , Peñarrubia Maria Jesús , Zato Esther , Barez Abelardo , Davila Julio , Caballero Juan Carlos , Orfao Alberto , González Marcos , Garcia-Sanz Ramon
Published: October 2019

CONTEXT: Microenvironment of Hodgkin and Reed Sternberg(H-RS) cells is a current focus of interest to define risk and predict evolution of Hodgkin′s Lymphoma.

OBJECTIVES: To determine the role of infiltrating CD8+ cells, using flow cytometry (FCM), in the evolution of patients with HL.

DESIGN: Cell suspensions obtained after mechanical disintegration of ganglion biopsies of patients with newly diagnosed HL were analyzed using FCM. Definitive diagnosis was made by conventional histology and immunohistochemistry. Clinical data were collected from medical records. Statistical analysis was performed using SPSS 20.0 software.

SETTING: In the University Hospital of Salamanca, we consecutively analyzed by FCM all lymph nodes with suspected lymphoma at diagnosis. There was no selection bias when collecting patients, except for some cases with inadequate quality(insufficient cells).

PATIENTS OR OTHER PARTICIPANTS: From 1996(earliest available FCM data) to 2014, 104 of that samples had a definitive diagnosis of HL. Treatment depended on stage: a)early diagnosis received ABVD(x3) & local radiotherapy(RT) (20-30 Gy), b)advanced: ABVD(x6 to 8), plus RT in selected cases. Median follow-up was 10 years.

INTERVENTIONS :This was a retrospective observational study.

MAIN OUTCOMES MEASURES: Primary end points were overall survival(OS) and freedom from treatment failure(FFTF), considered from diagnosis to progression or relapse.

RESULTS: Most cells obtained were lymphocytes (Median17.1±86.9%) with a T/B/NK distribution of 72%/27%/1.6%(median) and predominance of CD4+ (median CD4:51%-CD8:12%). Median CD8+ cells(12%) was used to divide patients into 2 groups.FFTF was longer in patients with more than 12% of CD8+cells(93% vs. 71%, p=0.01). When analyzed separately patients with early/advanced disease, the clinical benefit remained in the group with advanced disease (p=0.006), whereas the statistical significance was lost in the group with early disease, possibly due to the excellent prognosis for those patients(FFTF 10 years > 95%). No differences were observed in the OS, because second line therapy was highly effective. In the multivariate analysis using Cox regression, advanced stage (HR=9.6 with 95% CI 1.2 to 73.9) and>12% CD8+ tumor infiltrating T-cells were independent variables(HR=0.26, 95% CI 0.07 to 0.9).

CONCLUSIONS: Increased number of CD8+ in the H-RSC microenvironment of HL is associated with better FFTF, particularly in the advanced-disease group. This should be considered as a new biomarker to identify high-risk patients.

Figure 1. Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Glasgow Prognostic Score Is Superior to Other Inflammation-Based Scores in Predicting Survival of Diffuse Large B-Cell Lymphoma
Authors: Hao Xiaoxiao , Wei Yongqiang , Huang Fen , Wei Xiaolei , Zhang Yuankun , Feng Ru
Published: October 2019

Inflammation-based prognostic scores, such as the glasgow prognostic score (GPS), prognostic index(PI), prognostic nutritional index(PNI), neutrophil lymphocyte ratio(NLR), platelet lymphocyte ratio(PLR) was related to survival in many solid tumors. Recent study showed that GPS can be used to predict outcome in diffuse large B-cell lymphoma(DLBCL). However other inflammation related scores had not been reported in DLBCL, and it also remained unknown which of them was more useful to evaluate the survival in DLBCL. In this retrospective study, a number of 252 newly diagnosed and histologically proven DLBCL patients from January 2003 to December 2014 were included. An elevated GPS, PI, NLR, PNI and PLR were all associated with decreased overall survival(p=0.000, p=0.000, p=0.006, p=0.001 and p=0.001, respectively) and event-free survival (p=0.000, p=0.000, p=0.011, p=0.001 and p=0.009, respectively) in univariate analysis. Multivariate analysis indicated that GPS(RR=1.768, 95%CI=1.043-3.000, p =0.034) remained an independent prognostic predictor in DLBCL. The area under the curve of GPS (0.735, 95%CI=0.645-0.824) was greater than that of PI(0.710, 95%CI=0.621-0.799), PNI(0.600, 95%CI=0.517-0.683), NLR(0.572, 95%CI=0.503-0.642), and PLR(0.599, 95%CI=0.510-0.689) by Harrell's C-statistics. Especially in the DLBCL patients treated with R-CHOP, GPS also remained the most powerful inflammation-based prognostic score when comparing with PI, NLR, PNI and PLR (p=0.004, p=0.000, respectively for OS and EFS). In conclusion, these results indicate that Inflammation-based prognostic scores such as GPS, PI, NLR, PNI and PLR can be used to evaluate the outcome in DLBCL patients. Among them, GPS is the most powerful tool in predicting survival in DLBCL patients, even in the rituximab era.

