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In spite of therapeutic improvements in the treatment of different hematologic malignancies, the prognosis of acute myeloid leukemia (AML) treated solely with conventional induction and consolidation chemotherapy remains poor, especially in association with high risk chromosomal or molecular aberrations. Recent discoveries describe the complex interaction of immune effector cells, as well as the role of the bone marrow microenvironment in the development, maintenance and progression of AML. Lipids, and in particular omega-3 as well as omega-6 polyunsaturated fatty acids (PUFAs) have been shown to play a vital role as signaling molecules of immune processes in numerous benign and malignant conditions. While the majority of research in cancer has been focused on the role of lipid mediators in solid tumors, some data are showing their involvement also in hematologic malignancies. There is a considerable amount of evidence that AML cells are targetable by innate and adaptive immune mechanisms, paving the way for immune therapy approaches in AML. In this article we review the current data showing the lipid mediator and lipidome patterns in AML and their potential links to immune mechanisms.
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Leucemia Mieloide Aguda/tratamento farmacológico , Lipídeos/uso terapêutico , Imunidade Adaptativa/efeitos dos fármacos , Medula Óssea , Progressão da Doença , Ácidos Graxos Ômega-3/imunologia , Ácidos Graxos Ômega-3/uso terapêutico , Ácidos Graxos Ômega-6/imunologia , Ácidos Graxos Ômega-6/uso terapêutico , Ácidos Graxos Insaturados , Neoplasias Hematológicas/tratamento farmacológico , Hematopoese , Humanos , Imunidade Inata/efeitos dos fármacos , Imunoterapia , Inflamação , Leucemia Mieloide Aguda/imunologia , Lipídeos/imunologia , Neoplasias/tratamento farmacológico , Prognóstico , Microambiente TumoralRESUMO
The modified Matutes score has been the basis for the diagnosis of chronic lymphocytic leukaemia (CLL) by flow cytometry for the past 15 years. To increase the specificity of the current score we systematically evaluated the diagnostic value of established as well as novel markers, such as CD200, in a large cohort of patients with untreated B-cell malignancies (n = 370). Double positivity for CD5 and CD23 was of very high value to differentiate between CLL and non-CLL cases. In addition, lack of FMC7 expression as well as CD79b expression intensity showed high sensitivity (90·4% and 92·3%) with acceptable specificity (74·4% and 76·9%). For surface IgM, low or absent expression displayed poor specificity in distinguishing CLL from non-CLL cases (51,3%; sensitivity 83,7%). Finally, CD200 positivity showed high sensitivity and specificity. Therefore, CD5/CD23, FMC7, CD79b and CD200 were included in our new CLLflow score, which retained high sensitivity (97·1% vs. 98·6% for the Matutes score, P = 0·38), but showed markedly increased specificity (87·2% vs. 53·8%, P < 0·001). These results were confirmed in our validation cohort (sensitivity 97·0% vs. 100%, P = not applicable; specificity 86·4% vs. 59·1%, P = 0·03). Our data support the use of our new CLLflow score for the diagnosis of CLL with significantly higher specificity.
Assuntos
Antígenos CD/sangue , Biomarcadores Tumorais/sangue , Leucemia Linfocítica Crônica de Células B/diagnóstico , Antígenos CD5/sangue , Antígenos CD79/sangue , Diagnóstico Diferencial , Glicoproteínas/sangue , Humanos , Imunoglobulina M/sangue , Imunofenotipagem , Receptores de IgE/sangue , Sensibilidade e EspecificidadeRESUMO
Antibody-based immunotherapy represents a promising strategy to target and eliminate chemoresistant leukemic cells. Here, we evaluated the CD33/CD3-bispecific T cell engaging (BiTE) antibody (AMG 330) for its suitability as a therapeutic agent in acute myeloid leukemia (AML). We first assessed CD33 expression levels by flow cytometry and found expression in >99% of patient samples (n = 621). CD33 was highest expressed in AMLs with NPM1 mutations (P < .001) and lower in AMLs with complex karyotypes and t(8;21) translocations (P < .001). Furthermore, leukemic stem cells within the CD34(+)/CD38(-) compartment displayed CD33 at higher levels than healthy donor stem cells (P = .047). In MS-5 feeder cell-based long-term cultures that supported the growth of primary AML blasts for up to 36 days, AMG 330 efficiently recruited and expanded residual CD3(+)/CD45RA(-)/CCR7(+) memory T cells within the patient sample. Even at low effector to target ratios, the recruited T cells lysed autologous blasts completely in the majority of samples and substantially in the remaining samples in a time-dependent manner. This study provides the first correlation of CD33 expression levels with AML genotype in a comprehensive analysis of adult patients. Targeting CD33 ex vivo using AMG 330 in primary AML samples led to T cell recruitment and expansion and remarkable antibody-mediated cytotoxicity, suggesting efficient therapeutic potential in vivo.
