Molecular subgroups of T-cell acute lymphoblastic leukemia in adults treated according to pediatric-based GMALL protocols.

In contrast to B-cell precursor acute lymphoblastic leukemia (ALL), molecular subgroups are less well defined in T-lineage ALL. Comprehensive studies on molecular T-ALL subgroups have been predominantly performed in pediatric ALL patients. Currently, molecular characteristics are rarely considered for risk stratification. Herein, we present a homogenously treated cohort of 230 adult T-ALL patients characterized on transcriptome, and partly on DNA methylation and gene mutation level in correlation with clinical outcome. We identified nine molecular subgroups based on aberrant oncogene expression correlating to four distinct DNA methylation patterns. The subgroup distribution differed from reported pediatric T-ALL cohorts with higher frequencies of prognostic unfavorable subgroups like HOXA or LYL1/LMO2. A small subset (3%) of HOXA adult T-ALL patients revealed restricted expression of posterior HOX genes with aberrant activation of lncRNA HOTTIP. With respect to outcome, TLX1 (n = 44) and NKX2-1 (n = 4) had an exceptionally favorable 3-year overall survival (3y-OS) of 94%. Within thymic T-ALL, the non TLX1 patients had an inferior but still good prognosis. To our knowledge this is the largest cohort of adult T-ALL patients characterized by transcriptome sequencing with meaningful clinical follow-up. Risk classification based on molecular subgroups might emerge and contribute to improvements in outcome.

Somatic epimutations enable single-cell lineage tracing in native hematopoiesis across the murine and human lifespan.

Current approaches to lineage tracing of stem cell clones require genetic engineering or rely on sparse somatic DNA variants, which are difficult to capture at single-cell resolution. Here, we show that targeted single-cell measurements of DNA methylation at single-CpG resolution deliver joint information about cellular differentiation state and clonal identities. We develop EPI-clone, a droplet-based method for transgene-free lineage tracing, and apply it to study hematopoiesis, capturing hundreds of clonal trajectories across almost 100,000 single-cells. Using ground-truth genetic barcodes, we demonstrate that EPI-clone accurately identifies clonal lineages throughout hematopoietic differentiation. Applied to unperturbed hematopoiesis, we describe an overall decline of clonal complexity during murine ageing and the expansion of rare low-output stem cell clones. In aged human donors, we identified expanded hematopoietic clones with and without genetic lesions, and various degrees of clonal complexity. Taken together, EPI-clone enables accurate and transgene-free single-cell lineage tracing at scale.

The IL-7R antagonist Lusvertikimab reduces leukemic burden in xenograft-ALL via antibody-dependent cellular phagocytosis.

Acute lymphoblastic leukemia (ALL) arises from the uncontrolled proliferation of precursor B or T cells (BCP- or T-ALL). Current treatment protocols obtain high cure rates in children but are based on toxic polychemotherapy. Novel therapies are urgently needed, especially in relapsed/refractory (r/r) disease, high-risk leukemias and T-ALL, where immunotherapy approaches remain scarce. While the Interleukin-7 receptor (IL-7R) plays a pivotal role in ALL development, no IL-7R-targeting immunotherapy has yet reached clinical application in ALL. The IL-7Rα chain (CD127)-targeting IgG4 antibody Lusvertikimab (formerly OSE-127) is a full antagonist of the IL-7R pathway showing a good safety profile in healthy volunteers. Here, we show that ~85% of ALL cases express surface CD127. We demonstrate significant in vivo efficacy of Lusvertikimab immunotherapy in a heterogeneous cohort of BCP- and T-ALL patient-derived xenografts (PDX) in minimal residual disease (MRD) and overt leukemia models, including r/r and high-risk leukemias. Importantly, Lusvertikimab was particularly effective when combined with polychemotherapy in a phase 2-like PDX study with CD127high samples leading to MRD-negativity in >50% of mice treated with combination therapy. Mechanistically, Lusvertikimab targeted ALL cells via a dual mode of action comprising direct IL-7R antagonistic activity and induction of macrophage-mediated antibody-dependent cellular phagocytosis (ADCP). Lusvertikimab-mediated in vitro ADCP levels significantly correlated with CD127 expression levels and the reduction of leukemia burden upon treatment of PDX animals in vivo. Altogether, through its dual mode of action and good safety profile, Lusvertikimab may represent a novel immunotherapy option for any CD127-positive ALL, particularly in combination with standard-of-care polychemotherapy.