Disclosures

No relevant conflicts of interest to declare.
Switch to Subsequent Line of Treatment in Children and Adolescents with Chronic Myeloid Leukemia (CML) Treated with Imatinib: Experience of the International Registry for Chronic Myeloid Leukemia in Children and Adolescents (I-CML-Ped Study)
Authors: Millot Frédéric , Guilhot Joelle , Suttorp Meinolf , Meunier Anne Sophie , Adalet Meral Gunes , Sedlacek Petr , de Bont Eveline , Li Chi Kong , Kalwak Krzysztof , Lausen Birgitte , Culic Srdjana , De Moerloose Barbara , Biondi Andrea , Baruchel Andre
Published: October 2019

Aims: To determine in children and adolescents with chronic myeloid leukemia (CML) in chronic phase (CP) treated with imatinib front line, (i) the probability of switch to a second line therapy, (ii) to characterize the reasons and the type of switch and (iii) to determine the impact of the switch on outcome.

Methods: Children and adolescents (<18 years) with CML diagnosed later than the year 2000 and enrolled in the international registry for childhood CML (I-CML-Ped Study) were the subjects of the study.

Results: The I-CML-Ped study enrolled 301 children and adolescents with CML in CP treated with imatinib front line. Among them 112 patients subsequently switched to a second line therapy (median duration of imatinib treatment before the switch: 16 months [range: 1-111]).The probability of switch at 38 months was 50% (95% CI: 29-60). Primary resistance was the cause of switch in 47% of the patients: failure to achieve complete hematologic response (CHR, 1%), complete cytogenetic response (CCR, 20%) or major molecular response (MMR, 24%); not detailed (2%). A loss of response (CHR loss [2%] or CCR loss [4%] or MMR loss [13%]) or progression were the cause of switch in 19% and 4% of the patients, respectively. Occurrence of non hematologic toxicity (mainly muscle-skeleton pain) was the cause of switch in 10% of the patients. The reason of switch was the physician's choice in 20% of the patients (switch to hematopoietic stem cell transplantation [HSCT] while the patients were in MMR or deeper molecular response). The second line therapy consisted of second generation tyrosine kinase inhibitors (63%), chemotherapy (4%) or HSCT (33%). With a median follow up of 38 months (range: 2-150), overall, 8 deaths were recorded among switching patients: all were patients transplanted for acute phase (4 patients), hematologic resistance (1 patient), loss of hematologic response (1 patient) or physician's choice (2 patients). The causes of death were treatment related mortality (7 patients) and relapse (1 patient). One death only was recorded among the non switching patients. The probability of overall survival at 48 months was 90% (95% CI: 81% - 95%) for switching patients and 98% (95% CI: 89% - 100%) (p=0.005) for the non switching patients.

Conclusion: Treatment failure is the main reason for a switch to a second line therapy in children and adolescents treated with imatinib front line. Efficacy of second line therapy still needs improvement.