Assuntos
Anticorpos Biespecíficos/imunologia , Imunoterapia/métodos , Leucemia Mieloide Aguda/imunologia , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Linfócitos T/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Biespecíficos/uso terapêutico , Técnicas de Cultura de Células , Técnicas de Cocultura , Feminino , Citometria de Fluxo , Genótipo , Humanos , Cariotipagem , Leucemia Mieloide Aguda/terapia , Masculino , Pessoa de Meia-Idade , Nucleofosmina , Fatores de TempoRESUMO
We retrospectively compared the incidence of virus infections and outcome in the context of immune reconstitution in two different HLA-haploidentical transplantation (haplo-HSCT) settings. The first was a combined T-cell-replete and T-cell-deplete approach using antithymocyte globulin (ATG) prior to transplantation in patients with hematological diseases (cTCR/TCD group, 28 patients; median age 31 years). The second was a T-cell-replete (TCR) approach using high-dose posttransplantation cyclophosphamide (TCR/PTCY group, 27 patients; median age 43 years). The incidence of herpesvirus infection was markedly lower in the TCR/PTCY (22 %) than in the cTCR/TCD group (93 %). Recovery of CD4+ T cells on day +100 was faster in the TCR/PTCY group. CMV reactivation was 30 % in the TCR/PTCY compared to 57 % in the cTCR/TCD group, and control with antiviral treatment was superior after TCR/PTCY transplantation (100 vs 50 % cTCR/TCD). Twenty-five percent of the patients in the cTCR/TCD group but no patient in the TCR/PTCY group developed PTLD. While 1-year OS was not different (TCR/PTCY 59 % vs cTCR/TCD 39 %; p = 0.28), virus infection-related mortality (VIRM) was significantly lower after TCR/PTCY transplantation (1-year VIRM, 0 % TCR/PTCY vs 29 % cTCR/TCD; p = 0.009). On day +100, predictors of better OS were lymphocytes >300/µl, CD3+ T cells >200/µl, and CD4+ T cells >150/µl, whereas the application of steroids >1 mg/kg was correlated with worse outcome. Our results suggest that by presumably preserving antiviral immunity and allowing fast immune recovery of CD4+ T cells, the TCR approach using posttransplantation cyclophosphamide is well suited to handle the important issue of herpesvirus infection after haplo-HSCT.
Assuntos
Antígenos HLA/imunologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Infecções por Herpesviridae/epidemiologia , Infecções por Herpesviridae/imunologia , Recuperação de Função Fisiológica/imunologia , Adolescente , Adulto , Linfócitos T CD4-Positivos/imunologia , Feminino , Doença Enxerto-Hospedeiro/diagnóstico , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/imunologia , Haplótipos , Infecções por Herpesviridae/diagnóstico , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Adulto JovemAssuntos
Medula Óssea/patologia , Leucemia Mieloide Aguda/diagnóstico , Neoplasia Residual/diagnóstico , Adulto , Idoso , Biomarcadores , Feminino , Citometria de Fluxo , Humanos , Imunofenotipagem , Leucemia Mieloide Aguda/complicações , Leucemia Mieloide Aguda/etiologia , Leucemia Mieloide Aguda/terapia , Masculino , Pessoa de Meia-Idade , Prognóstico , Indução de Remissão , Análise de SobrevidaRESUMO
Monitoring minimal residual disease is an important way to identify patients with acute myeloid leukemia at high risk of relapse. In this study we investigated the prognostic potential of minimal residual disease monitoring by quantitative real-time polymerase chain reaction analysis of NPM1 mutations in patients treated in the AMLCG 1999, 2004 and 2008 trials. Minimal residual disease was monitored - in aplasia, after induction therapy, after consolidation therapy, and during follow-up - in 588 samples from 158 patients positive for NPM1 mutations A, B and D (with a sensitivity of 10(-6)). One hundred and twenty-seven patients (80.4%) achieved complete remission after induction therapy and, of these, 56 patients (44.1%) relapsed. At each checkpoint, minimal residual disease cut-offs were calculated. After induction therapy a cut-off NPM1 mutation ratio of 0.01 was associated with a high hazard ratio of 4.26 and the highest sensitivity of 76% for the prediction of relapse. This was reflected in a cumulative incidence of relapse after 2 years of 77.8% for patients with ratios above the cut-off versus 26.4% for those with ratios below the cut-off. In the favorable subgroup according to European LeukemiaNet, the cut-off after induction therapy also separated the cohort into two prognostic groups with a cumulative incidence of relapse of 76% versus 6% after 2 years. Our data demonstrate that in addition to pre-therapeutic factors, the course of minimal residual disease in an individual is an important prognostic factor and could be included in clinical trials for the guidance of post-remission therapy. The trials from which data were obtained were registered at www.clinicaltrials.gov (#NCT01382147, #NCT00266136) and at the European Leukemia Trial Registry (#LN_AMLINT2004_230).
