Reactive Oxygen Species in Acute Lymphoblastic Leukaemia: Reducing Radicals to Refine Responses

Acute lymphoblastic leukaemia (ALL) is the most common cancer diagnosed in children and adolescents. Approximately 70% of patients survive >5-years following diagnosis, however, for those that fail upfront therapies, survival is poor. Reactive oxygen species (ROS) are elevated in a range of cancers and are emerging as significant contributors to the leukaemogenesis of ALL. ROS modulate the function of signalling proteins through oxidation of cysteine residues, as well as promote genomic instability by damaging DNA, to promote chemotherapy resistance. Current therapeutic approaches exploit the pro-oxidant intracellular environment of malignant B and T lymphoblasts to cause irreversible DNA damage and cell death, however these strategies impact normal haematopoiesis and lead to long lasting side-effects. Therapies suppressing ROS production, especially those targeting ROS producing enzymes such as the NADPH oxidases (NOXs), are emerging alternatives to treat cancers and may be exploited to improve the ALL treatment. Here, we discuss the roles that ROS play in normal haematopoiesis and in ALL. We explore the molecular mechanisms underpinning overproduction of ROS in ALL, and their roles in disease progression and drug resistance. Finally, we examine strategies to target ROS production, with a specific focus on the NOX enzymes, to improve the treatment of ALL.

An Autoantigen Profile from Jurkat T-Lymphoblasts Provides a Molecular Guide for Investigating Autoimmune Sequelae of COVID-19

In order to understand autoimmune phenomena contributing to the pathophysiology of COVID-19 and post-COVID syndrome, we have been profiling autoantigens (autoAgs) from various cell types. Although cells share numerous autoAgs, each cell type gives rise to unique COVID-altered autoAg candidates, which may explain the wide range of symptoms experienced by patients with autoimmune sequelae of SARS-CoV-2 infection. Based on the unifying property of affinity between autoantigens (autoAgs) and the glycosaminoglycan dermatan sulfate (DS), this paper reports 140 candidate autoAgs identified from proteome extracts of human Jurkat T-cells, of which at least 105 (75%) are known targets of autoantibodies. Comparison with currently available multi-omic COVID-19 data shows that 125 (89%) of DS-affinity proteins are altered at protein and/or RNA levels in SARS-CoV-2-infected cells or patients, with at least 94 being known autoAgs in a wide spectrum of autoimmune diseases and cancer. Protein alterations by ubiquitination and phosphorylation in the viral infection are major contributors of autoAgs. The autoAg protein network is significantly associated with cellular response to stress, apoptosis, RNA metabolism, mRNA processing and translation, protein folding and processing, chromosome organization, cell cycle, and muscle contraction. The autoAgs include clusters of histones, CCT/TriC chaperonin, DNA replication licensing factors, proteasome and ribosome proteins, heat shock proteins, serine/arginine-rich splicing factors, 14-3-3 proteins, and cytoskeletal proteins. AutoAgs such as LCP1 and NACA that are altered in the T cells of COVID patients may provide insight into T-cell responses in the viral infection and merit further study. The autoantigen-ome from this study contributes to a comprehensive molecular map for investigating acute, subacute, and chronic autoimmune disorders caused by SARS-CoV-2.

The Earliest T-Precursors in the Mouse Embryo Are Susceptible to Leukemic Transformation

Acute lymphoblastic leukemia (ALL) is the most common malignancy in pediatric patients. About 10–15% of pediatric ALL belong to T-cell ALL (T-ALL), which is characterized by aggressive expansion of immature T-lymphoblasts and is categorized as high-risk leukemia. Leukemia initiating cells represent a reservoir that is responsible for the initiation and propagation of leukemia. Its perinatal origin has been suggested in some childhood acute B-lymphoblastic and myeloblastic leukemias. Therefore, we hypothesized that child T-ALL initiating cells also exist during the perinatal period. In this study, T-ALL potential of the hematopoietic precursors was found in the para-aortic splanchnopleura (P-Sp) region, but not in the extraembryonic yolk sac (YS) of the mouse embryo at embryonic day 9.5. We overexpressed the Notch intracellular domain (NICD) in the P-Sp and YS cells and transplanted them into lethally irradiated mice. NICD-overexpressing P-Sp cells rapidly developed T-ALL while YS cells failed to display leukemia propagation despite successful NICD induction. These results suggest a possible role of fetal-derived T-cell precursors as leukemia-initiating cells.

In vitro dexamethasone treatment does not induce alternative ATM transcripts in cells from Ataxia–Telangiectasia patients

Short term treatment with low doses of glucocorticoid analogues has been shown to ameliorate neurological symptoms in Ataxia–Telangiectasia (A–T), a rare autosomal recessive multisystem disease that mainly affects the cerebellum, immune system, and lungs. Molecular mechanisms underlying this clinical observation are unclear. We aimed at evaluating the effect of dexamethasone on the induction of alternative ATM transcripts (ATMdexa1). We showed that dexamethasone cannot induce an alternative ATM transcript in control and A–T lymphoblasts and primary fibroblasts, or in an ATM-knockout HeLa cell line. We also demonstrated that some of the reported readouts associated with ATMdexa1 are due to cellular artifacts and the direct induction of γH2AX by dexamethasone via DNA-PK. Finally, we suggest caution in interpreting dexamethasone effects in vitro for the results to be translated into a rational use of the drug in A–T patients.

