Caspase-2 protects against ferroptotic cell death.

Caspase-2, one of the most evolutionarily conserved members of the caspase family, is an important regulator of the cellular response to oxidative stress. Given that ferroptosis is suppressed by antioxidant defense pathways, such as that involving selenoenzyme glutathione peroxidase 4 (GPX4), we hypothesized that caspase-2 may play a role in regulating ferroptosis. This study provides the first demonstration of an important and unprecedented function of caspase-2 in protecting cancer cells from undergoing ferroptotic cell death. Specifically, we show that depletion of caspase-2 leads to the downregulation of stress response genes including SESN2, HMOX1, SLC7A11, and sensitizes mutant-p53 cancer cells to cell death induced by various ferroptosis-inducing compounds. Importantly, the canonical catalytic activity of caspase-2 is not required for its role and suggests that caspase-2 regulates ferroptosis via non-proteolytic interaction with other proteins. Using an unbiased BioID proteomics screen, we identified novel caspase-2 interacting proteins (including heat shock proteins and co-chaperones) that regulate cellular responses to stress. Finally, we demonstrate that caspase-2 limits chaperone-mediated autophagic degradation of GPX4 to promote the survival of mutant-p53 cancer cells. In conclusion, we document a novel role for caspase-2 as a negative regulator of ferroptosis in cells with mutant p53. Our results provide evidence for a novel function of caspase-2 in cell death regulation and open potential new avenues to exploit ferroptosis in cancer therapy.

Transketolase regulates sensitivity to APR-246 in p53-null cells independently of oxidative stress modulation.

The prevalence and dire implications of mutations in the tumour suppressor, p53, highlight its appeal as a chemotherapeutic target. We recently showed that impairing cellular antioxidant systems via inhibition of SLC7A11, a component of the system xc- cystine-glutamate antiporter, enhances sensitivity to mutant-p53 targeted therapy, APR-246. We investigated whether this synergy extends to other genes, such as those encoding enzymes of the pentose phosphate pathway (PPP). TKT, one of the major enzymes of the PPP, is allegedly regulated by NRF2, which is in turn impaired by accumulated mutant-p53 protein. Therefore, we investigated the relationship between mutant-p53, TKT and sensitivity to APR-246. We found that mutant-p53 does not alter expression of TKT, nor is TKT modulated directly by NRF2, suggesting a more complex mechanism at play. Furthermore, we found that in p53null cells, knockdown of TKT increased sensitivity to APR-246, whilst TKT overexpression conferred resistance to the drug. However, neither permutation elicited any effect on cells overexpressing mutant-p53 protein, despite mediating oxidative stress levels in a similar fashion to that in p53-null cells. In sum, this study has unveiled TKT expression as a determinant for sensitivity to APR-246 in p53-null cells.

A thiol-bound drug reservoir enhances APR-246-induced mutant p53 tumor cell death.

The tumor suppressor gene TP53 is the most frequently mutated gene in cancer. The compound APR-246 (PRIMA-1Met/Eprenetapopt) is converted to methylene quinuclidinone (MQ) that targets mutant p53 protein and perturbs cellular antioxidant balance. APR-246 is currently tested in a phase III clinical trial in myelodysplastic syndrome (MDS). By in vitro, ex vivo, and in vivo models, we show that combined treatment with APR-246 and inhibitors of efflux pump MRP1/ABCC1 results in synergistic tumor cell death, which is more pronounced in TP53 mutant cells. This is associated with altered cellular thiol status and increased intracellular glutathione-conjugated MQ (GS-MQ). Due to the reversibility of MQ conjugation, GS-MQ forms an intracellular drug reservoir that increases availability of MQ for targeting mutant p53. Our study shows that redox homeostasis is a critical determinant of the response to mutant p53-targeted cancer therapy.

Impaired haematopoietic stem cell differentiation and enhanced skewing towards myeloid progenitors in aged caspase-2-deficient mice.

The apoptotic cysteine protease caspase-2 has been shown to suppress tumourigenesis in mice and its reduced expression correlates with poor prognosis in some human malignancies. Caspase-2-deficient mice develop normally but show ageing-related traits and, when challenged by oncogenic stimuli or certain stress, show enhanced tumour development, often accompanied by extensive aneuploidy. As stem cells are susceptible to acquiring age-related functional defects because of their self-renewal and proliferative capacity, we examined whether loss of caspase-2 promotes such defects with age. Using young and aged Casp2-/- mice, we demonstrate that deficiency of caspase-2 results in enhanced aneuploidy and DNA damage in bone marrow (BM) cells with ageing. Furthermore, we demonstrate for the first time that caspase-2 loss results in significant increase in immunophenotypically defined short-term haematopoietic stem cells (HSCs) and multipotent progenitors fractions in BM with a skewed differentiation towards myeloid progenitors with ageing. Caspase-2 deficiency leads to enhanced granulocyte macrophage and erythroid progenitors in aged mice. Colony-forming assays and long-term culture-initiating assay further recapitulated these results. Our results provide the first evidence of caspase-2 in regulating HSC and progenitor differentiation, as well as aneuploidy, in vivo.

Multifunctional and multitargeted nanoparticles for drug delivery to overcome barriers of drug resistance in human cancers.

The recurrence and metastatic spread of cancer are major drawbacks in cancer treatment. Although chemotherapy is one of the most effective methods for the treatment of metastatic cancers, it is nonspecific and causes significant toxic damage. The development of drug resistance to chemotherapeutic agents through various mechanisms also limits their therapeutic potential. However, as we discuss here, the use of nanodelivery systems that are a combination of diagnostics and therapeutics (theranostics) is as relatively novel concept in the treatment of cancer. Such systems are likely to improve the therapeutic benefits of encapsulated drugs and can transit to the desired site, maintaining their pharmaceutical properties. The specific targeting of malignant cells using multifunctional nanoparticles exploits theranostics as an improved agent for delivering anticancer drugs and as a new solution for overriding drug resistance.