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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.

Transcriptomic and functional characterization of LSC-like cells

Cell line sensitivity and additional patient data

Comparison of MAC-Score and BH-3 profiling based response prediction

MAC-Score predict response within mutational subgroups

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.

Subpopulation specific Bcl-2 family levels and time on HMA/Venetoclax

Subpopulation specific Bcl-2 family levels and response to HMA/Venetoclax

Bcl-2 family gene expression in LSCs predicts in vitro response to HMA/VEN

7 Tables showing: molecular and clinical patient data, cell line info, antibody panels and overview of assays used

Supplementary Figure S1 shows enrichment of FBL in LSC, correlation of FBL with LSC signature and rRNA 2'-O-Me in healthy hematopoietic cells. Supplementary Figure S2 shows the association of rRNA 2’-O-Me with LSC and hematopoietic differentiation signatures. Supplementary Figure S3 shows strategy for LSC sorting and distribution of LSC methylation sites on ribosomes. Supplementary Figure S4 shows the effect of FBL knockdown on rRNA 2’-O-methylation and in vitro proliferation of leukemia cells. Supplementary Figure S5 show the effect of FBL overexpression on in vivo engraftment of primary AML cells. Supplementary Figure S6 shows the effect of FBL knockdown on nascent proteome and metabolism of AML cells. Supplementary Figure S7 shows the ribosome footprinting analysis after FBL knockdown. Supplementary Figure S8 shows the association of Gm1447 with LSC signature and the effect of Gm1447 supression on cellular amino acid levels. Supplementary Figure S9 shows regulatory effect of Gm1447 on in vivo leukemic self-renewal.

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

Supplementary table S1 shows all rRNA 2’-O-methylation sites identified in human AMLs. Supplementary table S2 shows dynamic rRNA 2’-O-methylation sites in each DyMeC cluster. Supplementary table S3 shows the location of rRNA 2’-O-methylation static sites in ribosome regions. Supplementary table S4 shows proteins with altered translation after FBL knockdown.

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.

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., Cell Stem Cell, 30 (5), ISSN:1934-5909, ISSN:1875-9777

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.

Inter-patient variability and the similarity of healthy and leukemic stem cells have impeded the characterization of leukemic stem cells (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. While residual healthy cells dominated the dormant stem cell compartment, active leukemic stem cells resembled their healthy counterpart and retained erythroid capacity. By contrast, downstream myeloid progenitors were highly aberrant and constituted the disease-defining compartment: Their gene expression and differentiation state determined both chemotherapy response and the leukemia’s ability to differentiate to transcriptomically normal monocytes. Finally, we demonstrated the potential of CloneTracer to identify surface markers mis-regulated specifically in leukemic cells by intra-patient comparisons. Taken together, CloneTracer revealed a differentiation landscape that mimics its healthy counterpart and determines biology and therapy response in AML.

Acute lymphoblastic leukemia (ALL) arises from the uncontrolled proliferation of precursor B or T cells (B- or T-ALL). Novel immunotherapies are urgently needed to reduce polychemotherapy-related toxicities and to target relapsed/refractory disease. The Interleukin 7 receptor (IL7R) plays a pivotal role in the development of ALL through mutations leading to constitutive activation of the oncogenic IL7R pathway or through overexpression of the receptor. The IL7Rα chain (CD127)-targeting antibody OSE-127 is a full antagonist of the IL7R pathway with an excellent safety profile (NCT03980080), currently evaluated in phase 2 trials in inflammatory and autoimmune diseases (NCT04882007, NCT04605978). We previously reported in vivo efficacy of OSE-127 in CD127+ ALL patient-derived xenografts (PDXs) and synergy with standard of care polychemotherapy, but we had not evaluated CD127 protein expression in ALL patient cells. Moreover, the mechanism of action underlying OSE-127’s anti-leukemic activity remained to be fully elucidated. Here we show that CD127 is expressed by >84% ALL patients and that OSE-127 efficiently targets CD127 not only via its IL7R antagonist activity but also through macrophage-mediated antibody dependent phagocytosis (ADCP). First, CD127 surface expression was prospectively measured by flow cytometry in 372 diagnostic ALL patient samples. We detected CD127-positivity (defined as ≥10% CD127+ blasts) in 84.4% (314/372) cases, of which 39.5% (147/372) were CD127hi (≥ 50% CD127+ blasts). CD127 expression was higher in T- than in B-ALL. Among B-ALL, highest expression was detected in E2A-PBX1+ and BCR-ABL-like ALLs. Mechanistically, OSE-127 blocked STAT5 phosphorylation in IL7-responsive ALL-PDX cells, leading to decreased cell survival and proliferation. Surprisingly, we detected high in vivo efficacy of OSE-127 therapy in IL7-unresponsive PDXs (e.g., unable to induce phosphorylation of STAT5 upon ex vivo IL7 stimulation or with IL7R mutation-induced constitutive phosphorylation of STAT5). We uncovered that OSE-127 induced macrophage-mediated ADCP of CD127+ ALL cells in vitro. Accordingly, treatment of IL7-unresponsive, but OSE-127-sensitive models with an OSE-127-LALAPG variant (conserving IL7R-antagonistic properties but lacking ADCP activity due to Fc-inactivating mutations) did not impact leukemia development, thereby substantiating that ADCP contributes to the anti-leukemic efficacy of OSE-127. In fact, ADCP levels induced by OSE-127 treatment in vitro correlated with its capacity to reduce ALL blasts in the blood of PDX mice in vivo. Altogether, through its dual mode of action, OSE-127 may represent a powerful novel immunotherapy option for ALL patients, including cases with dysregulated IL7R signaling, particularly in combination with standard of care polychemotherapy. Citation Format: Irene Baccelli, Lennart Lenk, Anna Laqua, Dorothee Winterberg, Anna Dietterle, Frederique Corallo, Julien Taurelle, Emma Narbeburu, Beat Bornhauser, Jean-Pierre Bourquin, Fotini Vogiatzi, Simon Raffel, Martin Schrappe, Gunnar Cario, Monika Brüggemann, Denis M Schewe, Nicolas Poirier. CD127 is expressed by acute lymphoblastic leukemias and is efficiently targeted by the IL7R-antagonist OSE-127 through macrophage-mediated antibody dependent phagocytosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2957.

