RESUMO
Non-hematopoietic cells are essential contributors to hematopoiesis. However, heterogeneity and spatial organization of these cells in human bone marrow remain largely uncharacterized. We used single-cell RNA sequencing (scRNA-seq) to profile 29,325 non-hematopoietic cells and discovered nine transcriptionally distinct subtypes. We simultaneously profiled 53,417 hematopoietic cells and predicted their interactions with non-hematopoietic subsets. We employed co-detection by indexing (CODEX) to spatially profile over 1.2 million cells. We integrated scRNA-seq and CODEX data to link predicted cellular signaling with spatial proximity. Our analysis revealed a hyperoxygenated arterio-endosteal neighborhood for early myelopoiesis, and an adipocytic localization for early hematopoietic stem and progenitor cells (HSPCs). We used our CODEX atlas to annotate new images and uncovered mesenchymal stromal cell (MSC) expansion and spatial neighborhoods co-enriched for leukemic blasts and MSCs in acute myeloid leukemia (AML) patient samples. This spatially resolved, multiomic atlas of human bone marrow provides a reference for investigation of cellular interactions that drive hematopoiesis.
Assuntos
Medula Óssea , Células-Tronco Hematopoéticas , Células-Tronco Mesenquimais , Proteômica , Análise de Célula Única , Transcriptoma , Humanos , Análise de Célula Única/métodos , Medula Óssea/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Proteômica/métodos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Hematopoese , Nicho de Células-Tronco , Células da Medula Óssea/metabolismo , Células da Medula Óssea/citologiaRESUMO
The transformed state in acute leukemia requires gene regulatory programs involving transcription factors and chromatin modulators. Here, we uncover an IRF8-MEF2D transcriptional circuit as an acute myeloid leukemia (AML)-biased dependency. We discover and characterize the mechanism by which the chromatin "reader" ZMYND8 directly activates IRF8 in parallel with the MYC proto-oncogene through their lineage-specific enhancers. ZMYND8 is essential for AML proliferation in vitro and in vivo and associates with MYC and IRF8 enhancer elements that we define in cell lines and in patient samples. ZMYND8 occupancy at IRF8 and MYC enhancers requires BRD4, a transcription coactivator also necessary for AML proliferation. We show that ZMYND8 binds to the ET domain of BRD4 via its chromatin reader cassette, which in turn is required for proper chromatin occupancy and maintenance of leukemic growth in vivo. Our results rationalize ZMYND8 as a potential therapeutic target for modulating essential transcriptional programs in AML.
Assuntos
Fatores Reguladores de Interferon/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Cromatina/genética , Elementos Facilitadores Genéticos/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Fatores Reguladores de Interferon/genética , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , Proto-Oncogene Mas , Fatores de Transcrição/metabolismo , Transcrição Gênica/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Myeloid malignancies, including acute myeloid leukaemia (AML), arise from the expansion of haematopoietic stem and progenitor cells that acquire somatic mutations. Bulk molecular profiling has suggested that mutations are acquired in a stepwise fashion: mutant genes with high variant allele frequencies appear early in leukaemogenesis, and mutations with lower variant allele frequencies are thought to be acquired later1-3. Although bulk sequencing can provide information about leukaemia biology and prognosis, it cannot distinguish which mutations occur in the same clone(s), accurately measure clonal complexity, or definitively elucidate the order of mutations. To delineate the clonal framework of myeloid malignancies, we performed single-cell mutational profiling on 146 samples from 123 patients. Here we show that AML is dominated by a small number of clones, which frequently harbour co-occurring mutations in epigenetic regulators. Conversely, mutations in signalling genes often occur more than once in distinct subclones, consistent with increasing clonal diversity. We mapped clonal trajectories for each sample and uncovered combinations of mutations that synergized to promote clonal expansion and dominance. Finally, we combined protein expression with mutational analysis to map somatic genotype and clonal architecture with immunophenotype. Our findings provide insights into the pathogenesis of myeloid transformation and how clonal complexity evolves with disease progression.
