Single-cell gene expression analyses reveal distinct self-renewing and proliferating subsets in the leukemia stem cell compartment in acute myeloid leukemia

Karen Sachs; Aaron L. Sarver; Klara E. Noble-Orcutt; Rebecca S. LaRue; Marie Lue Antony; Daniel Chang; Yoonkyu Lee; Connor M. Navis; Alexandria L. Hillesheim; Ian R. Nykaza; Ngoc A. Ha; Conner J. Hansen; Fatma K. Karadag; Rachel J. Bergerson; Michael R. Verneris; Matthew M. Meredith; Matthew L. Schomaker; Michael A. Linden; Chad L. Myers; David A. Largaespada; Zohar Sachs

doi:10.1158/0008-5472.CAN-18-2932

Single-cell gene expression analyses reveal distinct self-renewing and proliferating subsets in the leukemia stem cell compartment in acute myeloid leukemia

Karen Sachs, Aaron L. Sarver, Klara E. Noble-Orcutt, Rebecca S. LaRue, Marie Lue Antony, Daniel Chang, Yoonkyu Lee, Connor M. Navis, Alexandria L. Hillesheim, Ian R. Nykaza, Ngoc A. Ha, Conner J. Hansen, Fatma K. Karadag, Rachel J. Bergerson, Michael R. Verneris, Matthew M. Meredith, Matthew L. Schomaker, Michael A. Linden, Chad L. Myers, David A. LargaespadaZohar Sachs

Research output: Contribution to journal › Article › peer-review

47 Scopus citations

Abstract

Standard chemotherapy for acute myeloid leukemia (AML) targets proliferative cells and efficiently induces complete remission; however, many patients relapse and die of their disease. Relapse is caused by leukemia stem cells (LSC), the cells with self-renewal capacity. Self-renewal and proliferation are separate functions in normal hematopoietic stem cells (HSC) in steady-state conditions. If these functions are also separate functions in LSCs, then antiproliferative therapies may fail to target self-renewal, allowing for relapse. We investigated whether proliferation and self-renewal are separate functions in LSCs as they often are in HSCs. Distinct transcriptional profiles within LSCs of Mll-AF9/ NRAS G12V murine AML were identified using single-cell RNA sequencing. Single-cell qPCR revealed that these genes were also differentially expressed in primary human LSCs and normal human HSPCs. A smaller subset of these genes was upregulated in LSCs relative to HSPCs; this subset of genes constitutes "LSC-specific" genes in human AML. To assess the differences between these profiles, we identified cell surface markers, CD69 and CD36, whose genes were differentially expressed between these profiles. In vivo mouse reconstitution assays resealed that only CD69 High LSCs were capable of self-renewal and were poorly proliferative. In contrast, CD36 High LSCs were unable to transplant leukemia but were highly proliferative. These data demonstrate that the transcriptional foundations of self-renewal and proliferation are distinct in LSCs as they often are in normal stem cells and suggest that therapeutic strategies that target self-renewal, in addition to proliferation, are critical to prevent relapse and improve survival in AML. SIGNIFICANCE: These findings define and functionally validate a self-renewal gene profile of leukemia stem cells at the single-cell level and demonstrate that self-renewal and proliferation are distinct in AML. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/3/458/F1.large.jpg.

Original language	English (US)
Pages (from-to)	458-470
Number of pages	13
Journal	Cancer Research
Volume	80
Issue number	3
DOIs	https://doi.org/10.1158/0008-5472.CAN-18-2932
State	Published - Feb 1 2020

