Loss of G0S2 in Kinase-Independent TKI Resistance and Blastic Transformation of CML

Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1 have turned chronic myeloid leukemia (CML) from a fatal to a chronic condition. Despite impressive results, resistance is a clinical problem, and TKIs do not target the CML leukemic stem cell (LSC) population, which is independent of BCR-ABL1 kinase activity. To identify mechanisms associated with BCR-ABL1 kinase-independent TKI resistance, we compared the transcriptional profile of TKI-naïve CD34⁺ cells from newly diagnosed CML patients who subsequently either responded (n=41) or did not respond (n=18) to imatinib over a twelve-month period (McWeeney et al. Blood 2010). The transcriptional profile of imatinib resistance in non-responders revealed striking overlap with reported signatures of CML blastic transformation and also implicated pathways involved in LSC survival. These findings suggest that similar biological mechanisms may be driving three different scenarios of BCR-ABL1-independent resistance: 1) primary kinase-independent TKI resistance, 2) transformation of CML from the chronic phase (CP-CML) to the blastic phase (BP-CML), and 3) persistence of residual leukemia in TKI-responsive patients.

Among the genes most profoundly downregulated in patients destined to fail imatinib therapy was G0S2 (>3-fold, p<0.02), encoding a small protein that regulates multiple cellular functions, including apoptosis, quiescence, lipid metabolism, and oxidative phosphorylation. We compared G0S2 mRNA expression in CD34⁺ cells from normal cord blood (n=5) and from patient specimens lacking BCR-ABL1 kinase domain mutations, including newly diagnosed CP-CML (n=6), TKI-resistant CP-CML (n=2), and BP-CML (n=5). G0S2 was 4-fold downregulated in newly diagnosed CP-CML compared to normal cord blood CD34⁺ cells (p<0.01), and further downregulated by >3-fold in TKI-resistant samples and BP-CML (p<0.01), consistent with published microarray data (McWeeney et al. Blood 2010). Retrospective analysis of survival data for patients whose samples were evaluated in the transcriptional profile revealed that low G0S2 expression correlated with a shorter overall survival (n=35, p=0.036). Consistently, G0S2 mRNA and protein were downregulated in TKI-resistant K562^R cells lacking BCR-ABL1 kinase domain mutations compared to TKI-sensitive controls. Ectopic expression of Flag-tagged G0S2 (G0S2-Flag) significantly reduced colony formation and increased apoptosis of TKI-sensitive and TKI-resistant K562 cells, and also CD34⁺ cells from CP-CML (n=5, p<0.05) and BP-CML (n=3, p<0.05) patients, having a more pronounced effect in BP-CML in the presence of imatinib. G0S2-Flag did not affect colony formation or apoptosis of normal cord blood CD34⁺ cells (n=2), which already express high levels of G0S2. Importantly, qRT-PCR revealed that G0S2 mRNA was significantly lower in primitive CD34⁺38^- stem cells compared to more committed CD34⁺38⁺ progenitor cells in CP-CML (n=6, p<0.01) but not normal cord blood CD34⁺ cells (n=4, p=ns). These data suggest that loss of G0S2 plays a role not only in primary TKI resistance and blastic transformation of CML, but also in TKI resistance of the CML LSC. Consistently, CFSE staining revealed that G0S2 mRNA levels in CP-CML CD34⁺ cells are lowest in cell populations with the least number of cell divisions.

Altogether, these findings suggest that G0S2 plays a role in regulating leukemic stem and progenitor cell survival and TKI response in vitro, and that restoring G0S2 expression combined with BCR-ABL1 inhibition may be a novel strategy to induce treatment-free remission in CML patients with kinase-independent TKI resistance and BP-CML, or to eradicate the CML LSC population. Future studies will assess the functional role of G0S2 in LSC survival and BCR-ABL1 leukemogenesis in vivo, and determine the mechanism by which G0S2 is downregulated in TKI resistance.