RESUMO
CML is readily treatable with tyrosine kinase inhibitors (TKIs); however, resistance occurs, with the disease curable in only â¼15%-20% of patients. Using integrated functional genomics, Adnan Awad et al.1 identify agents effective against CML stem cells and describe mechanisms underlying TKI resistance.
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Resistencia a Medicamentos Antineoplásicos , Genômica , Leucemia Mielogênica Crônica BCR-ABL Positiva , Inibidores de Proteínas Quinases , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Genômica/métodosRESUMO
Acute myeloid leukemia (AML) and myeloid neoplasms develop through acquisition of somatic mutations that confer mutation-specific fitness advantages to hematopoietic stem and progenitor cells. However, our understanding of mutational effects remains limited to the resolution attainable within immunophenotypically and clinically accessible bulk cell populations. To decipher heterogeneous cellular fitness to preleukemic mutational perturbations, we performed single-cell RNA sequencing of eight different mouse models with driver mutations of myeloid malignancies, generating 269,048 single-cell profiles. Our analysis infers mutation-driven perturbations in cell abundance, cellular lineage fate, cellular metabolism, and gene expression at the continuous resolution, pinpointing cell populations with transcriptional alterations associated with differentiation bias. We further develop an 11-gene scoring system (Stem11) on the basis of preleukemic transcriptional signatures that predicts AML patient outcomes. Our results demonstrate that a single-cell-resolution deep characterization of preleukemic biology has the potential to enhance our understanding of AML heterogeneity and inform more effective risk stratification strategies.
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Leukaemia stem cells (LSCs) are the critical seed for the growth of haematological malignancies, driving the clonal expansion that enables disease initiation, relapse and often resistance. Specifically, they display inherent phenotypic and epigenetic plasticity resulting in complex heterogenic diseases. In this review, we discuss the key principles of deregulation of epigenetic processes that shape this disease evolution. We consider measures to define and quantify clonal heterogeneity, combining information from recent studies assessing mutational, transcriptional and epigenetic landscapes at single cell resolution in myeloid neoplasms (MN). We highlight the importance of integrating epigenetic and genetic information to better understand inter- and intra-patient heterogeneity and discuss how this understanding further informs evolution and progression trajectories and subsequent clinical response in MN. Under this topic, we also discuss efforts to identify mechanisms of resistance, by longitudinal analyses of patient samples. Finally, we highlight how we might target these aberrant epigenetic processes for better therapeutic outcomes and to potentially eradicate LSCs.
Assuntos
Leucemia Mieloide Aguda , Humanos , Leucemia Mieloide Aguda/genética , Epigênese Genética , Mutação , Células-Tronco , Células-Tronco Neoplásicas/patologiaRESUMO
Haematopoietic stem cells (HSC) reside in the bone marrow microenvironment (BMM), where they respond to extracellular calcium [eCa2+] via the G-protein coupled calcium-sensing receptor (CaSR). Here we show that a calcium gradient exists in this BMM, and that [eCa2+] and response to [eCa2+] differ between leukaemias. CaSR influences the location of MLL-AF9+ acute myeloid leukaemia (AML) cells within this niche and differentially impacts MLL-AF9+ AML versus BCR-ABL1+ leukaemias. Deficiency of CaSR reduces AML leukaemic stem cells (LSC) 6.5-fold. CaSR interacts with filamin A, a crosslinker of actin filaments, affects stemness-associated factors and modulates pERK, ß-catenin and c-MYC signaling and intracellular levels of [Ca2+] in MLL-AF9+ AML cells. Combination treatment of cytarabine plus CaSR-inhibition in various models may be superior to cytarabine alone. Our studies suggest CaSR to be a differential and targetable factor in leukaemia progression influencing self-renewal of AML LSC via [eCa2+] cues from the BMM.
