SETD2 deficiency in chronic myeloid leukemia (CML) contributes to tyrosine kinase inhibitor (TKI) resistance and disease acceleration by enhancing genetic instability and rewiring cellular metabolism and might be a novel biomarker of high risk disease since diagnosis

Abstract

Abstract There is an active quest for biomarkers of high-risk disease in CML. ASXL1 and other cancer gene mutations, some additional cytogenetic alterations and Philadelphia-associated rearrangements (collectively, additional genetic abnormalities or AGAs) appear to have a negative impact on response and survival outcomes, but they are likely to mirror a more genetically unstable disease. SETD2 is a histone methyltransferase responsible for the deposition of the trimethyl mark at histone H3 lysine 36 (H3K36me3), a key epigenetic modification implicated in transcriptional elongation, chromatin architecture, DNA damage repair. SETD2 loss-of-function (LOF) due to inactivating mutations or, more frequently, accelerated proteasomal degradation, has recently been reported in patients (pts) with multi-TKI-resistant chronic phase (CP) or with blast phase (BP) CML, but it can be observed as early as at diagnosis, particularly in CD34+ hematopoietic progenitors. In this study, we aimed to dissect the impact of SETD2 LOF in primary CD34+ progenitors and in CML cellular models where SETD2 was alternatively silenced or overexpressed, using an integrated approach combining liquid chromatography-tandem mass spectrometry (LC-MS/MS), RNA sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-seq) and single nucleotide polymorphism arrays (SNP-arrays) for discovery, and Western blotting (WB), immunofluorescence (IF), and co-immunoprecipitation (co-IP) for validation. In SETD2-deficient primary CD34+ progenitors from newly diagnosed CP CML pts, nucleofection of a SETD2-expressing construct reduced clonogenic potential of >50%, indicating that SETD2 LOF enhances leukemic cell propagation. Differential transcriptomic profiling in cell line models revealed SETD2-dependent transcriptional regulation of genes involved in DNA repair (MSH2, MSH6), cell cycle control (CDK1), and metabolic homeostasis (PFKP, LDHA, PDK1). Differential interactome profiling by LC-MS/MS identified SETD2 interactions with proteins critically involved in mismatch repair (MSH2, MSH6), cell division (α-/β-tubulin), and glycolysis (PFKP, PFKFB3, PD, LDHA). Notably, SETD2 was also found to interact with key kinases regulating proliferation and stress response, including ERK1/2 and p38 MAPK. All MS-identified interactions were experimentally validated by IF and co-IP in nuclear, cytoplasmic, or cytoskeletal fractions. Furthermore, IF imaging demonstrated the nuclear colocalization of SETD2 with γ-H2AX foci upon hydrogen peroxide and UV-induced genotoxic stress and its recruitment to DNA damage sites, where it spatially overlapped with MSH2/MSH6 complexes. Integration of SNP-array analysis after chronic exposure to DNA damaging agents with ChIP-seq-based genomic mapping of H3K36me3 showed enrichment of breakpoints at SETD2 target sites (SETD2 knocked-down cell line: 29/45 vs 4/45 regions enriched in genomic breakpoints had loss vs gain of H3K36me3, respectively [p=0.0002]; SETD2-deficient cell line: 25/33 vs 1/33 regions enriched in genomic breakpoints had H3K36me3 loss vs gain, respectively [p=0.009]). Notably, we uncovered a novel role for SETD2 LOF in rewiring cellular metabolism, since SETD2 re-expression attenuated the glycolytic shift observed in SETD2-deficient cells, as evidenced by downregulation of glycolytic enzymes and mitochondrial oxidative phosphorylation complexes used by SETD2-deficient cells as compensation of TCA down-regulation. This was functionally validated in both total lysates and isolated mitochondrial fractions. In contrast, SETD2-deficient cells displayed hyperactivation of hypoxia-associated pathways, consistent with pseudohypoxic reprogramming. Finally, SETD2/H3K36me3 deficiency as assessed by a simple WB assay in total leukocytes could be detected in CP CML pts with AGAs at diagnosis, and, importantly, could discriminate non-optimal vs optimal responders to subsequent imatinib therapy (tx). Our findings point to SETD2 LOF as a key cooperating event in CML, that may act since diagnosis to set the stage for TKI resistance and disease acceleration by i) sustaining BCR::ABL1-independent genomic instability that fuels acquisition of AGAs before and despite TKI tx, and ii) inducing metabolic reprogramming towards glycolysis; ultimately enhancing leukemogenicity of CML progenitors. SETD2 LOF may serve as a biomarker of high-risk disease at diagnosis, and its impact on response to 2nd-gen TKIs or asciminib vs imatinib is worth to be explored further.