Assuntos
Células Clonais/patologia , Análise Mutacional de DNA , Mutação , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Análise de Célula Única , Separação Celular , Células Clonais/metabolismo , Humanos , ImunofenotipagemRESUMO
The control of cell fate is an epigenetic process initiated by transcription factors (TFs) that recognize DNA motifs and recruit activator complexes and transcriptional machineries to chromatin. Lineage specificity is thought to be provided solely by TF-motif pairing, while the recruited activators are passive. Here, we show that INTS13, a subunit of the Integrator complex, operates as monocytic/macrophagic differentiation factor. Integrator is a general activator of transcription at coding genes and is required for eRNA maturation. Here, we show that INTS13 functions as an independent sub-module and targets enhancers through Early Growth Response (EGR1/2) TFs and their co-factor NAB2. INTS13 binds poised monocytic enhancers eliciting chromatin looping and activation. Independent depletion of INTS13, EGR1, or NAB2 impairs monocytic differentiation of cell lines and primary human progenitors. Our data demonstrate that Integrator is not functionally homogeneous and has TF-specific regulatory potential, revealing a new enhancer regulatory axis that controls myeloid differentiation.
Assuntos
Diferenciação Celular , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Elementos Facilitadores Genéticos , Monócitos/metabolismo , Células Progenitoras Mieloides/metabolismo , Proteínas Repressoras/metabolismo , Linhagem Celular , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 2 de Resposta de Crescimento Precoce/genética , Humanos , Células Progenitoras Mieloides/citologia , Proteínas Repressoras/genéticaRESUMO
Older patients with acute myeloid leukemia (AML) have high relapse risk and poor survival after allogeneic hematopoietic cell transplantation (HCT). Younger patients may receive myeloablative conditioning to mitigate relapse risk associated with high-risk genetics or measurable residual disease (MRD), but older adults typically receive reduced-intensity conditioning (RIC) to limit toxicity. To identify factors that drive HCT outcomes in older patients, we performed targeted mutational analysis (variant allele fraction ≥2%) on diagnostic samples from 295 patients with AML aged ≥60 years who underwent HCT in first complete remission, 91% of whom received RIC, and targeted duplex sequencing at remission in a subset comprising 192 patients. In a multivariable model for leukemia-free survival (LFS) including baseline genetic and clinical variables, we defined patients with low (3-year LFS, 85%), intermediate (55%), high (35%), and very high (7%) risk. Before HCT, 79.7% of patients had persistent baseline mutations, including 18.3% with only DNMT3A or TET2 (DT) mutations and 61.4% with other mutations (MRD positive). In univariable analysis, MRD positivity was associated with increased relapse and inferior LFS, compared with DT and MRD-negative mutations. However, in a multivariable model accounting for baseline risk, MRD positivity had no independent impact on LFS, most likely because of its significant association with diagnostic genetic characteristics, including MDS-associated gene mutations, TP53 mutations, and high-risk karyotype. In summary, molecular associations with MRD positivity and transplant outcomes in older patients with AML are driven primarily by baseline genetics, not by mutations present in remission. In this group of patients, where high-intensity conditioning carries substantial risk of toxicity, alternative approaches to mitigating MRD-associated relapse risk are needed.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Leucemia Mieloide Aguda , Idoso , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Neoplasia Residual/diagnóstico , Neoplasia Residual/genética , Recidiva , Estudos Retrospectivos , Condicionamento Pré-Transplante , Transplante HomólogoRESUMO
Most genes associated with acute myeloid leukemia (AML) are mutated in less than 10% of patients, suggesting that alternative mechanisms of gene disruption contribute to this disease. Here, we find a set of splicing events that alter the expression of a subset of AML-associated genes independent of known somatic mutations. In particular, aberrant splicing triples the number of patients with reduced functional EZH2 compared with that predicted by somatic mutation alone. In addition, we unexpectedly find that the nonsense-mediated decay factor DHX34 exhibits widespread alternative splicing in sporadic AML, resulting in a premature stop codon that phenocopies the loss-of-function germline mutations observed in familial AML. Together, these results demonstrate that classical mutation analysis underestimates the burden of functional gene disruption in AML and highlight the importance of assessing the contribution of alternative splicing to gene dysregulation in human disease.
