Anne Kathrin Merbach

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.

Somatic mosaicism in blood cells, referred to as clonal hematopoiesis (CH), arises when hematopoietic stem cells (HSCs) acquire somatic mutations that provide a substrate for clonal selection and subsequent clonal outgrowth over time. This condition becomes common with aging and is associated with adverse clinical outcomes related to mutated progenitor cells and increased inflammation. It has been shown that mutated clones have growth rates ranging from 5% to more than 50% per year and that they are acquired decades before they reach a substantial clone size. In CH, the most frequently mutated gene is DNMT3A encoding a de novo methyltransferase enzyme and the mutations are predominantly heterozygous, scattered throughout the three functional domains. In contrast, more than half of DNMT3A mutations in acute myeloid leukemia are missense alterations within the catalytic domain of the enzyme at residue R882. The high frequency of mutations at this specifc site suggests a gain-of-function activity. Beyond haploinsufficiency, the common heterozygous R882H allele creates an altered protein with dominant negative activity. Despite the increasing knowledge of how CH develops over time, the question regarding which mechanisms lead to clonal selection of mutant clones remains unresolved. Here, we performed single-cell multi-omics profiling of FACS-enriched CD34+ hematopoietic stem and progenitor cells (HSPCs) and CD34- mature blood cells from n=9 DNMT3Amut peripheral blood samples with G-CSF mobilized HSPCs (autologous stem cell grafts) collected from multiple myeloma patients in remission. In total, 140,000 single cells were sequenced. Simultaneous mutation analysis enabled intra-sample comparison between DNMT3Amut and wild-type cells. Differential gene- and surface protein expression analysis (CITEseq) revealed downregulated MHC-II molecules in DNMT3AR882mut compared to wild-type HSCs. This observation was restricted to DNMT3AR882mut comparted to DNMT3ANon-R882mut samples. To validate this phenotype, we established a FACS-sorting strategy using HLA-DR, an MHC-II cell surface receptor involved in presenting peptides to CD4+ T cells, and compared mutant fractions (VAF) in HLA-DRlow vs. HLA-DRhigh FACS-sorted CD34+ HSPCs by digital-droplet PCR, thereby confirming an up to 2.5-fold higher DNMT3AR882mut fraction in HLA-DRlow sorted HSPCs. We have previously shown that HSPCs are capable of activating CD4+ T cells upon presentation of both, endogenous and exogenous antigens via MHC-II and that the presentation of immunogenic antigens via MHC-II by HSPCs mediates bidirectional interactions with antigen-specific CD4+ T cells. Therefore, we hypothesize that the observed downregulation of MHC-II expression in DNMT3AR882mut HSCs can alter the activation of CD4+ T cells, which can functionally impact the development of CH over time. To explore mechanisms linking DNMT3AR882 mutations to the MHC-II phenotype in a model system, we studied interactions between DNMT3AR882Hmut Lin-Sca1+c-Kit+ cells and CD4+ T cells using an (pI:pC)-inducible humanized mouse model for CH that conditionally expresses human DNMT3A cDNA carrying the R882H hotspot mutation (Mx1-Cre+:DNMT3AWT/R882H). We performed multi-parameter flow cytometry to characterize MHC-II expression on HSPCs and progenitor cell populations. Consistent with our human single-cell data, we observed that MHC-II (I-A/I-E) expression on HSPC subpopulations collected from mice with monoallelic DNMT3AR882H expression was downregulated compared to wild-type mice. In contrast, there was no difference at the progenitor cell level. To understand whether the observed downregulation of MHC-II molecules impacts the antigen-specific activation of CD4+ T cells, we co-cultured FACS-sorted HSPCs with CD4+ T cells from OT-II mice that express transgenic T cell receptors specifically recognizing the chicken ovalbumin (OVA329-339 peptide), when presented via MHC-II. Our preliminary analysis revealed that despite lower MHC-II expression, DNMT3AR882H HSPCs are still capable of activating CD4+ T cells in vitro. Together, our data provide evidence in primary human CH samples and in an engineered mouse model for DNMT3AR882Hmut CH that HSCs carrying DNMT3AR882 hotspot mutations in CH down-regulate MHC-II molecules, which potentially leads to altered immunosurveillance mechanisms in DNMT3AR882 mutant CH.

