ABSTRACT
The implementation of targeted therapies for acute myeloid leukaemia (AML) has been challenging because of the complex mutational patterns within and across patients as well as a dearth of pharmacologic agents for most mutational events. Here we report initial findings from the Beat AML programme on a cohort of 672 tumour specimens collected from 562 patients. We assessed these specimens using whole-exome sequencing, RNA sequencing and analyses of ex vivo drug sensitivity. Our data reveal mutational events that have not previously been detected in AML. We show that the response to drugs is associated with mutational status, including instances of drug sensitivity that are specific to combinatorial mutational events. Integration with RNA sequencing also revealed gene expression signatures, which predict a role for specific gene networks in the drug response. Collectively, we have generated a dataset-accessible through the Beat AML data viewer (Vizome)-that can be leveraged to address clinical, genomic, transcriptomic and functional analyses of the biology of AML.
Subject(s)
Gene Expression Regulation, Neoplastic/genetics , Genome, Human/genetics , Genomics , Leukemia, Myeloid, Acute/genetics , Core Binding Factor Alpha 2 Subunit/genetics , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA Methyltransferase 3A , Datasets as Topic , Exome/genetics , Female , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/metabolism , Male , Molecular Targeted Therapy , Nuclear Proteins/genetics , Nucleophosmin , Proto-Oncogene Proteins/genetics , Repressor Proteins/genetics , Sequence Analysis, RNA , Serine-Arginine Splicing Factors/geneticsABSTRACT
Acute myeloid leukemia (AML) is the most common acute leukemia in adults, with approximately four new cases per 100,000 persons per year. Standard treatment for AML consists of induction chemotherapy with remission achieved in 50 to 75% of cases. Unfortunately, most patients will relapse and die from their disease, as 5-y survival is roughly 29%. Therefore, other treatment options are urgently needed. In recent years, immune-based therapies have led to unprecedented rates of survival among patients with some advanced cancers. Suppression of T cell function in the tumor microenvironment is commonly observed and may play a role in AML. We found that there is a significant association between T cell infiltration in the bone marrow microenvironment of newly diagnosed patients with AML and increased overall survival. Functional studies aimed at establishing the degree of T cell suppression in patients with AML revealed impaired T cell function in many patients. In most cases, T cell proliferation could be restored by blocking the immune checkpoint molecules PD-1, CTLA-4, or TIM3. Our data demonstrate that AML establishes an immune suppressive environment in the bone marrow, in part through T cell checkpoint function.
Subject(s)
Bone Marrow/metabolism , Leukemia, Myeloid, Acute/metabolism , T-Lymphocytes/metabolism , Tumor Microenvironment/physiology , Bone Marrow/immunology , CTLA-4 Antigen/metabolism , Cell Proliferation , Cytokines/metabolism , Hepatitis A Virus Cellular Receptor 2/metabolism , Humans , Leukemia, Myeloid, Acute/immunology , Leukemia, Myeloid, Acute/therapy , Programmed Cell Death 1 Receptor/metabolism , T-Lymphocytes/immunologySubject(s)
Alemtuzumab/administration & dosage , Leukemia, Prolymphocytic, T-Cell , Aged , Aged, 80 and over , Disease-Free Survival , Female , Humans , Leukemia, Prolymphocytic, T-Cell/drug therapy , Leukemia, Prolymphocytic, T-Cell/metabolism , Leukemia, Prolymphocytic, T-Cell/mortality , Leukemia, Prolymphocytic, T-Cell/pathology , Male , Middle Aged , Survival RateABSTRACT
Luxeptinib (CG-806) simultaneously targets FLT3 and select other kinase pathways operative in myeloid malignancies. We investigated the range of kinases it inhibits, its cytotoxicity landscape ex vivo with acute myeloid leukemia (AML) patient samples, and its efficacy in xenograft models. Luxeptinib inhibits wild-type (WT) and many of the clinically relevant mutant forms of FLT3 at low nanomolar concentrations. It is a more potent inhibitor of the activity of FLT3-internal tandem duplication, FLT3 kinase domain and gatekeeper mutants than against WT FLT3. Broad kinase screens disclosed that it also inhibits other kinases that can drive oncogenic signaling and rescue pathways, but spares kinases known to be associated with clinical toxicity. In vitro profiling of luxeptinib against 186 AML fresh patient samples demonstrated greater potency relative to other FLT3 inhibitors, including cases with mutations in FLT3, isocitrate dehydrogenase-1/2, ASXL1, NPM1, SRSF2, TP53, or RAS, and activity was documented in a xenograft AML model. Luxeptinib administered continuously orally every 12 hours at a dose that yielded a mean Cmin plasma concentration of 1.0 ± 0.3 µmol/L (SEM) demonstrated strong antitumor activity but no myelosuppression or evidence of tissue damage in mice or dogs in acute toxicology studies. On the basis of these studies, luxeptinib was advanced into a phase I trial for patients with AML and myelodysplastic/myeloproliferative neoplasms.