Assuntos
Processamento Alternativo , Leucemia Mieloide Aguda/genética , Mutação , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Genótipo , Humanos , Degradação do RNAm Mediada por Códon sem Sentido , RNA Helicases/genética , RNA Helicases/metabolismoRESUMO
B- and T-cell acute lymphoblastic leukemia (B/T-ALL) may be refractory or recur after therapy by suppressing host anticancer immune surveillance mediated specifically by natural killer (NK) cells. We delineated the phenotypic and functional defects in NK cells from high-risk patients with B/T-ALL using mass cytometry, flow cytometry, and in silico cytometry, with the goal of further elucidating the role of NK cells in sustaining acute lymphoblastic leukemia (ALL) regression. We found that, compared with their normal counterparts, NK cells from patients with B/T-ALL are less cytotoxic but exhibit an activated signature that is characterized by high CD56, high CD69, production of activated NK cell-origin cytokines, and calcium (Ca2+) signaling. We demonstrated that defective maturation of NK cells into cytotoxic effectors prevents NK cells from ALL from lysing NK cell-sensitive targets as efficiently as do normal NK cells. Additionally, we showed that NK cells in ALL are exhausted, which is likely caused by their chronic activation. We found that increased frequencies of activated cytokine-producing NK cells are associated with increased disease severity and independently predict poor clinical outcome in patients with ALL. Our studies highlight the benefits of developing NK cell profiling as a diagnostic tool to predict clinical outcome in patients with ALL and underscore the clinical potential of allogeneic NK cell infusions to prevent ALL recurrence.
Assuntos
Células Matadoras Naturais/imunologia , Ativação Linfocitária , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígeno CD56/imunologia , Células Cultivadas , Citocinas/imunologia , Citotoxicidade Imunológica , Humanos , Lectinas Tipo C/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , PrognósticoRESUMO
B-cell lymphoma 6 (BCL6) is a transcription repressor and proto-oncogene that plays a crucial role in the innate and adaptive immune system and lymphoid neoplasms. However, its role in myeloid malignancies remains unclear. Here, we explored the role of BCL6 in acute myeloid leukemia (AML). BCL6 was expressed at variable and often high levels in AML cell lines and primary AML samples. AMLs with higher levels of BCL6 were generally sensitive to treatment with BCL6 inhibitors, with the exception of those with monocytic differentiation. Gene expression profiling of AML cells treated with a BCL6 inhibitor revealed induction of BCL6-repressed target genes and transcriptional programs linked to DNA damage checkpoints and downregulation of stem cell genes. Ex vivo treatment of primary AML cells with BCL6 inhibitors induced apoptosis and decreased colony-forming capacity, which correlated with the levels of BCL6 expression. Importantly, inhibition or knockdown of BCL6 in primary AML cells resulted in a significant reduction of leukemia-initiating capacity in mice, suggesting ablation of leukemia repopulating cell functionality. In contrast, BCL6 knockout or inhibition did not suppress the function of normal hematopoietic stem cells. Treatment with cytarabine further induced BCL6 expression, and the levels of BCL6 induction were correlated with resistance to cytarabine. Treatment of AML patient-derived xenografts with BCL6 inhibitor plus cytarabine suggested enhanced antileukemia activity with this combination. Hence, pharmacologic inhibition of BCL6 might provide a novel therapeutic strategy for ablation of leukemia-repopulating cells and increased responsiveness to chemotherapy.
Assuntos
Leucemia Mieloide Aguda/patologia , Proteínas de Neoplasias/fisiologia , Proteínas Proto-Oncogênicas c-bcl-6/fisiologia , Animais , Antineoplásicos/farmacologia , Apoptose , Autorrenovação Celular , Citarabina/uso terapêutico , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células-Tronco Hematopoéticas/citologia , Humanos , Indóis/farmacologia , Indóis/uso terapêutico , Leucemia Mieloide Aguda/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/citologia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-bcl-6/antagonistas & inibidores , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , RNA-Seq , Quimera por Radiação , Tiazolidinedionas/farmacologia , Tiazolidinedionas/uso terapêutico , Ensaio Tumoral de Célula-Tronco , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Intensive chemotherapy with cytarabine and anthracycline (7&3) remains the standard therapy for patients medically fit for induction, but the assessment of fitness remains controversial. Venetoclax and hypomethylating agent (ven/HMA) combination therapy has improved outcomes in unfit patients but no prospective study has assessed ven/HMA versus 7&3 as initial therapy in older, fit patients. Given no studies and expectation of ven/HMA use in patients outside of trial criteria, we evaluated retrospective outcomes among newly diagnosed patients. A nationwide electronic health record (EHR)-derived database and the University of Pennsylvania EHR identified 312 patients receiving 7&3 and 488 receiving ven/HMA who were 60-75 years old without history of organ failure. Ven/HMA patients were older and more likely to have secondary AML, adverse cytogenetics, and adverse mutations. Median overall survival (OS) for patients receiving intensive chemotherapy was 22 versus 10 months for ven/HMA (HR 0.53, 95% CI 0.40-0.60). Controlling for measured baseline characteristic imbalances reduced survival advantage by half (HR 0.71, 95% CI 0.53-0.94). A sub-group of patients with equipoise, likelihood at least 30%-70% of receiving either treatment, had similar OS outcomes (HR 1.10, 95% CI 0.75-1.6). Regarding safety outcomes, 60-day mortality was higher for ven/HMA (15% vs. 6% at 60 days) despite higher documented infections and febrile neutropenia for 7&3. In this multicenter real-word dataset, patients selected for intensive chemotherapy had superior OS but a large group had similar outcomes with ven/HMA. Prospective randomized studies, controlling for both measured and unmeasured confounders, must confirm this outcome.