Disclosures

No relevant conflicts of interest to declare.
Evaluating Frequency of Anti-Xa Monitoring in Treatment of Pediatric Venous Thromboembolism
Authors: Roberts Laura E , Fan Jennifer L , Lee-Kim YoungNa J , Shah Mona D
Published: October 2019

BACKGROUND/INTRODUCTION: With the rising incidence of pediatric venous thromboembolism (VTE), it is imperative that anticoagulation treatment protocols are standardized. While unfractionated heparin and warfarin were considered the standards of care, enoxaparin has now become the drug of choice in many pediatric institutions, given improved efficacy, fewer bleeding complications, and a more favorable pharmacokinetic profile. The increased bioavailability, predictable dose response, and less frequent drug monitoring have made enoxaparin popular in the adult population. In pediatrics, there are insufficient guidelines regarding frequency of enoxaparin monitoring. At Texas Children's Hospital (TCH), we titrate the enoxaparin dose during initial hospitalization to a therapeutic Anti-Xa level of 0.5 - 1.0 units/mL. Patients are instructed to return for outpatient follow-up levels within 9 days of discharge, and monthly thereafter. It is unclear whether this frequency of monitoring is optimal.

OBJECTIVES: In an effort to reduce overall patient morbidity and healthcare costs, we evaluated current Anti-Xa level monitoring practices in children with VTEs at TCH to develop recommendations for standardizing Anti-Xa level monitoring.

METHODS: Using the TCH DVT Registry, we retrospectively identified 107 subjects diagnosed with uncomplicated VTE and treated with enoxaparin. Demographic information, Anti-Xa levels, dose modifications, and outcomes were abstracted using the electronic medical record (Epic®) and analyzed.

RESULTS: 103 patients had therapeutic Anti-Xa levels at the time of hospital discharge. As instructed, 82 (80%) patients returned for follow-up within 9 days of discharge, with only 59 (72%) patients having therapeutic Anti-Xa levels. Of these 59 subjects, only 34 patients returned to clinic for their 1-month follow-up visit, of which 27 (80%) had therapeutic levels requiring no dose adjustments. Of the original 59 subjects, 29 patients returned for their 2-month follow-up, of which 26 (90%) patients continued to have therapeutic levels.

CONCLUSIONS: Initial therapeutic levels immediately post-discharge are critical to ensuring therapeutic dosing throughout the course of therapy. Once confirmed, the majority of subjects continued to have therapeutic levels at their 1-month (80%) and 2-month follow-ups (90%). Given the discomfort of laboratory testing, costs, and time to families, it may be reasonable to decrease the frequency of laboratory monitoring at these monthly visits.

Disclosures

Off Label Use: Enoxaparin for anticoagulation of pediatric patients with venous thromboembolism.
Enhancer Landscape Dynamics and the Role of IRF8 in Mononuclear Phagocyte Development
Authors: Kurotaki Daisuke , Nakabayashi Jun , Nishiyama Akira , Sasaki Haruka , Kaneko Naofumi , Koizumi Shin-ichi , Ozato Keiko , Suzuki Yutaka , Tamura Tomohiko
Published: October 2019

Monocytes and dendritic cells (DCs) are critical mononuclear phagocytes that regulate innate and adaptive immune responses. Hematopoietic stem cells give rise to monocytes and DCs via intermediate myeloid progenitor populations, such as granulocyte-monocyte progenitors (GMPs), monocyte-DC progenitors (MDPs), and common monocyte progenitors (cMoPs) or common DC progenitors (CDPs). However, the molecular mechanism underlying their lineage determination is poorly understood. Recently, promoter-distal enhancers have been found to be key for cell fate decision. In this study, we performed chromatin immunoprecipitation-sequencing (ChIP-seq) analysis of three histone modifications (H3K4me1, H3K27ac, and H3K4me3, representing primed enhancers, active enhancers, and transcriptional intiation, respectively) in seven myeloid populations (GMPs, MDPs, cMoPs, CDPs, monocytes, DCs, and neutrophils), and identified approximately sixty thousand putative enhancer regions. We found that a majority of monocyte- and DC-specific active enhancers were gradually established at progenitor stages prior to gene expression and terminal differentiation. Computational DNA motif analysis implicated that these enhancers were regulated by combinations of lineage-determining transcription factors such as PU.1, RUNX, C/EBP, and IRF. Indeed, ChIP-seq of PU.1 confirmed that the myeloid master regulator PU.1 was the common transcription factors bound to enhancers at all the stages examined. IRF binding motifs were enriched at the enhancer regions in MDPs, cMoPs and CDPs, but not GMPs. Among IRFs, IRF8, a partner of PU.1, has been shown to be highly expressed in MDPs and their descendants, and to be required for MDP-to-CDP and cMoP-to-monocyte transitions. Indeed, ChIP-seq analysis in Irf8-/- progenitors demonstrated that the enhancer landscapes of Irf8-/- GMPs, MDPs, and cMoPs all remained similar to that of wild-type GMPs. Moreover, ChIP-seq for IRF8 binding revealed that IRF8 directly promoted the priming and activation of many enhancers in MDPs and cMoPs. These results contribute to a comprehensive understanding of how transcription factors govern the enhancer landscape dynamics during mononuclear phagocyte development.

