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CRISPR Screens Identify Candidate Therapeutic Targets in Leukemia

Rodriguez Zabala, Maria LU (2023) In Lund University, Faculty of Medicine Doctoral Dissertation Series
Abstract
Acute myeloid leukemia (AML) is a complex hematological malignancy marked by proliferation of immature myeloid cells with a dismal 5-year survival. A major challenge is the persistence of leukemia stem cells (LSCs) after standard treatment, leading to relapse. This thesis employs in vivo CRISPR/Cas9 screening to investigate critical AML and LSC molecular mechanisms, interrogating the dependancies on cell surface receptors for use as potential therapies.

In our initial study (Article I), we identify the chemokine receptor CXCR4 as a crucial dependency of AML cell growth and survival. Using a murine model of AML driven by MLL::AF9, we find that CXCR4 loss triggers oxidative stress and differentiation in vivo, with CXCL12 ligand... (More)
Acute myeloid leukemia (AML) is a complex hematological malignancy marked by proliferation of immature myeloid cells with a dismal 5-year survival. A major challenge is the persistence of leukemia stem cells (LSCs) after standard treatment, leading to relapse. This thesis employs in vivo CRISPR/Cas9 screening to investigate critical AML and LSC molecular mechanisms, interrogating the dependancies on cell surface receptors for use as potential therapies.

In our initial study (Article I), we identify the chemokine receptor CXCR4 as a crucial dependency of AML cell growth and survival. Using a murine model of AML driven by MLL::AF9, we find that CXCR4 loss triggers oxidative stress and differentiation in vivo, with CXCL12 ligand signaling being non-essential for leukemia development.
Expanding our study to interrogate nearly one thousand cell surface receptors, we identify three additional AML dependencies. Among these, GLUT1, a primary cellular glucose transporter, emerges as a key regulator of energy metabolism, driving MLL::AF9 LSC survival (Article II). Inhibition of GLUT1 suppresses cellular bioenergetics, prompting autophagy as a metabolic adaptation. Notably, dual inhibition of GLUT1 and oxidative phosphorylation effectively eliminates human AML cells, especially for the RUNX1-mutated AML subtype.

Furthermore, our research also reveals iron metabolism as another critical AML dependency (Article III). We found that disrupting iron uptake through genetic knockdown of Tfrc, encoding the transferrin receptor (TFR1), suppresses leukemia development in a p53-dependent manner, leading to transcriptional repression of antioxidant defense and mitochondrial respiration pathways. Patient-derived AML cells were selectively targeted upon iron chelation treatment.

Additionally, our work uncovers the role of H2K1 in evading NK cell-mediated immune surveillance in vivo through disruption of NK cell maturation and activation (Article IV). Consistent with this finding, H2k1 disruption alone suffices to reverse this immune evasion, restoring NK cell-mediated anti-leukemic effects.

In conclusion, this thesis highlights the value of CRISPR/Cas9 screens in identifying physiologically relevant AML dependencies. It offers insights into targetable LSC vulnerabilities, emphasizing the potential of metabolic targeting and combined treatments for improved AML therapies.
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author
supervisor
opponent
  • Professor Schuringa, Jan J., University Medical Center Groningen
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Acute myeloid leukemia, CRISPR screen, leukemia stem cell, cell surface receptor, metabolism, immunotherapy, CXCR4, GLUT1, TFR1, H2-K1
in
Lund University, Faculty of Medicine Doctoral Dissertation Series
issue
2023:129
pages
138 pages
publisher
Lund University, Faculty of Medicine
defense location
Lundmarksalen, Astronomicentrum, Sölvegatan 27 i Lund
defense date
2023-11-10 13:00:00
ISSN
1652-8220
ISBN
978-91-8021-471-1
language
English
LU publication?
yes
id
4e430138-13a1-4f52-833a-1f8ec7efaa23
date added to LUP
2023-10-13 12:11:07
date last changed
2023-10-20 11:58:38
@phdthesis{4e430138-13a1-4f52-833a-1f8ec7efaa23,
  abstract     = {{Acute myeloid leukemia (AML) is a complex hematological malignancy marked by proliferation of immature myeloid cells with a dismal 5-year survival. A major challenge is the persistence of leukemia stem cells (LSCs) after standard treatment, leading to relapse. This thesis employs in vivo CRISPR/Cas9 screening to investigate critical AML and LSC molecular mechanisms, interrogating the dependancies on cell surface receptors for use as potential therapies. <br/><br/>In our initial study (Article I), we identify the chemokine receptor CXCR4 as a crucial dependency of AML cell growth and survival. Using a murine model of AML driven by MLL::AF9, we find that CXCR4 loss triggers oxidative stress and differentiation in vivo, with CXCL12 ligand signaling being non-essential for leukemia development.<br/>Expanding our study to interrogate nearly one thousand cell surface receptors, we identify three additional AML dependencies. Among these, GLUT1, a primary cellular glucose transporter, emerges as a key regulator of energy metabolism, driving MLL::AF9 LSC survival (Article II). Inhibition of GLUT1 suppresses cellular bioenergetics, prompting autophagy as a metabolic adaptation. Notably, dual inhibition of GLUT1 and oxidative phosphorylation effectively eliminates human AML cells, especially for the RUNX1-mutated AML subtype.<br/><br/>Furthermore, our research also reveals iron metabolism as another critical AML dependency (Article III). We found that disrupting iron uptake through genetic knockdown of Tfrc, encoding the transferrin receptor (TFR1), suppresses leukemia development in a p53-dependent manner, leading to transcriptional repression of antioxidant defense and mitochondrial respiration pathways. Patient-derived AML cells were selectively targeted upon iron chelation treatment. <br/><br/>Additionally, our work uncovers the role of H2K1 in evading NK cell-mediated immune surveillance in vivo through disruption of NK cell maturation and activation (Article IV). Consistent with this finding, H2k1 disruption alone suffices to reverse this immune evasion, restoring NK cell-mediated anti-leukemic effects.<br/><br/>In conclusion, this thesis highlights the value of CRISPR/Cas9 screens in identifying physiologically relevant AML dependencies. It offers insights into targetable LSC vulnerabilities, emphasizing the potential of metabolic targeting and combined treatments for improved AML therapies.<br/>}},
  author       = {{Rodriguez Zabala, Maria}},
  isbn         = {{978-91-8021-471-1}},
  issn         = {{1652-8220}},
  keywords     = {{Acute myeloid leukemia; CRISPR screen; leukemia stem cell; cell surface receptor; metabolism; immunotherapy; CXCR4; GLUT1; TFR1; H2-K1}},
  language     = {{eng}},
  number       = {{2023:129}},
  publisher    = {{Lund University, Faculty of Medicine}},
  school       = {{Lund University}},
  series       = {{Lund University, Faculty of Medicine Doctoral Dissertation Series}},
  title        = {{CRISPR Screens Identify Candidate Therapeutic Targets in Leukemia}},
  url          = {{https://lup.lub.lu.se/search/files/161841255/Maria_Rodriguez_PhD_thesis_spikning.pdf}},
  year         = {{2023}},
}