LOSS OF G0/G1 SWITCH GENE 2 (G0S2) IN CML PROMOTES TKI RESISTANCE BY DEREGULATING THE MITOCHONDRIAL ELECTRON TRANSPORT CHAIN.
Author(s): ,
Mayra Gonzalez
Affiliations:
Molecular and Translational Medicine,Texas Tech University Health Sciences Center,El Paso,United States
,
Alfonso Bencomo-Alvarez
Affiliations:
Molecular and Translational Medicine,Texas Tech University Health Sciences Center,El Paso,United States
,
Andres Rubio
Affiliations:
Molecular and Translational Medicine,Texas Tech University Health Sciences Center,El Paso,United States
,
Sara Dang
Affiliations:
Graduate School of Biomedical Sciences,Texas Tech University Health Sciences Center,El Paso,United States
,
Christian Barreto-Vargas
Affiliations:
Immunology Division,University of Guadalajara,Guadalajara,Mexico
,
Idaly Olivas
Affiliations:
Graduate School of Biomedical Sciences,Texas Tech University Health Sciences Center,El Paso,United States
,
Steven Glovier
Affiliations:
Graduate School of Biomedical Sciences,Texas Tech University Health Sciences Center,El Paso,United States
,
Joshua Lara
Affiliations:
Paul L. Foster School of Medicine,Texas Tech University Health Sciences Center,El Paso,United States
,
Dongqing Yan
Affiliations:
Huntsman Cancer Institute,The University of Utah,Salt Lake City,United States
,
Anna Senina
Affiliations:
Huntsman Cancer Institute,The University of Utah,Salt Lake City,United States
,
Jonathan Ahman
Affiliations:
Huntsman Cancer Institute,The University of Utah,Salt Lake City,United States
,
Katherine Varley
Affiliations:
Huntsman Cancer Institute,The University of Utah,Salt Lake City,United States
,
Clinton Mason
Affiliations:
Huntsman Cancer Institute,The University of Utah,Salt Lake City,United States
,
Christopher Eide
Affiliations:
Knight Cancer Institute,Oregon Health & Science University,Portland,United States
,
Brian Druker
Affiliations:
Knight Cancer Institute,Oregon Health & Science University,Portland,United States
Anna Eiring
Affiliations:
Molecular and Translational Medicine,Texas Tech University Health Sciences Center,El Paso,United States;Graduate School of Biomedical Sciences,Texas Tech University Health Sciences Center,El Paso,United States;Paul L. Foster School of Medicine,Texas Tech University Health Sciences Center,El Paso,United States
EHA Library. Gonzalez M. 06/09/21; 324558; S150
Mayra Gonzalez
Mayra Gonzalez
Contributions
Abstract
Presentation during EHA2021: All Oral presentations will be made available as of Friday, June 11, 2021 (09:00 CEST) and will be accessible for on-demand viewing until August 15, 2021 on the Virtual Congress platform.

Abstract: S150

Type: Oral Presentation

Session title: Response, resistance and treatment-free remission in CML

Background

Chronic myeloid leukemia (CML) was turned from a fatal to a chronic disease with the use of tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1. Despite these advances, clinical resistance is a problem, as TKIs do not target the CML leukemic stem cell (LSC), which are independent from BCR-ABL1 kinase activity. Microarray data on TKI-naïve versus TKI-resistant CML patients, after 12 months of therapy, revealed that mRNA encoding G0/G1 switch gene 2 (G0S2) is downregulated in TKI resistance (>3-fold, p<0.02).

Aims
Identify the mechanism by which G0S2 is downregulated in CML and how it contributes to TKI resistance. 

Methods

To assess the functional role of G0S2 in CML and TKI resistance, we used ectopic G0S2 expression and shRNA-mediated G0S2 knockdown in CML cell lines and CD34+ cells from chronic phase (CP-CML) and blast phase (BP-CML) CML patients. The consequence of altered G0S2 expression was measured using colony formation assays, apoptosis assays, and subcutaneous injection into immunocompromised mice. The effect of G0S2 on mitochondrial respiration was identified with RNA sequencing (RNAseq) and metabolomics analyses. 

Results
G0S2 mRNA and protein were downregulated in CML CD34+ cells compared with normal cord blood CD34+ cells (p=0.0043), and further downregulated in samples from BP-CML (p=0.0079) and TKI-resistant (p=0.0357) patients. Low levels of G0S2 correlated with a worse overall survival (n=35, p=0.036) in CP-CML. We observed lower G0S2 mRNA expression in primitive, TKI-resistant CD34+38- LSCs compared with more committed CD34+38+ progenitor cells in CML (n=6, p<0.01), and in granulocyte-macrophage progenitors (GMPs) from CML versus normal CD34+ cells (p=0.044). Loss of G0S2 expression in CML was not affected by TKI treatment, and thus occurs in a BCR-ABL1-independent manner. G0S2 ectopic expression significantly reduced colony formation of TKI-sensitive and TKI-resistant K562 cells, and in CD34+ cells from both CP-CML and BP-CML patients. While ectopic G0S2 alone did not alter apoptosis, it restored imatinib-mediated apoptosis in BP-CML CD34+ cells (p<0.05). Consistently, ectopic G0S2 reduced in vivo subcutaneous tumor growth of K562 cells injected into nude mice. ShRNA-mediated G0S2 knockdown, conversely, enhanced survival of CML cell lines in the presence and absence of imatinib. DNA bisulfite conversion and patch PCR sequencing on primary CML CD34+ cells revealed that G0S2 downregulation was not due to promoter hypermethylation. Rather, ChIP-PCR revealed the presence of MYC/MAX at the G0S2 promoter in CML, suggesting MYC-mediated transcriptional repression. Accordingly, ectopic MYC resulted in rapid downregulation of G0S2 mRNA and protein in CML cell lines. G0S2 has been shown to inhibit adipocyte triglyceride lipase (ATGL), the rate-limiting enzyme in intracellular lipolysis. However, ectopic G0S2 impaired survival even in the presence of ATGL knockdown. RNAseq and metabolomics data suggest that reduced G0S2 expression in CML results in deregulation of the mitochondrial ETC. This has implications for TKI response, as CML LSCs were shown to be more dependent on oxidative phosphorylation than their normal counterparts, which could serve as a novel target for therapy.

