The NOTCH1 gene encodes a transmembrane receptor protein with activating mutations observed in many T‐cell acute lymphoblastic leukemias (T‐ALLs) and lymphomas, as well as in other tumor types, which has led to interest in inhibiting NOTCH1 signaling as a therapeutic target in cancer. Several classes of Notch inhibitors have been developed, including monoclonal antibodies against NOTCH receptors or ligands, decoys, blocking peptides, and γ‐secretase inhibitors (GSIs). GSIs block a critical proteolytic step in NOTCH activation and are the most widely studied. Current treatments with GSIs have not successfully passed clinical trials because of side effects that limit the maximum tolerable dose. Multiple γ‐secretase–cleavage substrates may be involved in carcinogenesis, indicating that there may be other targets for GSIs. Resistance mechanisms may include PTEN inactivation, mutations involving FBXW7, or constitutive MYC expression conferring independence from NOTCH1 inactivation. Recent studies have suggested that selective targeting γ‐secretase may offer an improved efficacy and toxicity profile over the effects caused by broad‐spectrum GSIs. Understanding the mechanism of GSI‐induced cell death and the ability to accurately identify patients based on the activity of the pathway will improve the response to GSI and support further investigation of such compounds for the rational design of anti‐NOTCH1 therapies for the treatment of T‐ALL. Implications for Practice γ‐secretase has been proposed as a therapeutic target in numerous human conditions, including cancer. A better understanding of the structure and function of the γ‐secretase inhibitor (GSI) would help to develop safe and effective γ‐secretase–based therapies. The ability to accurately identify patients based on the activity of the pathway could improve the response to GSI therapy for the treatment of cancer. Toward these ends, this study focused on γ‐secretase inhibitors as a potential therapeutic target for the design of anti‐NOTCH1 therapies for the treatment of T‐cell acute lymphoblastic leukemias and lymphomas.

Summary: Despite the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway being frequently altered in T‐ALL/LBL, no specific therapy has been approved for T‐ALL/LBL patients with constitutive signalling by JAK/STAT, so there is an urgent need to identify pathway members that may be potential therapeutic targets. In the present study, we searched for JAK/STAT pathway members potentially modulated through aberrant methylation and identified SOCS3 hypermethylation as a recurrent event in T‐ALL/LBL. Additionally, we explored the implications of SOCS3 deregulation in T‐ALL/LBL and demonstrated that SOCS3 counteracts the constitutive activation of the JAK/STAT pathway through different molecular mechanisms. Therefore, SOCS3 emerges as a potential therapeutic target in T‐ALL/LBL.

Precursor T-cell lymphoblastic neoplasms are aggressive malignancies in need for more effective and specific therapeutic treatments. A significant fraction of these neoplasms harbor deletions on the locus 9p21, targeting the tumor suppressor CDKN2A but also deleting the aconitase 1 (ACO1) gene, a neighboring housekeeping gene involved in cytoplasm and mitochondrial metabolism. Here we show that reducing the aconitase activity with fluorocitrate decreases the viability of T-cell lymphoblastic neoplasia cells in correlation to the differential aconitase expression. The consequences of the treatment were evidenced in vitro using T-cell lymphoblastic neoplasia cell lines exhibiting 9p21 deletions and variable levels of ACO1 expression or activity. Similar results were observed in melanoma cell lines, suggesting a true potential for fluorocitrate in different cancer types. Notably, ectopic expression of ACO1 alleviated the susceptibility of cell lines to fluorocitrate and, conversely, knockdown experiments increased susceptibility of resistant cell lines. These findings were confirmed in vivo on athymic nude mice by using tumor xenografts derived from two T-cell lines with different levels of ACO1. Taken together, our results indicate that the non-targeted ACO1 deficiency induced by common deletions exerts a collateral cellular lethality that can be used as a novel therapeutic strategy in the treatment of several types of cancer.

In the quest for more effective radiation treatment options that can improve both cell killing and healthy tissue recovery, combined radiation therapies are lately in the spotlight. The molecular response to a combined radiation regime where exposure to an initial low dose (priming dose) of ionizing radiation is administered prior to a subsequent higher radiation dose (challenging dose) after a given latency period have not been thoroughly explored. In this study we report on the differential response to either a combined radiation regime or a single challenging dose both in mouse in vivo and in human ex vivo thymocytes. A differential cell cycle response including an increase in the subG1 fraction on cells exposed to the combined regime was found. Together with this, a differential protein expression profiling in several pathways including cell cycle control (ATM, TP53, p21^CDKN1A), damage response (γH2AX) and cell death pathways such as apoptosis (Cleaved Caspase-3, PARP1, PKCδ and H3T45ph) and ferroptosis (xCT/GPX4) was demonstrated. This study also shows the epigenetic regulation following a combined regime that alters the expression of chromatin modifiers such as DNMTs (DNMT1, DNMT2, DNMT3A, DNMT3B, DNMT3L) and glycosylases (MBD4 and TDG). Furthermore, a study of the underlying cellular status six hours after the priming dose alone showed evidence of retained modifications on the molecular and epigenetic pathways suggesting that the priming dose infers a “radiation awareness phenotype” to the thymocytes, a sensitization key to the differential response seen after the second hit with the challenging dose. These data suggest that combined-dose radiation regimes could be more efficient at making cells respond to radiation and it would be interesting to further investigate how can these schemes be of use to potential new radiation therapies.

The standard-of-care treatment of T-cell acute lymphoblastic leukaemia (T-ALL) with chemotherapy usually achieves reasonable rates of initial complete response. However, patients who relapse or do not respond to conventional therapy show dismal outcomes, with cure rates below 10% and limited therapeutic options. To ameliorate the clinical management of these patients, it is urgent to identify biomarkers able to predict their outcomes. In this work, we investigate whether NRF2 activation constitutes a biomarker with prognostic value in T-ALL. Using transcriptomic, genomic, and clinical data, we found that T-ALL patients with high NFE2L2 levels had shorter overall survival. Our results demonstrate that the PI3K-AKT-mTOR pathway is involved in the oncogenic signalling induced by NRF2 in T-ALL. Furthermore, T-ALL patients with high NFE2L2 levels displayed genetic programs of drug resistance that may be provided by NRF2-induced biosynthesis of glutathione. Altogether, our results indicate that high levels of NFE2L2 may be a predictive biomarker of poor treatment response in T-ALL patients, which would explain the poor prognosis associated with these patients. This enhanced understanding of NRF2 biology in T-ALL may allow a more refined stratification of patients and the proposal of targeted therapies, with the ultimate goal of improving the outcome of relapsed/refractory T-ALL patients.