Publications by authors named "Giles Best"

44 Publications

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.

Br J Haematol 2021 May 13;193(3):556-560. Epub 2021 Apr 13.

Department of Molecular Medicine and Genetics, Flinders Health and Medical Research Institute, College of Medicine and Public Health, Flinders University, Adelaide, SA.

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.
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http://dx.doi.org/10.1111/bjh.17394DOI Listing
May 2021

IBL-202 is synergistic with venetoclax in CLL under in vitro conditions that mimic the tumor microenvironment.

Blood Adv 2020 10;4(20):5093-5106

Northern Blood Research Centre, Kolling Institute, Royal North Shore Hospital, St Leonards, NSW, Australia.

The B-cell receptor signaling pathway and dysregulation of the Bcl-2 family of proteins play crucial roles in the pathogenesis of chronic lymphocytic leukemia (CLL). Despite significant advances in the treatment of the disease, relapse and drug resistance are not uncommon. In the current study, we investigated the dual PI3/PIM kinase inhibitor IBL-202 in combination with venetoclax as a treatment option for CLL using both primary CLL cells and TP53-deficient OSU-CLL cells generated using the CRISPR-Cas9 system. IBL-202 and venetoclax were highly synergistic against primary CLL cells cocultured with CD40L fibroblasts (combination index [CI], 0.4, at a fractional effect of 0.9) and TP53-knockout (KO) OSU-CLL cells (CI, 0.5, at a fractional effect of 0.9). Synergy between the drugs was consistent, with a significant (P < .05) reduction in the 50% inhibitory concentration for both drugs. IBL-202 and venetoclax in combination induced cell-cycle arrest and slowed the proliferation of both wild-type and TP53-KO cell lines. The drug combination inhibited AKT phosphorylation, reduced expression of Bcl-xL and NF-κB, and increased the Noxa/Mcl-1 ratio. Downregulation of CXCR4 was consistent with inhibition of the SDF-1α-induced migratory capacity of CLL cells. Synergy between IBL-202 and venetoclax against primary CLL cells cultured under conditions that mimic the tumor microenvironment suggests this drug combination may be effective against CLL cells within the lymph nodes and bone marrow. Furthermore, the efficacy of the combination against the TP53-KO OSU-CLL cell line suggests the combination may be a highly effective treatment strategy for high-risk CLL.
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http://dx.doi.org/10.1182/bloodadvances.2019001369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7594376PMC
October 2020

Combination of the dual PIM/PI3-kinase inhibitor IBL-202 and venetoclax is effective in diffuse large B-cell lymphoma.

Leuk Lymphoma 2020 12 29;61(13):3165-3176. Epub 2020 Jul 29.

Northern Blood Research Centre, Kolling Institution of Medical Research, The University of Sydney. St Leonards, Australia.

Current chemoimmunotherapy is unable to cure up to 40% of patients diagnosed with diffuse large B-cell lymphoma (DLBCL). Targeting the mechanisms by which DLBCL evades apoptosis is crucial to overcoming treatment failure in this heterogeneous disease as both current and novel treatments depend on the apoptosis of malignant cells. Despite the common overexpression of BCL-2, venetoclax is ineffective in DLBCL due to MCL-1 co-expression. This is driven by pro-growth PI3-kinase signaling, which is promoted in turn by PIM kinases. In this study, the novel dual-kinase inhibitor, IBL-202, was combined with venetoclax against a panel of DLBCL cell lines that have variable expression of pro-survival proteins. The results support the efficacy of simultaneously targeting inter-related molecules to overcome apoptotic escape in this biologically heterogeneous disease. As venetoclax, pan-PIM-kinase and pan-PI3-kinase inhibitors have, or are currently being studied in clinical trials, it may be rational to consider these drugs in combination for the treatment of DLBCL.
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http://dx.doi.org/10.1080/10428194.2020.1795156DOI Listing
December 2020

Molecular pathogenesis of chronic lymphocytic leukaemia.

Br J Haematol 2019 09 31;186(5):668-684. Epub 2019 Jul 31.

Northern Blood Research Centre, Kolling Institute of Medical Research, Sydney, NSW, Australia.

Chronic lymphocytic leukaemia (CLL) is characterised by the clonal expansion of mature, CD5 positive, B lymphocytes in the blood, marrow, lymph nodes and spleen. For the majority of patients, CLL follows an indolent clinical course, while a proportion of patients experience rapid disease progression. Despite the strong correlation between certain genetic defects and prognosis, there remains no single unifying pathogenic lesion in CLL. With recent advances in therapy it is increasingly important to stratify CLL patients according to risk. This has been highlighted by two recent studies, the first showing that immunoglobulin heavy chain mutational status predicts a durable response to frontline chemoimmunotherapy and the second showing that complex karyotype is a stronger predictor of poor response to ibrutinib and venetoclax therapy than TP53 deletion. In this review we discuss the molecular features of CLL and how technological advances can identify patient subsets and stratify them according to risk.
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http://dx.doi.org/10.1111/bjh.16102DOI Listing
September 2019

Dual inhibition of MEK1/2 and AKT by binimetinib and MK2206 induces apoptosis of chronic lymphocytic leukemia cells under conditions that mimic the tumor microenvironment.

