A primary hierarchically organized patient-derived model enables in depth interrogation of stemness driven by the coding and non-coding genome

Abstract

Many cancers are organized as cellular hierarchies sustained by cancer stem cells (CSC), whose eradication is crucial for achieving long-term remission. Difficulties to isolate and undertake in vitro and in vivo experimental studies of rare CSC under conditions that preserve their original properties currently constitute a bottleneck for identifying molecular mechanisms involving coding and non-coding genomic regions that govern stemness. We focussed on acute myeloid leukemia (AML) as a paradigm of the CSC model and developed a patient-derived system termed OCI-AML22 that recapitulates the cellular hierarchy driven by leukemia stem cells (LSC). Through classical flow sorting and functional analyses, we established that a single phenotypic population is highly enriched for LSC. The LSC fraction can be easily isolated and serially expanded in culture or in xenografts while faithfully recapitulating functional, transcriptional and epigenetic features of primary LSCs. A novel non-coding regulatory element was identified with a new computational approach using functionally validated primary AML LSC fractions and its role in LSC stemness validated through efficient CRISPR editing using methods optimized for OCI-AML22 LSC. Collectively, OCI-AML22 constitutes a valuable resource to uncover mechanisms governing CSC driven malignancies.

Identification of leukemia stem cell subsets with distinct transcriptional, epigenetic and functional properties

“…To characterize in depth the pool of LSC in OCI-AML22, we performed single cell (sc) multiome analysis (scATAC-Seq/scRNA-Seq) on 7,160 cells isolated from the LSC-enriched CD34+CD38-fraction ( in vivo LSC frequency is 1 in 200) 34 (Figure 1A). The majority of these cells were enriched for stem cell programs when mapped onto a single cell transcriptomic map of stem and progenitor populations from healthy human bone marrow (Supplementary Figure 1A) 41 .…”

“…These findings suggest that core stemness properties of LSC that drive clinical outcomes and treatment sensitivity are shared across AML patients, and support studies to understand whether the basis for the observed functional heterogeneity within the LSC compartment may also be shared. The gold standard for studies of functional properties of LSC has been AML patient-derived xenograft (PDX) assays, as most AML cell lines do not exhibit a LSC-driven hierarchy that captures patient biology 34 . However, PDX assays are laborious, difficult to standardize and cannot be tailored for high-throughput studies.…”

“…However, PDX assays are laborious, difficult to standardize and cannot be tailored for high-throughput studies. Recently, we developed a patient-derived AML model (OCI-AML22) that retains biological properties with broad clinical relevance 34 . The OCI-AML22 LSC population exhibits hallmark stemness properties that are highly prognostic across diverse independent cohorts of AML patient samples 1,2,29–32,35–40 .…”

Abemaciclib drives the therapeutic differentiation of acute myeloid leukaemia stem cells

“…The AML LSCs mainly reside within populations of CD34 + CD38 − AML cells and can reconstitute human AML in immunodeficient mice. Although recent studies have suggested that LSCs also exist in other AML fractions, 34 immature CD34 + CD38 − cells remain the best characterised and most potent population initiating leukaemia in various xenograft mouse models and re‐transplantation experiments 5,35–37 . Here, we assessed the therapeutic effects of abemaciclib in two clinically relevant AML PDX models established by xenotransplantation of primary LSC populations from relapsed patients (PDX‐1 and PDX‐2) into NCG mice (Figure 2A).…”

“…Although recent studies have suggested that LSCs also exist in other AML fractions, 34 immature CD34 + CD38 − cells remain the best characterised and most potent population initiating leukaemia in various xenograft mouse models and re-transplantation experiments. 5,[35][36][37] Here, we assessed the therapeutic effects of abemaciclib in two clinically relevant AML PDX models established by xenotransplantation of primary LSC populations from relapsed patients (PDX-1 and PDX-2) into NCG mice (Figure 2A). In this model, abemaciclib treatment suppressed the growth of primary human AML cells (Figure 2B) and led to a lower average size and weight of the spleen in abemaciclib-treated xenografted mice (Figure 2C).…”