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 (

Patients with acute myeloid leukaemia (AML) often achieve remission after therapy, but subsequently die of relapse1 that is driven by chemotherapy-resistant leukaemic stem cells (LSCs)2,3. LSCs are defined by their capacity to initiate leukaemia in immunocompromised mice4. However, this precludes analyses of their interaction with lymphocytes as components of anti-tumour immunity5, which LSCs must escape to induce cancer. Here we demonstrate that stemness and immune evasion are closely intertwined in AML. Using xenografts of human AML as well as syngeneic mouse models of leukaemia, we show that ligands of the danger detector NKG2D—a critical mediator of anti-tumour immunity by cytotoxic lymphocytes, such as NK cells6–9—are generally expressed on bulk AML cells but not on LSCs. AML cells with LSC properties can be isolated by their lack of expression of NKG2D ligands (NKG2DLs) in both CD34-expressing and non-CD34-expressing cases of AML. AML cells that express NKG2DLs are cleared by NK cells, whereas NKG2DL-negative leukaemic cells isolated from the same individual escape cell killing by NK cells. These NKG2DL-negative AML cells show an immature morphology, display molecular and functional stemness characteristics, and can initiate serially re-transplantable leukaemia and survive chemotherapy in patient-derived xenotransplant models. Mechanistically, poly-ADP-ribose polymerase 1 (PARP1) represses expression of NKG2DLs. Genetic or pharmacologic inhibition of PARP1 induces NKG2DLs on the LSC surface but not on healthy or pre-leukaemic cells. Treatment with PARP1 inhibitors, followed by transfer of polyclonal NK cells, suppresses leukaemogenesis in patient-derived xenotransplant models. In summary, our data link the LSC concept to immune escape and provide a strong rationale for targeting therapy-resistant LSCs by PARP1 inhibition, which renders them amenable to control by NK cells in vivo. Leukaemic stem cells in acute myeloid leukaemia are defined by a lack of expression of NKG2D ligands, which mediates their ability to evade surveillance by NK cells.

Single-cell genomics has transformed our understanding of complex cellular systems. However, excessive costs 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 major 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 measuring the functional capacities of precisely mapped cell states at the single-cell level. Our study serves as an accessible resource and paves the way for a data-driven era in cytometry.

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., Nature Communications, 12 (1), ISSN:2041-1723

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 (

Enrichment of leukemic blasts with a stem cell phenotype correlates with poor survival in acute myeloid leukemia (AML). In this context, measurement of the stem cell marker aldehyde-dehydrogenase (ALDH) activity can distinguish poor prognosis cases with increased fractions of ALDH-positive cells (ALDH-numerous AML) and favorable outcome cases with low percentages (ALDH-rare AML). It has been shown that ALDH-numerous AML favor leukemic engraftment in xenotransplantation assays which suggests increased leukemic stem cell (LSC) potential. To test if this reflects an immature cell of origin, comparative gene-expression studies of CD34+ leukemic blasts were performed. This analysis revealed increased expression of LSC and HSC signatures in ALDH-numerous AML, whereas ALDH-rare AML were enriched for a progenitor signature. The enrichment of stemness-associated transcriptional programs suggests that ALDH-numerous AML derive from immature hematopoietic progenitors and offers an explanation for the poor prognosis and therapy resistance of this subgroup which is likely caused by inherited stem cell properties.

