The Role of Cholesterol in Chronic Lymphocytic Leukemia Development and Pathogenesis.

Cholesterol has many critical functions in cells. It is a key component of membranes and cell-signalling processes, and it functions as a chemical precursor in several biochemical pathways, such as Vitamin D and steroid synthesis. Cholesterol has also been implicated in the development and progression of various cancers, in which it is thought to promote cell proliferation, migration, and invasion. Chronic lymphocytic leukemia (CLL) is an example of a lipid-avid cancer that relies on lipid metabolism, rather than glycolysis, to fuel cell proliferation. However, data regarding the role of cholesterol in CLL are conflicting. Studies have shown that dyslipidaemia is more common among CLL patients than age-matched healthy controls, and that CLL patients who take cholesterol-lowering drugs, such as statins, appear to have improved survival rates. Therefore, defining the roles of cholesterol in CLL may highlight the importance of monitoring and managing hyperlipidaemia as part of the routine management of patients with CLL. In this review, we discuss the roles of cholesterol in the context of CLL by examining the literature concerning the trafficking, uptake, endogenous synthesis, and intracellular handling of this lipid. Data from clinical trials investigating various classes of cholesterol and lipid-lowering drugs in CLL are also discussed.

Ferroptosis in Haematological Malignancies and Associated Therapeutic Nanotechnologies.

Haematological malignancies are heterogeneous groups of cancers of the bone marrow, blood or lymph nodes, and while therapeutic advances have greatly improved the lifespan and quality of life of those afflicted, many of these cancers remain incurable. The iron-dependent, lipid oxidation-mediated form of cell death, ferroptosis, has emerged as a promising pathway to induce cancer cell death, particularly in those malignancies that are resistant to traditional apoptosis-inducing therapies. Although promising findings have been published in several solid and haematological malignancies, the major drawbacks of ferroptosis-inducing therapies are efficient drug delivery and toxicities to healthy tissue. The development of tumour-targeting and precision medicines, particularly when combined with nanotechnologies, holds potential as a way in which to overcome these obstacles and progress ferroptosis-inducing therapies into the clinic. Here, we review the current state-of-play of ferroptosis in haematological malignancies as well as encouraging discoveries in the field of ferroptosis nanotechnologies. While the research into ferroptosis nanotechnologies in haematological malignancies is limited, its pre-clinical success in solid tumours suggests this is a very feasible therapeutic approach to treat blood cancers such as multiple myeloma, lymphoma and leukaemia.

Lipid uptake in chronic lymphocytic leukemia.

Many cancers rely on glucose as an energy source, but it is becoming increasingly apparent that some cancers use alternate substrates to fuel their proliferation. Chronic lymphocytic leukaemia (CLL) is one such cancer. Through the use of flow cytometry and confocal microscopy, low levels of glucose uptake were observed in the OSU-CLL and HG3 CLL cell lines relative to highly glucose-avid Raji cells (Burkitt's lymphoma). Glucose uptake in CLL cells correlated with low expression of the GLUT1 and GLUT3 receptors. In contrast, both CLL cell lines and primary CLL cells, but not healthy B cells, were found to rapidly internalise medium- and long-chain, but not short-chain, fatty acids (FAs). Differential FA uptake was also observed in primary cells taken from patients with unmutated immunoglobulin heavy variable chain usage (IGHV) compared with patients with mutated IGHV. Delipidation of serum in the culture medium slowed the proliferation and significantly reduced the viability of OSU-CLL and HG3 cells, effects that were partially reversed by supplementation with a chemically defined lipid concentrate. These observations highlight the potential importance of FAs in the pathogenesis of CLL and raise the possibility that targeting FA utilisation may represent a novel therapeutic and prognostic approach in this disease.

The future of laboratory testing in chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) is a malignant lymphoproliferative disorder characterised by the accumulation of dysfunctional B-lymphocytes in the blood and lymphoid tissues. It is a clonally complex disease with a high degree of both intra-tumoural and inter-patient heterogeneity. This variability leads to a wide range of clinical outcomes and highlights the critical need for accurate prognostic tests in CLL. With the advent of a range of new targeted agents for CLL in recent years, there is also a clinical need for improved predictive tests to therapy. This review of laboratory testing in CLL focuses on emerging technologies for prognostication including single nucleotide polymorphism microarray for karyotypic analysis, targeted next generation sequencing analysis of the immunoglobulin heavy chain variable region gene as well as genes recurrently mutated in the disease such as TP53, and detection of minimal residual disease.

Importance of between and within Subject Variability in Extracellular Vesicle Abundance and Cargo when Performing Biomarker Analyses.

