Patients with acute myeloid leukemia (AML) frequently relapse after chemotherapy, yet the mechanism by which AML reemerges is not fully understood. Herein, we show that primary AML cells enter a ...senescence-like phenotype following chemotherapy
and
. This is accompanied by induction of senescence/inflammatory and embryonic diapause transcriptional programs, with downregulation of
and leukemia stem cell genes. Single-cell RNA sequencing suggested depletion of leukemia stem cells
and
, and enrichment for subpopulations with distinct senescence-like cells. This senescence effect was transient and conferred superior colony-forming and engraftment potential. Entry into this senescence-like phenotype was dependent on ATR, and persistence of AML cells was severely impaired by ATR inhibitors. Altogether, we propose that AML relapse is facilitated by a senescence-like resilience phenotype that occurs regardless of their stem cell status. Upon recovery, these post-senescence AML cells give rise to relapsed AMLs with increased stem cell potential. SIGNIFICANCE: Despite entering complete remission after chemotherapy, relapse occurs in many patients with AML. Thus, there is an urgent need to understand the relapse mechanism in AML and the development of targeted treatments to improve outcome. Here, we identified a senescence-like resilience phenotype through which AML cells can survive and repopulate leukemia.
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The most aggressive B cell lymphomas frequently manifest extranodal distribution and carry somatic mutations in the poorly characterized gene TBL1XR1. Here, we show that TBL1XR1 mutations skew the ...humoral immune response toward generating abnormal immature memory B cells (MB), while impairing plasma cell differentiation. At the molecular level, TBL1XR1 mutants co-opt SMRT/HDAC3 repressor complexes toward binding the MB cell transcription factor (TF) BACH2 at the expense of the germinal center (GC) TF BCL6, leading to pre-memory transcriptional reprogramming and cell-fate bias. Upon antigen recall, TBL1XR1 mutant MB cells fail to differentiate into plasma cells and instead preferentially reenter new GC reactions, providing evidence for a cyclic reentry lymphomagenesis mechanism. Ultimately, TBL1XR1 alterations lead to a striking extranodal immunoblastic lymphoma phenotype that mimics the human disease. Both human and murine lymphomas feature expanded MB-like cell populations, consistent with a MB-cell origin and delineating an unforeseen pathway for malignant transformation of the immune system.
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•TBL1XR1 mutation skews the humoral immune response toward producing memory B cells•TBL1XR1 mutant memory cells feature aberrant cyclic reentry to new germinal centers•Mutant TBL1XR1 acts by triggering aberrant targeting of SMRT complex to BACH2•TBL1XR1 mutation gives rise to extranodal ABC-DLBCLs derived from memory B cells
A subset of B cell lymphomas is driven by mutations that impair plasma cell differentiation and instead bias cell fate toward immature memory B cells, which preferentially re-enter germinal center reactions to drive lymphomagenesis.
Disruption of epigenetic regulation is a hallmark of acute myeloid leukemia (AML), but epigenetic therapy is complicated by the complexity of the epigenome. Herein, we developed a long-term primary ...AML
platform to determine whether targeting different epigenetic layers with 5-azacytidine and LSD1 inhibitors would yield improved efficacy. This combination was most effective in
AML, where it extinguished leukemia stem cells and particularly induced genes with both LSD1-bound enhancers and cytosine-methylated promoters. Functional studies indicated that derepression of genes such as
contributes to drug efficacy. Mechanistically, combination therapy increased enhancer-promoter looping and chromatin-activating marks at the
locus. CRISPRi of the LSD1-bound enhancer in patient-derived
AML was associated with dampening of therapeutic
induction.
knockdown in human hematopoietic stem/progenitor cells induced loss of enhancer 5-hydroxymethylation and facilitated LSD1-mediated enhancer inactivation. Our data provide a basis for rational targeting of cooperating aberrant promoter and enhancer epigenetic marks driven by mutant epigenetic modifiers. SIGNIFICANCE: Somatic mutations of genes encoding epigenetic modifiers are a hallmark of AML and potentially disrupt many components of the epigenome. Our study targets two different epigenetic layers at promoters and enhancers that cooperate to aberrant gene silencing, downstream of the actions of a mutant epigenetic regulator.