Bibliographical note

Funding Information:
The authors thank Michael Franklin for editorial assistance. The authors thank Katy Richards-Hrdlicka and Jason McKinney from Fluidigm for helping us to assess and ensure the quality of the single-cell qPCR data. This work utilized the resources of the University of Minnesota Hematological Malignancies Tissue Bank, which is supported by the NCI (5P30CA077598-18), Minnesota Masonic Charities, and the Killebrew-Thompson Memorial Fund through the Cancer Experimental Therapeutics Initiative (CETI); the Flow Cytometry Resource and other services of the Masonic Cancer Center (which is supported by NIH 2P30CA077598-21) at the University of Minnesota; Jerry Daniel and Darrell Johnson and the University of Minnesota Genomics Center Fluidigm C1 single-cell capture and BioMark qPCR and next-generation sequencing services; the Mass Cytometry Shared Resource at the University of Minnesota (which is supported by the Office of the Vice President for Research, University of

Funding Information:
The authors thank Michael Franklin for editorial assistance. The authors thank Katy Richards-Hrdlicka and Jason McKinney from Fluidigm for helping us to assess and ensure the quality of the single-cell qPCR data. This work utilized the resources of the University of Minnesota Hematological Malignancies Tissue Bank, which is supported by the NCI (5P30CA077598-18), Minnesota Masonic Charities, and the Killebrew-Thompson Memorial Fund through the Cancer Experimental Therapeutics Initiative (CETI); the Flow Cytometry Resource and other services of the Masonic Cancer Center (which is supported by NIH 2P30CA077598-21) at the University of Minnesota; Jerry Daniel and Darrell Johnson and the University of Minnesota Genomics Center Fluidigm C1 single-cell capture and BioMark qPCR and next-generation sequencing services; the Mass Cytometry Shared Resource at the University of Minnesota (which is supported by the Office of the Vice President for Research, University of Minnesota) and the Minnesota Supercomputing Institute. This work and Z. Sachs were supported by the American Cancer Society, Frederick A. DeLuca Foundation, Mentored Research Scholar Grant (MRSG-16-195-01-DDC); the Clinical and Translational Science Institute at the University of Minnesota KL2 Career Development and K to R01 awards (NIH's National Center for Advancing Translational Sciences, grants KL2TR002492 and UL1TR002494); the Lois and Richard King Assistant Professorship in Medicine, the American Cancer Society Institutional Research Grant at the University of Minnesota (124166-IRG-58-001-55-IRG12); the Masonic Cancer Center at the University of Minnesota Translational Working Group Award and Genetic Mechanisms of Cancer Award; the University of Minnesota Department of Medicine Women's Early Research Career Award; the University of Minnesota Genomic Center Pilot Award; the Randy Shaver Cancer Research and Community Fund, the Masonic Cancer Center, the Division of Hematology, Oncology, and Transplantation, Department of Medicine; and the University of Minnesota Foundation donors. K. Sachs was supported in part by an award from the Muscular Dystrophy Association (MDA Award #574137). D. Largaespada was supported by an American Cancer Society Research Professorship.

Publisher Copyright:
© 2020 American Association for Cancer Research.

Keywords

Animals
Biomarkers, Tumor/genetics
Cell Proliferation/genetics
Cell Self Renewal/genetics
Gene Expression Profiling
Gene Expression Regulation, Leukemic
Hematopoietic Stem Cells/cytology
Humans
Leukemia, Myeloid, Acute/genetics
Mice
Neoplastic Stem Cells/metabolism
Single-Cell Analysis/methods

PubMed: MeSH publication types

Research Support, Non-U.S. Gov't
Journal Article
Research Support, N.I.H., Extramural

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1158/0008-5472.CAN-18-2932

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Sachs, K., Sarver, A. L., Noble-Orcutt, K. E., LaRue, R. S., Antony, M. L., Chang, D., Lee, Y., Navis, C. M., Hillesheim, A. L., Nykaza, I. R., Ha, N. A., Hansen, C. J., Karadag, F. K., Bergerson, R. J., Verneris, M. R., Meredith, M. M., Schomaker, M. L., Linden, M. A., Myers, C. L., ... Sachs, Z. (2020). Single-cell gene expression analyses reveal distinct self-renewing and proliferating subsets in the leukemia stem cell compartment in acute myeloid leukemia. Cancer Research, 80(3), 458-470. https://doi.org/10.1158/0008-5472.CAN-18-2932