Leuk Lymphoma 2019 07 16;60(7):1632-1643. Epub 2019 Jan 16.

a Northern Blood Research Centre, Kolling Institute , Royal North Shore Hospital , St Leonards , NSW , Australia.

Several key pathways mediate signaling via the B-cell receptor, including the mitogen-activated protein kinase-ERK1/2 pathway. However, inhibition of MEK1/2, a key component of the MAPK-ERK1/2 signaling cascade, results in paradoxical activation of AKT in chronic lymphocytic leukemia (CLL) cells. In the current study we demonstrate synergy between the MEK1/2 inhibitor binimetinib and the AKT inhibitor MK2206, which combined induce apoptosis of primary CLL cells and restrict the cell cycle progression and proliferation of the OSU-CLL cell line. The mechanisms of action of the drug combination involve dual inhibition of MAPK-ERK1/2 and AKT signaling and down-regulation of Mcl-1 expression. Collectively, these data suggest that dual inhibition of MEK1/2 and AKT may represent a therapeutic option for CLL, capable of overcoming the pro-survival effects of the lymph node and bone marrow microenvironments.
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http://dx.doi.org/10.1080/10428194.2018.1542148DOI Listing
July 2019

The dual inhibitor of the phosphoinositol-3 and PIM kinases, IBL-202, is effective against chronic lymphocytic leukaemia cells under conditions that mimic the hypoxic tumour microenvironment.

Br J Haematol 2018 09 5;182(5):654-669. Epub 2018 Jul 5.

Northern Blood Research Centre, Kolling Institute of Medical Research, Royal North Shore Hospital, St Leonards, Sydney, Australia.

Despite significant advances in treatment, chronic lymphocytic leukaemia (CLL) remains an incurable disease. Ibrutinib and idelalisib, which inhibit Bruton Tyrosine kinase (BTK) and phosphoinositol-3 (PI3) kinase-δ respectively, have become important treatment options for the disease and demonstrate the potential of targeting components of the B-cell receptor-signalling pathway. IBL-202 is a dual inhibitor of the PIM and PI3 kinases. Synergy between the pan-PIM inhibitor, pPIMi, and idelalisib against a range of haematological cell lines and primary CLL cells supports the rationale for preclinical studies of IBL-202 in CLL. Importantly, IBL-202, but not idelalisib, was cytotoxic against CLL cells under in vitro conditions that mimic the hypoxic tumour microenvironment. The significant effects of IBL-202 on CD49d and CXCR4 expression and migration, cycle and proliferation of CLL cells suggest the drug may also interfere with the migratory and proliferative capacity of the leukaemic cells. Collectively, these data demonstrate that dual inhibition of the PIM and PI3 kinases by IBL-202 may be an effective strategy for targeting CLL cells, particularly within the environmental niches known to confer drug-resistance.
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http://dx.doi.org/10.1111/bjh.15447DOI Listing
September 2018

Immune failure, infection and survival in chronic lymphocytic leukemia.

Haematologica 2018 07;103(7):e329

Department of Haematology, Royal North Shore Hospital, Kolling Institute, University of Sydney, St Leonards, Australia.

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http://dx.doi.org/10.3324/haematol.2018.196543DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6029549PMC
July 2018

Expression of Intracellular Reactive Oxygen Species in Hematopoietic Stem Cells Correlates with Time to Neutrophil and Platelet Engraftment in Patients Undergoing Autologous Bone Marrow Transplantation.

Biol Blood Marrow Transplant 2018 10 19;24(10):1997-2002. Epub 2018 Jun 19.

Department of Haematology and Transfusion Medicine, Royal North Shore Hospital, Sydney, New South Wales, Australia; Cellular Therapeutic Laboratory, Northern Blood Research Centre, Kolling Research Institute, Sydney, New South Wales, Australia.

Reactive oxygen species (ROS) play important roles in hematopoiesis and regulate the self-renewal, migration, and myeloid differentiation of hematopoietic stem cells (HSCs). This study was conducted to determine whether ROS levels in donor HSCs correlate with neutrophil and platelet engraftment in patients after bone marrow transplantation. Cryopreserved HSC samples from 51 patients who underwent autologous transplantation were studied. Levels of intracellular ROS were assessed by flow cytometry using 2',7'-dichlorodihydrofluorescein diacetate (HDCFDA) in the CD45/CD34 HSC population. Colony forming unit assays were performed on HSCs isolated from the ROS and ROS populations to assess the differentiation potential of these 2 cell subsets. Distinct populations of ROS and ROS cells were evident in all patient samples. The median percentage of ROS expressing HSCs in the study cohort was 75.8% (range, 2% to 95.2%). A significant correlation was identified between the percentage of ROS stem cells present in the hematopoietic progenitor cells collected by apheresis product infused and the time to neutrophil engraftment (P < .001, r = -.54), as well as time to plt20, plt50, and plt100 (P < 0.001; r = -.55, -.59, and -.56 respectively). The dose of CD34/ROS/kg infused also inversely correlated with a shorter time to neutrophil engraftment; time to engraftment for patients receiving > or ≤3 × 10 cells/kg was 11.5 days (range, 9 to 23) versus 14 days (range, 10 to 28), respectively (P = .02). The dose of ROS HSCs delivered did not correlate with platelet engraftment. Collectively, these data suggest that the dose of ROS stem cells delivered to patients may predict time to neutrophil engraftment after autologous transplantation.
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http://dx.doi.org/10.1016/j.bbmt.2018.06.014DOI Listing
October 2018

