The CIpP activator, TR-57, is highly effective as a single agent and in combination with venetoclax against CLL cells in vitro.

Despite advances in treatment, a significant proportion of patients with chronic lymphocytic leukemia (CLL) will relapse with drug-resistant disease. The imipridones, ONC-201 and ONC-212, are effective against a range of different cancers, including acute myeloid leukemia (AML) and tumors of the brain, breast, and prostate. These drugs induce cell death through activation of the mitochondrial protease, caseinolytic protease (CIpP), and the unfolded protein response (UPR). Here we demonstrate that the novel imipridone analog, TR-57, has efficacy as a single agent and synergises with venetoclax against CLL cells under in vitro conditions that mimic the tumor microenvironment. Changes in protein expression suggest TR-57 activates the UPR, inhibits the AKT and ERK1/2 pathways and induces pro-apoptotic changes in the expression of proteins of the BCL-2 family. The study suggests that TR-57, as a single agent and in combination with venetoclax, may represent an effective treatment option for CLL.

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.

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.

Insight into del17p low-frequency subclones in chronic lymphocytic leukaemia (CLL): data from the Australasian Leukaemia and Lymphoma Group (ALLG)/CLL Australian Research Consortium (CLLARC) CLL5 trial.

The clinical significance of low-frequency deletions of 17p13 [tumour protein p53 (TP53)] in patients with chronic lymphocytic leukaemia (CLL) is currently unclear. Low-frequency del17p clones (<25%) were identified in 15/95 patients in the Australasian Leukaemia and Lymphoma Group (ALLG)/CLL Australian Research Consortium (CLLARC) CLL5 trial. Patients with low del17p, without tumour protein p53 (TP53) mutation, had significantly longer progression-free survival and overall survival durations than patients with high del17p clones. In 11/15 cases with low-frequency del17p, subclones solely with del17p or del13q were also noted. These data suggest that low-frequency del17p does not necessarily confer a poor outcome in CLL and challenges the notion of del13q as a founding event in CLL.

Therapeutic approaches and drug-resistance in chronic lymphocytic leukaemia.

The treatment of chronic lymphocytic leukaemia has been revolutionised in recent years, first by the introduction of chemoimmunotherapy regimens and subsequently by the development of drugs, including ibrutinib, idelalisib and venetoclax, that target components of the B-cell receptor signalling pathway or B-cell lymphoma 2 family of proteins. Despite high initial response rates in patients treated with chemoimmunotherapy or targeted agents, a significant proportion of patients relapse with progressive and refractory disease. In a subset of these patients, drug resistance has been associated with specific genetic lesions or activation of alternate pro-survival pathways. However, the mechanisms that confer drug resistance in the remainder of the patients with refractory disease have yet to be fully elucidated. In this review, we discuss our current understanding of the mechanics of drug resistance in chronic lymphocytic leukaemia and describe how this knowledge may aid in rationalising future treatment strategies to prevent the development of refractory or aggressive transformation of the disease.

The impact of aneuploidy on cellular homeostasis.

Genomic instability is a common feature of tumours that has a wide range of disruptive effects on cellular homeostasis. In this review we briefly discuss how instability comes about, then focus on the impact of gain or loss of DNA (aneuploidy) on oxidative stress. We discuss several mechanisms that lead from aneuploidy to the production of reactive oxygen species, including the effects on protein complex stoichiometry, endoplasmic reticulum stress and metabolic disruption. Each of these are involved in positive feedback loops that amplify relatively minor genetic changes into major cellular disruption or cell death, depending on the capacity of the cell to induce antioxidants or processes such as mitophagy that can moderate the disruption. Finally we examine the direct effects of reactive oxygen species on mitosis and how oxidative stress can compromise centrosome number, cytoskeletal integrity and signalling processes that are vital for mitotic fidelity.

Altered expression of metabolic pathways in CLL detected by unlabelled quantitative mass spectrometry analysis.

