Periodic Reporting for period 1 - MRDAML (Deciphering the mechanistic basis of minimal residual disease and treatment resistance in acute myeloid leukemia)
Reporting period: 2023-09-01 to 2025-08-31
Acute myeloid leukemia (AML) is a highly aggressive cancer with frequent relapse and very poor clinical outcomes for most patients. AML is a genetically heterogenous disease and can be driven by various gene lesions. Currently, genetic classification of AML are routinely used as prognostic markers for risk assessment, therapy responses and patient outcomes. However, the ability to clinically predict resistance and relapse based on genetic subtypes remains limited, where patients with harmonized mutational backgrounds can have distinct outcomes (i.e. early relapse vs long-term remission). This is likely due to an additional level of non-genetically driven intra-patient heterogeneity including transcriptional dynamics, epigenetic regulation, metabolic adaptation or microenvironmental cues. In addition, different cell of origin further contributes to the heterogeneity and complex response to therapies in AML.
AML is usually an age-associated disease. This is because AML transformation occurs when DNA damages accumulate in hematopoietic stem and progenitor cells (HSPC) during their continuous cell proliferation and differentiation over an individual’s life time. HSPC proliferation and differentiation is essential, as this enables continuous supply of new red and white blood cells to maintain normal homeostasis. The HSPC compartment is known to be highly heterogeneous, containing distinct cell populations such as hematopoietic stem cell (HSC) and multipotent progenitor (MPP) cells. Within these populations, some are known to exhibit preferential tendency to develop into myeloid cells, while the others have a bias toward a different lineage such as lymphocytes. Such differential preference could contribute to the pathogenesis of leukemia of different subtype and origins.
The overarching goal of this proposal is to tackle the fundamental lack of understanding in how HPSC heterogeneity contribute to the non-genetic heterogeneity in AML. From the scientific point of view, this project will answer the following questions: Are HSPC populations with a bias towards myeloid differentiation more likely to develop into AML? If so, can we utilize the other population to compensate for such transformation, to rejuvenate the aging process and to delay the onset of AML? Can we understand the molecular mechanism regulating lineage bias, stem cell maintenance and aging? From the translational point of view, given that the global population is developing toward a more aged population overtime, it is of high importance to understand stem cell heterogeneity in normal, aged and leukemic conditions and such knowledge gain can contribute to the potential development of new preventative strategy of age-associated AML onset or therapies targeting different subsets of AMLs.
AML is usually an age-associated disease. This is because AML transformation occurs when DNA damages accumulate in hematopoietic stem and progenitor cells (HSPC) during their continuous cell proliferation and differentiation over an individual’s life time. HSPC proliferation and differentiation is essential, as this enables continuous supply of new red and white blood cells to maintain normal homeostasis. The HSPC compartment is known to be highly heterogeneous, containing distinct cell populations such as hematopoietic stem cell (HSC) and multipotent progenitor (MPP) cells. Within these populations, some are known to exhibit preferential tendency to develop into myeloid cells, while the others have a bias toward a different lineage such as lymphocytes. Such differential preference could contribute to the pathogenesis of leukemia of different subtype and origins.
The overarching goal of this proposal is to tackle the fundamental lack of understanding in how HPSC heterogeneity contribute to the non-genetic heterogeneity in AML. From the scientific point of view, this project will answer the following questions: Are HSPC populations with a bias towards myeloid differentiation more likely to develop into AML? If so, can we utilize the other population to compensate for such transformation, to rejuvenate the aging process and to delay the onset of AML? Can we understand the molecular mechanism regulating lineage bias, stem cell maintenance and aging? From the translational point of view, given that the global population is developing toward a more aged population overtime, it is of high importance to understand stem cell heterogeneity in normal, aged and leukemic conditions and such knowledge gain can contribute to the potential development of new preventative strategy of age-associated AML onset or therapies targeting different subsets of AMLs.