Inotuzumab Ozogamicin as Induction Therapy for Patients Older Than 55 Years With Philadelphia Chromosome-Negative B-Precursor ALL.

PURPOSE: Despite recent advances in adapting the intensity of treatment for older patients with ALL, current protocols are associated with high rates of early deaths, treatment-related toxicity, and dismal prognosis. We evaluated inotuzumab ozogamicin and dexamethasone (Dex) as induction therapy in older patients with ALL within the German Multicenter Study Group for Adult ALL (GMALL). PATIENTS AND METHODS: The open-label, multicenter, phase II, INITIAL-1 trial enrolled 45 patients older than 55 years with newly diagnosed, CD22-positive, BCR::ABL-negative B-precursor ALL (B-ALL). Patients received up to three cycles of inotuzumab ozogamicin/Dex and up to six cycles of age-adapted GMALL consolidation and maintenance therapy. RESULTS: Forty-three evaluable patients with common/pre-B (n = 38) and pro-B ALL (n = 5), with a median age of 64 years (range, 56-80), received at least two cycles of inotuzumab ozogamicin induction therapy. All patients achieved complete remission (CR/CR with incomplete hematologic recovery). Twenty-three (53%) and 30 (71%) patients had no evidence of molecularly assessed measurable residual disease (minimum 10e-4 threshold) after the second and third inductions, respectively. After a median follow-up of 2.7 years, event-free survival at one (primary end point) and 3 years was 88% (95% CI, 79 to 98) and 55% (95% CI, 40 to 71), while overall survival (OS) was 91% (95% CI, 82 to 99) and 73% (95% CI, 59 to 87), respectively. None of the patients died during 6 months after the start of induction. Most common adverse events having common toxicity criteria grade ≥3 during induction were leukocytopenia, neutropenia, thrombocytopenia, anemia, and elevated liver enzymes. One patient developed nonfatal veno-occlusive disease after induction II. CONCLUSION: Inotuzumab ozogamicin-based induction followed by age-adapted chemotherapy was well tolerated and resulted in high rates of remission and OS. These data provide a rationale for integrating inotuzumab ozogamicin into first-line regimens for older patients with B-ALL.

The ribosomal protein S6 kinase alpha-1 (RPS6KA1) induces resistance to venetoclax/azacitidine in acute myeloid leukemia.

Venetoclax/azacitidine combination therapy is effective in acute myeloid leukemia (AML) and tolerable for older, multimorbid patients. Despite promising response rates, many patients do not achieve sustained remission or are upfront refractory. Identification of resistance mechanisms and additional therapeutic targets represent unmet clinical needs. By using a genome-wide CRISPR/Cas9 library screen targeting 18,053 protein- coding genes in a human AML cell line, various genes conferring resistance to combined venetoclax/azacitidine treatment were identified. The ribosomal protein S6 kinase A1 (RPS6KA1) was among the most significantly depleted sgRNA-genes in venetoclax/azacitidine- treated AML cells. Addition of the RPS6KA1 inhibitor BI-D1870 to venetoclax/azacitidine decreased proliferation and colony forming potential compared to venetoclax/azacitidine alone. Furthermore, BI-D1870 was able to completely restore the sensitivity of OCI-AML2 cells with acquired resistance to venetoclax/azacitidine. Analysis of cell surface markers revealed that RPS6KA1 inhibition efficiently targeted monocytic blast subclones as a potential source of relapse upon venetoclax/azacitidine treatment. Taken together, our results suggest RPS6KA1 as mediator of resistance towards venetoclax/azacitidine and additional RPS6KA1 inhibition as strategy to prevent or overcome resistance.

Clonally resolved single-cell multi-omics identifies routes of cellular differentiation in acute myeloid leukemia.

Inter-patient variability and the similarity of healthy and leukemic stem cells (LSCs) have impeded the characterization of LSCs in acute myeloid leukemia (AML) and their differentiation landscape. Here, we introduce CloneTracer, a novel method that adds clonal resolution to single-cell RNA-seq datasets. Applied to samples from 19 AML patients, CloneTracer revealed routes of leukemic differentiation. Although residual healthy and preleukemic cells dominated the dormant stem cell compartment, active LSCs resembled their healthy counterpart and retained erythroid capacity. By contrast, downstream myeloid progenitors constituted a highly aberrant, disease-defining compartment: their gene expression and differentiation state affected both the chemotherapy response and leukemia's ability to differentiate into transcriptomically normal monocytes. Finally, we demonstrated the potential of CloneTracer to identify surface markers misregulated specifically in leukemic cells. Taken together, CloneTracer reveals a differentiation landscape that mimics its healthy counterpart and may determine biology and therapy response in AML.

