C067 | SETD2 DEFICIENCY IN CHRONIC MYELOID LEUKEMIA DRIVES METABOLIC REPROGRAMMING PROMOTING TYROSINE KINASE INHIBITOR RESISTANCE AND DISEASE PROGRESSION

Despite the success of TKIs, some CML patients still experience resistance and progression. We have reported that non-genomic loss of function of the SETD2 tumor suppressor is a recurrent event in multi-TKI-resistant and advanced-phase CML, where it fosters genetic instability by impairing DNA repair. We have more recently found that SETD2 deficiency may be observed as early as at the time of diagnosis in CML patients who will not achieve an optimal response to TKI therapy. To better elucidate the effects of SETD2 deficiency in CML, we employed an integrated approach of Mass Spectrometry (MS) and RNA-Seq to analyze the SETD2 interactome, target genes, and transcriptional programs in SETD2-proficient and -deficient cellular models of CML (LAMA84 and KCL22) where we used RNA interference (RNAi) and nucleofection to modulate SETD2 expression. Findings were validated by co-immunoprecipitation (co-IP), Western blotting (WB) and immunofluorescence (IF). We found that SETD2 loss induces metabolic reprogramming, driving CML cells towards glycolysis. Overexpression of key glycolytic enzymes, including HK2 (Hexokinase II), PDK1 (Pyruvate dehydrogenase kinase isozyme 1), and LDHA (lactate dehydrogenase) in SETD2-deficient cells (KCL22) as compared to -proficient cells (KCL22tsSED2) were found by RNA-seq. WB performed both in total protein extracts and in cytoplasmatic or mitochondrial compartments confirmed a reduction in the expression of these enzymes following SETD2 knock-in (Fig.1). IF confirmed WB findings, suggesting that SETD2 re-expression in KCL22 cells inhibits hypoxic signaling. Additionally, MS and co-IP identified novel SETD2 interactors involved in cell division and glycolytic metabolism, including α-tubulin and β-tubulin (checked in total protein extracts and in the cytoskeletal subfraction); PFKP, PFKFB3, PD, and LDHA (checked in total protein extracts, in the cytoplasmatic and in the mitochondrial subfraction). These findings underscore a critical role for SETD2 in maintaining cellular homeostasis. Our multi-omic approach highlighted a novel role for SETD2 deficiency in TKI-resistance and disease acceleration via metabolic reprogramming, that can be reversed by SETD2 re-expression. SETD2 thus represents a promising biomarker and interfering with its proteasome-mediated degradation or with SETD2-induced metabolic rewiring may represent a novel strategy to address TKI-resistant disease. Supported by AIRC IG 2019 grant (23001) and AILBologna ODV.