EHA Library - The official digital education library of European Hematology Association (EHA)

SOD2 PROMOTES ACUTE LEUKEMIA ADAPTATION TO AMINO ACID STARVATION THROUGH THE N-DEGRON PATHWAY
Author(s): ,
Nurul Khalida Ibrahim
Affiliations:
Department of Pediatric Hematology and Oncology,Hannover Medical School,Hannover,Allemagne;Department of Pediatric Hematology and Oncology,Hannover Medical School,Hannover,Deutschland;Department of Pediatric Hematology and Oncology,Hannover Medical School,Hannover,Germania;Department of Pediatric Hematology and Oncology,Hannover Medical School,Hannover,Germany;Department of Pediatric Hematology an
,
Sabine Schreek
Affiliations:
Department of Pediatric Hematology and Oncology,Hannover Medical School,Hannover,Allemagne;Department of Pediatric Hematology and Oncology,Hannover Medical School,Hannover,Deutschland;Department of Pediatric Hematology and Oncology,Hannover Medical School,Hannover,Germania;Department of Pediatric Hematology and Oncology,Hannover Medical School,Hannover,Germany;Department of Pediatric Hematology an
,
Buesra Cinar
Affiliations:
Department of Pediatric Hematology and Oncology,Hannover Medical School,Hannover,Allemagne;Department of Pediatric Hematology and Oncology,Hannover Medical School,Hannover,Deutschland;Department of Pediatric Hematology and Oncology,Hannover Medical School,Hannover,Germania;Department of Pediatric Hematology and Oncology,Hannover Medical School,Hannover,Germany;Department of Pediatric Hematology an
,
Lorent Loxha
Affiliations:
Department of Pediatric Hematology and Oncology,Hannover Medical School,Hannover,Allemagne;Department of Pediatric Hematology and Oncology,Hannover Medical School,Hannover,Deutschland;Department of Pediatric Hematology and Oncology,Hannover Medical School,Hannover,Germania;Department of Pediatric Hematology and Oncology,Hannover Medical School,Hannover,Germany;Department of Pediatric Hematology an
,
Beate Fehlhaber
Affiliations:
Department of Pediatric Hematology and Oncology,Hannover Medical School,Hannover,Allemagne;Department of Pediatric Hematology and Oncology,Hannover Medical School,Hannover,Deutschland;Department of Pediatric Hematology and Oncology,Hannover Medical School,Hannover,Germania;Department of Pediatric Hematology and Oncology,Hannover Medical School,Hannover,Germany;Department of Pediatric Hematology an
,
Jean-Pierre Bourquin
Affiliations:
Department of Pediatric Hematology and Oncology,University Children's Hospital Zurich,Zurich,Suisse;Department of Pediatric Hematology and Oncology,University Children's Hospital Zurich,Zurich,Schweiz;Department of Pediatric Hematology and Oncology,University Children's Hospital Zurich,Zurich,Svizzera;Department of Pediatric Hematology and Oncology,University Children's Hospital Zurich,Zurich,Swit
,
Beat Bornhauser
Affiliations:
Department of Pediatric Hematology and Oncology,University Children's Hospital Zurich,Zurich,Suisse;Department of Pediatric Hematology and Oncology,University Children's Hospital Zurich,Zurich,Schweiz;Department of Pediatric Hematology and Oncology,University Children's Hospital Zurich,Zurich,Svizzera;Department of Pediatric Hematology and Oncology,University Children's Hospital Zurich,Zurich,Swit
,
Cornelia Eckert
Affiliations:
Department of Pediatric Hematology and Oncology,Charité Universitätsmedizin,Berlin,Allemagne;Department of Pediatric Hematology and Oncology,Charité Universitätsmedizin,Berlin,Deutschland;Department of Pediatric Hematology and Oncology,Charité Universitätsmedizin,Berlin,Germania;Department of Pediatric Hematology and Oncology,Charité Universitätsmedizin,Berlin,Germany;Department of Pediatric Hemat
,
Gunnar Cario
Affiliations:
Department of Pediatrics I,Christian-Albrecht University Kiel and University Medical Center Schleswig-Holstein,Kiel,Allemagne;Department of Pediatrics I,Christian-Albrecht University Kiel and University Medical Center Schleswig-Holstein,Kiel,Deutschland;Department of Pediatrics I,Christian-Albrecht University Kiel and University Medical Center Schleswig-Holstein,Kiel,Germania;Department of Pediatr
,
Michael Forster
Affiliations:
Institute of Clinical Molecular Biology,Kiel,Allemagne;Institute of Clinical Molecular Biology,Kiel,Deutschland;Institute of Clinical Molecular Biology,Kiel,Germania;Institute of Clinical Molecular Biology,Kiel,Germany;Institute of Clinical Molecular Biology,Kiel,Alemania;Institute of Clinical Molecular Biology,Kiel,Duitsland;Institute of Clinical Molecular Biology,Kiel,Alemanha;Institute of Clini
,
Martin Stanulla
Affiliations:
Department of Pediatric Hematology and Oncology,Hannover Medical School,Hannover,Allemagne;Department of Pediatric Hematology and Oncology,Hannover Medical School,Hannover,Deutschland;Department of Pediatric Hematology and Oncology,Hannover Medical School,Hannover,Germania;Department of Pediatric Hematology and Oncology,Hannover Medical School,Hannover,Germany;Department of Pediatric Hematology an
,
Alejandro Gutierrez
Affiliations:
Division of Pediatric Hematology and Oncology,Boston Children's Hospital,Boston,États-unis;Division of Pediatric Hematology and Oncology,Boston Children's Hospital,Boston,Vereinigte Staaten;Division of Pediatric Hematology and Oncology,Boston Children's Hospital,Boston,Stati Uniti;Division of Pediatric Hematology and Oncology,Boston Children's Hospital,Boston,United States;Division of Pediatric He
Laura Hinze
Affiliations:
Department of Pediatric Hematology and Oncology,Hannover Medical School,Hannover,Allemagne;Department of Pediatric Hematology and Oncology,Hannover Medical School,Hannover,Deutschland;Department of Pediatric Hematology and Oncology,Hannover Medical School,Hannover,Germania;Department of Pediatric Hematology and Oncology,Hannover Medical School,Hannover,Germany;Department of Pediatric Hematology an
(Abstract release date: 05/12/22) EHA Library. Khalida Ibrahim N. 06/11/22; 356972; S107
Nurul Khalida Ibrahim
Nurul Khalida Ibrahim
Contributions
Abstract
Presentation during EHA2022: All Oral presentations will be presented between Friday, June 10 and Sunday, June 12 and will be accessible for on-demand viewing from Monday, June 20 until Monday, August 15, 2022 on the Congress platform.