Assuntos
Quimioterapia de Indução/métodos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Mutação/genética , Proteínas Nucleares/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Seguimentos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Nucleofosmina , Estudos Prospectivos , Recidiva , Estudos Retrospectivos , Fatores de Risco , Taxa de Sobrevida/tendências , Resultado do Tratamento , Adulto JovemRESUMO
Mutations in additional sex combs like 1 (ASXL1) confer poor prognosis both in myeloid malignancies and in premalignant clonal hematopoiesis (CH). However, the mechanisms by which these mutations contribute to disease initiation remain unresolved, and mutation-specific targeting has remained elusive. To address this, we developed a human disease model that recapitulates the disease trajectory from ASXL1-mutant CH to lethal myeloid malignancy. We demonstrate that mutations in ASXL1 lead to the expression of a functional, truncated protein and determine that truncated ASXL1 leads to global redistribution of the repressive chromatin mark H2AK119Ub, increased transposase-accessible chromatin, and activation of both myeloid and stem cell gene-expression programs. Finally, we demonstrate that H2AK119Ub levels are tied to truncated ASXL1 expression levels and leverage this observation to demonstrate that inhibition of the PRC1 complex might be an ASXL1-mutant-specific therapeutic vulnerability in both premalignant CH and myeloid malignancy. SIGNIFICANCE: Mutant ASXL1 is a common driver of CH and myeloid malignancy. Using primary human HSPCs, we determine that truncated ASXL1 leads to redistribution of H2AK119Ub and may affect therapeutic vulnerability to PRC1 inhibition.
Assuntos
Mutação , Proteínas Repressoras , Humanos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Ubiquitinação , Histonas/metabolismo , Histonas/genética , Hematopoese/genética , Hematopoiese Clonal/genéticaRESUMO
Rare preleukemic hematopoietic stem cells (pHSC) harboring only the initiating mutations can be detected at the time of acute myeloid leukemia (AML) diagnosis. pHSCs are the origin of leukemia and a potential reservoir for relapse. Using primary human samples and gene editing to model isocitrate dehydrogenase 1 (IDH1) mutant pHSCs, we show epigenetic, transcriptional, and metabolic differences between pHSCs and healthy hematopoietic stem cells (HSC). We confirm that IDH1-driven clonal hematopoiesis is associated with cytopenia, suggesting an inherent defect to fully reconstitute hematopoiesis. Despite giving rise to multilineage engraftment, IDH1-mutant pHSCs exhibited reduced proliferation, blocked differentiation, downregulation of MHC class II genes, and reprogramming of oxidative phosphorylation metabolism. Critically, inhibition of oxidative phosphorylation resulted in the complete eradication of IDH1-mutant pHSCs but not IDH2-mutant pHSCs or wild-type HSCs. Our results indicate that IDH1-mutant preleukemic clones can be targeted with complex I inhibitors, offering a potential strategy to prevent the development and relapse of leukemia. SIGNIFICANCE: A high burden of pHSCs is associated with worse overall survival in AML. Using single-cell sequencing, metabolic assessment, and gene-edited human models, we find human pHSCs with IDH1 mutations to be metabolically vulnerable and sensitive to eradication by complex I inhibition. See related commentary by Steensma.
