Abstract
Diffuse large B cell lymphoma (DLBCL) is an aggressive genetically heterogenous haematological malignancy and the most common histological subtype of aggressive lymphomas. The standard of care for DLBCL typically involves multi-drug chemoimmunotherapy regimens, which, despite high curative rates, are often poorly tolerated by patients, with drug resistance remaining a persistent challenge.
CREBBP, a histone acetyltransferase, is one of the most commonly mutated genes in DLBCL, with an alteration frequency of approximately 10-15% in overall cases and 25-35% in germinal centre B-cell (GCB) subtype. These mutations are predominantly missense, leading to loss of CREBBP function and contributing to lymphomagenesis. Previously, our research group and others have demonstrated that CREBBP functions as a 3D-specific tumour suppressor in other cancer types, promoting cell growth under nutrient stress conditions.
In this study, we set out to identify novel and robust metabolic vulnerabilities in CREBBP-mutant DLBCL at subcellular resolution that could be exploited therapeutically.
Utilising a panel of DLBCL cell lines stratified on their CREBBP status, we performed targeted small molecule metabolic inhibitor drug screens under physiologically relevant 3D culture conditions and identified Disulfiram, a known inhibitor of aldehyde dehydrogenase (ALDH) enzymes and clinically approved agent for the treatment of chronic alcoholism, as an agent that selectively killed CREBBP-mutant DLBCL cells. Interestingly, the cytotoxic impact of Disulfiram was observed to be independent of its ability to inhibit ALDH, due to it being a copper ionophore and potent inducer of the recently identified mode of cell death, Cuproptosis. Following up on these findings to confirm CREBBP specificity, we generated CREBBP isogenic lines containing the recurrent hotspot mutation R1446C as well as CREBBP knockout lines and observed again following targeted small molecule metabolic inhibitor drug screens that Disulfiram was a top candidate for inducing cytotoxicity in our CREBBP knockout lines.
By employing RNA-sequencing omics analysis together with time-course Western blotting, qPCR and flow cytometry, we demonstrated that Disulfiram treatment induced significant alterations in mitochondrial function; reducing the expression of lipoylated DLAT, DLAT and LIAS, key regulators of Cuproptotic cell death; perturbing mitochondrial membrane potential; activating cellular responses to metal ion detoxification, as well as upregulating the transport of Copper intracellularly via ZnT1 (SLC30A1) transporter expression. Alterations in mitochondrial biology and function were observed to be CREBBP-mutant-specific, with Disulfiram-induced cytotoxicity found to be completely rescued following pre-treatment with the copper chelator TTM, but not pre-treatment with apoptosis (Z-VAD-FMK) and ferroptosis inhibitors (Ferrostatin-1 and Liproxstatin-1), further confirming Disulfiram-induced Cuproptosis in our models.
To test Disulfiram's safety and efficacy in vivo, we performed subcutaneous injection of CREBBP wild type and mutant cells in NSG mice and observed no adverse effects on body weight, as well significant impairment of tumour growth in our CREBBP-mutant model only. In human DLBCL patient data we identified a common mechanism of ROS detoxification downregulation in CREBBP-mutant DLBCL, with the enzymes GPX1 and SOD2 expression being significantly downregulated. These findings implicate the possibility of patients with CREBBP-mutant DLBCL being more vulnerable to mitochondrial-targeting ROS-inducing agents, such as copper ionophores like Disulfiram. We are currently exploring further the exact mechanisms of this dependency, particularly intracellular copper homeostasis.In sum, we have identified Disulfiram to be synthetically lethal in CREBBP-mutant DLBCL cells grown under in vivo-like conditions. Our data identifies Disulfiram's mode of action to be associated with Cuproptotic induction and dependent on intracellular copper accumulation, and that CREBBP-mutant DLBCL cells and human cancers have impaired ROS detoxifying capacity. Finally, we validated the therapeutic benefit of Disulfiram in vivo, showing that it was safe and effective at reducing tumour growth in CREBBP-mutant DLBCL xenografts. Overall, this data supports the provocative hypothesis that Disulfiram could represent a novel, repurposed treatment option for patients with CREBBP-mutant DLBCL.