Comprehensive proteomic studies of HSC derived from bone marrow of healthy human subjects (n = 59) in different age groups (range: 20 - 72 years) showed that aging HSCs are characterized not only by myeloid lineage skewing, senescence associated secretory phenotype (SASP), accumulation of reactive oxygen species (ROS), anti-apoptosis, but prominently by elevated glycolysis, glucose uptake, and accumulation of glycogen. This is caused by a subset of HSC that has become more glycolytic than others and not on a per cell basis. Subsequent comparative transcriptome studies of HSCs from human subjects >60 years versus those from Provided with this background and based on glucose metabolism levels, we have developed a method to isolate human HSCs (CD34+ cells) from bone marrow into three distinct subsets with high, intermediate, and low glucose uptake (GU) capacity (GU high, GU inter, GU low). For human subjects >60 years old (n=9), the proportions of these subsets are: GU high= 5.4+3.5 %, GU inter= 66.4+22.5 %, GU low= 28.2+21.7 %. For subjects 60 years) subjects, as well as the scRNA-seq data from GU high subset versus GU inter and GU lowsubsets from each individual subject (n = 6). The results are shown in Figure 1. In analogy to the comparison between old (>60 years) versus young ( Conclusion: In a series of multi-omics studies, we have demonstrated that the GU high subset is identical to the senescent cells (SCs) in human HSC compartment. Studies in animal models have shown that SCs in murine bone marrow are responsible for driving the aging process, and elimination of this subset by inhibitors of anti-apoptotic factors is able to rejuvenate hematopoiesis in mice. Our present results have provided cellular and molecular evidence that SCs in human HSC compartment are also dependent on anti-apoptotic factors, elevated MTORC1 as well as increased glycolysis for survival. Inhibition of MTORC1 or glycolysis, either by specific inhibitors or by CR, may eliminate senescent HSCs and promote rejuvenation of human hematopoiesis. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction: Acute lymphoblastic leukemia (ALL) represents approx. 5% of all newly diagnosed leukemias in patients between 55 and 70 years of age. Despite recent advances especially in younger patients, the prognosis of elderly patients with ALL remains dismal, even after moderately intensive chemotherapy. Incorporation of antibody-based therapies in the first line induction therapy bears the potential of a significant increased response rate and survival with the reduction of treatment toxicity. Methods: This open label phase II study of the German Multicenter Study Group on Adult Acute Lymphoblastic Leukemia was conducted at 13 centers. Patients aged >55 years with newly diagnosed Ph/BCR-ABL negative acute B-precursor ALL were eligible. Leukemic blasts had CD22 surface expression of at least 20%. The 1st induction cycle consisted of InO 0.8mg/m2 on day1 and 0.5mg/m2 on d8 and d15 together with dexamethasone 10 mg/m2 (day7-8, day14-17) and one intrathecal injection (i.th.) of methotrexate (MTX), cytarabine (AraC) and dexamethasone (Dex). The 2nd and 3rd induction cycle consisted of InO 0.5mg/m2 on day 1, 8 and 15 plus i.th. MTX/AraC/Dex. Patients achieving a complete remission (CR) were offered to receive 5 conventional consolidation therapies (3 x ID-MTX/asparaginase; 2 x ID-AraC) and one reinduction therapy (idarubicine/ AraC/ cyclophosphamide / Dex.) in combination with rituximab (for CD20+ ALL), followed by a maintenance therapy with 6-MP/MTX. The primary endpoint was event free survival (EFS) at 12-months follow-up. An event was defined as any of the following: persisting bone marrow blasts after 2 cycles of InO, relapse or death. An event rate of ≤40% at 12 months follow-up was considered to qualify the experimental treatment as very promising for additional testing. Under the assumption of one-sided type I error of 5% and 80% power, 42 evaluable patients were needed for primary endpoint analysis (registered with ClinicalTrials.gov, identifier: NCT03460522). Results: As of July 2021, 45 patients were included, with induction results available for 43 patients. Two patients were excluded per protocol from further analysis, as they received only one induction: 1 prolonged toxicity (CR after induction), 1 withdrawn consent. Median age at initial diagnosis was 64 years (range 56-80 years). 38 patients were diagnosed with a common- and 5 with a pro-B ALL. Median CD22 expression on leukemic blasts was 69% (range 21-99%). Due to suspected therapy related liver toxicities, a single patient received only 2 induction cycles (complete remission after 1st induction). All other patients completed 3 cycles of induction therapy and achieved complete remission (CR/CRi), mainly after the 1st induction. Results of minimal residual disease (MRD), measured by real-time quantitative PCR, are available for all 43 patients, with 23/43 (53%) and 31/42 (74%) patients being MRD negative after 2nd and 3rd induction therapy, respectively. Four patients with MRD failure (≥10 -4) after the 3rd induction therapy are still in CR (2 were treated with conventional chemotherapy per this protocol, one proceeded to allogeneic stem cell transplantation (aSCT), one received blinatumomab). MRD below the threshold of 10 -4 was detected in 15 patients after the 2nd induction (no relapse occurred so far). 8 of these 15 patients were MRD negative after the 3rd induction. So far, 7 events have been reported (during year 1: 3 deaths in CR and 1 relapsed ALL; during year 2: 1 death in CR and 2 patients with relapse). With a median follow-up of 422 days, the probability of OS at 1 and 2 years is 91% (95% C.I. 80-100%) and 77% (95% C.I. 57-96%), respectively. 4 patients received an aSCT in 1st CR, 1 patient in 2nd CR. Regarding adverse events (AEs) during induction cycles 1, 2 and 3, most common AEs ≥CTC grade 3 reported were leukocytopenia (in 60%, 12% and 3% of all cases, respectively), anemia (28%, 2%, 0%), thrombocytopenia (35%, 7%, 3%) and elevation of liver enzymes (14%, 5%, 0%). So far, one patient has been reported with suspected veno occlusive disease (after 2nd induction therapy). Conclusion: Three cycles of InO as induction therapy for the treatment of elderly patients with acute B-cell ALL results in very high remission rates with MRD-response in up to 90% (MRD negativity or MRD-levels Figure 1 Figure 1. Disclosures Stelljes: Pfizer: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Medac: Speakers Bureau; MSD: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Celgene/BMS: Consultancy, Speakers Bureau; Kite/Gilead: Consultancy, Speakers Bureau. Wäsch: Sanofi: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Pfizer: Consultancy; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Novartis: Consultancy; BMS/Celgene: Consultancy; Gilead: Consultancy. Nachtkamp: Jazz: Honoraria; Bsh medical: Honoraria; Celgene: Other: Travel Support. Haenel: Jazz: Consultancy, Honoraria; GSK: Consultancy; Bayer Vital: Honoraria; Takeda: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Amgen: Consultancy; Celgene: Consultancy, Honoraria. Berdel: Philogen S.p.A.: Consultancy, Current equity holder in publicly-traded company, Honoraria, Membership on an entity's Board of Directors or advisory committees. Goekbuget: Amgen: Consultancy, Other: Invited talks for company sponsored symposia (with honoraria); Research funding for institution; Astra Zeneca: Other: Invited talk for company sponsored symposia (with honor); Erytech: Consultancy; Cellestia: Consultancy; Incyte: Other: Research funding for Institution; Jazz Pharmaceuticals: Other: Research funding for institution; Novartis: Consultancy, Other: Research funding for Institution; Gilead/Kite: Consultancy; Morphosys: Consultancy; Pfizer: Consultancy, Other: Research funding for institution; Servier: Consultancy, Other; Abbvie: Other. OffLabel Disclosure: Inotuzumab Ozogamicin