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Hematopoietic multipotent progenitors (MPPs) regulate blood cell production to appropriately meet the biological demands of the human body. Human MPPs remain ill-defined whereas mouse MPPs have been well characterized with distinct immunophenotypes and lineage potencies. Using multiomic single cell analyses and complementary functional assays, we identified new human MPPs and oligopotent progenitor populations within Lin-CD34+CD38dim/lo adult bone marrow with distinct biomolecular and functional properties. These populations were prospectively isolated based on expression of CD69, CLL1, and CD2 in addition to classical markers like CD90 and CD45RA. We show that within the canonical Lin-CD34+CD38dim/loCD90CD45RA-MPP population, there is a CD69+ MPP with long-term engraftment and multilineage differentiation potential, a CLL1+ myeloid-biased MPP, and a CLL1-CD69-erythroid-biased MPP. We also show that the canonical Lin-CD34+CD38dim/loCD90-CD45RA+ LMPP population can be separated into a CD2+ LMPP with lymphoid and myeloid potential, a CD2-LMPP with high lymphoid potential, and a CLL1+ GMP with minimal lymphoid potential. We used these new HSPC profiles to study human and mouse bone marrow cells and observe limited cell type specific homology between humans and mice and cell type specific changes associated with aging. By identifying and functionally characterizing new adult MPP sub-populations, we provide an updated reference and framework for future studies in human hematopoiesis.
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Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional properties such as proliferation and differentiation, thereby promoting the development of hematologic malignancies. Efficient and precise genetic manipulation of HSPCs is required to model, characterize, and better understand the functional consequences of recurrent somatic mutations. Mutations can have a deleterious effect on a gene and result in loss-of-function (LOF) or, in stark contrast, may enhance function or even lead to novel characteristics of a particular gene, termed gain-of-function (GOF). In contrast to LOF mutations, GOF mutations almost exclusively occur in a heterozygous fashion. Current genome-editing protocols do not allow for the selective targeting of individual alleles, hampering the ability to model heterozygous GOF mutations. Here, we provide a detailed protocol on how to engineer heterozygous GOF hotspot mutations in human HSPCs by combining CRISPR/Cas9-mediated homology-directed repair and recombinant AAV6 technology for efficient DNA donor template transfer. Importantly, this strategy makes use of a dual fluorescent reporter system to allow for the tracking and purification of successfully heterozygously edited HSPCs. This strategy can be employed to precisely investigate how GOF mutations affect HSPC function and their progression toward hematological malignancies.
Assuntos
Mutação com Ganho de Função , Edição de Genes , Humanos , Edição de Genes/métodos , Células-Tronco Hematopoéticas , Mutação , Sistemas CRISPR-CasRESUMO
Relapse of acute myeloid leukemia (AML) is highly aggressive and often treatment refractory. We analyzed previously published AML relapse cohorts and found that 40% of relapses occur without changes in driver mutations, suggesting that non-genetic mechanisms drive relapse in a large proportion of cases. We therefore characterized epigenetic patterns of AML relapse using 26 matched diagnosis-relapse samples with ATAC-seq. This analysis identified a relapse-specific chromatin accessibility signature for mutationally stable AML, suggesting that AML undergoes epigenetic evolution at relapse independent of mutational changes. Analysis of leukemia stem cell (LSC) chromatin changes at relapse indicated that this leukemic compartment underwent significantly less epigenetic evolution than non-LSCs, while epigenetic changes in non-LSCs reflected overall evolution of the bulk leukemia. Finally, we used single-cell ATAC-seq paired with mitochondrial sequencing (mtscATAC) to map clones from diagnosis into relapse along with their epigenetic features. We found that distinct mitochondrially-defined clones exhibit more similar chromatin accessibility at relapse relative to diagnosis, demonstrating convergent epigenetic evolution in relapsed AML. These results demonstrate that epigenetic evolution is a feature of relapsed AML and that convergent epigenetic evolution can occur following treatment with induction chemotherapy.