Sirolimus for the Treatment of Airway Obstruction due to Indolent T-Lymphoblastic Proliferation

Introduction. Indolent T-lymphoblastic proliferation (iT-LBP) is a rare nonmalignant entity that presents as a proliferation of T-lymphoblasts. We report a first such case with a recurrent laryngeal obstruction presentation that was successfully controlled with Sirolimus. Case presentation. This is the case of a 29-year-old female who presented with a recurrent significant lymphoid hyperplasia in the adenoid and tongue base region as well as a right cervical lymph node. After repeated adenoidectomies and tonsillectomies, and based on pathological and clinical findings she was diagnosed with iT-LBP. Trials of radiotherapy and immunotherapy with cyclosporine and rituximab all failed to control the progression of the disease. Sirolimus was finally able to restrict the growth and improve her symptoms. Conclusion. While It-LBP does not usually require treatment, it is important to report cases in which treatment was crucial for the survival of the patient, and the effective role of Sirolimus in doing so, without any major adverse effects.

Strain-Dependent Porcine Circovirus Type 2 (PCV2) Entry and Replication in T-Lymphoblasts

Porcine circovirus type 2 (PCV2) is the etiological agent of PCV2-associated diseases (PCVAD). PCV2 targets lymphoblasts, and pigs suffering from PCVAD display lymphocyte depletion in lymphoid tissues. PCV2 infection of lymphoblasts has not been studied. Here, the replication cycle of PCV2 (abortion strain 1121 and PMWS strain Stoon1010) in T-lymphoblasts was examined. The expression of Rep and Cap were found for both viral strains, while progeny virus was detected for Stoon1010 but not for 1121. PCV2 attached to 11–26% (1121-Stoon1010) of the T-lymphoblasts while 2.6–12.7% of cells showed virus internalization. Chondroitin sulfate (CS) was present on 25% of T-lymphoblasts, and colocalized with PCV2 on 31–32% of the PCV2+ cells. Enzymatic removal of CS reduced PCV2 infection. PCV2 infection was decreased by chlorpromazine, cytochalasin D and Clostridium difficile toxin B for both viral strains and by amiloride for 1121 but not for Stoon1010. Inhibiting either endosome acidification or serine proteases strongly reduced PCV2 infection. Three-dimensional analysis of Cap structure demonstrated a better Cap-nucleic acid affinity for Stoon1010 than for 1121. Taken together, PCV2 binds to T-lymphoblasts partially via CS, enters via clathrin-mediated endocytosis, and disassembles under functions of a pH-drop and serine proteases. Strain Stoon1010 displayed an enhanced viral binding, a specific receptor-mediated endocytosis, an increased Cap-nucleic acid affinity, and a more productive infection in T-lymphoblasts than 1121 did, indicating an evolution from 1121 to Stoon1010.

Integrin Activity Reduces Immunogenic Cell Death By Inhibiting Cell Surface Presentation of ERp57 and Calreticulin

Calreticulin presentation on the cell surface is an important hallmark of immunogenic cell death (ICD), serving as the 'eat me' engulfment signal for professional phagocytes. ERp57 is an interacting and co-translocating protein with calreticulin, but unlike surface calreticulin, surface ERp57 is not known to be immunogenic. Since calreticulin interacts with α-integrins via the conserved GFFKR motif within the integrin cytoplasmic domain, we assessed whether integrin function can modulate cell surface calreticulin and ERp57 levels in ICD. When stimulated to engage integrin substrates, leukemic T-lymphoblasts treated with an ICD-inducer exhibited decreased surface calreticulin and ERp57 compared with cells under non-adherent conditions. The inhibitory effect on surface calreticulin and ERp57 was recapitulated for cells in suspension and treated with various agents that stimulate integrin activation, including Mn2+ and the LIBS antibodies 9EG7 and HUTS21. Similarly, cells expressing a mutant truncated α-integrin bearing only GFFKR as the cytosolic tail, also exhibited low surface calreticulin and ERp57 when treated with ICD-inducers under non-adherent conditions. Furthermore, integrin β1 null cells with overall reduced α-integrin expression exhibited enhanced surface calreticulin and ERp57, consistent with an inhibitory role for integrins in ICD. We generated calreticulin or ERp57 null strains by CRISPR-Cas9, and show that ICD-induced surface calreticulin is ERp57-dependent, while surface ERp57 is not calreticulin-dependent. Using permeabilization techniques that distinguish between cytosolic and ER staining, we found that ICD-inducers promoted the accumulation of cytosolic calreticulin and ERp57 that originated from the ER. In similar fashion, ER to cytosolic trafficking for calreticulin is ERp57-dependent, while for ERp57, it is not calreticulin-dependent. We also showed that integrin-mediated inhibition of surface calreticulin was due to reduced cytosolic to surface translocation coupled with normal ER-cytosolic release, suggesting that activated integrins acts to sequester calreticulin within the cytosol. T-lymphoblasts co-treated with an ICD-inducer and integrin activators exhibited reduced phagocytosis by macrophages when compared with treatment with only ICD-inducer. Our study reveals a previously uncharacterized function of integrins as negative regulators of immunogenic cell death by suppressing the presentation of cell surface calreticulin. Disclosures No relevant conflicts of interest to declare.