CRISPR-Cas9 screen-driven identification of metabolic vulnerabilities reveals synthetic lethality via synergistic targeting of mitochondrial energy pathways and lactate export in oxphos-dependent T-ALL

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive hematologic malignancy driven by uncontrolled proliferation of immature T-cell precursors. Despite recent therapeutic advances, relapse remains frequent, underscoring the need for novel targeted strategies. Our previous work revealed that T-ALL cells exhibit a profound reliance on oxidative phosphorylation (OXPHOS), which supports energy production necessary for rapid proliferation and confers resistance to conventional therapies (Baran et al., Nat Commun 2022). While blockade of OXPHOS via inhibition of mitochondrial Complex I shows initial efficacy, T-ALL adapts by upregulating glutaminolysis and glycolysis, exporting lactate via monocarboxylate transporters (MCTs), and acidifying the microenvironment, thereby limiting durable responses. Understanding these intricate metabolic dependencies is crucial for identifying new intervention points aimed at disrupting energy pathways and overcoming therapy resistance. To systematically uncover these adaptive vulnerabilities, we performed genome-wide CRISPR-Cas9-based synthetic lethality screens in PF382 T-ALL cells treated with OXPHOS and MCT1 inhibitors. These screens particularly following MCT1 inhibition, identified key mitochondrial dependencies, including electron transport chain components (NDUF, UQCRC, COX), mitochondrial ribosomal proteins, mitochondrial translation factors, TCA cycle enzymes, mitochondrial genome regulators, and cofactors critical for mitochondrial function. Additionally, significant hits involved stress response pathways: sensors of apoptosis, chromatin- and nuclear membrane regulators, as well as lipid metabolism, lipid biosynthesis, and membrane trafficking genes. These findings suggest that dual OXPHOS/MCT1 inhibition triggers extensive metabolic reprogramming involving mitochondrial dysfunction, oxidative stress, and chromatin remodeling, which collectively enable cell survival. Combined OXPHOS/MCT1 targeting resulted in potent synthetic lethality (SL) by disrupting critical mitochondrial energy generation and lactate export. Consistent with these findings, Seahorse and GEA analyses indicated that MCT1 blockade increases OXPHOS activity, unveiling SL relationships involving mitochondrial biosynthesis and bioenergetics pathways, highlighting OXPHOS inhibition or downstream targeting as a promising potent therapeutic approach. We further validated these mechanisms utilizing multi-omics (GEA, targeted and untargeted metabolomics, in-silico METAFlux), functional assays (Seahorse, flow cytometry (FL), western blotting (WB)), and advanced imaging (confocal-, electron (EM)-, high-resolution (HRM)- microscopy in-vitro, hyperpolarized MRI in-vitro and in-vivo). In vitro, MCT1/OXPHOS inhibition caused irreversible mitochondrial damage, disrupted fusion/fission dynamics (EM, HRM), impaired enzymatic activity of mitochondrial complexes, perturbed transmembrane traffic of metabolites (Mass spectrometry, METAFlux), perturbed oxidative and anaerobic respiration (Seahorse), depleted ATP, disrupted redox homeostasis (Mass spectrometry), elevated ROS leading to DNA damage, and induced apoptosis (FL, WB), while sparing healthy hematopoietic cells. MCT1/OXPHOS blockade, in line with results of our screen, induced intracellular acidification and triggered lipophagy, rendering cells additionally vulnerable to inhibitors of lipid metabolism, as indicated in our in vitro screen. In vivo, hyperpolarized MRI in T-ALL PDX models, supported by an ex-vivo metabolites analysis (HPLC), confirmed the therapeutic effect, demonstrated by on-target reduced pyruvate-to-lactate ratios and increased lactate trapping post-MCT1 and MCT1/OXPHOS inhibitors treatment, with the latter leading to disease eradication and significantly prolonged overall survival. In summary, our CRISPR-Cas9 screens reveal critical mitochondrial dependencies and adaptive metabolic pathways in T-ALL. Targeting OXPHOS and MCT1, or their downstream signaling, simultaneously induces synthetic lethality toward T-ALL cells, offering a promising therapeutic strategy to eradicate T-ALL cells, providing therapeutic window to spare healthy hematopoietic cells, and ultimately warranting further in vitro and in vivo investigations.