In patients with acute myeloid leukemia and low percentages of aldehyde-dehydrogenase-positive cells, non-leukemic hematopoietic stem cells can be separated from leukemic cells. By relating hematopoietic stem cell frequencies to outcome we detected poor overall- and disease-free survival of patients with low hematopoietic stem cell frequencies. Serial analysis of matched diagnostic and follow-up samples further demonstrated that hematopoietic stem cells increased after chemotherapy in patients who achieved durable remissions. However, in patients who eventually relapsed, hematopoietic stem cell numbers decreased dramatically at the time of molecular relapse demonstrating that hematopoietic stem cell levels represent an indirect marker of minimal residual disease, which heralds leukemic relapse. Upon transplantation in immune-deficient mice cases with low percentages of hematopoietic stem cells of our cohort gave rise to leukemic or no engraftment, whereas cases with normal hematopoietic stem cell levels mostly resulted in multi-lineage engraftment. Based on our experimental data, we propose that leukemic stem cells have increased niche affinity in cases with low percentages of hematopoietic stem cells. To validate this hypothesis, we developed new mathematical models describing the dynamics of healthy and leukemic cells under different regulatory scenarios. These models suggest that the mechanism leading to decreases in hematopoietic stem cell frequencies before leukemic relapse must be based on expansion of leukemic stem cells with high niche affinity and the ability to dislodge hematopoietic stem cells. Thus, our data suggest that decreasing numbers of hematopoietic stem cells indicate leukemic stem cell persistence and the emergence of leukemic relapse.