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Isocitrate dehydrogenase 1 and 2 (IDH) are mutated in multiple cancers and drive production of (R)-2-hydroxyglutarate (2HG). We identified a lipid synthesis enzyme [acetyl CoA carboxylase 1 (ACC1)] as a synthetic lethal target in mutant IDH1 (mIDH1), but not mIDH2, cancers. Here, we analyzed the metabolome of primary acute myeloid leukemia (AML) blasts and identified an mIDH1-specific reduction in fatty acids. mIDH1 also induced a switch to b-oxidation indicating reprogramming of metabolism toward a reliance on fatty acids. Compared with mIDH2, mIDH1 AML displayed depletion of NADPH with defective reductive carboxylation that was not rescued by the mIDH1-specific inhibitor ivosidenib. In xenograft models, a lipid-free diet markedly slowed the growth of mIDH1 AML, but not healthy CD34+ hematopoietic stem/progenitor cells or mIDH2 AML. Genetic and pharmacologic targeting of ACC1 resulted in the growth inhibition of mIDH1 cancers not reversible by ivosidenib. Critically, the pharmacologic targeting of ACC1 improved the sensitivity of mIDH1 AML to venetoclax. SIGNIFICANCE: Oncogenic mutations in both IDH1 and IDH2 produce 2-hydroxyglutarate and are generally considered equivalent in terms of pathogenesis and targeting. Using comprehensive metabolomic analysis, we demonstrate unexpected metabolic differences in fatty acid metabolism between mutant IDH1 and IDH2 in patient samples with targetable metabolic interventions. See related commentary by Robinson and Levine, p. 266. This article is highlighted in the In This Issue feature, p. 247.
Assuntos
Isocitrato Desidrogenase , Leucemia Mieloide Aguda , Humanos , Glutaratos/metabolismo , Inibidores Enzimáticos/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , MutaçãoRESUMO
Disease-initiating mutations in the transcription factor RUNX1 occur as germline and somatic events that cause leukemias with particularly poor prognosis. However, the role of RUNX1 in leukemogenesis is not fully understood, and effective therapies for RUNX1-mutant leukemias remain elusive. Here, we used primary patient samples and a RUNX1-KO model in primary human hematopoietic cells to investigate how RUNX1 loss contributes to leukemic progression and to identify targetable vulnerabilities. Surprisingly, we found that RUNX1 loss decreased proliferative capacity and stem cell function. However, RUNX1-deficient cells selectively upregulated the IL-3 receptor. Exposure to IL-3, but not other JAK/STAT cytokines, rescued RUNX1-KO proliferative and competitive defects. Further, we demonstrated that RUNX1 loss repressed JAK/STAT signaling and rendered RUNX1-deficient cells sensitive to JAK inhibitors. Our study identifies a dependency of RUNX1-mutant leukemias on IL-3/JAK/STAT signaling, which may enable targeting of these aggressive blood cancers with existing agents.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core , Interleucina-3 , Leucemia , Humanos , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Regulação da Expressão Gênica , Interleucina-3/genética , Interleucina-3/farmacologia , Leucemia/tratamento farmacológico , Leucemia/genética , Transdução de SinaisRESUMO
Rare preleukemic hematopoietic stem cells (pHSCs) harboring only the initiating mutations can be detected at the time of AML diagnosis. pHSCs are the origin of leukemia and a potential reservoir for relapse. Using primary human samples and gene-editing to model isocitrate dehydrogenase 1 (IDH1) mutant pHSCs, we show epigenetic, transcriptional, and metabolic differences between pHSCs and healthy hematopoietic stem cells (HSCs). We confirm that IDH1 driven clonal hematopoiesis is associated with cytopenia, suggesting an inherent defect to fully reconstitute hematopoiesis. Despite giving rise to multilineage engraftment, IDH1-mutant pHSCs exhibited reduced proliferation, blocked differentiation, downregulation of MHC Class II genes, and reprogramming of oxidative phosphorylation metabolism. Critically, inhibition of oxidative phosphorylation resulted in complete eradication of IDH1-mutant pHSCs but not IDH2-mutant pHSCs or wildtype HSCs. Our results indicate that IDH1-mutant preleukemic clones can be targeted with complex I inhibitors, offering a potential strategy to prevent development and relapse of leukemia.