Background: The BCL-2 inhibitor Venetoclax (VEN) with hypomethylating agents (HMA) has become the standard treatment for unfit AML patients. Recently, we reported that therapy outcome depends on leukemic stem cells (LSCs). By developing the “Mediators-of-Apoptosis-Combinatorial Score” (MAC-Score) linking the ratio of protein expression of BCL-2, BCL-xL, and MCL-1 in LSCs, we demonstrated that BCL-2 apoptotic dependency in LSCs is currently the most accurate therapy response predictor. Aims: In this study we aimed to identify the molecular mechanisms of VEN resistance and apoptotic dependency of LSCs. Methods: Our study cohort comprises samples from 87 AML patients who underwent HMA/VEN therapy including 19 diagnostic/relapse pairs. We aimed to decipher causes of VEN response in FACS-sorted GPR56+ LSCs using transcriptome and methylome analysis as well as functional xenotransplantation and apoptotic dependency assays. Results: To account for heterogeneous patterns of therapy resistance we mapped the gene expression profiles of LSCs onto a single-cell reference map of healthy hematopoiesis. 54 % of LSCs resembled lymphoid-myeloid progenitors (LMPP), 34 % megakaryocytic progenitors (MkP) and 12 % of LSCs showed transcriptomes most similar to hematopoietic stem cells (HSC). Importantly, VEN response and event-free survival (EFS) was associated with LMPP LSCs while VEN refractory patients predominantly resembled other LSC subtypes. The favorable EFS of LMPP LSCs correlated with higher BCL-2 and lower MCL1/BCL-xL expression suggesting a higher BCL-2 dependency for survival compared to other LSC subtypes. Our data further suggest that the LSC subtype is determined by cytogenetic/mutational drivers. AMLs harboring VEN therapy favorable mutations like IDH1/2, splicing factor or RUNX1 mutations were associated to LSCs with an LMPP subtype, while TP53, RAS and JAK2/CALR preferentially result in LSCs with MkP or HSC phenotypes. Further evidence for the LSC-subtype as key determinant of VEN response was found in the differentially expressed genes between responders and non-responders. Non-responders showed higher expression of cell cycle regulators also highly expressed in healthy MkP. We functionally validated these data by performing NSG xenotransplantation assays using AML cells from >40 patients. These results show that VEN refractory patients rapidly induce AML in NSG mice while responders largely fail to engraft even after 8 months, connecting LSC engraftment potential with VEN resistance. Furthermore, LSC potency was significantly associated with reduced EFS and differentiation stage of LSCs, cytogenetic/mutational drivers and apoptotic dependency. In line with these results, paired diagnosis – relapse samples showed that LSC activity is acquired at relapse, while simultaneously BCL-2 dependency decreases. Last, we identified a rare functional LSC population displaying markers of monocytic differentiation (Mono-LSCs) with predominantly KMT2A-rearrangements. These Mono-LSC were distinct from our previously described LSCs as well as from monocytic AML cells, and displayed a unique transcriptomic signature with both monocytic and stem cell features making them poor clinical responders to VEN. Summary/Conclusion: In summary, our data show that LSCs resemble several mutation driven differentiation stages, which shape apoptotic dependencies, LSC potential and ultimately determine the upfront response to HMA/VEN. At time of relapse both apoptotic dependencies and stemness are reshaped and resemble LSCs from refractory patients.Keywords: Leukemic stem cell, Venetoclax, Acute myeloid leukemia, BCL2