Chronic lymphocytic leukaemia (CLL) remains the most common incurable malignancy of B cells in the western world. Patient outcomes are heterogeneous and can be difficult to predict with current prognostic markers. Here, we used a quantitative label-free proteomic technique to ascertain differences in the B-cell proteome from healthy donors and CLL patients with either mutated (M-CLL) or unmutated (UM-CLL) IGHV to identify new prognostic markers. In peripheral B-CLL cells, 349 (22%) proteins were differentially expressed between normal B cells and B-CLL cells and 189 (12%) were differentially expressed between M-CLL and UM-CLL. We also examined the proteome of proliferating CLL cells in the lymph nodes, and identified 76 (~8%) differentially expressed proteins between healthy and CLL lymph nodes. B-CLL cells show over-expression of proteins involved in lipid and cholesterol metabolism. A comprehensive lipidomic analysis highlighted large differences in glycolipids and sphingolipids. A shift was observed from the pro-apoptotic lipid ceramide towards the anti-apoptotic/chemoresistant lipid, glucosylceramide, which was more evident in patients with aggressive disease (UM-CLL). This study details a novel quantitative proteomic technique applied for the first time to primary patient samples in CLL and highlights that primary CLL lymphocytes display markers of a metabolic shift towards lipid synthesis and breakdown.

Quantitative mass spectrometry to identify protein markers for diagnosis of malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is a devastating malignancy with a prognosis of <12 months. Even with bans on the use of asbestos in most Western countries, the incidence is still increasing due to the long latency periods between exposure and development of the disease. Diagnosis is often delayed due to invasive biopsies and lack of distinguishable markers. Patients frequently present with pleural effusions months to years before a radiologically detectable mass appears. This study aimed to investigate the proteome of pleural effusions taken from patients with MPM, adenocarcinoma and benign conditions in an attempt to identify a biomarker for early diagnosis. We identified several proteins that may be possible targets and warrant further investigation. Due to the predominance of up regulated proteins involved in VEGF signalling in MPM, we analysed VEGFA levels in effusions and found a strong correlation between VEGFA levels and survival in MPM.

Aberrant determination of phenotypic markers in chronic lymphocytic leukemia (CLL) lymphocytes after cryopreservation.

The cryopreservation of peripheral blood mononuclear cells (PBMCs) is a routine research laboratory process, enabling long-term storage of primary patient blood samples. Retrospective analysis of these samples has the potential to identify markers that may be associated with prognosis and response to treatment. To draw valid biological conclusions from this type of analysis, it is essential to ensure that any observed changes are directly related to the pathology of the disease rather than the preservation process itself. Therefore, we have investigated 15 cell surface markers that are relevant to chronic lymphocytic leukemia (CLL) on matched fresh and thawed samples to determine the effect of cryopreservation on their detection. We found that the number of CLL cells positive for the markers CD22, CD40, CD49d, CD54, CD69, and CXCR3 was decreased significantly after cryopreservation. In addition, the mean fluorescence intensity of 10 of the 15 markers changed significantly after cryopreservation. These findings demonstrate that care must be taken when interpreting this type of analysis on thawed samples.

Development of locus specific sub-clone separation by fluorescence in situ hybridization in suspension in chronic lymphocytic leukemia.

Intra-tumor genetic heterogeneity is a hallmark of cancer. The ability to monitor and analyze these sub-clonal cell populations can be considered key to successful treatment, particularly in the modern era of targeted therapies. Although advances in sequencing technologies have significantly improved our ability to analyze the mutational landscape of tumors, this utility is reduced when considering small, but clinically significant sub-clones, that is, those representing <10% of the tumor burden. We have developed a high-throughput method that utilizes a 17-probe labeled bacterial artificial chromosome contig to quantify sub-clonal populations of cells based on deletion of a single locus. Chronic lymphocytic leukemia (CLL) cells harboring deletion of the short arm of chromosome 17 (del17p), an important prognostic marker for CLL were used to demonstrate the technique. Sub-clones of del17p cells were quantified and isolated from heterogeneous CLL populations using fluorescence in situ hybridization in suspension (FISH-IS) and the locus specific probe set. Using the combination of FISH-IS with the locus-specific probe set enables automated analysis of tens of thousands of cells, accurately quantifying and isolating cells carrying a del17p. Based on the fluorescence intensity of 17p probes, 17p (TP53) deleted cells were identified and sorted using flow cytometric techniques, and enrichment was demonstrated using single nucleotide polymorphism analysis. The ability to separate sub-clones of cells based on genetic heterogeneity, independent of the clone size, highlights the potential application of this method not only in the diagnostic and prognostic setting, but also as an unbiased approach to enable further detailed genetic analysis of the sub-clone with deep sequencing approaches. © 2017 International Society for Advancement of Cytometry.