Conclusion
Altogether, our data suggest that MYC-mediated G0S2 downregulation contributes to TKI resistance and blastic transformation of CML. Restoring G0S2 expression could have clinical utility by reestablishing TKI sensitivity in TKI-resistant patients and altering mitochondrial metabolism.

Keyword(s): Chronic myeloid leukemia, Cord blood CD34+ Cells, Leukemic stem cell, Tyrosine kinase inhibitor

Presentation during EHA2021: All Oral presentations will be made available as of Friday, June 11, 2021 (09:00 CEST) and will be accessible for on-demand viewing until August 15, 2021 on the Virtual Congress platform.

Abstract: S150

Type: Oral Presentation

Session title: Response, resistance and treatment-free remission in CML

Background

Chronic myeloid leukemia (CML) was turned from a fatal to a chronic disease with the use of tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1. Despite these advances, clinical resistance is a problem, as TKIs do not target the CML leukemic stem cell (LSC), which are independent from BCR-ABL1 kinase activity. Microarray data on TKI-naïve versus TKI-resistant CML patients, after 12 months of therapy, revealed that mRNA encoding G0/G1 switch gene 2 (G0S2) is downregulated in TKI resistance (>3-fold, p<0.02).

Aims
Identify the mechanism by which G0S2 is downregulated in CML and how it contributes to TKI resistance. 

Methods

To assess the functional role of G0S2 in CML and TKI resistance, we used ectopic G0S2 expression and shRNA-mediated G0S2 knockdown in CML cell lines and CD34+ cells from chronic phase (CP-CML) and blast phase (BP-CML) CML patients. The consequence of altered G0S2 expression was measured using colony formation assays, apoptosis assays, and subcutaneous injection into immunocompromised mice. The effect of G0S2 on mitochondrial respiration was identified with RNA sequencing (RNAseq) and metabolomics analyses. 

Results
G0S2 mRNA and protein were downregulated in CML CD34+ cells compared with normal cord blood CD34+ cells (p=0.0043), and further downregulated in samples from BP-CML (p=0.0079) and TKI-resistant (p=0.0357) patients. Low levels of G0S2 correlated with a worse overall survival (n=35, p=0.036) in CP-CML. We observed lower G0S2 mRNA expression in primitive, TKI-resistant CD34+38- LSCs compared with more committed CD34+38+ progenitor cells in CML (n=6, p<0.01), and in granulocyte-macrophage progenitors (GMPs) from CML versus normal CD34+ cells (p=0.044). Loss of G0S2 expression in CML was not affected by TKI treatment, and thus occurs in a BCR-ABL1-independent manner. G0S2 ectopic expression significantly reduced colony formation of TKI-sensitive and TKI-resistant K562 cells, and in CD34+ cells from both CP-CML and BP-CML patients. While ectopic G0S2 alone did not alter apoptosis, it restored imatinib-mediated apoptosis in BP-CML CD34+ cells (p<0.05). Consistently, ectopic G0S2 reduced in vivo subcutaneous tumor growth of K562 cells injected into nude mice. ShRNA-mediated G0S2 knockdown, conversely, enhanced survival of CML cell lines in the presence and absence of imatinib. DNA bisulfite conversion and patch PCR sequencing on primary CML CD34+ cells revealed that G0S2 downregulation was not due to promoter hypermethylation. Rather, ChIP-PCR revealed the presence of MYC/MAX at the G0S2 promoter in CML, suggesting MYC-mediated transcriptional repression. Accordingly, ectopic MYC resulted in rapid downregulation of G0S2 mRNA and protein in CML cell lines. G0S2 has been shown to inhibit adipocyte triglyceride lipase (ATGL), the rate-limiting enzyme in intracellular lipolysis. However, ectopic G0S2 impaired survival even in the presence of ATGL knockdown. RNAseq and metabolomics data suggest that reduced G0S2 expression in CML results in deregulation of the mitochondrial ETC. This has implications for TKI response, as CML LSCs were shown to be more dependent on oxidative phosphorylation than their normal counterparts, which could serve as a novel target for therapy.

Conclusion
Altogether, our data suggest that MYC-mediated G0S2 downregulation contributes to TKI resistance and blastic transformation of CML. Restoring G0S2 expression could have clinical utility by reestablishing TKI sensitivity in TKI-resistant patients and altering mitochondrial metabolism.

Keyword(s): Chronic myeloid leukemia, Cord blood CD34+ Cells, Leukemic stem cell, Tyrosine kinase inhibitor

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