Small extracellular vesicles (sEV) have emerged as a potential rich source of biomarkers in human blood and present the intriguing potential for a 'liquid biopsy' to track disease and the effectiveness of interventions. Recently, we have further demonstrated the potential for EV derived biomarkers to account for variability in drug exposure. This study sought to evaluate the variability in abundance and cargo of global and liver-specific circulating sEV, within (diurnal) and between individuals in a cohort of healthy subjects (n = 10). We present normal ranges for EV concentration and size and expression of generic EV protein markers and the liver-specific asialoglycoprotein receptor 1 (ASGR1) in samples collected in the morning and afternoon. EV abundance and cargo was generally not affected by fasting, except CD9 which exhibited a statistically significant increase (p = 0.018). Diurnal variability was observed in the expression of CD81 and ASGR1, which significantly decreased (p = 0.011) and increased (p = 0.009), respectively. These results have potential implications for study sampling protocols and normalisation of biomarker data when considering the expression of sEV derived cargo as a biomarker strategy. Specifically, the novel finding that liver-specific EVs exhibit diurnal variability in healthy subjects should have broad implications in the study of drug metabolism and development of minimally invasive biomarkers for liver disease.

The ClpP activator ONC-212 (TR-31) inhibits BCL2 and B-cell receptor signaling in CLL.

Despite advances in therapy, a significant proportion of patients with chronic lymphocytic leukemia (CLL) relapse with drug resistant disease. Novel treatment approaches are required, particularly for high risk disease. The imipridones represent a new class of cancer therapy that has been investigated in pre-clinical and clinical trials against a range of different cancers. We investigated the effects of the imipridone, ONC-212, against CLL cells cultured under conditions that mimic aspects of the tumour microenvironment and a TP53ko CLL cell line (OSU-CLL-TP53ko). ONC-212 induced dose-dependent apoptosis, cell cycle arrest and reduced the migration of CLL cells in vitro, including cells from patients with TP53 lesions and OSU-CLL-TP53ko cells. The effects of ONC-212 were associated with protein changes consistent with activation of the mitochondrial protease, CIpP, and the integrated stress response. We also observed inhibition of pathways downstream of the B-cell receptor (BCR) (AKT and MAPK-ERK1/2) and a pro-apoptotic shift in the balance of proteins of the BCL2 family of proteins (BCL2, MCL1, BCLxL, BAX and NOXA). In conclusion, the study suggests ONC-212 may represent an effective treatment for high risk CLL disease by inhibiting multiple facets of the BCR signaling pathway and the pro-survival effects of the BCL2-family proteins.

Assessing cross-reactivity to neuromuscular blocking agents by skin and basophil activation tests in patients with neuromuscular blocking agent anaphylaxis.

BACKGROUND: Following diagnosis of neuromuscular blocking agent (NMBA) anaphylaxis, identifying safe alternatives for subsequent anaesthesia is critical. A patient with anaphylaxis to one NMBA can also have an allergic reaction to other NMBAs (cross-reactivity). Whilst drug provocation testing is standard for identifying or excluding allergy, there is significant risk. In vitro, after an allergen activates basophils, basophils express surface activation markers that can be measured by basophil activation testing (BAT). We compared cross-reactivity between NMBAs assessed by BAT against that by skin testing. METHODS: All patients attending an anaesthetic allergy clinic in Sydney, Australia between May 2017 and July 2018 diagnosed with NMBA anaphylaxis qualified for this study comparing intradermal skin tests and BAT with a panel of NMBAs (rocuronium, vecuronium, pancuronium, suxamethonium, cisatracurium). RESULTS: Of the 61 patients participating, sensitisation on skin testing and on BAT completely matched in only nine patients (15%). Sensitisation was not in agreement for pancuronium, cisatracurium and rocuronium, but was in agreement for vecuronium and suxamethonium. Nine patients with negative skin tests subsequently tolerated cisatracurium, and one false positive on BAT to cisatracurium was detected. CONCLUSIONS: The utility of BAT in identifying safe NMBAs for subsequent anaesthesia needs further evaluation. BAT detects a different cross-reactivity profile to skin tests. Negative skin testing and BAT might increase confidence in performing drug provocation testing, but this and follow-up of subsequent anaesthesia in our cohort is necessary to determine the clinical significance of BAT sensitisation.

Integrating basophil activation tests into evaluation of perioperative anaphylaxis to neuromuscular blocking agents.

