Single-cell Sequencing of Acute Leukemia

Abstract

Acute leukemia affects individuals of all ages and encompasses a heterogeneous group of malignancies driven by genetic alterations that give rise to leukemia stem cells (LSCs), which sustain self-renewal and drive the expansion of abnormal cells. Based on how these alterations affect maturation, acute leukemia is classified into acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). However, leukemia is influenced not only by mutations but also by altered transcriptional programs and dynamic cellular ecosystems that shape disease evolution and treatment response. In this thesis, the aim was to investigate the cellular and molecular landscape of acute leukemia to enhance disease understanding, improve diagnostics, and identify novel therapeutic targets. Taken together, more than 800,000 single cells from 130 patients diagnosed with acute leukemia and 20 healthy donors were analyzed.
In Article I, a multimodal single-cell approach provided novel insights into the pathogenesis of childhood ALL, revealing distinct maturation patterns across subtypes and highlighting the particular vulnerability of DUX4-rearranged ALL to targeted therapies, including phosphoinositide 3-kinase (PI3K) inhibitors and CD3,72 chimeric antigen receptor (CAR) T cell therapy.

In Article II, the fine-grained detail of the single-cell data led to the identification of a novel stem cell marker, C3AR, on NPM1-mutated AML, the most common subtype of AML. We demonstrated that targeting C3AR with antibodies effectively eliminated AML cells while sparing normal progenitor cells, making it a promising therapeutic strategy.

Article III demonstrates that bulk AML gene expression is shaped by diverse cellular signatures better resolved with single-cell technologies, which also uncover unexpected heterogeneity beyond current genomic classification systems. Notably, NPM1-mutated AML could be stratified into two novel, clinically relevant subclasses, with different immune evasion properties.

The aim of Article IV was to investigate age-related characteristics in adult and pediatric AML using single-cell RNA-sequencing data. The analysis revealed that cellular maturation did not differ between the age groups but was strongly correlated with genetic profiles. We successfully identified putative LSC populations, and while no differences were found between adult and pediatric AML in terms of maturation, distinct transcriptional programs were observed, including higher levels of inflammatory signaling and bone marrow remodeling in pediatric AML. Preliminary findings suggest that incorporating maturation status could improve AML classification and highlight potential therapeutic opportunities for both adult and pediatric patients.

In conclusion this thesis demonstrates the power of single-cell technologies in unraveling the cellular and molecular landscape of acute leukemias, leading to the identification of novel leukemia subtypes, deeper insights into LSC biology, and the discovery of potential therapeutic targets.

Abstract (Swedish)

Acute leukemia affects individuals of all ages and encompasses a heterogeneous group of malignancies driven by genetic alterations that give rise to leukemia stem cells (LSCs), which sustain self-renewal and drive the expansion of abnormal cells. Based on how these alterations affect maturation, acute leukemia is classified into acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). However, leukemia is influenced not only by mutations but also by altered transcriptional programs and dynamic cellular ecosystems that shape disease evolution and treatment response. In this thesis, the aim was to investigate the cellular and molecular landscape of acute leukemia to enhance disease understanding, improve diagnostics, and identify novel therapeutic targets. Taken together, more than 800,000 single cells from 130 patients diagnosed with acute leukemia and 20 healthy donors were analyzed.
In Article I, a multimodal single-cell approach provided novel insights into the pathogenesis of childhood ALL, revealing distinct maturation patterns across subtypes and highlighting the particular vulnerability of DUX4-rearranged ALL to targeted therapies, including phosphoinositide 3-kinase (PI3K) inhibitors and CD3,72 chimeric antigen receptor (CAR) T cell therapy.

In Article II, the fine-grained detail of the single-cell data led to the identification of a novel stem cell marker, C3AR, on NPM1-mutated AML, the most common subtype of AML. We demonstrated that targeting C3AR with antibodies effectively eliminated AML cells while sparing normal progenitor cells, making it a promising therapeutic strategy.

Article III demonstrates that bulk AML gene expression is shaped by diverse cellular signatures better resolved with single-cell technologies, which also uncover unexpected heterogeneity beyond current genomic classification systems. Notably, NPM1-mutated AML could be stratified into two novel, clinically relevant subclasses, with different immune evasion properties.

The aim of Article IV was to investigate age-related characteristics in adult and pediatric AML using single-cell RNA-sequencing data. The analysis revealed that cellular maturation did not differ between the age groups but was strongly correlated with genetic profiles. We successfully identified putative LSC populations, and while no differences were found between adult and pediatric AML in terms of maturation, distinct transcriptional programs were observed, including higher levels of inflammatory signaling and bone marrow remodeling in pediatric AML. Preliminary findings suggest that incorporating maturation status could improve AML classification and highlight potential therapeutic opportunities for both adult and pediatric patients.

In conclusion this thesis demonstrates the power of single-cell technologies in unraveling the cellular and molecular landscape of acute leukemias, leading to the identification of novel leukemia subtypes, deeper insights into LSC biology, and the discovery of potential therapeutic targets.

• author: Thorsson, Hanna
• supervisor: Thoas Fioretos; Henrik Lilljebjörn; Christina Orsmark Pietras
• opponent: Professor, MD Huntly, Brian, University of Cambridge, England
• organization: Translational Genomic and Functional Studies of Leukemia (research group); Division of Clinical Genetics; LUCC: Lund University Cancer Centre
• publishing date: 2025
• type: Thesis
• publication status: published
• subject: Clinical Medicine
• keywords: acute lymphoblastic leukemia, acute myeloid leukemia, pediatric leukemia, NPM1-mutated AML, DUX4-rearranged ALL, single-cell sequencing, bioinformatics, cellular hierarchies, leukemia stem cell, target therapies, immunotherapies, CD371, C3AR, PI3K
• in: Lund University, Faculty of Medicine Doctoral Dissertation Series
• issue: 2025:42
• pages: 105 pages
• defense location: Belfragesalen, BMC D15, Klinikgatan 32 i Lund
• defense date: 2025-05-02 09:00:00
• ISSN: 1652-8220
• ISBN: 978-91-8021-695-1
• language: English
• LU publication?: yes
• id: da0c8a82-4157-4f8c-bc2f-b6e1811434ae
• date added to LUP: 2025-04-02 13:32:55
• date last changed: 2025-04-12 10:15:07