MRD in ALL is defined as the detection of leukemic cells in bone marrow below the microscopic threshold in complete remission (CR). Patients (pts) with molecular failure (MolFail) or molecular relapse (MolRel) after induction/consolidation therapy are at a high risk for hematologic relapse. Targeted therapies should prevent hematologic relapse, reduce MRD load and provide a bridging strategy to allogeneic stem cell transplantation (SCT) and thereby improve overall outcome of these pts. In pts without (wo) SCT option reduction of MRD load is an essential goal as well. Blinatumomab is an antibody construct that redirects CD3+ T cells to CD19+ target cells, resulting in a serial lysis of CD19+ B cells. In a study in pts with MRD ≥10-3, 78% achieved complete MRD response (Gökbuget N et al., Blood 2018). The MolAct1 trial was initiated by the GMALL study group to evaluate the efficacy and tolerability of Blinatumomab in MRD+ ALL including those with MRD below 10-3 and pts with MRD after SCT. Adults (≥18 yrs) with CD19+, Ph-negative BCP ALL in CR after ≥ 3 chemotherapies with MRD ≥10-4 were eligible (NCT03109093). Recruitment of pts with MRD ≥10-3 was stopped after marketing authorization for this entity. After an amendment, which became effective after 44 recruited pts, pts with MRD below 10-4 or non-quantifiable (nq) MRD were eligible. Blinatumomab 28 μg/day was given as 4-wk infusion, followed by a 2-wk break (1 cycle). Responders could receive up to 4 cycles or undergo HSCT after ≥ 1 cycle. MRD after 1 cycle was the primary endpoint. MRD was centrally assessed by allele-specific quantitative real-time PCR of clonal rearrangements of immunoglobulin or T-cell receptor genes. For definition of MRD at inclusion and at response assessment see table 1. 64 pts with a median age of 44 (18-83) yrs were included from 19 centers and 60 were evaluable. 63 pts were treated in first CR (5 after SCT). Overall, 67% achieved MolCR, 10% had MolFail, 23% MolNE. MolNE identifies an intermediate response with different options clarified table 1. 81% of the pts included with MRD ≥10-4 had a molecular response i.e. MolCR or MRD < 10-4. No significant differences in terms of MRD response were observed according to MRD level at inclusion or other patient characteristics (table 1). 60 pts have completed study treatment (40 HSCT, 8 relapses during treatment, 4 completed 4 cycles wo SCT, 2 stopped earlier due to toxicities - 1 with subsequent SCT, 1 due to GvHD, 1 due to physicians' decision and 4 pts returned to standard treatment after 2 cycles). SCT pts had a median age of 42 (18-66) yrs and follow-up is available in 37 / 41 pts (29 CCR, 3 relapse, 5 death in CR). 16 relapses occurred: 8 during treatment (1 after MolCR, 5 MolNE1-3 and 2 with MolFail resp); after SCT in 3/41 pts; 5/11 with CR at end of treatment wo subsequent SCT. The median observation time of surviving pts is 12 (1-38) mo and the median survival is not reached. At 2 yrs the survival probability (OS) was 64%. OS was 70%, 64% and 43% in pts with MRD between 10-4-10-3, 10-3-10-2 and >10-2 at inclusion, resp (p>.05; Fig.1). OS was 71% vs 54% in pts MolFail vs MolRel at inclusion (p>.05). OS was 72%, 40% and 56% in pts with MolCR, MolFail and MolNE after cycle 1 resp (P=0.02; Fig.2). Overall, the results from previous trials were confirmed. In addition, it was demonstrated that pts with MRD between 10-4 and 10-3 had a similar response and a trend towards better outcome compared to pts with higher MRD levels >10-2. So far only 7 pts with MRD below 10-4 were included; more data are needed to evaluate the impact of Blinatumomab in this population; GMALL does currently not recommend SCT for these pts unless there is an indication due to other risk factors. Interestingly, a significant proportion of pts (23%) had an incomplete MRD response and the outcome results indicate that these pts together with those with MolFail may have an inferior survival compared to those with MolCR. The results underline that the clear definition of MRD categories and consideration of level and sensitivity is essential for interpretation, which is possible due to well defined standards for the PCR method used here. 68% of the pts received SCT in CR after Blinatumomab with a so far limited mortality (13%). Further follow-up is certainly required. The GMALL will continue to recruit patients with MRD levels below 10-3 in order to improve their chances for long-term survival This study was supported by Amgen Inc. Disclosures Goekbuget: Servier: Consultancy, Research Funding; Jazz: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Erytech: Consultancy; Kite: Consultancy; Gilead: Consultancy. Schwartz:Pfizer: Other: personal fees ; AMGEN: Other: personal fees and non-financial support; BTG Intl Inc: Other: personal fees ; Gilead Sciences: Other: personal fees and non-financial support ; MSD Sharp & Dohme: Other: personal fees ; Basilea: Other: non-financial suppor; Novartis: Other: personal fees and non-financial support; Jazz Pharmaceuticals: Other: personal fees and non-financial support. Hüttmann:Lead Discovery Center GmbH: Consultancy; University Hospital Essen, University of Duisburg-Essen, Essen, Germany: Current Employment; Celgene: Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Seattle Genetics: Research Funding; Roche: Other: Travel expenses; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Gilead: Honoraria. Viardot:Roche: Honoraria, Other: advisory board; Kite/Gilead: Honoraria, Other: advisory board; Novartis: Honoraria, Other: advisory board; Amgen: Honoraria, Other: advisory board. Fiedler:Daiichi Sankyo: Other: support for meeting attendance; Gilead: Other: support for meeting attendance; Jazz Pharmaceuticals: Honoraria, Other: support for meeting attendance; Abbvie: Membership on an entity's Board of Directors or advisory committees; Morphosys: Consultancy, Honoraria; Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria; ARIAD/Incyte: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Other: support for meeting attendance, Patents & Royalties, Research Funding. Alakel:Pfizer: Consultancy. Stelljes:Pfizer: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Speakers Bureau. Subklewe:Seattle Genetics: Research Funding; AMGEN: Consultancy, Honoraria, Research Funding; Morphosys: Research Funding; Gilead Sciences: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Honoraria; Janssen: Consultancy; Roche AG: Consultancy, Research Funding. Wäsch:Pfizer: Consultancy; Amgen: Consultancy; Janssen: Consultancy. Vucinic:Celgene: Honoraria. Fransecky:Amgen: Consultancy. Bargou:Amgen: Consultancy, Honoraria, Patents & Royalties; Gemoab: Consultancy, Honoraria; Cellex: Consultancy, Honoraria; Catalym: Consultancy, Honoraria. Topp:Amgen, Boehringer Ingelheim, KITE, Regeneron, Roche: Research Funding; Amgen, KITE, Novartis, Regeneron, Roche: Consultancy. Brüggemann:Regeneron: Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy; Incyte: Consultancy; Roche: Consultancy; Affimed: Research Funding; Janssen: Consultancy, Honoraria. OffLabel Disclosure: Blinatumomab in MRD positive disease below 10-3