Disclosures

No relevant conflicts of interest to declare.
Molecular Classification of Bone Marrow Failure Syndromes: Protein Signatures As Surrogate Biomarker for Accurate Diagnosis of Severe Aplastic Anemia, Hypoplastic Myelodysplastic Syndrome and Paroxysmal Nocturnal Hemoglobinuria Patients
Authors: Alaiya Ayodele , Alzahrani Hazza A , Shinwari Zakia , Owaidah Tarek , Al Mohareb Fahad , Al Sharif Fahad , Al Nounou Randa , Chaudhri Naeem A. , Aldawsari Ghuzayel , Hanbali Amr , Shaheen Marwan , Alfraih Feras , Ahmed Syed Osman , Aljurf Mahmoud
Published: October 2019

Background/Purpose: Bone marrow failure syndrome is an example of disease entity where accurate diagnosis of Severe Aplastic Anemia (SAA), Paroxysmal Nocturnal Hemoglobinuria (PNH) and Hypoplastic Myelodysplastic Syndrome (MDS) is very challenging. The aim of this study was to identify panels of disease-specific /disease-associated proteins biomarkers to be used for more objective diagnosis and better prediction of disease prognosis of patients presenting with features of bone marrow failure syndromes.

Methodology:

Bone marrow plasma (MBP) and peripheral blood plasma (PBP) samples from 20 patients with bone marrow hypoplasia; including AA/MDS/PNH were subjected to expression proteome analysis using label-free quantitative liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS).

Results: Approximately 300 unique protein species were identified of which 107 and 218 were significantly differentially expressed (> 2- ∞- fold change & p < 0.05) in BMP and PBP respectively. These protein fingerprints independently discriminates patients into three distinct clusters; AA/MDS/PNH. Furthermore, only approx. 25% of the proteins were common between the two datasets from BMP and PBP. Some of the identified proteins were filtered and mapped using Ingenuity Pathway Analysis, and were associated with five different networks. The top two of these networks involved cell-to-cell signaling interaction, hematological system development and function, and immune cell trafficking. Only three of the differentially expressed proteins were uniquely expressed in SAA and MDS but absent in PNH, thus making these proteins potential biomarkers.

The probable diagnostic utility of these proteins would be validated in large archival clinical samples. Our data indicates the utility of multivariate analysis of quantitative proteome data as a means of discovery of disease related or disease specific biomarkers for bone marrow syndromes.

Conclusions:

We have identified protein signatures capable of objective classification of bone marrow failure syndromes patients. Our expression proteomics strategy is very promising for identification of clinically useful biomarkers. These proteins once validated, on a larger cohort of patients, might be valuable to complement the currently existing parameters for reliable and objective disease diagnosis, monitoring treatment response and clinical outcome of bone marrow failure syndrome patients.

