Actionable cancer vulnerability due to translational arrest, p53 aggregation and ribosome biogenesis stress evoked by the disulfiram metabolite CuET.

Drug repurposing is a versatile strategy to improve current therapies. Disulfiram has long been used in the treatment of alcohol dependency and multiple clinical trials to evaluate its clinical value in oncology are ongoing. We have recently reported that the disulfiram metabolite diethyldithiocarbamate, when combined with copper (CuET), targets the NPL4 adapter of the p97VCP segregase to suppress the growth of a spectrum of cancer cell lines and xenograft models in vivo. CuET induces proteotoxic stress and genotoxic effects, however important issues concerning the full range of the CuET-evoked tumor cell phenotypes, their temporal order, and mechanistic basis have remained largely unexplored. Here, we have addressed these outstanding questions and show that in diverse human cancer cell models, CuET causes a very early translational arrest through the integrated stress response (ISR), later followed by features of nucleolar stress. Furthermore, we report that CuET entraps p53 in NPL4-rich aggregates leading to elevated p53 protein and its functional inhibition, consistent with the possibility of CuET-triggered cell death being p53-independent. Our transcriptomics profiling revealed activation of pro-survival adaptive pathways of ribosomal biogenesis (RiBi) and autophagy upon prolonged exposure to CuET, indicating potential feedback responses to CuET treatment. The latter concept was validated here by simultaneous pharmacological inhibition of RiBi and/or autophagy that further enhanced CuET's tumor cytotoxicity, using both cell culture and zebrafish in vivo preclinical models. Overall, these findings expand the mechanistic repertoire of CuET's anti-cancer activity, inform about the temporal order of responses and identify an unorthodox new mechanism of targeting p53. Our results are discussed in light of cancer-associated endogenous stresses as exploitable tumor vulnerabilities and may inspire future clinical applications of CuET in oncology, including combinatorial treatments and focus on potential advantages of using certain validated drug metabolites, rather than old, approved drugs with their, often complex, metabolic profiles.

A drug repurposing strategy for overcoming human multiple myeloma resistance to standard-of-care treatment.

Despite several approved therapeutic modalities, multiple myeloma (MM) remains an incurable blood malignancy and only a small fraction of patients achieves prolonged disease control. The common anti-MM treatment targets proteasome with specific inhibitors (PI). The resulting interference with protein degradation is particularly toxic to MM cells as they typically accumulate large amounts of toxic proteins. However, MM cells often acquire resistance to PIs through aberrant expression or mutations of proteasome subunits such as PSMB5, resulting in disease recurrence and further treatment failure. Here we propose CuET-a proteasome-like inhibitor agent that is spontaneously formed in-vivo and in-vitro from the approved alcohol-abuse drug disulfiram (DSF), as a readily available treatment effective against diverse resistant forms of MM. We show that CuET efficiently kills also resistant MM cells adapted to proliferate under exposure to common anti-myeloma drugs such as bortezomib and carfilzomib used as the first-line therapy, as well as to other experimental drugs targeting protein degradation upstream of the proteasome. Furthermore, CuET can overcome also the adaptation mechanism based on reduced proteasome load, another clinically relevant form of treatment resistance. Data obtained from experimental treatment-resistant cellular models of human MM are further corroborated using rather unique advanced cytotoxicity experiments on myeloma and normal blood cells obtained from fresh patient biopsies including newly diagnosed as well as relapsed and treatment-resistant MM. Overall our findings suggest that disulfiram repurposing particularly if combined with copper supplementation may offer a promising and readily available treatment option for patients suffering from relapsed and/or therapy-resistant multiple myeloma.

Post-transplant cyclophosphamide versus antithymocyte globulin in patients with acute myeloid leukemia in first complete remission undergoing allogeneic stem cell transplantation from 10/10 HLA-matched unrelated donors.