Sachs, K, Sarver, AL , Noble-Orcutt, KE , LaRue, RS, Antony, ML, Chang, D, Lee, Y, Navis, CM, Hillesheim, AL, Nykaza, IR, Ha, NA, Hansen, CJ, Karadag, FK, Bergerson, RJ, Verneris, MR, Meredith, MM, Schomaker, ML, Linden, MA , Myers, CL , Largaespada, DA & Sachs, Z 2020, 'Single-cell gene expression analyses reveal distinct self-renewing and proliferating subsets in the leukemia stem cell compartment in acute myeloid leukemia', Cancer Research, vol. 80, no. 3, pp. 458-470. https://doi.org/10.1158/0008-5472.CAN-18-2932

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title = "Single-cell gene expression analyses reveal distinct self-renewing and proliferating subsets in the leukemia stem cell compartment in acute myeloid leukemia",

abstract = "Standard chemotherapy for acute myeloid leukemia (AML) targets proliferative cells and efficiently induces complete remission; however, many patients relapse and die of their disease. Relapse is caused by leukemia stem cells (LSC), the cells with self-renewal capacity. Self-renewal and proliferation are separate functions in normal hematopoietic stem cells (HSC) in steady-state conditions. If these functions are also separate functions in LSCs, then antiproliferative therapies may fail to target self-renewal, allowing for relapse. We investigated whether proliferation and self-renewal are separate functions in LSCs as they often are in HSCs. Distinct transcriptional profiles within LSCs of Mll-AF9/ NRAS G12V murine AML were identified using single-cell RNA sequencing. Single-cell qPCR revealed that these genes were also differentially expressed in primary human LSCs and normal human HSPCs. A smaller subset of these genes was upregulated in LSCs relative to HSPCs; this subset of genes constitutes {"}LSC-specific{"} genes in human AML. To assess the differences between these profiles, we identified cell surface markers, CD69 and CD36, whose genes were differentially expressed between these profiles. In vivo mouse reconstitution assays resealed that only CD69 High LSCs were capable of self-renewal and were poorly proliferative. In contrast, CD36 High LSCs were unable to transplant leukemia but were highly proliferative. These data demonstrate that the transcriptional foundations of self-renewal and proliferation are distinct in LSCs as they often are in normal stem cells and suggest that therapeutic strategies that target self-renewal, in addition to proliferation, are critical to prevent relapse and improve survival in AML. SIGNIFICANCE: These findings define and functionally validate a self-renewal gene profile of leukemia stem cells at the single-cell level and demonstrate that self-renewal and proliferation are distinct in AML. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/3/458/F1.large.jpg. ",

keywords = "Animals, Biomarkers, Tumor/genetics, Cell Proliferation/genetics, Cell Self Renewal/genetics, Gene Expression Profiling, Gene Expression Regulation, Leukemic, Hematopoietic Stem Cells/cytology, Humans, Leukemia, Myeloid, Acute/genetics, Mice, Neoplastic Stem Cells/metabolism, Single-Cell Analysis/methods",

author = "Karen Sachs and Sarver, {Aaron L.} and Noble-Orcutt, {Klara E.} and LaRue, {Rebecca S.} and Antony, {Marie Lue} and Daniel Chang and Yoonkyu Lee and Navis, {Connor M.} and Hillesheim, {Alexandria L.} and Nykaza, {Ian R.} and Ha, {Ngoc A.} and Hansen, {Conner J.} and Karadag, {Fatma K.} and Bergerson, {Rachel J.} and Verneris, {Michael R.} and Meredith, {Matthew M.} and Schomaker, {Matthew L.} and Linden, {Michael A.} and Myers, {Chad L.} and Largaespada, {David A.} and Zohar Sachs",