Disclosures

Owaidah: King abdulaziz city for science, Novo Nordisk, Bayer: Honoraria, Research Funding.
PKR Inhibits Hematopoietic Stem Cell Differentiation
Authors: Bennett Richard L , Byrne Michael T. , Cheng Xiaodong , Patel Mohini , Patel Zeel , Asous Amalin , May W. Stratford
Published: October 2019

Recent work from our laboratory has demonstrated that increased expression and activation of the interferon-inducible, dsRNA-activated kinase (PKR) is associated with inferior survival for many hematologic malignancies including AML. Mice expressing a PKR transgene (TgPKR) specifically in hematopoietic cells develop a preleukemic/myelodysplastic syndrome (MDS)-like disease consisting of peripheral blood (PB) cytopenias and bone marrow (BM) dysplasia with an increased frequency of BM blasts. In addition, TgPKR mice have a reduced number of BM Lin-, Sca+, cKit+ hematopoietic stem and progenitor cells (HSPCs) and an increased percentage of these cells are quiescent (G0) compared to WT mice. New data now reveals that BM of TgPKR mice have a significantly reduced frequency of multipotent progenitor cells (MPPs) compared to wild type (WT) mice. Furthermore, MPPs isolated from BM of TgPKR mice display a partial block of the cell cycle in G0/G1 compared to MPPs from WT mice. In contrast, PKR knockout mice (PKRKO) have an increased number of HSPCs that are more frequently proliferating (S+G2/M) than WT mice. While the BM of PKRKO mice have a significantly increased frequency of MPPs there is a decreased frequency of both phenotypic LT- and ST-HSCs compared to either WT or TgPKR mice. Thus, in long term culture initiating cell (LTC-IC) assays BM of TgPKR mice has nearly double the colony forming capacity after >4 weeks in culture compared to BM of PKRKO mice, indicating that the TgPKR expressing mice have increased numbers of HSCs that are functional in vitro but not in vivo. Significantly, K562 cells with reduced PKR expression by siRNA knockdown display an increased rate and extent of PMA-induced megakaryocyte differentiation compared to control cells. Taken together these results suggest that increased PKR expression/activation as observed in high-risk MDS, AML and other hematologic malignancies may have a previously unrecognized function to block hematopoietic cell differentiation. This may account, at least in part, for why TgPKR mice accumulate quiescent HSPCs that are unable to terminally differentiate in vivo. Since we recently discovered that nuclear PKR inhibits DNA damage response signaling that results in an increased frequency of somatic mutations, we propose that increased PKR may contribute to leukemogenesis by promoting both genomic instability and a differentiation block in the HSPC populations.

Disclosures

No relevant conflicts of interest to declare.
Oncogenic Nras Grants Apoptosis Resistance to Hematopoietic Stem Cells
Authors: Yang Kevin B , Ng Victor , Ney Gina , Liu Lu , Jin Xi , Li Qing
Published: October 2019

Genetic lesions hijack the regulatory programs of hematopoietic stem cells and progenitors (HSC/P) to transform them into leukemic stem cells (LSC). These initiating mutations confer a competitive advantage to promote clonal expansion of pre-leukemic stem cells (pre-LSC). Pre-LSCs are believed to serve as a reservoir for leukemia relapses and a cure for leukemia likely depends on the eliminating of pre-LSCs. Better understanding of how mutations dysregulate HSCs to transform them into pre-LSCs will identify new therapeutic targets to eliminate pre-LSCs.

Activating RAS mutations are highly prevalent in hematopoietic malignancies. We have previously shown that a single allele of oncogenic NrasG12D promotes clonal expansion in hematopoietic stem cells (HSCs) through increased proliferation and self-renewal, but the role apoptosis plays in the clonal dominance of NrasG12D HSCs remains unclear. Here we report that oncogenic NRasG12D protects HSCs from cellular stress. Upon cytokine starvation and γ-irradiation, HSCs fromMx1-cre; LSL-NrasG12D/+ mice display reduced apoptosis as measured by Annexin V staining and Caspase 3/7 activation. This NRasG12D-mediated HSC survival is not dependent on autophagy since Bafilomycin A, an inhibitor previously shown to inhibit autophagy in HSCs, did not rescue the phenotype. Moreover, NrasG12D HSCs exhibits decreased levels of cellular reactive oxygen species (ROS), and restoration of ROS levels with buthionine sulfoximine significantly blocked the survival of NrasG12D HSCs.