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BACKGROUND
Psoralea corylifolia L. (PC) was commonly used to treat miscarriages clinically. The aim of this study was to examine its embryotoxicity in mice and embryonic stem cells (ESCs).
METHODS
...Quality control of PC extract including reference marker compounds, pesticide residues, and heavy metals was authenticated with HPLC, Gas chromatography‐mass spectrometry (GC‐MS), and inductively coupled plasma‐mass spectrometry. Pregnant mice were randomly assigned into five groups and dosed with distilled water (G1), PC extract of 2 (G2), 4 (G3), or 8 g/kg/day (G4), and vitamin A (G5). Meanwhile, half maximal inhibitory concentration values for ESCs and 3T3 cells were identified in a cytotoxicity assay, and apoptosis in neuroepithelium was assessed by transmission electron microscopy.
RESULTS
In the G4 group, a statistically significant decrease in the total fetus, live fetus, and gravid uterine weight, and increase in the resorbed fetus, postimplantation loss, and neuroepithelial apoptosis as well as maternal liver‐weight were found (p < 0.05).
CONCLUSIONS
PC extracts at 8 g/kg/day might cause fetal toxicity and maternal liver damage in mice, although it did not cause typical malformation and ESC's cytotoxicity in this experiment. Our data suggested that high dosage and long‐term administration of PC preparations may not be safe for pregnant women.
It is common for the airlines to sell a pool of identical seats at different prices according to different booking classes to improve revenues in a very competitive market. Under this practice, a ...complex yet very crucial problem is to determine whether a booking request for seats in a certain booking class occurring at some point in time during the booking period should be accepted or denied. This paper develops a discrete-time dynamic programming model for finding an optimal booking policy, which can be reduced to a set of critical values. Unlike many existing models, this model does not require any assumptions about the arrival pattern for the various booking classes. Furthermore, multiple seat bookings, which are a practical issue in airline seat inventory control, are also incorporated into the model. In this paper, the basic properties of the model are studied. Numerical examples are presented. Computational issues, including the computational efficiency of the model, are also discussed.
We have previously reported that hypermethylationof the GADD45A promoter (GADD45AmeHI) occurs frequently in AML at a specific CpG residue (CpG1) and associates with poor overall survival for patients ...on standard chemotherapy (Perugini et al, Leukemia 2013). Sequenom multiplex analysis of 195 AML patients revealed a co-occurrence of GADD45AmeHI with recurrent mutations at conserved residues in IDH1 and IDH2 (p<0.0001, Fisher’s exact test). These mutations in IDH1 and IDH2 result in enzyme isoforms that produce high levels of the onco-metabolite 2-hydroxyglutarate with a wide-range of effects including inhibition of α-KG-dependent dioxygenases and association with a profound DNA hypermethylation phenotype in AML (Figueroa et al, Cancer Cell 2010). Furthermore these mutations are found in pre-leukemic AML clones (Shlush et al, Nature 2014) and lead to pre-leukaemic phenotypes in mouse models (Sasaki et al, Nature 2012, Kats et al, Cell Stem Cell 2014, Ogawara et al, Cancer Research 2015). Here we investigated the relationship between hypermethylation at GADD45A CpG1, IDH1/2 mutation status, global methylation patterns and patient survival.