note = "Funding Information: The authors thank Michael Franklin for editorial assistance. The authors thank Katy Richards-Hrdlicka and Jason McKinney from Fluidigm for helping us to assess and ensure the quality of the single-cell qPCR data. This work utilized the resources of the University of Minnesota Hematological Malignancies Tissue Bank, which is supported by the NCI (5P30CA077598-18), Minnesota Masonic Charities, and the Killebrew-Thompson Memorial Fund through the Cancer Experimental Therapeutics Initiative (CETI); the Flow Cytometry Resource and other services of the Masonic Cancer Center (which is supported by NIH 2P30CA077598-21) at the University of Minnesota; Jerry Daniel and Darrell Johnson and the University of Minnesota Genomics Center Fluidigm C1 single-cell capture and BioMark qPCR and next-generation sequencing services; the Mass Cytometry Shared Resource at the University of Minnesota (which is supported by the Office of the Vice President for Research, University of Funding Information: The authors thank Michael Franklin for editorial assistance. The authors thank Katy Richards-Hrdlicka and Jason McKinney from Fluidigm for helping us to assess and ensure the quality of the single-cell qPCR data. This work utilized the resources of the University of Minnesota Hematological Malignancies Tissue Bank, which is supported by the NCI (5P30CA077598-18), Minnesota Masonic Charities, and the Killebrew-Thompson Memorial Fund through the Cancer Experimental Therapeutics Initiative (CETI); the Flow Cytometry Resource and other services of the Masonic Cancer Center (which is supported by NIH 2P30CA077598-21) at the University of Minnesota; Jerry Daniel and Darrell Johnson and the University of Minnesota Genomics Center Fluidigm C1 single-cell capture and BioMark qPCR and next-generation sequencing services; the Mass Cytometry Shared Resource at the University of Minnesota (which is supported by the Office of the Vice President for Research, University of Minnesota) and the Minnesota Supercomputing Institute. This work and Z. Sachs were supported by the American Cancer Society, Frederick A. DeLuca Foundation, Mentored Research Scholar Grant (MRSG-16-195-01-DDC); the Clinical and Translational Science Institute at the University of Minnesota KL2 Career Development and K to R01 awards (NIH's National Center for Advancing Translational Sciences, grants KL2TR002492 and UL1TR002494); the Lois and Richard King Assistant Professorship in Medicine, the American Cancer Society Institutional Research Grant at the University of Minnesota (124166-IRG-58-001-55-IRG12); the Masonic Cancer Center at the University of Minnesota Translational Working Group Award and Genetic Mechanisms of Cancer Award; the University of Minnesota Department of Medicine Women's Early Research Career Award; the University of Minnesota Genomic Center Pilot Award; the Randy Shaver Cancer Research and Community Fund, the Masonic Cancer Center, the Division of Hematology, Oncology, and Transplantation, Department of Medicine; and the University of Minnesota Foundation donors. K. Sachs was supported in part by an award from the Muscular Dystrophy Association (MDA Award #574137). D. Largaespada was supported by an American Cancer Society Research Professorship. Publisher Copyright: {\textcopyright} 2020 American Association for Cancer Research.",

year = "2020",

month = feb,

day = "1",

doi = "10.1158/0008-5472.CAN-18-2932",

language = "English (US)",

volume = "80",

pages = "458--470",

journal = "Cancer Research",

issn = "0008-5472",

publisher = "American Association for Cancer Research Inc.",

number = "3",

}

TY - JOUR

T1 - Single-cell gene expression analyses reveal distinct self-renewing and proliferating subsets in the leukemia stem cell compartment in acute myeloid leukemia

AU - Sachs, Karen

AU - Sarver, Aaron L.

AU - Noble-Orcutt, Klara E.

AU - LaRue, Rebecca S.

AU - Antony, Marie Lue

AU - Chang, Daniel

AU - Lee, Yoonkyu

AU - Navis, Connor M.

AU - Hillesheim, Alexandria L.

AU - Nykaza, Ian R.