We next sought to identify the signaling activated by NRasG12D to promote HSC survival. Although our previous studies show that STAT5 is required for NRasG12D-mediated HSC proliferation, STAT5 is dispensable for NRasG12D-mediated HSC survival. We then determined whether inhibition of the canonical Ras effector pathways blocks NRasG12D-mediated HSC survival. To our surprise, inhibition of PI3K/AKT, MEK/ERK, or mTOR signaling did not abrogate the pro-survival effect of NRasG12D in HSCs. However, inhibition of protein kinase C (PKC) with two structurally independent inhibitors rescued the pro-survival phenotype of NrasG12D/+ HSCs. PKC inhibition also led to increased ROS levels in NrasG12D mutant HSCs, suggesting that NRasG12D reduces ROS and protects HSCs from stress through activation of PKC signaling. Taken together, we discover that in addition to increasing HSC proliferation, oncogenic NRasG12D promotes HSC survival under stress conditions. Furthermore, we reveal a pathway NRasG12D relies on to evade apoptosis and manage reactive oxygen species in HSCs independent of the STAT5 signaling that governs cell proliferation. Targeting PKC signaling, alone or in combination with STAT5 signaling, may have therapeutic benefit in eliminating pre-LSCs.

Disclosures

No relevant conflicts of interest to declare.
A Novel Feed-Forward Loop Involving the High Mobility Group A1 (HMGA1) Chromatin Remodeling Protein and cMYC in Acute Myeloid Leukemia Is Targeted By JQ1
Authors: Resar Linda , Rushworth Stuart A , Xian Lingling , Bowles Kristian
Published: October 2019

Acute myeloid leukemia (AML) comprises a heterogeneous group of highly lethal, clonal disorders of blood cell progenitors for which more effective therapies are urgently needed. The underlying genetic abnormalities transform hematopoietic progenitors into AML blasts that are resistant to apoptosis and fail to undergo normal differentiation. While conventional chemotherapy regimens frequently ablate actively cycling leukemic blasts and induce remissions, most patients relapse with abysmal outcomes. Recent studies highlight an important role for stem cell transcriptional networks in supporting survival and resistance to chemotherapy in leukemic blasts, although druggable targets that maintain these networks have remained elusive. We previously discovered that the cMYC oncoprotein activates HMGA1 expression in experimental models of lymphoid leukemia, while HMGA1 directly induces cMYC in embryonic stem cells. Here, we demonstrate for the first time that the HMGA1-cMYC network is a therapeutic target that is critical for leukemic cell survival and resistance to chemotherapy.

In primary AML blasts, we found that both HMGA1 and cMYC gene expression are enriched relative to levels in normal CD34+ hematopoietic stem and progenitor cells and their expression is positively correlated. Silencing HMGA1 or cMYC in AML blasts dramatically halts cell growth, leading to rapid apoptotic cell death. HMGA1 transcriptionally activates cMYC in leukemic blasts, while the cMYC oncoprotein, in turn, induces HMGA1, resulting in potent feed-forward loop that maintains survival in AML blasts.We also discovered that AML blasts exposed to AraC (cytosine arabinoside), a standard cytotoxic agent used in AML, become resistant by up-regulating both HMGA1 and cMYC. Moreover, silencing either HMGA1 or cMYC restores sensitivity to AraC, demonstrating that induction of HMGA1 and cMYC is functionally important. Next, we sought to determine whether the inhibitor of the BET family of bromodomain proteins, JQ1, has anti-leukemic effects in AML blasts resistant to AraC because it functions, at least in part, by disrupting cMYC transcriptional complexes. We found that cMYC is repressed within 4 hours, while HMGA1 requires 24 hours for down-regulation following exposure to JQ1. Moreover, JQ1 restores sensitivity to AraC in the resistant AML blasts.

Together, these studies reveal a novel role for HMGA1 and cMYC in a feed-forward loop that is critical for AML blast survival and chemoresistance. These findings also suggest that JQ1 will be effective therapy in AML blasts that are resistant to AraC.