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Leucemia Mieloide Aguda , Receptores de Detecção de Cálcio , Humanos , Receptores de Detecção de Cálcio/genética , Proteínas Proto-Oncogênicas c-myc , Cálcio , Proteínas de Fusão Oncogênica/metabolismo , Transdução de Sinais , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Citarabina , Microambiente TumoralRESUMO
Cellular differentiation requires extensive alterations in chromatin structure and function, which is elicited by the coordinated action of chromatin and transcription factors. By contrast with transcription factors, the roles of chromatin factors in differentiation have not been systematically characterized. Here, we combine bulk ex vivo and single-cell in vivo CRISPR screens to characterize the role of chromatin factor families in hematopoiesis. We uncover marked lineage specificities for 142 chromatin factors, revealing functional diversity among related chromatin factors (i.e. barrier-to-autointegration factor subcomplexes) as well as shared roles for unrelated repressive complexes that restrain excessive myeloid differentiation. Using epigenetic profiling, we identify functional interactions between lineage-determining transcription factors and several chromatin factors that explain their lineage dependencies. Studying chromatin factor functions in leukemia, we show that leukemia cells engage homeostatic chromatin factor functions to block differentiation, generating specific chromatin factor-transcription factor interactions that might be therapeutically targeted. Together, our work elucidates the lineage-determining properties of chromatin factors across normal and malignant hematopoiesis.
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Cromatina , Leucemia , Humanos , Cromatina/genética , Linhagem da Célula/genética , Hematopoese/genética , Diferenciação Celular/genética , Fatores de Transcrição/genéticaRESUMO
Resistance to standard and novel therapies remains the main obstacle to cure in acute myeloid leukaemia (AML) and is often driven by metabolic adaptations which are therapeutically actionable. Here we identify inhibition of mannose-6-phosphate isomerase (MPI), the first enzyme in the mannose metabolism pathway, as a sensitizer to both cytarabine and FLT3 inhibitors across multiple AML models. Mechanistically, we identify a connection between mannose metabolism and fatty acid metabolism, that is mediated via preferential activation of the ATF6 arm of the unfolded protein response (UPR). This in turn leads to cellular accumulation of polyunsaturated fatty acids, lipid peroxidation and ferroptotic cell death in AML cells. Our findings provide further support to the role of rewired metabolism in AML therapy resistance, unveil a connection between two apparently independent metabolic pathways and support further efforts to achieve eradication of therapy-resistant AML cells by sensitizing them to ferroptotic cell death.
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Leucemia Mieloide Aguda , Manose , Humanos , Morte Celular , Citarabina/farmacologia , Linhagem Celular Tumoral , Leucemia Mieloide Aguda/metabolismo , Apoptose , Tirosina Quinase 3 Semelhante a fmsRESUMO
HOXA9 is commonly upregulated in acute myeloid leukemia (AML), in which it confers a poor prognosis. Characterizing the protein interactome of endogenous HOXA9 in human AML, we identified a chromatin complex of HOXA9 with the nuclear matrix attachment protein SAFB. SAFB perturbation phenocopied HOXA9 knockout to decrease AML proliferation, increase differentiation and apoptosis in vitro, and prolong survival in vivo. Integrated genomic, transcriptomic, and proteomic analyses further demonstrated that the HOXA9-SAFB (H9SB)-chromatin complex associates with nucleosome remodeling and histone deacetylase (NuRD) and HP1γ to repress the expression of factors associated with differentiation and apoptosis, including NOTCH1, CEBPδ, S100A8, and CDKN1A. Chemical or genetic perturbation of NuRD and HP1γ-associated catalytic activity also triggered differentiation, apoptosis, and the induction of these tumor-suppressive genes. Importantly, this mechanism is operative in other HOXA9-dependent AML genotypes. This mechanistic insight demonstrates the active HOXA9-dependent differentiation block as a potent mechanism of disease maintenance in AML that may be amenable to therapeutic intervention by targeting the H9SB interface and/or NuRD and HP1γ activity.