We performed survival analysis to determine disease-free survival (DFS) and relapse-free survival (RFS) for AML patients with GADD45AmeHI or IDH1/2-mutations. This showed that GADD45AmeHI is a significant independent predictor of poor DFS and RFS, particularly in normal karyotype AML (Cox regression analysis, NK-AML DFS, P=0.009 HR=2.55, RFS, P=0.003 HR=2.75). Despite the co-association of GADD45AmeHI with mutations in IDH1 and IDH2, the mutation status of IDH1/2 did not predict DFS or RFS in these patients. To examine further the relationship between GADD45AmeHI and IDH1/2-mutation, and to investigate how this might influence tumour cell biology in AML, we determined global methylation patterns for a panel of AML diagnosis (Dx) samples (base-pair-resolution analysis using enhanced reduced representation bisulfite sequencing; ERRBS) in which both GADD45AmeHI and IDH mutation status has been determined. Unsupervised analyses of global methylation patterns grouped the AML Dx samples into three clusters including cluster 1 (n=12) which was associated with GADD45AmeHI samples with IDH- mutations, cluster 2 (n=13) which was enriched for GADD45AmeHI lacking IDH- mutations, and cluster 3 (n=9) which was associated with GADD45AmeLO (low CpG1 methylation) IDH-WT AML. We propose that this CpG in the GADD45A promoter may be subject to alternative events affecting DNA methylation in AML pathogenesis, including events distinct from IDH1/2 mutation. Finally, in GADD45AmeHI AML we detected hypermethylated regions compared to CD34+ normal bone marrow controls within 2016 gene promoters, 848 of which were unique to the GADD45AmeHI samples and not present in IDH1/2-mutant samples. We hypothesize that these differentially methylated genes may contribute mechanistically to the poor survival observed for this subtype.
To determine how GADD45AmeHI status might associate with disease progression, DNA methylation assessment was performed on the patient panel-matched relapse samples (Rx). While GADD45AmeHI occurs frequently in both cluster 1 and 2 there is a significant difference in level of GADD45A CpG1 methylation at Dx and Rx for samples in cluster 1 vs cluster 2 and 3 (Figure 1), consistent with mutant IDH1/2 activity influencing methylation levels at this CpG site. Given that GADD45A has an established basal role in the maintenance of genomic stability (Liebermann & Hoffman, Springer 2013), and is a determinant of HSC self-renewal and response to genotoxic insult (Wingert et al, Stem Cells 2016, Chen et al, Blood 2014) we are also investigating whether GADD45A methylation and silencing plays a direct role in determining aggressiveness and response to chemotherapy for GADD45AmeHIAML.
In conclusion this data suggests that methylation at this specific CpGof the GADD45A promoter, in combination with IDH1/2 mutation status, associate with varying global methylation phenotypes. Importantly, we demonstrate that GADD45AmeHI better predicts poorer prognosis than IDH1/2 mutation status, despite the significant co-association of these characteristics in AML.
SES and FEGB contributed equally to this work.
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Guzman:Cellectis: Research Funding. Roboz:Agios, Amgen, Amphivena, Astex, AstraZeneca, Boehringer Ingelheim, Celator, Celgene, Genoptix, Janssen, Juno, MEI Pharma, MedImmune, Novartis, Onconova, Pfizer, Roche/Genentech, Sunesis, Teva: Consultancy; Cellectis: Research Funding. Levine:Qiagen: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy. Melnick:Janssen: Research Funding.
Epigenetic silencing of tumor suppressor genes, mediated by aberrant DNA hypermethylation and repressive histone modifications, is a hallmark of acute myeloid leukemia (AML). Novel epigenetic ...therapies are emerging, but single-agent approaches targeting only one category of epigenetic marks have limited therapeutic activity. We show that combinatorial treatment strategy targeting epigenetic modifications of both promoter and enhancer elements can improve therapeutic intervention.
As current cell lines do not accurately represent the heterogeneous nature of AML, we established an ex vivo culturing system enabling us to propagate primary AML specimens long enough to test epigenetic therapeutics (48 specimens expanded 4-6 weeks; 6 specimens expanded >12 months; 39 specimen fail to expand). We tested 52 expandable primary AMLs with a combination therapy utilizing the DNA demethylating agent 5-Azacytidine (5-Aza) and a novel small molecule inhibitor against LSD1, a histone demethylase that removes enhancer-associated marks (mono-/di-methylated histone H3 lysine 4; H3K4me1/2). In order to avoid DNA damage-induced cytotoxicity, we applied a non-DNA damaging dose of 5-Aza (≤ 200 nM). 5-Aza treatment had modest inhibition of cell growth for the majority of cases, but did not appreciably alter cell viability. Treatment with the LSD1 inhibitor (LSD1i) substantially impaired cell growth and survival of ~80% (p<0.001) of cultured primary AMLs. Notably, we found that dual combination therapy using LSD1i and 5-Aza demonstrated significantly improved inhibition of cell viability and growth in most LSD1i-resistant AML cases. Targeted resequencing analysis of 200 genes recurrently mutated in myeloid malignancies revealed an association of mutations with therapeutic activity; TET2mut AML cases without concurrent DNMT3A mutations were most substantially impaired by combinatorial treatment (p<0.001). Also, leukemia burden was significantly reduced after combination therapy in NSG mice transplanted with luciferase-labelled TET2mut patient-derived AML cells (p<0.0002).