Disclosures

No relevant conflicts of interest to declare.
A Therapeutic TCR Mimic Monoclonal Antibody for Intracellular PRAME Protein in Leukemias
Authors: Chang Aaron , Dao Tao , Scott Andrew , Dubrovsky Leonid , Liu Cheng , Scheinberg David A.
Published: October 2019

Preferentially expressed antigen in melanoma (PRAME) is a well-validated target for T cell-based immunotherapy in leukemias and solid tumors. PRAME is a retinoic acid receptor binding protein that prevents retinoic acid-mediated differentiation, proliferation arrest, and apoptosis. As a cancer-testis antigen, PRAME has limited expression in healthy adult tissue that is restricted to the testes, ovaries, and endometrium. However, PRAME is over-expressed in multiple cancers including ALL, AML, melanomas, and breast cancers, making it a specific and highly attractive therapeutic target. PRAME is an intracellular protein making it impossible to target using traditional antibodies and it is not currently druggable. After proteasomal processing, the PRAME300-309 peptide is presented on the cell surface in the context of HLA*A02:01 molecules, for recognition by CD8 T cells. We therefore hypothesized that a TCR-mimic (TCRm) monoclonal antibody that recognizes surface PRAME300-309 presented by HLA*A02:01 could have therapeutic activity. Here, we describe Pr20, a therapeutic TCRm antibody, specific for the PRAME300-309 peptide in complex with HLA*A02:01, identified through a human scFv phage display library screen. Pr20 was engineered into full length human IgG1. Pr20 exhibited specific binding to PRAME300-309 -pulsed TAP-deficient T2 cells and bound PRAME+/ HLA*A02:01+ Ph+ ALL and AML, demonstrating that endogenously presented PRAME300-309 could be recognized by Pr20. Pr20 was determined to have 4 nM binding affinity by scatchard plot analysis. The specific epitope was mapped using alanine substitutions of non-anchor residues in the PRAME300-309 peptide and determined to primarily require the C-terminal residues. Pr20M, an afucosylated form of the antibody with enhanced Fc binding, mediated antibody-dependent cellular cytotoxicity (ADCC) in-vitro in a PRAME+/ HLA*A02:01+ restricted manner. Pharmacokinetic studies in C57BL/6 mice indicated that Pr20M was stable in-vivo and biodistribution studies in HLA*A02:01 transgenic mice suggested that there was no significant antibody sink. Pr20M was therapeutically active in established xenograft leukemia models in NSG mice (T, B, and NK-deficient). Interestingly, Pr20 binding to PRAME+/HLA*A02:01+ melanomas was minimally detectable, but was dramatically increased upon treatment with IFNγ, which also led to an increased sensitivity to ADCC. The data provide rationale for developing TCRm antibodies against intracellular oncoproteins as therapeutics.

Disclosures

No relevant conflicts of interest to declare.
Plasmacytoid Dendritic Cell Burden in the Bone Marrow Predicts End of Induction Minimal Residual Disease Status in Adult Acute Myeloid Leukemia
Authors: Subramanian Papagudi Ganesan , Patkar Nikhil , Tembhare Prashant , Badrinath Yajamanam , Ghogale Sitaram G , Gujral Sumeet , Khattry Navin
Published: October 2019

Introduction:

Plasmacytoid dendritic cells (pDCs) are a subset of immune cells that secrete type 1 interferons and serve as antigen presenting cells. In many tumors increased pDC frequencies have been observed and are involved in tumor response initiation. However, there is not much data in acute myeloid leukemia especially in the context of minimal residual disease. Here we evaluated the frequencies of pDCs in the post induction bone marrow and found a significant correlation with MRD levels.

Methods:

All adult (>18 years ) of patients who were treated for AML [other than AML with t(15;17)] were accrued over a 2 year period. The presence of MRD was assessed using 8 colour flow cytometry on a post induction bone marrows using CD45, CD36, CD38, CD123, CD33, CD117, CD34, HLADR, CD7, CD56, CD13, CD19, CD16, CD11b, CD15 and CD14. Minimum of 500,000 events were acquired/tube on an 8 colour BD FACS Canto II or a 10 colour BC Navios instruments. Kaluza software (v1.3) was used to analyze the .fcs files. MRD was calculated as a percentage of abnormal leukemic cells per total viable cells as gated in forward scatter v side scatter plot. pDCs were calculated as CD123 bright population which expressed HLA-DR (while gating on the progenitors and monocytes based on their expression of CD45 and side scatter). The pDC percentages were counted as a fraction of all viable cells. Based on the results the levels of pDCs were divided into pDC High and pDC Low groups. Statistical analysis was done using Chi squared groups.