Assuntos
Citarabina , Leucemia Mieloide Aguda , Humanos , Idoso , Pessoa de Meia-Idade , Estudos Retrospectivos , Compostos Bicíclicos Heterocíclicos com Pontes/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversosRESUMO
Acute erythroleukemia (AEL or acute myeloid leukemia [AML]-M6) is a rare but aggressive hematologic malignancy. Previous studies showed that AEL leukemic cells often carry complex karyotypes and mutations in known AML-associated oncogenes. To better define the underlying molecular mechanisms driving the erythroid phenotype, we studied a series of 33 AEL samples representing 3 genetic AEL subgroups including TP53-mutated, epigenetic regulator-mutated (eg, DNMT3A, TET2, or IDH2), and undefined cases with low mutational burden. We established an erythroid vs myeloid transcriptome-based space in which, independently of the molecular subgroup, the majority of the AEL samples exhibited a unique mapping different from both non-M6 AML and myelodysplastic syndrome samples. Notably, >25% of AEL patients, including in the genetically undefined subgroup, showed aberrant expression of key transcriptional regulators, including SKI, ERG, and ETO2. Ectopic expression of these factors in murine erythroid progenitors blocked in vitro erythroid differentiation and led to immortalization associated with decreased chromatin accessibility at GATA1-binding sites and functional interference with GATA1 activity. In vivo models showed development of lethal erythroid, mixed erythroid/myeloid, or other malignancies depending on the cell population in which AEL-associated alterations were expressed. Collectively, our data indicate that AEL is a molecularly heterogeneous disease with an erythroid identity that results in part from the aberrant activity of key erythroid transcription factors in hematopoietic stem or progenitor cells.
Assuntos
Leucemia Eritroblástica Aguda/genética , Proteínas de Neoplasias/fisiologia , Fatores de Transcrição/fisiologia , Transcriptoma , Adulto , Animais , Transformação Celular Neoplásica/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Dioxigenases , Eritroblastos/metabolismo , Eritropoese/genética , Feminino , Fator de Transcrição GATA1/deficiência , Fator de Transcrição GATA1/genética , Técnicas de Introdução de Genes , Heterogeneidade Genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Pessoa de Meia-Idade , Mutação , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/metabolismo , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , RNA-Seq , Quimera por Radiação , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Fatores de Transcrição/genética , Regulador Transcricional ERG/genética , Regulador Transcricional ERG/fisiologia , Sequenciamento do Exoma , Adulto JovemRESUMO
Aberrant splicing is a hallmark of leukemias with mutations in splicing factor (SF)-encoding genes. Here we investigated its prevalence in pediatric B-cell acute lymphoblastic leukemias (B-ALL), where SFs are not mutated. By comparing these samples to normal pro-B cells, we found thousands of aberrant local splice variations (LSVs) per sample, with 279 LSVs in 241 genes present in every comparison. These genes were enriched in RNA processing pathways and encoded â¼100 SFs, e.g. hnRNPA1. HNRNPA1 3'UTR was most pervasively mis-spliced, yielding the transcript subject to nonsense-mediated decay. To mimic this event, we knocked it down in B-lymphoblastoid cells and identified 213 hnRNPA1-regulated exon usage events comprising the hnRNPA1 splicing signature in pediatric leukemia. Some of its elements were LSVs in DICER1 and NT5C2, known cancer drivers. We searched for LSVs in other leukemia and lymphoma drivers and discovered 81 LSVs in 41 additional genes. Seventy-seven LSVs out of 81 were confirmed using two large independent B-ALL RNA-seq datasets, and the twenty most common B-ALL drivers, including NT5C2, showed higher prevalence of aberrant splicing than of somatic mutations. Thus, post-transcriptional deregulation of SF can drive widespread changes in B-ALL splicing and likely contributes to disease pathogenesis.