Abstract: S107

Type: Oral Presentation

Session title: From biology to preclinical models in ALL

Background
The ability of cells to tolerate amino acid starvation is fundamental for survival under cellular stress conditions. Some cancer cells are vulnerable to asparagine depletion, which is exploited therapeutically using asparaginase. However, the mechanisms of adaptation to amino acid starvation in leukemia cells remain incompletely understood.

Aims
We recently performed a genome-wide CRISPR/Cas9 loss-of-function screen in the resistant T-ALL cell line CCRF-CEM to identify molecular pathways that promote asparaginase resistance. We found that Wnt-dependent stabilization of proteins (Wnt/STOP) induces a profound therapeutic vulnerability to asparaginase in acute leukemias and colorectal cancers (Hinze et. al., 2019; Hinze et al., 2020). Another unrelated gene on the top of the screen included SOD2, a mitochondrial superoxide dismutase. Intriguingly, to date, SOD2 activity has not been linked to a cellular amino acid starvation response, whose biologic basis we thus sought to further investigate.

Methods
To evaluate the significance of SOD2 in mediating an asparaginase response, we employed genetic epistasis experiments as well as phenotypic assays including short hairpin RNA (shRNA) mediated knockdowns, quantitative PCRs, Western blots, amino acid starvation, and viability assays.

Results

Knockdown of SOD2 (shSOD2) resulted in a profound sensitivity to asparaginase in several T-ALL and B-ALL cell lines (p<0.0001), and an increase in apoptosis, as assessed by caspase 3/7 activity (p<0.001). The sensitization was rescued by either overexpressing SOD2 cDNA (p<0.0001), or by adding the functional SOD2 mimetic MnTBAP (p<0.01). Of note, shSOD2 mediated sensitization was selective to asparaginase, as it could not be observed for other commonly used chemotherapeutic agents including vincristine, doxorubicin, dexamethasone, and 6-mercaptopurine (p=ns). Due to the selectivity to asparagine depletion, we then investigated whether SOD2 inhibition mediates a broader amino acid starvation response. Indeed, culturing SOD2-inhibited T-ALL cells in the absence of essential amino acids (EAA) or non-EAA, induced a significant decrease in cell viability (p<0.05). Sensitization appeared to be specific to the SOD2 isoform, and distinct from known SOD2-associated pathways including reactive oxygen species, cell cycle changes, alterations of mTOR signaling, or glutamine anaplerosis. To better understand the molecular underpinnings of SOD2 in regulating an amino acid starvation response, we leveraged the Bioplex Interactome database (Huttlin et al., 2020), and identified UBR2, an E3 ubiquitin ligase in the N-degron pathway, as a unique binding partner of SOD2. Ubiquitin E3 ligases target their substrates for ubiquitination, leading to proteasome-mediated degradation (Yang et al., 2010). Indeed, SOD2 and UBR2 were co-immunoprecipitated, suggesting the formation of a complex that can drive proteasome-dependent protein catabolism. In line, inhibition of SOD2 significantly decreased ubiquitin levels, suggesting that SOD2 positively regulates catabolic protein degradation through the N-degron pathway to promote cancer cell fitness in amino acid starved conditions.

