Overexpression of fructose transporter increases pro-survival signaling and confers venetoclax resistance in Ph-like B-cell acute lymphoblastic leukemia

Philadelphia chromosome-like (Ph-like) B-cell acute lymphoblastic leukemia (B-ALL) represents a high-risk subtype characterized by therapy resistance and an adverse clinical profile similar to Ph+ ALL. Recent evidence highlights the growing significance of metabolic reprogramming in drug resistance in B-ALL. We have identified increased fructose transporter GLUT5 expression in Ph-like and Ph+ primary samples and cell lines compared to other B-ALL subsets. In this study, we aim to investigate the novel mechanistic roles of GLUT5 in pro-survival signaling and drug resistance in Ph-like B-ALL. Firstly, to study the role of GLUT5 and fructose availability in cell proliferation, Ph-like B-ALL cell lines (MHH-cALL4 and MUT5Z) stably expressing GLUT5 or empty vector control, were cultured in glucose- or fructose-rich standard media. GLUT5-overexpressing B-ALL cell lines in glucose- or fructose-rich media showed increased proliferation (quantified by CFSE) when compared to control in glucose-rich media. GLUT5-overexpressing cells showed increased colony formation in glucose- or fructose-rich media, while control cells showed the same effect only in fructose-rich media when compared to those in glucose-rich media, indicating that GLUT5-mediated fructose uptake provided a proliferation advantage. To gain better insights on the significance of GLUT5 expression in high-risk B-ALL, bulk RNA-Seq was performed on control or GLUT5-overexpressing Ph-like B-ALL cell lines in glucose- or fructose-rich media. Pro-survival signaling pathways (such as PI3K/AKT, MAPK, NFKB, HIF-alpha and others) were significantly upregulated in GLUT5-overexpressing cell lines in glucose- or fructose-rich and control cells in fructose-rich conditions (vs. control in glucose-rich condition). We confirmed increased expression of p-AKT/AKT, MYC and NFKB in GLUT5-overexpressing Ph-like B-ALL cell lines in glucose- or fructose-rich conditions by immunofluorescence and immunoblotting. Further, increased BCL-2 expression was observed in GLUT5-overexpressing Ph-like cell lines in fructose-rich conditions only, indicative of the potential role of GLUT5 and fructose availability in conferring venetoclax resistance. Consistent with this, GLUT5-overexpressing B-ALL cell lines treated with venetoclax in fructose-rich media showed significantly increased resistance vs. control. A combination of venetoclax with serine synthesis pathway (SSP) inhibitor partially reversed venetoclax resistance. This suggests that GLUT5 expression and fructose availability may divert glycolytic pathway intermediates to SSP as an alternative cell survival mechanism. In summary, GLUT5 may have clinically relevant role in high-risk B-ALL where it modulates pro-survival signaling and confers therapy resistance. AD and VP - equally contributing senior authors