Assuntos
Processamento Alternativo , Linfócitos B/metabolismo , Regulação Leucêmica da Expressão Gênica , Ribonucleoproteína Nuclear Heterogênea A1/genética , Degradação do RNAm Mediada por Códon sem Sentido , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Regiões 3' não Traduzidas , 5'-Nucleotidase/genética , 5'-Nucleotidase/metabolismo , Adulto , Linfócitos B/patologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Linhagem Celular Tumoral , Criança , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Éxons , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Humanos , Íntrons , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Cultura Primária de Células , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Fatores de Processamento de Serina-Arginina/antagonistas & inibidores , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Transativadores/genética , Transativadores/metabolismoRESUMO
Philadelphia chromosome (Ph)-like B-cell acute lymphoblastic leukemia (Ph-like ALL) is associated with activated JAK/STAT, Abelson kinase (ABL), and/or phosphatidylinositol 3-kinase (PI3K) signaling and poor clinical outcomes. PI3K pathway signaling inhibitors have been minimally investigated in Ph-like ALL. We hypothesized that targeted inhibition of PI3Kα, PI3Kδ, PI3K/mTOR, or target of rapamycin complex 1/2 (TORC1/TORC2) would decrease leukemia proliferation and abrogate aberrant kinase signaling and that combined PI3K pathway and JAK inhibition or PI3K pathway and SRC/ABL inhibition would have superior efficacy compared to inhibitor monotherapy. We treated 10 childhood ALL patient-derived xenograft models harboring various Ph-like genomic alterations with 4 discrete PI3K pathway protein inhibitors and observed marked leukemia reduction and in vivo signaling inhibition in all models. Treatment with dual PI3K/mTOR inhibitor gedatolisib resulted in near eradication of ALL in cytokine receptor-like factor 2 (CRLF2)/JAK-mutant models with mean 92.2% (range, 86.0%-99.4%) reduction vs vehicle controls (P < .0001) and in prolonged animal survival. Gedatolisib also inhibited ALL proliferation in ABL/platelet-derived growth factor receptor (PDGFR)-mutant models with mean 66.9% (range, 42.0%-87.6%) reduction vs vehicle (P < .0001). Combined gedatolisib and ruxolitinib treatment of CRLF2/JAK-mutant models more effectively inhibited ALL proliferation than either inhibitor alone (P < .001) and further enhanced survival. Similarly, superior efficacy of combined gedatolisib and dasatinib was observed in ABL/PDGFR-mutant models (P < .001). Overall, PI3K/mTOR inhibition potently decreased ALL burden in vivo; antileukemia activity was further enhanced with combination inhibitor therapy. Clinical trials testing combinations of kinase inhibitors in Ph-like ALL patients are indicated.