Results:

After exclusion of induction deaths, a total of 94 patients of adult AML was assessed for the presence of MRD at the end of induction. Of these MRD was detected in 48 (51.1%, range 0.01-40%). pDC values ranged from <0.01% to 1.95% (median 0.15%). Median pDC value in the MRD positive group was lower (0.05%) as compared to the MRD negative group (0.23%). Out of 94 patients of 44.7% patients belonged to the pDC Low group of which majority patients were MRD positive (66.7% of the pDC Low group). Similarly majority of patients in the pDC High group were MRD negative (61.5% of the pDC High group). A statistical analysis of these categories was also found to be significant (p=0.008)

Conclusion:

These pilot data seem to indicate that the pDC burden in the bone marrow may have a role in influencing MRD clearance in the bone marrow. A detailed investigation of the pDC function in the bone marrow microenvironment is warranted.

Disclosures

No relevant conflicts of interest to declare.
Mitochondrial Protein Kinase A Regulates Heme Biosynthesis
Authors: Chung Jacky , Wittig Johannes G. , Bauer Daniel E. , Coon Joshua J. , Pagliarini Dave J. , Dailey Harry A. , Lodish Harvey F. , Paw Barry H.
Published: October 2019

During erythropoiesis, heme production becomes dramatically increased to support the production of oxygen-carrying hemoglobin. Heme biosynthesis requires an eight-step enzymatic cascade culminating with the insertion of ferrous iron into protoporphyrin IX by the mitochondrial enzyme ferrochelatase (FECH). This last step is the rate-limiting step in red blood cells (RBCs) and represents a critical regulatory juncture in RBC biology and human blood diseases. Most notably, in humans, FECH mutations are strongly associated with a disorder called erythropoietic protoporphyria (EPP) that typically presents with mild anemia and porphyria resulting from inadequate heme production and upstream accumulation of porphyrin intermediates, respectively. Disease severity can be highly heterogeneous, suggesting that additional modulators of FECH likely exist. However, despite its implications in EPP and other human blood disorders, very little is known regarding such regulatory mechanisms.

Here, using complementary biochemical and genetics approaches, we identify FECH as a direct physiologic target for protein kinase A (PKA) phosphorylation during erythroid maturation. Quantitative proteomics revealed that PKA becomes enriched in mitochondria of differentiating murine erythroid cells. Phosphorylation of a highly conserved Thr116 in the catalytic domain of human FECH by PKA increases its activity resulting in elevated hemoglobinization of erythroblasts. We examined the importance of this phosphorylation in vivo by taking advantage of CRISPR/Cas9-mediated genome editing to knock-in the analogous Thr115Ala substitution into the endogenous Fech gene in murine RBCs. This approach allowed us to examine FECH function in a more physiologic context. Murine erythroid cells harboring only Thr115Ala Fech show reduced ferrous iron incorporation into protoporphyrin IX and, consequently, compromised hemoglobinization. In primary murine erythroid cells, we demonstrate that a distinct PKA gene expression signature is induced early in erythropoiesis. This suggests that the PKA pathway is engaged by physiologic signaling mechanisms during RBC development.

Together, our results support a model where in maturing RBCs, PKA becomes enriched in the mitochondria where it phosphorylates FECH. Phosphorylation of FECH is required for full activity to support elevated heme biosynthesis and hemoglobin production. Our data also uncover PKA as a novel mechanism that links early erythroid signaling pathways to regulate heme biosynthesis and suggests that porphyrin accumulation can result not only from defective porphyrin transport but also aberrant cell signaling.

Figure 1. Figure 1.

Disclosures

Bauer: Biogen: Research Funding; Editas Medicine: Consultancy.