BACKGROUND: Neuromuscular blocking agents (NMBAs) remain the leading cause of perioperative anaphylaxis in Australia. Standard evaluation comprises history, skin tests, and in vitro specific immunoglobulin E tests. Drug provocation tests to suspected NMBA culprits are associated with a significant risk. Basophil activation testing (BAT) is a potentially useful in vitro test that is not commercially available in Australia or as part of standard evaluation. METHODS: All patients attending the Anaesthetic Allergy Clinic in Sydney, Australia between May 2017 and July 2018 exposed to an NMBA before the onset of anaphylaxis during their anaesthetic qualified for the study. We recruited 120 patients sequentially who received standard evaluation plus BAT using CD63, CD203c, and CD300a as surface activation markers. RESULTS: BAT results were expressed as % upregulation above the negative control and stimulation index (mean fluorescence index of stimulated sample divided by the negative control). We calculated cut-offs of 4.45% and 1.44 for CD63, and 8.80% and 1.49 for CD203c, respectively. Sensitivity was 77% with specificity of 76%. A subgroup of 10 patients with NMBA anaphylaxis had no sensitisation on skin tests. BAT using CD63 and CD203c showed sensitisation in six of these 10, and adding CD300a identified sensitisation in nine patients. BAT was positive in seven of nine patients with anaphylaxis of unknown aetiology. CONCLUSIONS: BAT may be a useful supplement to the standard evaluation in diagnosing NMBA anaphylaxis in patients with suggestive histories, but no sensitisation on skin tests. Ongoing study of this specific group of patients is required to clarify its utility in clinical practice.

Cell surface phenotype profiles distinguish stable and progressive chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is clinically heterogeneous. While some patients have indolent disease for many years, 20-30% will progress and ultimately die of their disease. CLL may be classified by the Rai or Binet staging system, mutational status of the immunoglobulin variable heavy-chain gene (IGVH), ZAP-70 overexpression, cytogenetic abnormalities (13q-, + 12, 11q-, 17p-) and expression of several cell surface antigens (CD38, CD49d) that correlate with risk of disease progression. However, none of these markers identify all cases of CLL at risk. In a recent review, we summarized those CD antigens known to correlate with the prognosis of CLL. The present study has identified surface profiles of CD antigens that distinguish clinically progressive CLL from slow-progressive and stable CLL. Using an extended DotScan(™) CLL antibody microarray (Version 3; 182 CD antibodies), and with refined analysis of purified CD19 + B-cells, the following 27 CD antigens were differentially abundant for progressive CLL: CD11a, CD11b, CD11c, CD18, CD19, CD20 (two epitopes), CD21, CD22, CD23, CD24, CD25, CD38, CD40, CD43, CD45, CD45RA, CD52, CD69, CD81, CD84, CD98, CD102, CD148, CD180, CD196 and CD270. The extensive surface profiles obtained provide disease signatures with an accuracy of 79.2%, a sensitivity of 83.9% and a specificity of 72.5% that could provide the basis for a rapid test to triage patients with CLL according to probability of clinical progression and potential earlier requirement for treatment.

Oxygen-evoked changes in transcriptional activity of the 5'-flanking region of the human amiloride-sensitive sodium channel (alphaENaC) gene: role of nuclear factor kappaB.

Expression of the alpha-subunit of the amiloride-sensitive sodium channel (alphaENaC) is regulated by a number of factors in the lung, including oxygen partial pressure (PO2). As transcriptional activation is a mechanism for raising cellular mRNA levels, we investigated the effect of physiological changes in PO2 on the activity of the redox-sensitive transcription factor nuclear factor kappaB (NF-kappaB) and transcriptional activity of 5'-flanking regions of the human alphaENaC gene using luciferase reporter-gene vectors transiently transfected into human adult alveolar carcinoma A549 cells. By Western blotting we confirmed the presence of NF-kappaB p65 but not p50 in these cells. Transiently increasing PO2 from 23 to 42 mmHg for 24 h evoked a significant increase in NF-kappaB DNA-binding activity and transactivation of a NF-kappaB-driven luciferase construct (pGLNF-kappaBpro), which was blocked by the NF-kappaB activation inhibitor sulphasalazine (5 mM). Transcriptional activity of alphaENaC-luciferase constructs containing 5'-flanking sequences (including the NF-kappaB consensus) were increased by raising PO2 from 23 to 142 mmHg if they contained transcriptional initiation sites (TIS) for exons 1A and 1B (pGL3E2.2) or the 3' TIS of exon 1B alone (pGL3E0.8). Sulphasalazine had no significant effect on the activity of these constructs, suggesting that the PO2-evoked rise in activity was not a direct consequence of NF-kappaB activation. Conversely, the relative luciferase activity of a construct that lacked the 3' TIS, a 3' intron and splice site but still retained the 5' TIS and NF-kappaB consensus sequence was suppressed significantly by raising PO2. This effect was reversed by sulphasalazine, suggesting that activation of NF-kappaB mediated PO2-evoked suppression of transcription from the exon 1A TIS of alphaENaC.