Introduction: Acute lymphoblastic leukemia (ALL) represents around 5% of all newly diagnosed leukemia in patients between 55 and 70 years of age. Despite recent advances especially in younger patients, the prognosis of older patients with ALL remains dismal, even after moderately intensive chemotherapy. Due to increasing toxicity and infection rates in older patients, dose intensification of induction treatment often is no justifiable option. Consequently, new treatment options are needed to improve the survival of older ALL patients. Methods: This open label phase II study of the German Multicenter Study Group on Adult Acute Lymphoblastic Leukemia (GMALL) is currently activated in 14 centers in Germany. Patients aged >55 years with newly diagnosed acute B lymphoblastic leukemia, with the exception of Philadelphia-chromosome or BCR-ABL positive ALL or Burkitt's or mixed phenotype acute leukemia, are eligible. Leukemic blasts must have CD22 surface expression of at least 20%. No previous ALL-specific treatment, with the exception of corticosteroids and/or single dose vincristine and/or up to 3 doses of cyclophosphamide (cycloph.) plus standard prephase treatment are allowed. The 1st induction cycle consists of inotuzumab ozogamicin (InO) 0.8 mg/m2 on day1 and 0.5 mg/m2 on days 8 and 15 together with dexamethasone 10 mg/m2 (days 7-8, days 14-17) and 1 intrathecal injection of methotrexate (MTX), cytarabine (AraC) and dexamethasone (Dexa). The 2nd and 3rd induction cycle consist of InO 0.5 mg/m2 on days 1, 8 and 15 plus intrathecal injection of MTX/AraC/Dexa. Response evaluation is scheduled after each cycle. Patients achieving a complete remission are offered to receive 5 conventional consolidation therapies (3 x ID-MTX/asparaginase; 2 x ID-AraC) and one reinduction therapy (idarubicine/AraC/cycloph./Dexa) in combination with rituximab (for CD20+ ALL), followed by a maintenance therapy with 6-mercaptopurine/MTX. The primary endpoint is is event free survival (EFS) at 12-months follow-up. An event is any of the following: persisting bone marrow blasts after 2 cycles of InO, relapse or death. An event rate of ≤40% at 12 months follow-up is considered to qualify the experimental treatment as very promising for additional testing. Under the assumption of one-sided type I error of 5% and 80% power, 42 evaluable patients were needed for primary endpoint analysis. The INITIAL-1 trial is registered with ClinicalTrials.gov, identifier: NCT03460522. Results: As of July 2020, 31 patients have been included, with induction results available for 29 patients. Median age at initial diagnosis was 64 years (range 56-80 years). Twenty-five patients were diagnosed with a common- and 4 with a pro-B lymphoblastic leukemia. Median CD22 expression on leukemic blasts was 70% (range 21-99%). Due to suspected therapy related liver toxicities, 1 patient received 1 induction cycle and 1 patient 2 induction cycles (both were in remission after the 1st induction). All other patients completed induction therapy and achieved complete remission (CR/CRi) mainly after the 1st induction. Results of minimal residual disease (MRD) measured by PCR are available for 23 patients, with 17 being MRD-negative after induction. So far, 4 events have been reported (2 deaths in remission and 1 relapsed ALL in the 1st year of treatment; one relapsed disease in the 2nd year). With a median follow-up of 242 days, the probability of OS at 1 year is 82.4 %. Two patients received an allogeneic stem cell transplantation in ongoing 1st remission. With regard to adverse events (AEs) during induction therapy 1, 2 and 3, most common AEs ≥CTC 3 reported were leukocytopenia (in 64%, 33% and 13% of all cases, respectively), anemia (54%, 28%, 13%), thrombocytopenia (68%, 17%, 26%) and elevation of liver enzymes (31%, 22%, 20%). Conclusion: Replacement of conventional induction chemotherapy by InO is feasible, results in promising remission rates, and may reduce the risk of early morbidity and lethality, particular in older patients with acute B lymphoblastic leukemia. Disclosures Stelljes: Amgen: Consultancy, Speakers Bureau; Pfizer: Consultancy, Honoraria, Research Funding, Speakers Bureau. Wäsch:Pfizer: Consultancy; Amgen: Consultancy; Janssen: Consultancy. Haenel:Amgen, Novartis, Roche, Celgene, Takeda, Bayer: Honoraria. Lenz:Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy; AstraZeneca: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding, Speakers Bureau; Agios: Research Funding; AQUINOX: Research Funding; Novartis: Consultancy; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Verastem: Research Funding; Morphosys: Consultancy, Honoraria, Research Funding. Brüggemann:Affimed: Research Funding; Regeneron: Research Funding; Celgene: Consultancy; Roche: Consultancy; Incyte: Consultancy; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding. Goekbuget:Gilead: Consultancy; Kite: Consultancy; Servier: Consultancy, Research Funding; Jazz: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Erytech: Consultancy; Amgen: Consultancy, Research Funding. Alakel:Pfizer: Consultancy. OffLabel Disclosure: Inotuzumab ozogamicin for induction therapy (1st line therapy)