Combinatorial BCL2 Family Expression in Acute Myeloid Leukemia Stem Cells Predicts Clinical Response to Azacitidine/Venetoclax.

The BCL2 inhibitor venetoclax (VEN) in combination with azacitidine (5-AZA) is currently transforming acute myeloid leukemia (AML) therapy. However, there is a lack of clinically relevant biomarkers that predict response to 5-AZA/VEN. Here, we integrated transcriptomic, proteomic, functional, and clinical data to identify predictors of 5-AZA/VEN response. Although cultured monocytic AML cells displayed upfront resistance, monocytic differentiation was not clinically predictive in our patient cohort. We identified leukemic stem cells (LSC) as primary targets of 5-AZA/VEN whose elimination determined the therapy outcome. LSCs of 5-AZA/VEN-refractory patients displayed perturbed apoptotic dependencies. We developed and validated a flow cytometry-based "Mediators of apoptosis combinatorial score" (MAC-Score) linking the ratio of protein expression of BCL2, BCL-xL, and MCL1 in LSCs. MAC scoring predicts initial response with a positive predictive value of more than 97% associated with increased event-free survival. In summary, combinatorial levels of BCL2 family members in AML-LSCs are a key denominator of response, and MAC scoring reliably predicts patient response to 5-AZA/VEN. SIGNIFICANCE: Venetoclax/azacitidine treatment has become an alternative to standard chemotherapy for patients with AML. However, prediction of response to treatment is hampered by the lack of clinically useful biomarkers. Here, we present easy-to-implement MAC scoring in LSCs as a novel strategy to predict treatment response and facilitate clinical decision-making. This article is highlighted in the In This Issue feature, p. 1275.

General condition and comorbidity of long-term survivors of adult acute lymphoblastic leukemia.

Cure rates in adult acute lymphoblastic leukemia (ALL) improved using pediatric-based chemotherapy and stem cell transplantation (SCT). However, limited data on the health condition of cured adults are available whereas pediatric data cannot be transferred. The GMALL analyzed the health status in survivors of adult ALL retrospectively. Physicians answered a questionnaire on general condition (Eastern Cooperative Oncology Group [ECOG] status) and comorbidity or syndrome occurrence observed after treatment. Five hundred and thirty-eight patients with a median age of 29 (range, 15-64) years at diagnosis were analyzed, median follow-up was 7 (range, 3-24) years. Thirty-one percent had received SCT. ECOG status was 0-1 in 94%, 34% had not developed significant comorbidities. Most frequent comorbidities involved the neurologic system (27%), endocrine system (20%), skin (18%), graft-versus-host-disease (15%), cardiac system (13%), fatigue (13%). SCT impacted ECOG status and comorbidity occurrence significantly. ECOG 0-1 was observed in 86% of SCT and 98% of non-SCT patients (P<0.0001); comorbidity was observed in 87% and 57% respectively (P<0.0001). Our analysis elucidates the spectrum of comorbidities in cured adult ALL patients, with higher risk for transplanted patients, providing stimulations for the design of adequate aftercare programs. Overall, a large proportion of non-SCT patients achieved unrestricted general condition. The data provide a reference for new patient-centered endpoints in future trials.

A Dynamic rRNA Ribomethylome Drives Stemness in Acute Myeloid Leukemia.

The development and regulation of malignant self-renewal remain unresolved issues. Here, we provide biochemical, genetic, and functional evidence that dynamics in ribosomal RNA (rRNA) 2'-O-methylation regulate leukemia stem cell (LSC) activity in vivo. A comprehensive analysis of the rRNA 2'-O-methylation landscape of 94 patients with acute myeloid leukemia (AML) revealed dynamic 2'-O-methylation specifically at exterior sites of ribosomes. The rRNA 2'-O-methylation pattern is closely associated with AML development stage and LSC gene expression signature. Forced expression of the 2'-O-methyltransferase fibrillarin (FBL) induced an AML stem cell phenotype and enabled engraftment of non-LSC leukemia cells in NSG mice. Enhanced 2'-O-methylation redirected the ribosome translation program toward amino acid transporter mRNAs enriched in optimal codons and subsequently increased intracellular amino acid levels. Methylation at the single site 18S-guanosine 1447 was instrumental for LSC activity. Collectively, our work demonstrates that dynamic 2'-O-methylation at specific sites on rRNAs shifts translational preferences and controls AML LSC self-renewal. SIGNIFICANCE: We establish the complete rRNA 2'-O-methylation landscape in human AML. Plasticity of rRNA 2'-O-methylation shifts protein translation toward an LSC phenotype. This dynamic process constitutes a novel concept of how cancers reprogram cell fate and function. This article is highlighted in the In This Issue feature, p. 247.

