Abstract 648: Role of G0S2 in chronic myeloid leukemia and TKI resistance
Authors: Mayra A. Gonzalez, Alfonso E. Bencomo, Christian Barreto-Vargas, Andres J. Rubio, Idaly M. Olivas, Joshua J. Lara, Anna Senina, Jonathan Ahmann, Katherine T. Varley, Luis F. Jave-Suarez, O'Hare Thomas, Michael W. Deininger, Anna M. Eiring
Journal: Cancer Research (2020)

Abstract Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1 have turned chronic myeloid leukemia (CML) from a fatal to a chronic disease. However, resistance is a clinical problem, and TKIs do not target CML leukemic stem cells (LSC), which are independent from BCR-ABL1 kinase activity. To understand mechanisms driving BCR-ABL1-independent resistance, we analyzed the transcriptional signature of TKI-naïve CD34+ cells from patients with or without a response to imatinib after 12 months of therapy (McWeeney et al. Blood 2010). Among the genes most profoundly downregulated in patients destined to fail imatinib was G0/G1 switch gene 2 (G0S2) (&amp;gt;3-fold, n=59, p&amp;lt;0.02). Retrospective analysis of survival data for patients whose samples were evaluated in a microarray revealed that high G0S2 expression (&amp;gt;50%) correlated with a longer overall survival (n=30 responders, n=16 non-responders, p=0.036). We next analyzed G0S2 mRNA expression in CD34+ cells from normal cord blood and from primary CD34+ cells lacking BCR-ABL1 kinase domain mutations. G0S2 was 4-fold downregulated in newly diagnosed CML (n=6) compared to normal cord blood (n=5, p&amp;lt;0.01), and further downregulated by &amp;gt;3-fold in TKI-resistant (n=2) and BP-CML samples (n=5, p&amp;lt;0.01).G0S2 mRNA was also lower in CD34+38- stem cells compared to committed CD34+38+ progenitor cells in CML (n=6, p&amp;lt;0.01) but not normal cord blood (n=4, p=ns). CFSE staining revealed that G0S2 mRNA levels in CP-CML CD34+ cells are lowest in cell populations with the least number of cell divisions. To assess the role of G0S2 in CML, we cloned G0S2 amplified from normal human mononuclear cells into a lentiviral expression system, and confirmed ectopic expression by qRT-PCR and immunoblot. Ectopic G0S2 reduced colony formation in both TKI-sensitive and TKI-resistant CML cell lines and CD34+ CML patient samples. While G0S2 has been reported to induce apoptosis by direct inhibition of BCL-2, reduced colony formation was not associated with an increase of apoptosis, but did restore imatinib-induced apoptosis. These data are consistent with a tumor suppressor role for G0S2 in CML. G0S2 has also been shown to inhibit adipocyte triglyceride lipase (ATGL), the rate-limiting enzyme in the conversion of triglycerides to free fatty acids (FFAs). While ATGL is indeed expressed at the protein level in CML cells, shRNAs targeting ATGL do not phenocopy ectopic G0S2 expression. Furthermore, the effects of ectopic G0S2 are not impaired in cells with simultaneous ATGL knockdown, and ectopic G0S2 has no effect on intracellular FFA or ATP levels. Finally, G0S2 gene expression has been shown to be regulated by promoter DNA hypermethylation. However, DNA bisulfite and patch PCR sequencing in primary CD34+ cells from CML patients did not reveal G0S2 promoter hypermethylation in CML. Rather, ChIP-PCR revealed the presence of MYC/MAX at the G0S2 promoter in CML, suggesting MYC-mediated transcriptional repression. Altogether, these data suggest that G0S2 downregulation plays a functional role in CML disease progression and TKI response, but is independent from its role as an inhibitor of BCL-2 or ATGL. These data unravel a new role for G0S2 as a regulator of TKI response in CML, and suggest that restoring G0S2 expression may have clinical utility. Citation Format: Mayra A. Gonzalez, Alfonso E. Bencomo, Christian Barreto-Vargas, Andres J. Rubio, Idaly M. Olivas, Joshua J. Lara, Anna Senina, Jonathan Ahmann, Katherine T. Varley, Luis F. Jave-Suarez, O'Hare Thomas, Michael W. Deininger, Anna M. Eiring. Role of G0S2 in chronic myeloid leukemia and TKI resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 648.