From genome to proteome: Looking beyond DNA and RNA in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) remains the most common leukemia in the Western world. Whilst its disease course is extremely heterogeneous (ranging from indolent to aggressive), current methods are unable to accurately predict the clinical journey of each patient. There is clearly a pressing need for both improved prognostication and treatment options for patients with this disease. Whilst molecular studies have analyzed both genetic mutations and gene expression profiles of these malignant B-cells, and as a result have shed light on the pathogenesis of CLL, proteomic studies have been largely overlooked to date. This review summarizes our current knowledge of the proteomics of CLL, and discusses some of the issues in CLL proteomic research, such as reproducibility and data interpretation. In addition, we look ahead to how proteomics may significantly help in the development of a successful treatment for this currently incurable disease.

Long-term Ro60 humoral autoimmunity in primary Sjögren's syndrome is maintained by rapid clonal turnover.

Long-term humoral autoimmunity to RNA-protein autoantigens is considered a hallmark of systemic autoimmune diseases. We use high resolution Orbitrap mass spectrometric autoantibody sequencing to track the evolution of a Ro60-specific public clonotypic autoantibody in 4 patients with primary Sjögren's syndrome. This clonotype is specified by a VH3-23/VK3-20 heavy and light chain pairing. Despite apparent stability by conventional immunoassay, analysis of V-region molecular signatures of clonotypes purified from serum samples collected retrospectively over 7years revealed sequential clonal replacement. Prospective longitudinal studies confirmed clonotype loss and replacement at approximately three-monthly intervals. Levels of secreted anti-Ro60 clonotypes fluctuated markedly over time, despite minimal changes in clonal affinity. Our novel findings indicate a relentless turnover of short-lived clonotypic variants, masquerading as long-lived Ro60 humoral autoimmunity.

Secreted human Ro52 autoantibody proteomes express a restricted set of public clonotypes.

Long-lived secreted autoantibody responses in systemic autoimmunity are generally regarded to be polyclonal and to express a diverse B-cell repertoire. Here, we have used a proteomic approach based on de novo sequencing to determine the clonality and V region structures of human autoantibodies directed against a prototypic systemic autoantigen, Ro52 (TRIM21). Remarkably, anti-Ro52 autoantibodies from patients with primary Sjögren's syndrome, systemic lupus erythematosus, systemic sclerosis or polymyositis were restricted to two IgG1 kappa clonotypes that migrated as a single species on isoelectric focusing; shared a common light chain paired with one of two closely-related heavy chains; and were public in unrelated patients. Targeted mass spectrometry using these uniquely mutated V region peptides as surrogates detected anti-Ro52 autoantibodies in human sera with high sensitivity and specificity compared with traditional ELISA. Mass spectrometry-based detection of specific autoantibody motifs provides a powerful new tool for analysis of humoral autoimmunity.

Pathogenicity and proteomic signatures of autoantibodies to Ro and La.

Ro/SSA and La/SSB comprise a linked set of autoantigens that are clinically important members of the extractable nuclear antigen family and key translational biomarkers for lupus and primary Sjögren's syndrome. Autoantibodies directed against the Ro60 and La polypeptide components of the Ro/La ribonucleoprotein complex, and the structurally unrelated Ro52 protein, mediate tissue damage in the neonatal lupus syndrome, a model of passively acquired autoimmunity in humans in which the most serious manifestation is congenital heart block (CHB). Recent studies have concentrated on two distinct pathogenic mechanisms by which maternal anti-Ro/La autoantibodies can cause CHB: by forming immune complexes with apoptotic cells in developing fetal heart; and/or by acting as functional autoantibodies that cross-react with and inhibit calcium channels. Although the precise role of the individual autoantibodies is yet to be settled, maternal anti-Ro60 and anti-Ro52 remain the most likely culprits. This article will discuss the molecular pathways that culminate in the development of CHB, including the recent discovery of ß2 glycoprotein I as a protective factor, and present a proteomic approach based on direct mass spectrometric sequencing, which may give a more representative snapshot of the idiotype repertoire of these autoantibodies than genomic-based technologies.

The effect of intracrystalline and surface-bound osteopontin on the degradation and dissolution of calcium oxalate dihydrate crystals in MDCKII cells.