Main scientific actions and achievements:
1. Identification of a new marker to further resolve the heterogeneity of normal HSPC.
The surface marker Endothelia Protein C Receptor (EPCR) is a well-known marker for HSC identification, which is conserved between species and different conditions such as homeostasis and stress. In the project, we identified that EPCR expression was not unique to the HSC population, but also found in subsets of different MPP populations. This raised the questions on whether EPCR-expressing MPP cells also marks functional HSCs. If so, are there any distinct roles of these EPCR-expressing HSC-like MPP in aging and AML transformation compared to previously known HSCs? The results of this part have been published in 2023 (DOI: 10.1016/j.cdev.2023.203843 ).
2. Development of an in vitro functional assay to assess lineage differentiation.
The gold standard assay to study HSC function is via the transplantation into animals so that they can differentiate into multiple mature blood lineages include red blood cells (RBC), platelets, myeloid cells and lymphocytes. In this project, we have developed an in vitro culture assay that allow the development of all of these four mature lineages in a dish. This provides a platform for comprehensive functional characterization of stem cell populations of interests before conducting large scale animal transplantation experiments, adhering to the Replace, Reduce, and Refine (3R) principles.
3. Comprehensive characterization of a new HSC population based on positive expression of this marker.
Using the EPCR marker to further interrogate HSPC heterogeneity, we identified a new population of HSC that has not been previously studies in depth. Thus, we performed comprehensive characterization of this population at the molecular and functional level. Some key experiments performed include:
• In vitro multi-lineage differentiation assay as introduced above.
• Bulk transplantation assay
• Single cell transplantation assay
• Bulk RNA-sequencing
• Single cell RNA and Methylome sequencing
• Bulk ATAC-sequencing to profile DNA accessibility
4. Identification of the key feature of this new HSC population and its potential impact in aging and AML transformation.
A key discovery from this project is the new HSC population is characterized by their deep dormancy status. This enables them to participate minimally in proliferation and differentiation during normal condition, and become resilient to aging-associated DNA damage and AML transformation.
1. Identification of a new marker to further resolve the heterogeneity of normal HSPC.
The surface marker Endothelia Protein C Receptor (EPCR) is a well-known marker for HSC identification, which is conserved between species and different conditions such as homeostasis and stress. In the project, we identified that EPCR expression was not unique to the HSC population, but also found in subsets of different MPP populations. This raised the questions on whether EPCR-expressing MPP cells also marks functional HSCs. If so, are there any distinct roles of these EPCR-expressing HSC-like MPP in aging and AML transformation compared to previously known HSCs? The results of this part have been published in 2023 (DOI: 10.1016/j.cdev.2023.203843 ).
2. Development of an in vitro functional assay to assess lineage differentiation.
The gold standard assay to study HSC function is via the transplantation into animals so that they can differentiate into multiple mature blood lineages include red blood cells (RBC), platelets, myeloid cells and lymphocytes. In this project, we have developed an in vitro culture assay that allow the development of all of these four mature lineages in a dish. This provides a platform for comprehensive functional characterization of stem cell populations of interests before conducting large scale animal transplantation experiments, adhering to the Replace, Reduce, and Refine (3R) principles.
3. Comprehensive characterization of a new HSC population based on positive expression of this marker.
Using the EPCR marker to further interrogate HSPC heterogeneity, we identified a new population of HSC that has not been previously studies in depth. Thus, we performed comprehensive characterization of this population at the molecular and functional level. Some key experiments performed include:
• In vitro multi-lineage differentiation assay as introduced above.
• Bulk transplantation assay
• Single cell transplantation assay
• Bulk RNA-sequencing
• Single cell RNA and Methylome sequencing
• Bulk ATAC-sequencing to profile DNA accessibility
4. Identification of the key feature of this new HSC population and its potential impact in aging and AML transformation.
A key discovery from this project is the new HSC population is characterized by their deep dormancy status. This enables them to participate minimally in proliferation and differentiation during normal condition, and become resilient to aging-associated DNA damage and AML transformation.
The results generated from this project identified and characterized a new HSC population with superior ability to stay highly dormant and are resilient to age-associated decline. This will have strong implication in developing strategy to improve rejuvenation process, which helps to reduce the DNA damage stress that occur during HSC proliferation and differentiation, and reduce the incidents of AML transformation. Thus, further exploitation should focus on in detail basic research on the molecular mechanism regulating this feature, identification of biomarkers and potential translation if such biomarkers and/or important regulators are identified.