In Extended Data Fig. 1a of this Letter, the flow cytometry plot depicting the surface phenotype of AML sample DD08 was a duplicate of the plot for AML sample DD06. Supplementary Data 4 has been added to the Supplementary Information of the original Letter to clarify the proteome data acquisition and presentation. The original Letter has been corrected online.

A new study reveals that leukemia stem cells (LSCs) in acute myeloid leukemia downregulate natural killer cell‐activating receptor ligands to evade immune surveillance via the transcriptional co‐factor PARP1. The inhibition of PARP1 sensitizes LSCs to immunotherapy, highlighting its potential as a therapeutic target.

To understand the precise disease driving mechanisms in acute myeloid leukemia (AML), comparison of patient matched hematopoietic stem cells (HSC) and leukemia stem cells (LSC) is essential. In this analysis, we have examined the value of aldehyde dehydrogenase (ALDH) activity in combination with CD34 expression for the separation of HSC from LSC in 104 patients with de novo AML. The majority of AML patients (80 out of 104) had low percentages of cells with high ALDH activity (ALDH+ cells, What's new? To understand the precise disease‐driving mechanisms in acute myeloid leukemia (AML), comparison of patient‐matched hematopoietic stem cells (HSC) and leukemia stem cells (LSC) is essential. This study demonstrates the relevance of aldehyde dehydrogenase (ALDH) for the prospective identification of AML cases in which separation of functionally normal HSC from LSC is possible. Increased activity of this biomarker also characterizes a subgroup of patients with adverse outcome, which might be helpful in risk stratification prior to therapy. Overall, this study demonstrates functional heterogeneity of leukemia cells and suggests divergent roles for ALDH activity in normal HSC versus leukemia‐initiating cells.