In vivo, urinary crystals are associated with proteins located within the mineral bulk as well as upon their surfaces. Proteins incarcerated within the mineral phase of retained crystals could act as a defence against urolithiasis by rendering them more vulnerable to destruction by intracellular and interstitial proteases. The aim of this study was to examine the effects of intracrystalline and surface-bound osteopontin (OPN) on the degradation and dissolution of urinary calcium oxalate dihydrate (COD) crystals in cultured Madin Darby canine kidney (MDCK) cells. [(14)C]-oxalate-labelled COD crystals with intracrystalline (IC), surface-bound (SB) and IC + SB OPN, were generated from ultrafiltered (UF) urine containing 0, 1 and 5 mg/L human milk OPN and incubated with MDCKII cells, using UF urine as the binding medium. Crystal size and degradation were assessed using field emission scanning electron microscopy (FESEM) and dissolution was quantified by the release of radioactivity into the culture medium. Crystal size decreased directly with OPN concentration. FESEM examination indicated that crystals covered with SB OPN were more resistant to cellular degradation than those containing IC OPN, whose degree of disruption appeared to be related to OPN concentration. Whether bound to the crystal surface or incarcerated within the mineral interior, OPN inhibited crystal dissolution in direct proportion to its concentration. Under physiological conditions OPN may routinely protect against stone formation by inhibiting the growth of COD crystals, which would encourage their excretion in urine and thereby perhaps partly explain why, compared with calcium oxalate monohydrate crystals, COD crystals are more prevalent in urine, but less common in kidney stones.

The effect of intracrystalline and surface-bound osteopontin on the attachment of calcium oxalate dihydrate crystals to Madin-Darby canine kidney (MDCK) cells in ultrafiltered human urine.

OBJECTIVE: To determine the effects of intracrystalline (IC), surface-bound (SB) and combined IC + SB osteopontin (OPN) on the binding of urinary calcium oxalate dihydrate (COD) crystals to Madin-Darby canine kidney (MDCK-II) cells in ultrafiltered (UF) human urine. MATERIALS AND METHODS: (14)C-oxalic acid-labelled urinary COD crystals containing IC OPN were generated in pooled UF human urine containing human milk OPN at concentrations of 0, 1.0 and 5.0 mg/L. Additional labelled crystals were nucleated from a separate sample of the same pooled UF urine, to which were later added the same amounts of protein to produce crystals with SB OPN. COD crystals with IC+SB OPN were prepared using a combination of both techniques. Control crystals were prepared in the absence of OPN. Crystals were incubated with MDCK-II cells for up to 180 min in UF urine adjusted to 8 mm Ca(2+). Binding values for individual concentrations at specific time points and overall differences between binding curves were compared using the Mann-Whitney U-test. Crystal morphology and attachment to the cells were confirmed using field emission scanning electron microscopy (FESEM). RESULTS: The sizes of crystals precipitated from UF urine in the presence of 0, 1 and 5 mg/L OPN were 21.9 µm, 19.3 µm and 16.5 µm, indicating that OPN had inhibited crystal growth in a dose-dependent fashion. Binding curves for control crystals were smooth, while those of the IC and IC+SB COD crystals associated with 1 and 5 mg/L OPN were bimodal, as were those of the 1 mg/L SB crystals. This suggests that OPN induces or potentiates a transient response that enables MDCK-II cells to release COD crystals after they have attached. Although OPN generally reduced the binding of urinary COD crystals to MDCK-II cells, at times it also appeared to mediate adhesion. It is possible therefore that OPN can reduce or increase crystal binding, and that our data represent the net effect of its opposing inhibitory or promotory properties. CONCLUSIONS: In UF urine, OPN inhibits the growth of COD crystals and reduces the binding of urinary COD crystals to MDCK-II cells, regardless of whether it is IC, SB, or IC+SB. Future studies aimed at clarifying the effects of OPN, or indeed any urinary component, on crystal-cell interaction, should use crystals precipitated from urine and be performed under urinary conditions.

Molecular signature of a public clonotypic autoantibody in primary Sjögren's syndrome: a "forbidden" clone in systemic autoimmunity.