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Therapeutic cancer vaccination seeks to elicit activation of tumor-reactive T cells capable of recognizing tumor-associated antigens (TAA) and eradicating malignant cells. Here, we present a cancer vaccination approach utilizing myeloid-lineage reprogramming to directly convert cancer cells into tumor-reprogrammed antigen-presenting cells (TR-APC). Using syngeneic murine leukemia models, we demonstrate that TR-APCs acquire both myeloid phenotype and function, process and present endogenous TAAs, and potently stimulate TAA-specific CD4+ and CD8+ T cells. In vivo TR-APC induction elicits clonal expansion of cancer-specific T cells, establishes cancer-specific immune memory, and ultimately promotes leukemia eradication. We further show that both hematologic cancers and solid tumors, including sarcomas and carcinomas, are amenable to myeloid-lineage reprogramming into TR-APCs. Finally, we demonstrate the clinical applicability of this approach by generating TR-APCs from primary clinical specimens and stimulating autologous patient-derived T cells. Thus, TR-APCs represent a cancer vaccination therapeutic strategy with broad implications for clinical immuno-oncology. SIGNIFICANCE: Despite recent advances, the clinical benefit provided by cancer vaccination remains limited. We present a cancer vaccination approach leveraging myeloid-lineage reprogramming of cancer cells into APCs, which subsequently activate anticancer immunity through presentation of self-derived cancer antigens. Both hematologic and solid malignancies derive significant therapeutic benefit from reprogramming-based immunotherapy. This article is highlighted in the In This Issue feature, p. 1027.
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Vacinas Anticâncer , Leucemia , Neoplasias , Animais , Camundongos , Células Apresentadoras de Antígenos , Neoplasias/terapia , Antígenos de Neoplasias , ImunoterapiaRESUMO
BACKGROUND: Immunotherapy of acute myeloid leukemia has experienced considerable advances, however novel target antigens continue to be sought after. To this end, unbiased approaches for surface protein detection are limited and integration with other data types, such as gene expression and somatic mutational burden, are poorly utilized. The Cell Surface Capture technology provides an unbiased, discovery-driven approach to map the surface proteins on cells of interest. Yet, direct utilization of primary patient samples has been limited by the considerable number of viable cells needed. METHODS: Here, we optimized the Cell Surface Capture protocol to enable direct interrogation of primary patient samples and applied our optimized protocol to a set of samples from patients with acute myeloid leukemia (AML) to generate the AML surfaceome. We then further curated this AML surfaceome to exclude antigens expressed on healthy tissues and integrated mutational burden data from hematologic cancers to further enrich for targets which are likely to be essential to leukemia biology. Finally, we validated our findings in a separate cohort of AML patient samples. RESULTS: Our protocol modifications allowed us to double the yield in identified proteins and increased the specificity from 54 to 80.4% compared to previous approaches. Using primary AML patient samples, we were able to identify a total of 621 surface proteins comprising the AML surfaceome. We integrated this data with gene expression and mutational burden data to curate a set of robust putative target antigens. Seventy-six proteins were selected as potential candidates for further investigation of which we validated the most promising novel candidate markers, and identified CD148, ITGA4 and Integrin beta-7 as promising targets in AML. Integrin beta-7 showed the most promising combination of expression in patient AML samples, and low or absent expression on healthy hematopoietic tissue. CONCLUSION: Taken together, we demonstrate the feasibility of a highly optimized surfaceome detection method to interrogate the entire AML surfaceome directly from primary patient samples and integrate this data with gene expression and mutational burden data to achieve a robust, multiomic target identification platform. This approach has the potential to accelerate the unbiased target identification for immunotherapy of AML.
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The conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) is a key step in DNA demethylation that is mediated by ten-eleven translocation (TET) enzymes, which require ascorbate/vitamin C. Here, we report the 5hmC landscape of normal hematopoiesis and identify cell type-specific 5hmC profiles associated with active transcription and chromatin accessibility of key hematopoietic regulators. We utilized CRISPR/Cas9 to model TET2 loss-of-function mutations in primary human hematopoietic stem and progenitor cells (HSPC). Disrupted cells exhibited increased colonies in serial replating, defective erythroid/megakaryocytic differentiation, and in vivo competitive advantage and myeloid skewing coupled with reduction of 5hmC at erythroid-associated gene loci. Azacitidine and ascorbate restored 5hmC abundance and slowed or reverted the expansion of TET2-mutant clones in vivo. These results demonstrate the key role of 5hmC in normal hematopoiesis and TET2-mutant phenotypes and raise the possibility of utilizing these agents to further our understanding of preleukemia and clonal hematopoiesis. SIGNIFICANCE: We show that 5-hydroxymethylation profiles are cell type-specific and associated with transcriptional abundance and chromatin accessibility across human hematopoiesis. TET2 loss caused aberrant growth and differentiation phenotypes and disrupted 5hmC and transcriptional landscapes. Treatment of TET2 KO HSPCs with ascorbate or azacitidine reverted 5hmC profiles and restored aberrant phenotypes. This article is highlighted in the In This Issue feature, p. 265.