Relapse after remission remains the primary cause of treatment failure in acute myeloid leukemia (AML), underscoring the need for strategies to eliminate residual leukemic cells. The bone marrow microenvironment, largely orchestrated by the CXCR4-CXCL12 axis, enables leukemia cell survival and chemoresistance by anchoring blasts in their protective bone marrow niche. Motixafortide, a selective CXCR4 antagonist, mobilizes leukemic cells and disrupts tumor-microenvironment interactions in preclinical models. In this randomized, double-blind, placebo-controlled phase II trial (NCT02502968; registered at ClinicalTrials.gov), 128 patients in first remission received high‑dose cytarabine (HiDAC) plus Motixafortide or placebo. Median relapse-free survival (RFS) did not substantially differ between groups: 10.3 months (95% CI, 8.0-12.0) for Motixafortide and 11.5 months (95% CI, 8.6-24.1) for placebo (log-rank p=0.98). But scMRD analysis, performed before consolidation, demonstrated heterogeneity of CXCR4 inhibition benefit: in the placebo group, higher CXCR4 expression was associated with increased relapse risk (p=0.02), whereas in the Motixafortide group, higher CXCR4 expression was linked to a reduced relapse rate (p=0.047). Exploratory analyses identified scMRD levels at which higher MRD burden was associated with inferior overall survival (OS). Taken together, combining functional MRD profiling with biomarker-driven patient selection, such as CXCR4 expression, may enable more precise and effective post-remission interventions in AML. This clinical trial is registered at EudraCT number: 2014-002702-21.

Measurable residual disease (MRD) assessment is important in acute myeloid leukemia (AML), as post-remission MRD indicates higher relapse risk. Current techniques, however, lack precision in individual outcome prediction, complicated by AML's clonal heterogeneity. Novel single cell MRD (scMRD) workflows based on DNA mutations offer a potential breakthrough, simultaneously capturing genotypic and immunophenotypic changes at a single-cell level. The Tapestri® platform (Mission Bio) incorporates a microfluidic droplet technology with cell barcoding beads and gene-specific primers that enables amplification and comprehensive identification of low-frequency subclones that may lead to overt disease or resistance. With this approach, complex heterogeneity and differentiation between leukemic clones and clonal hematopoiesis can be deconstructed. Objective: This study represents the first large-scale scMRD analysis in AML using this novel scMRD technology to deconvolute clonal heterogeneity to identify potential relapse markers and to differentiate between leukemic clones and clonal hematopoiesis. Methods: We analyzed a cohort of 69 AML patients in first complete remission (CR) as assessed by bone marrow morphology from the BLAST trial (NCT02502968). For 11 patients, we analyzed paired samples from both first CR and relapse. We performed single-cell DNA + protein sequencing on a total of 250,000 cells across 23 multiplexed pools. Our methodology included 40 selected mutation hotspot genes and a 19-plex antibody oligonucleotide cocktail for AML-specific surface markers as well as patient-specific hash tag antibodies for enhanced demultiplexing. We used magnetic activated cell sorting (MACS) to enrich CD34/CD117-positive blasts to ensure high-quality samples for subsequent analysis. Results: Our analyses revealed a diverse genetic landscape with 25 different mutated genes. DNMT3A and NPM1:p.W228Cfs mutations were the most common alterations. The genetic distribution included a predominance of preleukemic genes (51.5%), chromatin remodelers (12.1%), splice factors (9.1%), transcription factors (6.1%), cell cycle regulators (6.1%), and other functional categories (15.2%). Lower MRD (and clonal hematopoiesis) levels were consistently associated with improved overall survival (OS) and relapse-free survival (RFS) across different thresholds (<5%, <10%, <20%, <30%, <40% and <50% MRD burden per patient), with patients in the <5% MRD group showing a significant difference in RFS (p = 0.0013). The median overall survival in this group was 18.0 months, while the median relapse-free survival was 11,7 months. OS and RFS remained significantly different in all MRD groups even after exclusion of DNMT3A and TET2 mutations, which most likely represented clonal hematopoiesis. In paired samples, TP53 mutations were notably prominent during follow-up, remaining the dominant clone, indicating a mixed composition, and the persistence of treatment resistant clones. Overall, our study highlighted the dynamic nature of clonal evolution under therapeutic pressure by demonstrating continuous clonal diversity during clinical remission and notable changes in clonal composition during relapse. Conclusion: In this AML cohort, our study provides insights into the genetic landscape and clonal dynamics of AML in remission. Thus, our findings emphasize the importance and the potential of single-cell DNA + protein sequencing in refining MRD assessment compared to current techniques.