OBJECTIVE: This study was undertaken to determine the molecular characteristics of clonotypic autoantibodies in the sera of patients with primary Sjögren's syndrome (SS). This characterization is hampered by the presence of mixed anti-Ro/La specificities that may conceal clonotypic species. In order to narrow clonotypic diversity, a positive selection step was performed on a peg-like determinant of Ro 60 (termed Ro 60-peg) prior to analysis of the autoantibody proteome. METHODS: Monospecific anti-Ro 60-peg IgG were isolated by affinity purification from the sera of 7 patients with primary SS and anti-Ro/La and subjected to 2-dimensional gel electrophoresis and high-resolution orbitrap mass spectrometric sequencing. V regions of heavy and light chains were analyzed by combined database and de novo amino acid sequencing. RESULTS: Proteomic analysis revealed a Ro 60-peg-specific IgG1κ-restricted monoclonal autoantibody that was present in the sera of all patients and specified by a V(H) 3-23 heavy chain paired with a V(κ) 3-20 light chain. The public anti-Ro 60-peg clonotype was specified further by common mutations in the heavy-chain and light-chain complementarity-determining regions. Titers and relative affinities of clonotypic IgG did not vary over the course of the disease. CONCLUSION: The expression of a Ro 60-reactive public B cell clonotype in a subset of patients with primary SS as a long-lived, class-switched circulating autoantibody implies a common breach of B cell tolerance checkpoints in these patients. The unique heavy chain/light chain signature opens the possibility of tracking the development of a "forbidden" clone against a bona fide systemic autoantigen in human disease.

Comparison of the specific incorporation of intracrystalline proteins into urinary calcium oxalate monohydrate and dihydrate crystals.

The aim of this study was to compare the comprehensive intracrystalline protein profiles of calcium oxalate monohydrate (COM) and dihydrate (COD) crystals precipitated from the same human urine samples. Three separate batches of COM and COD crystals were precipitated from pooled healthy human urine by the addition of sodium oxalate at calcium concentrations of 2 and 8 mM, respectively. Proteins in whole extracts of demineralised COM and COD crystals, as well as in spots excised from 2D-PAGE gels of the extracts, were identified using liquid chromatography and tandem mass spectrometry (LC-MS/MS). The number and type of individual proteins differed between COM and COD: 14 substantive proteins were found inside COM crystal extracts and 34 inside COD, with 9 proteins occurring in both crystal types. Numerous keratins were detected. However, in line with consensus in the proteomics literature, as well as a lack of published evidence linking them to urolithiasis, they were excluded as contaminants, leaving very few consistently detected proteins. On the basis of their known association with stone disease or identification in multiple runs, the principal proteins in COM crystal extracts were prothrombin fragment 1, protein S100A9, and IGkappaV1-5, while those in extracts of COD crystals included osteopontin, IGkappaV1-5, protein S100A9, annexin A1, HMW kininogen-1, and inter-alpha-inhibitor (IalphaI). In general, proteins incorporated into both hydromorphs were acidic (pI<6), smaller than 55 kDa, and calcium binders. We concluded that the incorporation of proteins into urinary COM and COD crystals is selective and that only a few of the urinary proteins associated with the two hydromorphs are likely to play any significant role in stone pathogenesis.

Proteomic analysis of proteins selectively associated with hydroxyapatite, brushite, and uric acid crystals precipitated from human urine.

The aim of this study was to compare the intracrystalline protein profiles of hydroxyapatite (HA), brushite (BR), and uric acid (UA) crystals precipitated from the same urine samples. HA, BR, and UA crystals were precipitated on two different occasions from the same pooled healthy urine. Crystals were washed to remove surface-bound proteins, and their composition was confirmed using Fourier transform infrared spectroscopy (FTIR) and field emission scanning electron microscopy (FESEM) coupled with energy dispersive X-ray analysis (EDAX). SDS-PAGE was used for visual comparison of the protein content of the demineralised crystal extracts, which were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). HA comprised nanosized particles interspersed with organic material, which was absent from the BR and UA crystals. The number and type of individual proteins differed between the 3 minerals: 45 proteins were detected in the HA crystal extracts and 77 in the BR crystals, including a number of keratins, which were regarded as methodological contaminants. After excluding the keratins, 21 proteins were common to both HA and BR crystals. Seven nonkeratin proteins were identified in the UA extracts. Several proteins consistently detected in the HA and BR crystal extracts have been previously implicated in kidney stone disease, including osteopontin, prothrombin, protein S100A9 (calgranulin B), inter-α-inhibitor, α1-microglobulin bikunin (AMBP), heparan sulfate proteoglycan, and Tamm-Horsfall glycoprotein, all of which are strong calcium binders. We concluded that the association of proteins with HA, BR, and UA crystals formed in healthy urine is selective and that only a few of the numerous proteins present in healthy urine are likely to play any significant role in preventing stone pathogenesis.