AU - Ha, Ngoc A.

AU - Hansen, Conner J.

AU - Karadag, Fatma K.

AU - Bergerson, Rachel J.

AU - Verneris, Michael R.

AU - Meredith, Matthew M.

AU - Schomaker, Matthew L.

AU - Linden, Michael A.

AU - Myers, Chad L.

AU - Largaespada, David A.

AU - Sachs, Zohar

N1 - Funding Information: The authors thank Michael Franklin for editorial assistance. The authors thank Katy Richards-Hrdlicka and Jason McKinney from Fluidigm for helping us to assess and ensure the quality of the single-cell qPCR data. This work utilized the resources of the University of Minnesota Hematological Malignancies Tissue Bank, which is supported by the NCI (5P30CA077598-18), Minnesota Masonic Charities, and the Killebrew-Thompson Memorial Fund through the Cancer Experimental Therapeutics Initiative (CETI); the Flow Cytometry Resource and other services of the Masonic Cancer Center (which is supported by NIH 2P30CA077598-21) at the University of Minnesota; Jerry Daniel and Darrell Johnson and the University of Minnesota Genomics Center Fluidigm C1 single-cell capture and BioMark qPCR and next-generation sequencing services; the Mass Cytometry Shared Resource at the University of Minnesota (which is supported by the Office of the Vice President for Research, University of Funding Information: The authors thank Michael Franklin for editorial assistance. The authors thank Katy Richards-Hrdlicka and Jason McKinney from Fluidigm for helping us to assess and ensure the quality of the single-cell qPCR data. This work utilized the resources of the University of Minnesota Hematological Malignancies Tissue Bank, which is supported by the NCI (5P30CA077598-18), Minnesota Masonic Charities, and the Killebrew-Thompson Memorial Fund through the Cancer Experimental Therapeutics Initiative (CETI); the Flow Cytometry Resource and other services of the Masonic Cancer Center (which is supported by NIH 2P30CA077598-21) at the University of Minnesota; Jerry Daniel and Darrell Johnson and the University of Minnesota Genomics Center Fluidigm C1 single-cell capture and BioMark qPCR and next-generation sequencing services; the Mass Cytometry Shared Resource at the University of Minnesota (which is supported by the Office of the Vice President for Research, University of Minnesota) and the Minnesota Supercomputing Institute. This work and Z. Sachs were supported by the American Cancer Society, Frederick A. DeLuca Foundation, Mentored Research Scholar Grant (MRSG-16-195-01-DDC); the Clinical and Translational Science Institute at the University of Minnesota KL2 Career Development and K to R01 awards (NIH's National Center for Advancing Translational Sciences, grants KL2TR002492 and UL1TR002494); the Lois and Richard King Assistant Professorship in Medicine, the American Cancer Society Institutional Research Grant at the University of Minnesota (124166-IRG-58-001-55-IRG12); the Masonic Cancer Center at the University of Minnesota Translational Working Group Award and Genetic Mechanisms of Cancer Award; the University of Minnesota Department of Medicine Women's Early Research Career Award; the University of Minnesota Genomic Center Pilot Award; the Randy Shaver Cancer Research and Community Fund, the Masonic Cancer Center, the Division of Hematology, Oncology, and Transplantation, Department of Medicine; and the University of Minnesota Foundation donors. K. Sachs was supported in part by an award from the Muscular Dystrophy Association (MDA Award #574137). D. Largaespada was supported by an American Cancer Society Research Professorship. Publisher Copyright: © 2020 American Association for Cancer Research.