Assuntos
Antineoplásicos/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Humanos , Janus Quinases/antagonistas & inibidores , Camundongos , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Distribuição Aleatória , Serina-Treonina Quinases TOR/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We and others previously reported potent antileukemia efficacy of CD123-redirected chimeric antigen receptor (CAR) T cells in preclinical human acute myeloid leukemia (AML) models at the cost of severe hematologic toxicity. This observation raises concern for potential myeloablation in patients with AML treated with CD123-redirected CAR T cells and mandates novel approaches for toxicity mitigation. We hypothesized that CAR T-cell depletion with optimal timing after AML eradication would preserve leukemia remission and allow subsequent hematopoietic stem cell transplantation. To test this hypothesis, we compared 3 CAR T-cell termination strategies: (1) transiently active anti-CD123 messenger RNA-electroporated CART (RNA-CART123); (2) T-cell ablation with alemtuzumab after treatment with lentivirally transduced anti-CD123-4-1BB-CD3ζ T cells (CART123); and (3) T-cell ablation with rituximab after treatment with CD20-coexpressing CART123 (CART123-CD20). All approaches led to rapid leukemia elimination in murine xenograft models of human AML. Subsequent antibody-mediated depletion of CART123 or CART123-CD20 did not impair leukemia remission. Time-course studies demonstrated that durable leukemia remission required CAR T-cell persistence for 4 weeks prior to ablation. Upon CAR T-cell termination, we further demonstrated successful hematopoietic engraftment with a normal human donor to model allogeneic stem cell rescue. Results from these studies will facilitate development of T-cell depletion strategies to augment the feasibility of CAR T-cell therapy for patients with AML.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Imunoterapia Adotiva/métodos , Subunidade alfa de Receptor de Interleucina-3/imunologia , Leucemia Mieloide Aguda/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Alemtuzumab , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Antígenos CD20/genética , Antígenos CD20/imunologia , Complexo CD3/genética , Complexo CD3/imunologia , Feminino , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Subunidade alfa de Receptor de Interleucina-3/antagonistas & inibidores , Subunidade alfa de Receptor de Interleucina-3/genética , Lentivirus/genética , Lentivirus/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Depleção Linfocítica , Masculino , Camundongos , Camundongos Endogâmicos NOD , RNA Antissenso/genética , RNA Antissenso/imunologia , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Receptores de Antígenos de Linfócitos T/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Indução de Remissão , Rituximab/farmacologia , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/transplante , Transplante Heterólogo , Resultado do Tratamento , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
To improve the outcome of relapsed/refractory acute myeloid leukemia (AML), a randomized phase II trial of three novel regimens was conducted. Ninety patients were enrolled and were in first relapse or were refractory to induction/re-induction chemotherapy. They were randomized to the following regimens: carboplatin-topotecan (CT), each by continuous infusion for 5 days; alvocidib (formerly flavopiridol), cytarabine, and mitoxantrone (FLAM) in a timed sequential regimen; or sirolimus combined with mitoxantrone, etoposide, and cytarabine (S-MEC). The primary objective was attainment of a complete remission (CR). A Simon two-stage design was used for each of the three arms. The median age of the patients in the FLAM arm was older at 62 years compared with 55 years for the CT arm and the S-MEC arm. The overall response was 14% in the CT arm (5/35, 90% CI 7%-35%), 28% in the FLAM arm (10/36, 90% CI, 16%-43%), and 16% in the S-MEC arm (3/19, 90% CI, 4%-36%). There were nine treatment-related deaths, seven of which occurred in the FLAM arm with four of these in elderly patients. We conclude that the FLAM regimen had an encouraging response rate and should be considered for further clinical development but should be used with caution in elderly patients.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Terapia de Salvação , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Carboplatina/administração & dosagem , Carboplatina/efeitos adversos , Citarabina/administração & dosagem , Citarabina/efeitos adversos , Intervalo Livre de Doença , Etoposídeo/administração & dosagem , Etoposídeo/efeitos adversos , Feminino , Flavonoides/administração & dosagem , Flavonoides/efeitos adversos , Seguimentos , Gastroenteropatias/induzido quimicamente , Doenças Hematológicas/induzido quimicamente , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Mitoxantrona/administração & dosagem , Mitoxantrona/efeitos adversos , Piperidinas/administração & dosagem , Piperidinas/efeitos adversos , Recidiva , Indução de Remissão , Terapia de Salvação/efeitos adversos , Sirolimo/administração & dosagem , Sirolimo/efeitos adversos , Topotecan/administração & dosagem , Topotecan/efeitos adversos , Síndrome de Lise Tumoral/etiologiaRESUMO
Background Mammalian Target of Rapamycin Complex 1 (mTORC1) inhibitors enhance chemotherapy response in acute myelogenous leukemia (AML) cells in vitro. However whether inhibiting mTORC1 enhances clinical response to AML chemotherapy remains controversial. We previously optimized measurement of mTORC1's kinase activity in AML blasts during clinical trials using serial phospho-specific flow cytometry of formaldehyde-fixed whole blood or marrow specimens. To validate mTORC1 as a therapeutic target in AML, we performed two clinical trials combining an mTORC1 inhibitor (sirolimus) and MEC (mitoxantrone, etoposide, cytarabine) in patients with relapsed, refractory, or untreated high-risk AML. Methods Flow cytometric measurements of ribosomal protein S6 phosphorylation (pS6) were performed before and during sirolimus treatment to determine whether mTORC1 inhibition enriched for chemotherapy response. Results In 51 evaluable subjects, the overall response rate (ORR) to the combination regimen was 47% (95% confidence interval 33-61%, 33% CR, 2% CRi, 12% PR) and similar toxicity to historic experience with MEC alone. 37 subjects had baseline pS6 measured pre-sirolimus, of whom 27 (73%) exhibited mTORC1 activity. ORR was not significantly different between subjects with and without baseline mTORC1 activity (52% vs 40%, respectively, p = 0.20). The ORR among subjects with baseline target activation and mTORC1 inhibition during therapy was 71% (12/17) compared to 20% (2/10) in subjects without target inhibition. Conclusions Fixed, whole blood pS6 by flow cytometry may be a predictive biomarker for clinical response to mTORC1 inhibitor-based regimens. These data provide clinical confirmation that mTORC1 activation mediates chemotherapy resistance in patients with AML.