Cellular hierarchies predict drug response in acute myeloid leukemia.

In a recent Nature Medicine study, Zeng and colleagues integrate both genomic and stem cell models of acute myeloid leukemia (AML) by deconvoluting cellular hierarchies of more than 1,000 AML samples. This work introduces a framework capable of predicting drug responses to targeted therapies in future clinical trials.

Venetoclax synergizes with gilteritinib in FLT3 wild-type high-risk acute myeloid leukemia by suppressing MCL-1.

BCL-2 inhibition has been shown to be effective in acute myeloid leukemia (AML) in combination with hypomethylating agents or low-dose cytarabine. However, resistance and relapse represent major clinical challenges. Therefore, there is an unmet need to overcome resistance to current venetoclax-based strategies. We performed high-throughput drug screening to identify effective combination partners for venetoclax in AML. Overall, 64 antileukemic drugs were screened in 31 primary high-risk AML samples with or without venetoclax. Gilteritinib exhibited the highest synergy with venetoclax in FLT3 wild-type AML. The combination of gilteritinib and venetoclax increased apoptosis, reduced viability, and was active in venetoclax-azacitidine-resistant cell lines and primary patient samples. Proteomics revealed increased FLT3 wild-type signaling in specimens with low in vitro response to the currently used venetoclax-azacitidine combination. Mechanistically, venetoclax with gilteritinib decreased phosphorylation of ERK and GSK3B via combined AXL and FLT3 inhibition with subsequent suppression of the antiapoptotic protein MCL-1. MCL-1 downregulation was associated with increased MCL-1 phosphorylation of serine 159, decreased phosphorylation of threonine 161, and proteasomal degradation. Gilteritinib and venetoclax were active in an FLT3 wild-type AML patient-derived xenograft model with TP53 mutation and reduced leukemic burden in 4 patients with FLT3 wild-type AML receiving venetoclax-gilteritinib off label after developing refractory disease under venetoclax-azacitidine. In summary, our results suggest that combined inhibition of FLT3/AXL potentiates venetoclax response in FLT3 wild-type AML by inducing MCL-1 degradation. Therefore, the venetoclax-gilteritinib combination merits testing as a potentially active regimen in patients with high-risk FLT3 wild-type AML.

Antigen presentation safeguards the integrity of the hematopoietic stem cell pool.

Hematopoietic stem and progenitor cells (HSPCs) are responsible for the production of blood and immune cells. Throughout life, HSPCs acquire oncogenic aberrations that can cause hematological cancers. Although molecular programs maintaining stem cell integrity have been identified, safety mechanisms eliminating malignant HSPCs from the stem cell pool remain poorly characterized. Here, we show that HSPCs constitutively present antigens via major histocompatibility complex class II. The presentation of immunogenic antigens, as occurring during malignant transformation, triggers bidirectional interactions between HSPCs and antigen-specific CD4+ T cells, causing stem cell proliferation, differentiation, and specific exhaustion of aberrant HSPCs. This immunosurveillance mechanism effectively eliminates transformed HSPCs from the hematopoietic system, thereby preventing leukemia onset. Together, our data reveal a bidirectional interaction between HSPCs and CD4+ T cells, demonstrating that HSPCs are not only passive receivers of immunological signals but also actively engage in adaptive immune responses to safeguard the integrity of the stem cell pool.

Glucose Metabolism and Aging of Hematopoietic Stem and Progenitor Cells.