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Leucemia Mieloide Aguda , Proteínas de Ligação à Região de Interação com a Matriz , Humanos , Proteômica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Fatores de Transcrição/genética , Proteínas Associadas à Matriz Nuclear , Cromatina , Receptores de Estrogênio/genética , Receptores de Estrogênio/uso terapêutico , Proteínas de Ligação à Região de Interação com a Matriz/genéticaRESUMO
PURPOSE: Molibresib is a selective, small molecule inhibitor of the bromodomain and extra-terminal (BET) protein family. This was an open-label, two-part, Phase I/II study investigating molibresib monotherapy for the treatment of hematological malignancies (NCT01943851). PATIENTS AND METHODS: Part 1 (dose escalation) determined the recommended Phase 2 dose (RP2D) of molibresib in patients with acute myeloid leukemia (AML), Non-Hodgkin lymphoma (NHL), or multiple myeloma. Part 2 (dose expansion) investigated the safety and efficacy of molibresib at the RP2D in patients with relapsed/refractory myelodysplastic syndrome (MDS; as well as AML evolved from antecedent MDS) or cutaneous T-cell lymphoma (CTCL). The primary endpoint in Part 1 was safety and the primary endpoint in Part 2 was objective response rate (ORR). RESULTS: There were 111 patients enrolled (87 in Part 1, 24 in Part 2). Molibresib RP2Ds of 75 mg daily (for MDS) and 60 mg daily (for CTCL) were selected. Most common Grade 3+ adverse events included thrombocytopenia (37%), anemia (15%), and febrile neutropenia (15%). Six patients achieved complete responses [3 in Part 1 (2 AML, 1 NHL), 3 in Part 2 (MDS)], and 7 patients achieved partial responses [6 in Part 1 (4 AML, 2 NHL), 1 in Part 2 (MDS)]. The ORRs for Part 1, Part 2, and the total study population were 10% [95% confidence interval (CI), 4.8-18.7], 25% (95% CI, 7.3-52.4), and 13% (95% CI, 6.9-20.6), respectively. CONCLUSIONS: While antitumor activity was observed with molibresib, use was limited by gastrointestinal and thrombocytopenia toxicities. Investigations of molibresib as part of combination regimens may be warranted.
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Neoplasias Hematológicas , Leucemia Mieloide Aguda , Linfoma não Hodgkin , Trombocitopenia , Humanos , Linfoma não Hodgkin/tratamento farmacológico , Neoplasias Hematológicas/tratamento farmacológico , Leucemia Mieloide Aguda/tratamento farmacológicoRESUMO
Several studies have documented aberrant RNA editing patterns across multiple tumors across large patient cohorts from The Cancer Genome Atlas (TCGA). However, studies on understanding the role of RNA editing in acute myeloid leukemia (AML) have been limited to smaller sample sizes. Using high throughput transcriptomic data from the TCGA, we demonstrated higher levels of editing as a predictor of poor outcome within the AML patient samples. Moreover, differential editing patterns were observed across individual AML genotypes. We also could demonstrate a negative association between the degree of editing and mRNA abundance for some transcripts, identifying the potential regulatory potential of RNA-editing in altering gene expression in AML. Further edQTL analysis suggests potential cis-regulatory mechanisms in RNA editing variation. Our work suggests a functional and regulatory role of RNA editing in the pathogenesis of AML and we extended our analysis to gain insight into the factors influencing altered levels of editing.
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Recurrent gene mutations often cooperate in a predefined stepwise and synergistic manner to alter global transcription, through directly or indirectly remodeling epigenetic landscape on linear and three-dimensional (3D) scales. Here, we present a multiomics data integration approach to investigate the impact of gene mutational synergy on transcription, chromatin states, and 3D chromatin organization in a murine leukemia model. This protocol provides an executable framework to study epigenetic remodeling induced by cooperating gene mutations and to identify the critical regulatory network involved. For complete details on the use and execution of this protocol, please refer to Yun et al. (2021).
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Montagem e Desmontagem da Cromatina , Multiômica , Animais , Camundongos , Cromatina , MutaçãoRESUMO
Clinical recommendations for Acute Myeloid Leukemia (AML) classification and risk-stratification remain heavily reliant on cytogenetic findings at diagnosis, which are present in <50% of patients. Using comprehensive molecular profiling data from 3,653 patients we characterize and validate 16 molecular classes describing 100% of AML patients. Each class represents diverse biological AML subgroups, and is associated with distinct clinical presentation, likelihood of response to induction chemotherapy, risk of relapse and death over time. Secondary AML-2, emerges as the second largest class (24%), associates with high-risk disease, poor prognosis irrespective of flow Minimal Residual Disease (MRD) negativity, and derives significant benefit from transplantation. Guided by class membership we derive a 3-tier risk-stratification score that re-stratifies 26% of patients as compared to standard of care. This results in a unified framework for disease classification and risk-stratification in AML that relies on information from cytogenetics and 32 genes. Last, we develop an open-access patient-tailored clinical decision support tool.