Summary To investigate miRNA function in human acute myeloid leukemia (AML) stem cells (LSC), we generated a prognostic LSC-associated miRNA signature derived from functionally validated subpopulations of AML samples. For one signature miRNA, miR-126, high bioactivity aggregated all in vivo patient sample LSC activity into a single sorted population, tightly coupling miR-126 expression to LSC function. Through functional studies, miR-126 was found to restrain cell cycle progression, prevent differentiation, and increase self-renewal of primary LSC in vivo. Compared with prior results showing miR-126 regulation of normal hematopoietic stem cell (HSC) cycling, these functional stem effects are opposite between LSC and HSC. Combined transcriptome and proteome analysis demonstrates that miR-126 targets the PI3K/AKT/MTOR signaling pathway, preserving LSC quiescence and promoting chemotherapy resistance., Graphical Abstract, Highlights • Clinical outcome in AML correlates with LSC-associated miRNA expression • miR-126 targets multiple components of the PI3K/AKT/MTOR signaling pathway • miR-126 promotes chemotherapy resistance by preserving LSC in a quiescent state • miR-126 governs opposing self-renewal outcomes in normal and malignant stem cells, Lechman et al. show that miR-126 targets the PI3K/AKT/MTOR signaling pathway to preserve quiescence, increase self-renewal, and promote chemotherapy resistance of acute myeloid leukemia stem cells (LSC). Reducing the miR-126 level impairs LSC maintenance in contrast to expanding normal hematopoietic stem cells.

The branched-chain amino acid (BCAA) pathway and high levels of BCAA transaminase 1 (BCAT1) have recently been associated with aggressiveness in several cancer entities. However, the mechanistic role of BCAT1 in this process remains largely uncertain. Here, by performing high-resolution proteomic analysis of human acute myeloid leukaemia (AML) stem-cell and non-stem-cell populations, we find the BCAA pathway enriched and BCAT1 protein and transcripts overexpressed in leukaemia stem cells. We show that BCAT1, which transfers α-amino groups from BCAAs to α-ketoglutarate (αKG), is a critical regulator of intracellular αKG homeostasis. Further to its role in the tricarboxylic acid cycle, αKG is an essential cofactor for αKG-dependent dioxygenases such as Egl-9 family hypoxia inducible factor 1 (EGLN1) and the ten-eleven translocation (TET) family of DNA demethylases. Knockdown of BCAT1 in leukaemia cells caused accumulation of αKG, leading to EGLN1-mediated HIF1α protein degradation. This resulted in a growth and survival defect and abrogated leukaemia-initiating potential. By contrast, overexpression of BCAT1 in leukaemia cells decreased intracellular αKG levels and caused DNA hypermethylation through altered TET activity. AML with high levels of BCAT1 (BCAT1high) displayed a DNA hypermethylation phenotype similar to cases carrying a mutant isocitrate dehydrogenase (IDHmut), in which TET2 is inhibited by the oncometabolite 2-hydroxyglutarate. High levels of BCAT1 strongly correlate with shorter overall survival in IDHWTTET2WT, but not IDHmut or TET2mut AML. Gene sets characteristic for IDHmut AML were enriched in samples from patients with an IDHWTTET2WTBCAT1high status. BCAT1high AML showed robust enrichment for leukaemia stem-cell signatures, and paired sample analysis showed a significant increase in BCAT1 levels upon disease relapse. In summary, by limiting intracellular αKG, BCAT1 links BCAA catabolism to HIF1α stability and regulation of the epigenomic landscape, mimicking the effects of IDH mutations. Our results suggest the BCAA-BCAT1-αKG pathway as a therapeutic target to compromise leukaemia stem-cell function in patients with IDHWTTET2WT AML.

Similar to resting mature B cells, where the B-cell antigen receptor (BCR) controls cellular survival1-3, surface BCR expression is conserved in most mature B-cell lymphomas. The identification of activating BCR mutations and the growth disadvantage upon BCR knockdown of cells of certain lymphoma entities has led to the view that BCR signalling is required for tumour cell survival4-7. Consequently, the BCR signalling machinery has become an established target in the therapy of B-cell malignancies8,9. Here we study the effects of BCR ablation on MYC-driven mouse B-cell lymphomas and compare them with observations in human Burkitt lymphoma. Whereas BCR ablation does not, per se, significantly affect lymphoma growth, BCR-negative (BCR-) tumour cells rapidly disappear in the presence of their BCR-expressing (BCR+) counterparts in vitro and in vivo. This requires neither cellular contact nor factors released by BCR+ tumour cells. Instead, BCR loss induces the rewiring of central carbon metabolism, increasing the sensitivity of receptor-less lymphoma cells to nutrient restriction. The BCR attenuates glycogen synthase kinase 3 beta (GSK3ß) activity to support MYC-controlled gene expression. BCR- tumour cells exhibit increased GSK3ß activity and are rescued from their competitive growth disadvantage by GSK3ß inhibition. BCR- lymphoma variants that restore competitive fitness normalize GSK3ß activity after constitutive activation of the MAPK pathway, commonly through Ras mutations. Similarly, in Burkitt lymphoma, activating RAS mutations may propagate immunoglobulin-crippled tumour cells, which usually represent a minority of the tumour bulk. Thus, while BCR expression enhances lymphoma cell fitness, BCR-targeted therapies may profit from combinations with drugs targeting BCR- tumour cells.