Assuntos
Leucemia Mieloide Aguda/tratamento farmacológico , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Sirolimo/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Citarabina/uso terapêutico , Etoposídeo/uso terapêutico , Feminino , Humanos , Leucemia Mieloide Aguda/metabolismo , Masculino , Pessoa de Meia-Idade , Mitoxantrona/uso terapêutico , Fosforilação/efeitos dos fármacos , Projetos Piloto , Indução de Remissão/métodos , Transdução de Sinais/efeitos dos fármacosRESUMO
Patients with acute myeloid leukemia and a high white blood cell count are at increased risk of early death and relapse. Because mediators of inflammation contribute to leukostasis and chemoresistance, dexamethasone added to chemotherapy could improve outcomes. This retrospective study evaluated the impact of adding or not adding dexamethasone to chemotherapy in a cohort of 160 patients with at least 50×109 white blood cells. In silico studies, primary samples, leukemic cell lines, and xenograft mouse models were used to explore the antileukemic activity of dexamethasone. There was no difference with respect to induction death rate, response, and infections between the 60 patients in the dexamethasone group and the 100 patients in the no dexamethasone group. Multivariate analysis showed that dexamethasone was significantly associated with improved relapse incidence (adjusted sub-HR: 0.30; 95% CI: 0.14-0.62; P=0.001), disease-free survival (adjusted HR: 0.50; 95% CI: 0.29-0.84; P=0.010), event-free survival (adjusted HR: 0.35; 95% CI: 0.21-0.58; P<0.001), and overall survival (adjusted HR: 0.41; 95% CI: 0.22-0.79; P=0.007). In a co-culture system, dexamethasone reduced the frequency of leukemic long-term culture initiating cells by 38% and enhanced the cytotoxicity of doxorubicin and cytarabine. In a patient-derived xenograft model treated with cytarabine, chemoresistant cells were enriched in genes of the inflammatory response modulated by dexamethasone. Dexamethasone also demonstrated antileukemic activity in NPM1-mutated samples. Dexamethasone may improve the outcome of acute myeloid leukemia patients receiving intensive chemotherapy. This effect could be due to the modulation of inflammatory chemoresistance pathways and to a specific activity in acute myeloid leukemia with NPM1 mutation.