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Leucemia Mieloide Aguda , Humanos , Análise Citogenética , Citometria de Fluxo/métodos , Quimioterapia de Indução/métodos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Neoplasia ResidualRESUMO
Mutations in multiple cancers may synergize to alter the cellular epigenetic and transcriptional state and corrupt key signaling pathways. In this issue of Science Signaling, Pedicona et al. illustrate how the two processes intersect to regulate cellular differentiation in acute myeloid leukemia (AML) and show how inhibition of epigenetic regulators promotes sensitivity to kinase inhibitors.
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Leucemia Mieloide Aguda , Epigênese Genética , Epigenômica , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Mutação , Transdução de Sinais/genéticaRESUMO
The accessibility of cell surface proteins makes them tractable for targeting by cancer immunotherapy, but identifying suitable targets remains challenging. Here we describe plasma membrane profiling of primary human myeloma cells to identify an unprecedented number of cell surface proteins of a primary cancer. We used a novel approach to prioritize immunotherapy targets and identified a cell surface protein not previously implicated in myeloma, semaphorin-4A (SEMA4A). Using knock-down by short-hairpin RNA and CRISPR/nuclease-dead Cas9 (dCas9), we show that expression of SEMA4A is essential for normal myeloma cell growth in vitro, indicating that myeloma cells cannot downregulate the protein to avoid detection. We further show that SEMA4A would not be identified as a myeloma therapeutic target by standard CRISPR/Cas9 knockout screens because of exon skipping. Finally, we potently and selectively targeted SEMA4A with a novel antibody-drug conjugate in vitro and in vivo.
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Mieloma Múltiplo , Semaforinas , Membrana Celular/metabolismo , Humanos , Fatores Imunológicos , Imunoterapia , Proteínas de Membrana , Mieloma Múltiplo/genética , Mieloma Múltiplo/terapia , Proteômica , Semaforinas/genética , Semaforinas/metabolismoRESUMO
Epigenetic histone modifiers are key regulators of cell fate decisions in normal and malignant hematopoiesis. Their enzymatic activities are of particular significance as putative therapeutic targets in leukemia. In contrast, less is known about the contextual role in which those enzymatic activities are exercised and specifically how different macromolecular complexes configure the same enzymatic activity with distinct molecular and cellular consequences. We focus on KAT2A, a lysine acetyltransferase responsible for histone H3 lysine 9 acetylation, which we recently identified as a dependence in acute myeloid leukemia stem cells and that participates in 2 distinct macromolecular complexes: Ada two-A-containing (ATAC) and Spt-Ada-Gcn5-Acetyltransferase (SAGA). Through analysis of human cord blood hematopoietic stem cells and progenitors, and of myeloid leukemia cells, we identify unique respective contributions of the ATAC complex to regulation of biosynthetic activity in undifferentiated self-renewing cells and of the SAGA complex to stabilization or correct progression of cell type-specific programs with putative preservation of cell identity. Cell type and stage-specific dependencies on ATAC and SAGA-regulated programs explain multilevel KAT2A requirements in leukemia and in erythroid lineage specification and development. Importantly, they set a paradigm against which lineage specification and identity can be explored across developmental stem cell systems.