Comprehensive proteomics studies of human hematopoietic stem and progenitor cells (HSPC) have revealed that aging of the HSPC compartment is characterized by elevated glycolysis. This is in addition to deregulations found in murine transcriptomics studies, such as an increased differentiation bias towards the myeloid lineage, alterations in DNA repair, and a decrease in lymphoid development. The increase in glycolytic enzyme activity is caused by the expansion of a more glycolytic HSPC subset. We therefore developed a method to isolate HSPC into three distinct categories according to their glucose uptake (GU) levels, namely the GUhigh, GUinter and GUlow subsets. Single-cell transcriptomics studies showed that the GUhigh subset is highly enriched for HSPC with a differentiation bias towards myeloid lineages. Gene set enrichment analysis (GSEA) demonstrated that the gene sets for cell cycle arrest, senescence-associated secretory phenotype, and the anti-apoptosis and P53 pathways are significantly upregulated in the GUhigh population. With this series of studies, we have produced a comprehensive proteomics and single-cell transcriptomics atlas of molecular changes in human HSPC upon aging. Although many of the molecular deregulations are similar to those found in mice, there are significant differences. The most unique finding is the association of elevated central carbon metabolism with senescence. Due to the lack of specific markers, the isolation and collection of senescent cells have yet to be developed, especially for human HSPC. The GUhigh subset from the human HSPC compartment possesses all the transcriptome characteristics of senescence. This property may be exploited to accurately enrich, visualize, and trace senescence development in human bone marrow.

Prognostic value of low-level MRD in adult acute lymphoblastic leukemia detected by low- and high-throughput methods.

Persistence of minimal residual disease (MRD) after induction/consolidation therapy in acute lymphoblastic leukemia is the leading cause of relapse. The GMALL 07/2003 study used MRD detection by real-time quantitative polymerase chain reaction of clonal immune gene rearrangements with 1 × 10-4 as discriminating cutoff: levels ≥1 × 10-4 define molecular failure and MRD-negativity with an assay sensitivity of at least 1 × 10-4 defining complete molecular response. The clinical relevance of MRD results not fitting into these categories is unclear and termed "molecular not evaluable" (MolNE) toward MRD-based treatment decisions. Within the GMALL 07/03 study, 1019 consecutive bone marrow samples after first consolidation were evaluated for MRD. Patients with complete molecular response had significantly better outcome (5-year overall survival [OS] = 85% ± 2%, n = 603; 5-year disease-free survival [DFS] = 73% ± 2%, n = 599) compared with patients with molecular failure (5-year OS = 40% ± 3%, n = 238; 5-year DFS = 29% ± 3%, n = 208), with patients with MolNE in between (5-year OS = 66% ± 4%; 5-year DFS = 52% ± 4%, n = 178). Of MolNE samples reanalyzed using next-generation sequencing (NGS), patients with undetectable NGS-MRD (n = 44; 5-year OS = 88% ± 5%, 5-year DFS = 70% ± 7%) had significantly better outcome than those with positive NGS-MRD (n = 42; 5-year OS = 37% ± 8%; 5-year DFS = 33% ± 8%). MolNE MRD results not just are borderline values with questionable relevance but also form an intermediate-risk group, assignment of which can be further improved by NGS.

Single-cell proteo-genomic reference maps of the hematopoietic system enable the purification and massive profiling of precisely defined cell states.

Single-cell genomics technology has transformed our understanding of complex cellular systems. However, excessive cost and a lack of strategies for the purification of newly identified cell types impede their functional characterization and large-scale profiling. Here, we have generated high-content single-cell proteo-genomic reference maps of human blood and bone marrow that quantitatively link the expression of up to 197 surface markers to cellular identities and biological processes across all main hematopoietic cell types in healthy aging and leukemia. These reference maps enable the automatic design of cost-effective high-throughput cytometry schemes that outperform state-of-the-art approaches, accurately reflect complex topologies of cellular systems and permit the purification of precisely defined cell states. The systematic integration of cytometry and proteo-genomic data enables the functional capacities of precisely mapped cell states to be measured at the single-cell level. Our study serves as an accessible resource and paves the way for a data-driven era in cytometry.

Identification of leukemic and pre-leukemic stem cells by clonal tracking from single-cell transcriptomics.

Cancer stem cells drive disease progression and relapse in many types of cancer. Despite this, a thorough characterization of these cells remains elusive and with it the ability to eradicate cancer at its source. In acute myeloid leukemia (AML), leukemic stem cells (LSCs) underlie mortality but are difficult to isolate due to their low abundance and high similarity to healthy hematopoietic stem cells (HSCs). Here, we demonstrate that LSCs, HSCs, and pre-leukemic stem cells can be identified and molecularly profiled by combining single-cell transcriptomics with lineage tracing using both nuclear and mitochondrial somatic variants. While mutational status discriminates between healthy and cancerous cells, gene expression distinguishes stem cells and progenitor cell populations. Our approach enables the identification of LSC-specific gene expression programs and the characterization of differentiation blocks induced by leukemic mutations. Taken together, we demonstrate the power of single-cell multi-omic approaches in characterizing cancer stem cells.

Analysis of nonleukemic cellular subcompartments reconstructs clonal evolution of acute myeloid leukemia and identifies therapy-resistant preleukemic clones.