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Dioxigenases , Síndromes Mielodisplásicas , Pré-Leucemia , Azacitidina/farmacologia , Cromatina/genética , Proteínas de Ligação a DNA/genética , Dioxigenases/genética , Hematopoese/genética , Humanos , Proteínas Proto-Oncogênicas/genéticaRESUMO
Our knowledge of how clonal hematopoiesis relates to diverse health conditions has grown vastly over the past years, touching upon many specialties beyond cancer medicine. Given that clonal hematopoiesis can act as a precursor to overt disease in many settings, the promise of early intervention has garnered much attention. In this review, we discuss the state of clonal hematopoiesis research and outline the challenges in developing clinical trials of early interventions. We anticipate that incidental findings of clonal hematopoiesis will become more common in the near future, but evidence-based efforts of how to manage these findings is currently lacking. SIGNIFICANCE: Our knowledge regarding the relevance of clonal hematopoiesis has increased drastically over the past years. However, evidence of how to manage these findings is currently lacking. In this review, we summarize the current state of clonal hematopoiesis research and outline the challenges of developing clinical trials in this field. We anticipate that incidental findings of clonal hematopoiesis will become more common in the near future and argue that there is urgency to start designing and conducting prospective trials.
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Hematopoiese Clonal , Neoplasias , Evolução Clonal , Hematopoiese Clonal/genética , Hematopoese/genética , Humanos , Mutação , Neoplasias/genética , Neoplasias/terapia , Estudos ProspectivosRESUMO
Targeted DNA correction of disease-causing mutations in hematopoietic stem and progenitor cells (HSPCs) may enable the treatment of genetic diseases of the blood and immune system. It is now possible to correct mutations at high frequencies in HSPCs by combining CRISPR/Cas9 with homologous DNA donors. Because of the precision of gene correction, these approaches preclude clonal tracking of gene-targeted HSPCs. Here, we describe Tracking Recombination Alleles in Clonal Engraftment using sequencing (TRACE-Seq), a methodology that utilizes barcoded AAV6 donor template libraries, carrying in-frame silent mutations or semi-randomized nucleotides outside the coding region, to track the in vivo lineage contribution of gene-targeted HSPC clones. By targeting the HBB gene with an AAV6 donor template library consisting of ~20,000 possible unique exon 1 in-frame silent mutations, we track the hematopoietic reconstitution of HBB targeted myeloid-skewed, lymphoid-skewed, and balanced multi-lineage repopulating human HSPC clones in mice. We anticipate this methodology could potentially be used for HSPC clonal tracking of Cas9 RNP and AAV6-mediated gene targeting outcomes in translational and basic research settings.
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Alelos , Células Clonais , Marcação de Genes/métodos , Células-Tronco Hematopoéticas , Recombinação Genética , Animais , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Feminino , Edição de Genes/métodos , Terapia Genética/métodos , Humanos , Camundongos , Mutação , Reparo Gênico Alvo-Dirigido/métodosRESUMO
Although most patients with acute myeloid leukemia (AML) achieve clinical remission with induction chemotherapy, relapse rates remain high. Next-generation sequencing enables minimal/measurable residual disease (MRD) detection; however, clinical significance is limited due to difficulty differentiating between pre-leukemic clonal hematopoiesis and frankly malignant clones. Here, we investigated AML MRD using targeted single-cell sequencing (SCS) at diagnosis, remission, and relapse (n = 10 relapsed, n = 4 nonrelapsed), with a total of 310 737 single cells sequenced. Sequence variants were identified in 80% and 75% of remission samples for patients with and without relapse, respectively. Pre-leukemic clonal hematopoiesis clones were detected in both cohorts, and clones with multiple cooccurring mutations were observed in 50% and 0% of samples. Similar clonal richness was observed at diagnosis in both cohorts; however, decreasing clonal diversity at remission was significantly associated with longer relapse-free survival. These results show the power of SCS in investigating AML MRD and clonal evolution.