Subject(s)
Leukemia, Myeloid, Acute , Animals , Dogs , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Mice , Mutation , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Signal Transduction , fms-Like Tyrosine Kinase 3/genetics , fms-Like Tyrosine Kinase 3/metabolismABSTRACT
Acute myeloid leukemia (AML) is a cancer of myeloid-lineage cells with limited therapeutic options. We previously combined ex vivo drug sensitivity with genomic, transcriptomic, and clinical annotations for a large cohort of AML patients, which facilitated discovery of functional genomic correlates. Here, we present a dataset that has been harmonized with our initial report to yield a cumulative cohort of 805 patients (942 specimens). We show strong cross-cohort concordance and identify features of drug response. Further, deconvoluting transcriptomic data shows that drug sensitivity is governed broadly by AML cell differentiation state, sometimes conditionally affecting other correlates of response. Finally, modeling of clinical outcome reveals a single gene, PEAR1, to be among the strongest predictors of patient survival, especially for young patients. Collectively, this report expands a large functional genomic resource, offers avenues for mechanistic exploration and drug development, and reveals tools for predicting outcome in AML.
Subject(s)
Leukemia, Myeloid, Acute , Cell Differentiation , Cohort Studies , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Receptors, Cell Surface/genetics , TranscriptomeABSTRACT
Human cytomegalovirus (HCMV) is the most genetically and structurally complex human herpesvirus and is composed of an envelope, a tegument, and a dsDNA-containing capsid. HCMV tegument plays essential roles in HCMV infection and assembly. Using cryo electron tomography (cryoET), here we show that HCMV tegument compartment can be divided into two sub-compartments: an inner and an outer tegument. The inner tegument consists of densely-packed proteins surrounding the capsid. The outer tegument contains those components that are loosely packed in the space between the inner tegument and the pleomorphic glycoprotein-containing envelope. To systematically characterize the inner tegument proteins interacting with the capsid, we used chemical treatment to strip off the entire envelope and most tegument proteins to obtain a tegumented capsid with inner tegument proteins. SDS-polyacrylamide gel electrophoresis analyses show that only two tegument proteins, UL32-encoded pp150 and UL48-encoded high molecular weight protein (HMWP), remains unchanged in their abundance in the tegumented capsids as compared to their abundance in the intact particles. Three-dimensional reconstructions by single particle cryo electron microscopy (cryoEM) reveal that the net-like layer of icosahedrally-ordered tegument densities are also the same in the tegumented capsid and in the intact particles. CryoET reconstruction of the tegumented capsid labeled with an anti-pp150 antibody is consistent with the biochemical and cryoEM data in localizing pp150 within the ordered tegument. Taken together, these results suggest that pp150, a betaherpesvirus-specific tegument protein, is a constituent of the net-like layer of icosahedrally-ordered capsid-bound tegument densities, a structure lacking similarities in alpha and gammaherpesviruses.
Subject(s)
Capsid Proteins/chemistry , Capsid Proteins/ultrastructure , Capsid/chemistry , Capsid/ultrastructure , Cytomegalovirus/chemistry , Cytomegalovirus/ultrastructure , Protein Conformation , Cells, Cultured , Cryoelectron Microscopy/methods , Electron Microscope Tomography/methods , Humans , Models, MolecularSubject(s)
Dendritic Cells/metabolism , Dendritic Cells/virology , Endocytosis , HIV/physiology , T-Lymphocytes/virology , CD4 Antigens/immunology , CD4 Antigens/metabolism , Dendritic Cells/cytology , Humans , Receptors, CXCR4/immunology , Receptors, CXCR4/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolismABSTRACT
The smallest capsid proteins (SCPs) of the human herpesviruses differ substantially in size and sequence and are thought to impart some unique aspects of infection to their respective viruses. We used electron cryomicroscopy and antibody labeling to show that the 8-kDa SCP of human cytomegalovirus is attached only to major capsid protein subunits of the hexons, not the pentons. Thus, the SCPs of different herpesviruses illustrate that a protein can evolve significantly in sequence, structure, and function, while preserving its role in the architecture of the virus by binding to a specific partner in a specific oligomeric state.
Subject(s)
Capsid Proteins/analysis , Capsid/chemistry , Cytomegalovirus/chemistry , Amino Acid Sequence , Capsid Proteins/chemistry , Humans , Image Processing, Computer-Assisted , Microscopy, Electron , Molecular Sequence DataABSTRACT
Of the six herpesvirus capsid proteins, the smallest capsid proteins (SCPs) share the least sequence homology among herpesvirus family members and have been implicated in virus specificity during infection. The herpes simplex virus-1 (HSV-1) SCP was shown to be horn shaped and to specifically bind the upper domain of each major capsid protein in hexons but not in pentons. In Kaposi's sarcoma-associated herpesvirus (KSHV), the protein encoded by the ORF65 gene (pORF65) is the putative SCP but its location remains controversial due to the absence of such horn-shaped densities from both the pentons and hexons of the KSHV capsid reconstructions. To directly locate the KSHV SCP, we have used electron cryomicroscopy and three-dimensional reconstruction techniques to compare the three-dimensional structure of KSHV capsids to that of anti-pORF65 antibody-labeled capsids. Our difference map shows prominent antibody densities bound to the tips of the hexons but not to pentons, indicating that KSHV SCP is attached to the upper domain of the major capsid protein in hexons but not to that in pentons, similar to HSV-1 SCP. The lack of horn-shaped densities on the hexons indicates that KSHV SCP exhibits structural features that are substantially different from those of HSV-1 SCP. The location of SCP at the outermost regions of the capsid suggests a possible role in mediating capsid interactions with the tegument and cytoskeletal proteins during infection.