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Histona Acetiltransferases , Leucemia Mieloide Aguda , Acetilação , Hematopoese , Histonas/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismoRESUMO
BACKGROUND: Atherosclerosis is a chronic inflammatory disease of the artery wall. Regulatory T cells (Tregs) limit inflammation and promote tissue healing. Low doses of interleukin (IL)-2 have the potential to increase Tregs, but its use is contraindicated for patients with ischemic heart disease. METHODS: In this randomized, double-blind, placebo-controlled, dose-escalation trial, we tested low-dose subcutaneous aldesleukin (recombinant IL-2), given once daily for 5 consecutive days. In study part A, the primary end point was safety, and patients with stable ischemic heart disease were randomly assigned to receive placebo or to one of five dose groups (range, 0.3 to 3.0 × 106 IU daily). In study part B, patients with acute non-ST elevation myocardial infarction or unstable angina were randomly assigned to receive placebo or to one of two dose groups (1.5 and 2.5 × 106 IU daily). The coprimary end points were safety and the dose required to increase circulating Tregs by 75%. Single-cell RNA-sequencing of circulating immune cells was used to provide a mechanistic assessment of the effects of aldesleukin. RESULTS: Forty-four patients were randomly assigned to either study part A (n=26) or part B (n=18). In total, 3 patients withdrew before dosing, 27 received active treatment, and 14 received placebo. The majority of adverse events were mild. Two serious adverse events occurred, with one occurring after drug administration. In parts A and B, there was a dose-dependent increase in Tregs. In part B, the estimated dose to achieve a 75% increase in Tregs was 1.46 × 106 IU (95% confidence interval, 1.06 to 1.87). Single-cell RNA-sequencing demonstrated the engagement of distinct pathways and cellcell interactions. CONCLUSIONS: In this phase 1b/2a study, low-dose IL-2 expanded Tregs without adverse events of major concern. Larger trials are needed to confirm the safety and to further evaluate the efficacy of low-dose IL-2 as an anti-inflammatory therapy for patients with ischemic heart disease. (Funded by the Medical Research Council, the British Heart Foundation, and others; ClinicalTrials.gov number, NCT03113773)
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Interleucina-2 , Interleucina-2/análogos & derivados , Isquemia Miocárdica , Linfócitos T Reguladores , Humanos , Interleucina-2/administração & dosagem , Interleucina-2/uso terapêutico , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Isquemia Miocárdica/imunologia , Isquemia Miocárdica/tratamento farmacológico , Método Duplo-Cego , Masculino , Pessoa de Meia-Idade , Feminino , Proteínas RecombinantesRESUMO
Altered transcription is a cardinal feature of acute myeloid leukemia (AML); however, exactly how mutations synergize to remodel the epigenetic landscape and rewire three-dimensional DNA topology is unknown. Here, we apply an integrated genomic approach to a murine allelic series that models the two most common mutations in AML: Flt3-ITD and Npm1c. We then deconvolute the contribution of each mutation to alterations of the epigenetic landscape and genome organization, and infer how mutations synergize in the induction of AML. Our studies demonstrate that Flt3-ITD signals to chromatin to alter the epigenetic environment and synergizes with mutations in Npm1c to alter gene expression and drive leukemia induction. These analyses also allow the identification of long-range cis-regulatory circuits, including a previously unknown superenhancer of Hoxa locus, as well as larger and more detailed gene-regulatory networks, driven by transcription factors including PU.1 and IRF8, whose importance we demonstrate through perturbation of network members.
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Montagem e Desmontagem da Cromatina/genética , DNA de Neoplasias/química , Regulação Leucêmica da Expressão Gênica , Histonas/metabolismo , Leucemia Mieloide Aguda/genética , Mutação/genética , Processamento de Proteína Pós-Traducional , Animais , Sequência de Bases , Modelos Animais de Doenças , Elementos Facilitadores Genéticos/genética , Redes Reguladoras de Genes , Loci Gênicos , Humanos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/metabolismo , Nucleofosmina , Análise de Componente Principal , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
Histone acetyltransferases (HATs) catalyze the transfer of an acetyl group from acetyl-CoA to lysine residues of histones and play a central role in transcriptional regulation in diverse biological processes. Dysregulation of HAT activity can lead to human diseases including developmental disorders and cancer. Through genome-wide CRISPR-Cas9 screens, we identified several HATs of the MYST family as fitness genes for acute myeloid leukemia (AML). Here we investigate the essentiality of lysine acetyltransferase KAT7 in AMLs driven by the MLL-X gene fusions. We found that KAT7 loss leads to a rapid and complete loss of both H3K14ac and H4K12ac marks, in association with reduced proliferation, increased apoptosis, and differentiation of AML cells. Acetyltransferase activity of KAT7 is essential for the proliferation of these cells. Mechanistically, our data propose that acetylated histones provide a platform for the recruitment of MLL-fusion-associated adaptor proteins such as BRD4 and AF4 to gene promoters. Upon KAT7 loss, these factors together with RNA polymerase II rapidly dissociate from several MLL-fusion target genes that are essential for AML cell proliferation, including MEIS1, PBX3, and SENP6. Our findings reveal that KAT7 is a plausible therapeutic target for this poor prognosis AML subtype.