To gain insight into mechanism, we performed a genome-wide integrative analysis of the DNA methylome (Enhanced Reduced Representation Bisulfite Sequencing), LSD1 occupancy sites (ChIP-sequencing) and gene expression profiling (RNA-sequencing) in a TET2mut patient-derived AML sample. Single-agent LSD1i treatment led to induction of genes associated with myeloid differentiation. Even though 5-Aza induced substantial hypomethylation, there was only a minor effect on gene expression. However, combination therapy did induce greater de-repression of genes, in particular those where 5-Aza induced hypomethylation at promoters and at LSD1-occupied enhancers. Combination treatment especially potentiated induction of differentiation genes, suggesting potential anti-leukemia stem cell effects. Accordingly, combination therapy resulted in impaired colony formation potential (p=0.007). Moreover, limiting dilution analysis in NSG mice demonstrated significant impairment of leukemia-initiating cells (p=0.0025) after 5-Aza+LSD1i combination therapy when compared to either drug alone.
To determine what genes mediate the effect of combination therapy, we studied genes preferentially induced by 5-Aza+LSD1i. GATA2, a transcription factor involved in hematopoietic differentiation, was more potently induced by combination therapy in primary AML samples vs. either drug alone. We found LSD1 occupied at the GATA2 distal hematopoietic enhancer and verified by using quantitative ChIP analysis that inhibition of LSD1 induced an increase of H3K4me2 and H3K27ac, a mark that reflects enhancer activation. Overexpression of GATA2 using viral transduction in two patient-derived AML cases caused partial differentiation and attenuated leukemia growth. These results indicated that cellular responses to combination therapy are in part mediated by induction of GATA2.
In summary, our results provide a novel link for therapeutic approaches targeting redundant epigenetic silencing of tumor suppressors via cooperative enhancer and enhancer-promoter activation.
Duy:GlaxoSmithKline: Research Funding. Mohammad:GlaxoSmithKline: Employment. Smitheman:GlaxoSmithKline: Employment. Guzman:Cellectis: Research Funding. Kruger:GlaxoSmithKline: Employment. Creasy:GlaxoSmithKline: Employment. Levine:Novartis: Consultancy; Qiagen: Membership on an entity's Board of Directors or advisory committees. Melnick:Janssen: Research Funding.
Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. Unfortunately, a significant proportion of patients relapse after responding to initial treatment reflecting our poor ...understanding of the mechanisms mediating therapy resistance and relapse. We hypothesized that understanding the evolution of the mutational landscape between diagnosis and relapse is essential in order to identify mutational markers associated with sensitivity or resistance to treatment. To address this hypothesis we assembled a cohort of 53 clinically annotated, paired AML patient samples (diagnosis, relapse and patient-matched germline samples; mean age = 52 years). All patients achieved clinical remission after treatment with combination chemotherapy (cytarabine arabinoside and an anthracycline) during induction phase followed by consolidation chemotherapy treatment with or without a stem cell transplantation in first remission. Serial samples were collected at the time of initial diagnosis and within three months of relapse (mean time to relapse 455 days).