Conclusion
The interaction of SOD2 and the N-degron pathway represents a previously unknown molecular adaptation of cancer cells in response to amino acid starvation. These results serve as a strong proponent for an in-depth characterization of the N-degron pathway in mediating leukemia cell fitness upon amino acid starvation and thus provide a basis for therapeutic intervention in refractory leukemias.

Keyword(s): Acute leukemia, Asparaginase, Drug resistance, Proteasome

Presentation during EHA2022: All Oral presentations will be presented between Friday, June 10 and Sunday, June 12 and will be accessible for on-demand viewing from Monday, June 20 until Monday, August 15, 2022 on the Congress platform.

Abstract: S107

Type: Oral Presentation

Session title: From biology to preclinical models in ALL

Background
The ability of cells to tolerate amino acid starvation is fundamental for survival under cellular stress conditions. Some cancer cells are vulnerable to asparagine depletion, which is exploited therapeutically using asparaginase. However, the mechanisms of adaptation to amino acid starvation in leukemia cells remain incompletely understood.

Aims
We recently performed a genome-wide CRISPR/Cas9 loss-of-function screen in the resistant T-ALL cell line CCRF-CEM to identify molecular pathways that promote asparaginase resistance. We found that Wnt-dependent stabilization of proteins (Wnt/STOP) induces a profound therapeutic vulnerability to asparaginase in acute leukemias and colorectal cancers (Hinze et. al., 2019; Hinze et al., 2020). Another unrelated gene on the top of the screen included SOD2, a mitochondrial superoxide dismutase. Intriguingly, to date, SOD2 activity has not been linked to a cellular amino acid starvation response, whose biologic basis we thus sought to further investigate.

Methods
To evaluate the significance of SOD2 in mediating an asparaginase response, we employed genetic epistasis experiments as well as phenotypic assays including short hairpin RNA (shRNA) mediated knockdowns, quantitative PCRs, Western blots, amino acid starvation, and viability assays.

Results

Knockdown of SOD2 (shSOD2) resulted in a profound sensitivity to asparaginase in several T-ALL and B-ALL cell lines (p<0.0001), and an increase in apoptosis, as assessed by caspase 3/7 activity (p<0.001). The sensitization was rescued by either overexpressing SOD2 cDNA (p<0.0001), or by adding the functional SOD2 mimetic MnTBAP (p<0.01). Of note, shSOD2 mediated sensitization was selective to asparaginase, as it could not be observed for other commonly used chemotherapeutic agents including vincristine, doxorubicin, dexamethasone, and 6-mercaptopurine (p=ns). Due to the selectivity to asparagine depletion, we then investigated whether SOD2 inhibition mediates a broader amino acid starvation response. Indeed, culturing SOD2-inhibited T-ALL cells in the absence of essential amino acids (EAA) or non-EAA, induced a significant decrease in cell viability (p<0.05). Sensitization appeared to be specific to the SOD2 isoform, and distinct from known SOD2-associated pathways including reactive oxygen species, cell cycle changes, alterations of mTOR signaling, or glutamine anaplerosis. To better understand the molecular underpinnings of SOD2 in regulating an amino acid starvation response, we leveraged the Bioplex Interactome database (Huttlin et al., 2020), and identified UBR2, an E3 ubiquitin ligase in the N-degron pathway, as a unique binding partner of SOD2. Ubiquitin E3 ligases target their substrates for ubiquitination, leading to proteasome-mediated degradation (Yang et al., 2010). Indeed, SOD2 and UBR2 were co-immunoprecipitated, suggesting the formation of a complex that can drive proteasome-dependent protein catabolism. In line, inhibition of SOD2 significantly decreased ubiquitin levels, suggesting that SOD2 positively regulates catabolic protein degradation through the N-degron pathway to promote cancer cell fitness in amino acid starved conditions.

Conclusion
The interaction of SOD2 and the N-degron pathway represents a previously unknown molecular adaptation of cancer cells in response to amino acid starvation. These results serve as a strong proponent for an in-depth characterization of the N-degron pathway in mediating leukemia cell fitness upon amino acid starvation and thus provide a basis for therapeutic intervention in refractory leukemias.

Keyword(s): Acute leukemia, Asparaginase, Drug resistance, Proteasome

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