To acquire a better understanding of clonal evolution of acute myeloid leukemia (AML) and to identify the clone(s) responsible for disease recurrence, we have comparatively studied leukemia-specific mutations by whole-exome-sequencing (WES) of both the leukemia and the nonleukemia compartments derived from the bone marrow of AML patients. The T-lymphocytes, B-lymphocytes and the functionally normal hematopoietic stem cells (HSC), that is, CD34+ /CD38- /ALDH+ cells for AML with rare-ALDH+ blasts (<1.9% ALDH+ cells) were defined as the nonleukemia compartments. WES identified 62 point-mutations in the leukemia compartment derived from 12 AML-patients at the time of diagnosis and 73 mutations in 3 matched relapse cases. Most patients (8/12) showed 4 to 6 point-mutations per sample at diagnosis. Other than the mutations in the recurrently mutated genes such as DNMT3A, NRAS and KIT, we were able to identify novel point-mutations that have not yet been described in AML. Some leukemia-specific mutations and cytogenetic abnormalities including DNMT3A(R882H), EZH2(I146T) and inversion(16) were also detectable in the respective T-lymphocytes, B-lymphocytes and HSC in 5/12 patients, suggesting that preleukemia HSC might represent the source of leukemogenesis for these cases. The leukemic evolution was reconstructed for five cases with detectable preleukemia clones, which were tracked in follow-up and relapse samples. Four of the five patients with detectable preleukemic mutations developed relapse. The presence of leukemia-specific mutations in these nonleukemia compartments, especially after chemotherapy or after allogeneic stem cell transplantation, is highly relevant, as these could be responsible for relapse. This discovery may facilitate the identification of novel targets for long-term cure.

Glycogen accumulation, central carbon metabolism, and aging of hematopoietic stem and progenitor cells.

Inspired by recent proteomic data demonstrating the upregulation of carbon and glycogen metabolism in aging human hematopoietic stem and progenitor cells (HPCs, CD34+ cells), this report addresses whether this is caused by elevated glycolysis of the HPCs on a per cell basis, or by a subpopulation that has become more glycolytic. The average glycogen content in individual CD34+ cells from older subjects (> 50 years) was 3.5 times higher and more heterogeneous compared to younger subjects (< 35 years). Representative glycolytic enzyme activities in HPCs confirmed a significant increase in glycolysis in older subjects. The HPCs from older subjects can be fractionated into three distinct subsets with high, intermediate, and low glucose uptake (GU) capacity, while the subset with a high GU capacity could scarcely be detected in younger subjects. Thus, we conclude that upregulated glycolysis in aging HPCs is caused by the expansion of a more glycolytic HPC subset. Since single-cell RNA analysis has also demonstrated that this subpopulation is linked to myeloid differentiation and increased proliferation, isolation and mechanistic characterization of this subpopulation can be utilized to elucidate specific targets for therapeutic interventions to restore the lineage balance of aging HPCs.

Quantitative proteomics reveals specific metabolic features of acute myeloid leukemia stem cells.

Acute myeloid leukemia is characterized by the accumulation of clonal myeloid blast cells unable to differentiate into mature leukocytes. Chemotherapy induces remission in the majority of patients, but relapse rates are high and lead to poor clinical outcomes. Because this is primarily caused by chemotherapy-resistant leukemic stem cells (LSCs), it is essential to eradicate LSCs to improve patient survival. LSCs have predominantly been studied at the transcript level, thus information about posttranscriptionally regulated genes and associated networks is lacking. Here, we extend our previous report on LSC proteomes to healthy age-matched hematopoietic stem and progenitor cells (HSPCs) and correlate the proteomes to the corresponding transcriptomes. By comparing LSCs to leukemic blasts and healthy HSPCs, we validate candidate LSC markers and highlight novel and potentially targetable proteins that are absent or only lowly expressed in HSPCs. In addition, our data provide strong evidence that LSCs harbor a characteristic energy metabolism, adhesion molecule composition, as well as RNA-processing properties. Furthermore, correlating proteome and transcript data of the same individual samples highlights the strength of proteome analyses, which are particularly potent in detecting alterations in metabolic pathways. In summary, our study provides a comprehensive proteomic and transcriptomic characterization of functionally validated LSCs, blasts, and healthy HSPCs, representing a valuable resource helping to design LSC-directed therapies.

Publisher Correction: Absence of NKG2D ligands defines leukaemia stem cells and mediates their immune evasion.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.