We performed whole-exome and targeted capture followed by high-throughput sequencing. We aligned samples with BWA, recalibrated them with The Genome Analysis Toolkit (GATK) and then compiled integrated calls from substitution and indel callers (Mutect, Scalpel, Strelka, Varscan and Somatic Sniper). We performed several layers of post-processing filtering on these calls, including removing non-oncogenic mutations and previously documented non-somatic variants, and correcting for the variant allele fraction of indel calls. We filtered out the variants that were found to occur in non-copy number neutral re-arrangements using the clinically determined cytogenetic data. Furthermore, we assessed for copy number events, including loss of heterozygosity events, and for the presence and the variant allele frequency of the FLT3-ITD in our samples.
We observed a median of 4.5 and 5 mutations per patient at diagnosis and relapse, respectively, with 3.5 mutations being shared by paired diagnosis and relapse samples. When limiting our analysis to genes previously shown to contribute to leukemogenesis, we found a median of 1.5 and 2 mutations per patient at diagnosis and relapse, with 1 mutation being shared. FLT3, DNMT3A, IDH2, NRAS, RUNX1 and TET2 were among the most commonly mutated genes, with a detected presence rate of 28%, 25%, 19%, 19%, 11% and 11%, respectively, in the diagnosis samples and 39%, 23%, 19%, 4%, 13% and 11% in the relapse samples. We identified significant variation in the variant allele frequency (VAF) for several of the mutations related to these genes and others, denoting variations in the cellular prevalence of the related clones after adjustment for tumor content using the mutations with the highest VAF to delineate clonal architecture. Specifically, we observed that DNMT3A, IDH2, TET2 variants are most commonly present in the bulk AML clone, and persist after treatment. WT1, GATA2 and FLT3mutations are predicted to confer relative resistance to standard combination chemotherapy treatment based on their increased VAF at relapse, whereas KRAS and NRAS subclone(s) are more sensitive to chemotherapy since their VAFs decrease following multiagent chemotherapy. Fifteen patients presented new events in leukemogenesis-related genes at relapse.
Overall, our results support a model of AML as a disease with a complex mutational hierarchy and clonal architecture and provide further insight into how these change in response to standard induction therapy. Our data suggests that future efforts to develop targeted therapies with maximal clinical benefit in combination with standard induction treatments should be placed on mutated genes identified to be more strongly associated with disease relapse.
Authors contributed equally: F. Rapaport and M.R. De Massy
Authors contributed equally: A. al Hinai and M.A. Sanders
Guzman:Cellectis: Research Funding. Roboz:Cellectis: Research Funding; Agios, Amgen, Amphivena, Astex, AstraZeneca, Boehringer Ingelheim, Celator, Celgene, Genoptix, Janssen, Juno, MEI Pharma, MedImmune, Novartis, Onconova, Pfizer, Roche/Genentech, Sunesis, Teva: Consultancy. Melnick:Janssen: Research Funding. Levine:Qiagen: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.
Dear Editor Gene networks provide a system-level overview of genetic organizations and enable the dissection of functional modules underlying complex traits. Integration of diverse genomics data ...based on the Bayesian statistics framework has been successfully applied to the construction of genome-scale functional networks for major crop species such as rice （Lee et al., 2011）, soybean （Kim et al., 2017）, and tomato （Kim et al.,
ABO incompatibility is no longer considered a contraindication for adult living donor liver transplantation (ALDLT) due to various strategies to overcome the ABO blood group barrier. We report the ...largest single‐center experience of ABO‐incompatible (ABOi) ALDLT in 235 adult patients. The desensitization protocol included a single dose of rituximab and total plasma exchange. In addition, local graft infusion therapy, cyclophosphamide, or splenectomy was used for a certain time period, but these treatments were eventually discontinued due to adverse events. There were three cases (1.3%) of in‐hospital mortality. The cumulative 3‐year graft and patient survival rates were 89.2% and 92.3%, respectively, and were comparable to those of the ABO‐compatible group (n = 1301). Despite promising survival outcomes, 17 patients (7.2%) experienced antibody‐mediated rejection that manifested as diffuse intrahepatic biliary stricture; six cases required retransplantation, and three patients died. ABOi ALDLT is a feasible method for expanding a living liver donor pool, but the efficacy of the desensitization protocol in targeting B cell immunity should be optimized.
This article presents the clinical results of ABO‐incompatible adult living donor liver transplantation in a single institution.
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