Assuntos
Antineoplásicos Hormonais/uso terapêutico , Dexametasona/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Leucocitose/tratamento farmacológico , Leucocitose/patologia , Adolescente , Adulto , Idoso , Antineoplásicos Hormonais/administração & dosagem , Antineoplásicos Hormonais/efeitos adversos , Dexametasona/administração & dosagem , Dexametasona/efeitos adversos , Resistencia a Medicamentos Antineoplásicos , Feminino , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Estimativa de Kaplan-Meier , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Leucocitose/genética , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas Nucleares/genética , Nucleofosmina , Prognóstico , Recidiva , Indução de Remissão , Resultado do Tratamento , Adulto JovemRESUMO
Patient-derived xenotransplantation models of human myeloid diseases including acute myeloid leukemia, myelodysplastic syndromes and myeloproliferative neoplasms are essential for studying the biology of the diseases in pre-clinical studies. However, few studies have used these models for comparative purposes. Previous work has shown that acute myeloid leukemia blasts respond to human hematopoietic cytokines whereas myelodysplastic syndrome cells do not. We compared the engraftment of acute myeloid leukemia cells and myelodysplastic syndrome cells in NSG mice to that in NSG-S mice, which have transgene expression of human cytokines. We observed that only 50% of all primary acute myeloid leukemia samples (n=77) transplanted in NSG mice provided useful levels of engraftment (>0.5% human blasts in bone marrow). In contrast, 82% of primary acute myeloid leukemia samples engrafted in NSG-S mice with higher leukemic burden and shortened survival. Additionally, all of 5 injected samples from patients with myelodysplastic syndrome showed persistent engraftment on week 6; however, engraftment was mostly low (<2%), did not increase over time, and was only transiently affected by the use of NSG-S mice. Co-injection of mesenchymal stem cells did not enhance human myelodysplastic syndrome cell engraftment. Overall, we conclude that engraftment of acute myeloid leukemia samples is more robust compared to that of myelodysplastic syndrome samples and unlike those, acute myeloid leukemia cells respond positively to human cytokines, whereas myelodysplastic syndrome cells demonstrate a general unresponsiveness to them.
Assuntos
Citocinas/metabolismo , Sobrevivência de Enxerto/imunologia , Hospedeiro Imunocomprometido , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/metabolismo , Síndromes Mielodisplásicas/imunologia , Síndromes Mielodisplásicas/metabolismo , Animais , Transplante de Medula Óssea , Citocinas/sangue , Modelos Animais de Doenças , Feminino , Humanos , Leucemia Mieloide Aguda/terapia , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Síndromes Mielodisplásicas/terapia , Transplante HeterólogoRESUMO
Arabinosylcytosine (araC) is a mainstay in the initial treatment of acute myeloid leukemia (AML), although relapses are common. Given the recent recognition of altered DNA methylation patterns in relapsed AML, we considered whether araC, which acts by incorporation into DNA, could itself perturb methylation dynamics. To explore this possibility, we examined several DNA methyltransferases and find that araC embedded in DNA is consistently methylated with an efficiency diminished relative to that of deoxycytidine. Importantly, with the human maintenance methyltransferase DNMT1, the extent of araC methylation is reduced by more than â¼200-fold. These observations support a model whereby araC treatment may itself contribute to locus-specific, passive DNA demethylation in relapsed AML.
Assuntos
Antimetabólitos Antineoplásicos/metabolismo , Citarabina/metabolismo , Metilases de Modificação do DNA/metabolismo , Epigênese Genética , Leucemia Mieloide Aguda/genética , Antimetabólitos Antineoplásicos/uso terapêutico , Citarabina/uso terapêutico , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/enzimologiaRESUMO
Tyrosine kinase inhibitors (TKIs) represent transformative therapies for several malignancies. Two critical features necessary for maximizing TKI tolerability and response duration are kinase selectivity and invulnerability to resistance-conferring kinase domain (KD) mutations in the intended target. No prior TKI has demonstrated both of these properties. Aiming to maximize selectivity, medicinal chemists have largely sought to create TKIs that bind to an inactive (type II) kinase conformation. Here we demonstrate that the investigational type I TKI crenolanib is a potent inhibitor of Fms tyrosine kinase-3 (FLT3) internal tandem duplication, a validated therapeutic target in human acute myeloid leukemia (AML), as well as all secondary KD mutants previously shown to confer resistance to the first highly active FLT3 TKI quizartinib. Moreover, crenolanib is highly selective for FLT3 relative to the closely related protein tyrosine kinase KIT, demonstrating that simultaneous FLT3/KIT inhibition, a prominent feature of other clinically active FLT3 TKIs, is not required for AML cell cytotoxicity in vitro and may contribute to undesirable toxicity in patients. A saturation mutagenesis screen of FLT3-internal tandem duplication failed to recover any resistant colonies in the presence of a crenolanib concentration well below what has been safely achieved in humans, suggesting that crenolanib has the potential to suppress KD mutation-mediated clinical resistance. Crenolanib represents the first TKI to exhibit both kinase selectivity and invulnerability to resistance-conferring KD mutations, which is unexpected of a type I inhibitor. Crenolanib has significant promise for achieving deep and durable responses in FLT3-mutant AML, and may have a profound impact upon future medicinal chemistry efforts in oncology.