PY - 2020/2/1

Y1 - 2020/2/1

N2 - Standard chemotherapy for acute myeloid leukemia (AML) targets proliferative cells and efficiently induces complete remission; however, many patients relapse and die of their disease. Relapse is caused by leukemia stem cells (LSC), the cells with self-renewal capacity. Self-renewal and proliferation are separate functions in normal hematopoietic stem cells (HSC) in steady-state conditions. If these functions are also separate functions in LSCs, then antiproliferative therapies may fail to target self-renewal, allowing for relapse. We investigated whether proliferation and self-renewal are separate functions in LSCs as they often are in HSCs. Distinct transcriptional profiles within LSCs of Mll-AF9/ NRAS G12V murine AML were identified using single-cell RNA sequencing. Single-cell qPCR revealed that these genes were also differentially expressed in primary human LSCs and normal human HSPCs. A smaller subset of these genes was upregulated in LSCs relative to HSPCs; this subset of genes constitutes "LSC-specific" genes in human AML. To assess the differences between these profiles, we identified cell surface markers, CD69 and CD36, whose genes were differentially expressed between these profiles. In vivo mouse reconstitution assays resealed that only CD69 High LSCs were capable of self-renewal and were poorly proliferative. In contrast, CD36 High LSCs were unable to transplant leukemia but were highly proliferative. These data demonstrate that the transcriptional foundations of self-renewal and proliferation are distinct in LSCs as they often are in normal stem cells and suggest that therapeutic strategies that target self-renewal, in addition to proliferation, are critical to prevent relapse and improve survival in AML. SIGNIFICANCE: These findings define and functionally validate a self-renewal gene profile of leukemia stem cells at the single-cell level and demonstrate that self-renewal and proliferation are distinct in AML. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/3/458/F1.large.jpg.

AB - Standard chemotherapy for acute myeloid leukemia (AML) targets proliferative cells and efficiently induces complete remission; however, many patients relapse and die of their disease. Relapse is caused by leukemia stem cells (LSC), the cells with self-renewal capacity. Self-renewal and proliferation are separate functions in normal hematopoietic stem cells (HSC) in steady-state conditions. If these functions are also separate functions in LSCs, then antiproliferative therapies may fail to target self-renewal, allowing for relapse. We investigated whether proliferation and self-renewal are separate functions in LSCs as they often are in HSCs. Distinct transcriptional profiles within LSCs of Mll-AF9/ NRAS G12V murine AML were identified using single-cell RNA sequencing. Single-cell qPCR revealed that these genes were also differentially expressed in primary human LSCs and normal human HSPCs. A smaller subset of these genes was upregulated in LSCs relative to HSPCs; this subset of genes constitutes "LSC-specific" genes in human AML. To assess the differences between these profiles, we identified cell surface markers, CD69 and CD36, whose genes were differentially expressed between these profiles. In vivo mouse reconstitution assays resealed that only CD69 High LSCs were capable of self-renewal and were poorly proliferative. In contrast, CD36 High LSCs were unable to transplant leukemia but were highly proliferative. These data demonstrate that the transcriptional foundations of self-renewal and proliferation are distinct in LSCs as they often are in normal stem cells and suggest that therapeutic strategies that target self-renewal, in addition to proliferation, are critical to prevent relapse and improve survival in AML. SIGNIFICANCE: These findings define and functionally validate a self-renewal gene profile of leukemia stem cells at the single-cell level and demonstrate that self-renewal and proliferation are distinct in AML. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/3/458/F1.large.jpg.

KW - Animals

KW - Biomarkers, Tumor/genetics

KW - Cell Proliferation/genetics

KW - Cell Self Renewal/genetics

KW - Gene Expression Profiling

KW - Gene Expression Regulation, Leukemic

KW - Hematopoietic Stem Cells/cytology

KW - Humans

KW - Leukemia, Myeloid, Acute/genetics

KW - Mice

KW - Neoplastic Stem Cells/metabolism

KW - Single-Cell Analysis/methods

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SN - 0008-5472

VL - 80

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EP - 470

JO - Cancer Research

JF - Cancer Research

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ER -

Single-cell gene expression analyses reveal distinct self-renewing and proliferating subsets in the leukemia stem cell compartment in acute myeloid leukemia

Abstract

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Keywords

PubMed: MeSH publication types

UN SDGs

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