BACKGROUND: Graft-versus-host disease (GVHD) remains a major contributor to mortality and morbidity after allogeneic stem-cell transplantation (allo-HSCT). The updated recommendations suggest that rabbit antithymocyte globulin or anti-T-lymphocyte globulin (ATG) should be used for GVHD prophylaxis in patients undergoing matched-unrelated donor (MUD) allo-HSCT. More recently, using post-transplant cyclophosphamide (PTCY) in the haploidentical setting has resulted in low incidences of both acute (aGVHD) and chronic GVHD (cGVHD). Therefore, the aim of our study was to compare GVHD prophylaxis using either PTCY or ATG in patients with acute myeloid leukemia (AML) who underwent allo-HSCT in first remission (CR1) from a 10/10 HLA-MUD. METHODS: Overall, 174 and 1452 patients from the EBMT registry receiving PTCY and ATG were included. Cumulative incidence of aGVHD and cGVHD, leukemia-free survival, overall survival, non-relapse mortality, cumulative incidence of relapse, and refined GVHD-free, relapse-free survival were compared between the 2 groups. Propensity score matching was also performed in order to confirm the results of the main analysis RESULTS: No statistical difference between the PTCY and ATG groups was observed for the incidence of grade II-IV aGVHD. The same held true for the incidence of cGVHD and for extensive cGVHD. In univariate and multivariate analyses, no statistical differences were observed for all other transplant outcomes. These results were also confirmed using matched-pair analysis. CONCLUSION: These results highlight that, in the10/10 HLA-MUD setting, the use of PTCY for GVHD prophylaxis may provide similar outcomes to those obtained with ATG in patients with AML in CR1.

Targeting the NPL4 Adaptor of p97/VCP Segregase by Disulfiram as an Emerging Cancer Vulnerability Evokes Replication Stress and DNA Damage while Silencing the ATR Pathway.

Research on repurposing the old alcohol-aversion drug disulfiram (DSF) for cancer treatment has identified inhibition of NPL4, an adaptor of the p97/VCP segregase essential for turnover of proteins involved in multiple pathways, as an unsuspected cancer cell vulnerability. While we reported that NPL4 is targeted by the anticancer metabolite of DSF, the bis-diethyldithiocarbamate-copper complex (CuET), the exact, apparently multifaceted mechanism(s) through which the CuET-induced aggregation of NPL4 kills cancer cells remains to be fully elucidated. Given the pronounced sensitivity to CuET in tumor cell lines lacking the genome integrity caretaker proteins BRCA1 and BRCA2, here we investigated the impact of NPL4 targeting by CuET on DNA replication dynamics and DNA damage response pathways in human cancer cell models. Our results show that CuET treatment interferes with DNA replication, slows down replication fork progression and causes accumulation of single-stranded DNA (ssDNA). Such a replication stress (RS) scenario is associated with DNA damage, preferentially in the S phase, and activates the homologous recombination (HR) DNA repair pathway. At the same time, we find that cellular responses to the CuET-triggered RS are seriously impaired due to concomitant malfunction of the ATRIP-ATR-CHK1 signaling pathway that reflects an unorthodox checkpoint silencing mode through ATR (Ataxia telangiectasia and Rad3 related) kinase sequestration within the CuET-evoked NPL4 protein aggregates.

DNA damage signalling barrier, oxidative stress and treatment-relevant DNA repair factor alterations during progression of human prostate cancer.

The DNA damage checkpoints provide an anti-cancer barrier in diverse tumour types, however this concept has remained unexplored in prostate cancer (CaP). Furthermore, targeting DNA repair defects by PARP1 inhibitors (PARPi) as a cancer treatment strategy is emerging yet requires suitable predictive biomarkers. To address these issues, we performed immunohistochemical analysis of multiple markers of DNA damage signalling, oxidative stress, DNA repair and cell cycle control pathways during progression of human prostate disease from benign hyperplasia, through intraepithelial neoplasia to CaP, complemented by genetic analyses of TMPRSS2-ERG rearrangement and NQO1, an anti-oxidant factor and p53 protector. The DNA damage checkpoint barrier (γH2AX, pATM, p53) mechanism was activated during CaP tumorigenesis, albeit less and with delayed culmination compared to other cancers, possibly reflecting lower replication stress (slow proliferation despite cases of Rb loss and cyclin D1 overexpression) and progressive loss of ATM activator NKX3.1. Oxidative stress (8-oxoguanine lesions) and NQO1 increased during disease progression. NQO1 genotypes of 390 men did not indicate predisposition to CaP, yet loss of NQO1 in CaP suggested potential progression-opposing tumour suppressor role. TMPRSS2-ERG rearrangement and PTEN loss, events sensitizing to PARPi, occurred frequently along with heterogeneous loss of DNA repair factors 53BP1, JMJD1C and Rev7 (all studied here for the first time in CaP) whose defects may cause resistance to PARPi. Overall, our results reveal an unorthodox DNA damage checkpoint barrier scenario in CaP tumorigenesis, and provide novel insights into oxidative stress and DNA repair, with implications for biomarker guidance of future targeted therapy of CaP.