MEK1/2 inhibition by binimetinib is effective as a single agent and potentiates the actions of Venetoclax and ABT-737 under conditions that mimic the chronic lymphocytic leukaemia (CLL) tumour microenvironment.

Br J Haematol 2018 08 16;182(3):360-372. Epub 2018 May 16.

Northern Blood Research Centre, Kolling Institute of Medical Research, Royal North Shore Hospital, St Leonards, Sydney, Australia.

The survival and proliferation of chronic lymphocytic leukaemia (CLL) cells is driven by multiple signalling pathways, including those mediated by the B cell, Toll-like and chemokine receptors. Many of these pathways converge on the same signalling molecules, including those involved in the Raf-1/MEK/Erk1/2-MAPK pathway. We investigated the effects of the MEK1/2 (also termed MAP2K1/2) inhibitor, binimetinib, against CLL cells cultured under conditions that mimic aspects of the tumour microenvironment. Binimetinib blocked CLL cell survival induced by stroma-conditioned media and phorbol myristylate (PMA). Binimetinib was also significantly more toxic towards CLL cells cultured in the presence of either anti-IgM antibody or stroma-derived factor-1α (SDF-1α) and reduced CLL cell cycle progression and proliferation. Furthermore, binimetinib significantly increased the sensitivity of CLL cells co-cultured with CD40 ligand (CD40L)-expressing fibroblasts to the BH3-mimetics ABT-737 and Venetoclax (ABT-199) via a mechanism involving down-regulation of Mcl-1 (MCL1) activity and Bim (BCL2L11) and Bcl-xL (BCL2L1) expression. Collectively, these data suggest that binimetinib may have both cytotoxic and cytostatic effects on CLL cells by blocking microenvironment-derived signals known to drive survival and proliferation. The combination of binimetinib with a BH3 mimetic may be an effective treatment strategy for CLL, particularly against the proliferative fraction of the disease within the tumour microenvironment.
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http://dx.doi.org/10.1111/bjh.15282DOI Listing
August 2018

Humoral immune failure defined by immunoglobulin class and immunoglobulin G subclass deficiency is associated with shorter treatment-free and overall survival in Chronic Lymphocytic Leukaemia.

Br J Haematol 2018 04 21;181(1):97-101. Epub 2018 Feb 21.

Department of Haematology, Royal North Shore Hospital, Kolling Institute, University of Sydney, St Leonards, Sydney, Australia.

Immune dysfunction attributed to hypogammaglobulinaemia is common in chronic lymphocytic leukaemia (CLL) and infection is a major contributor to morbidity and mortality. A higher incidence of multiple immunoglobulin and immunoglobulin G (IgG) subclass deficiency was associated with more advanced disease (P < 0·001 and P < 0·001, respectively) in a cohort of 147 CLL patients. Multiple immunoglobulin and IgG subclass deficiency were significantly associated with shorter treatment-free survival (TFS) (P < 0·001 and P = 0·006, respectively). The association between disease stage and immune dysfunction demonstrated by these data suggest aspects of immune deficiency correlate with disease severity and may be associated with shorter TFS in CLL.
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http://dx.doi.org/10.1111/bjh.15146DOI Listing
April 2018

Ibrutinib and idelalisib block immunophenotypic changes associated with the adhesion and activation of CLL cells in the tumor microenvironment.

Leuk Lymphoma 2018 08 22;59(8):1927-1937. Epub 2017 Nov 22.

a School of Life and Environmental Sciences , University of Sydney , Sydney , Australia.

The lymph node and bone marrow microenvironments promote the survival and proliferation of CLL cells. Defining the immunophenotype of CLL cells from the tumor microenvironment may help to better understand the mechanisms of action of current therapies and identify novel drug targets. Significant changes in the levels of 25 CD antigens were identified using the DotScan™ antibody microarray following CLL-cell culture with CD40L-expressing fibroblasts. Ibrutinib or idelalisib countered the change in expression of 11 of these antigens (CD23, CD27, CD53, CD58, CD71, CD80, CD84, CD97, CD126, CD150, and FMC7), which have known roles in cell activation and adhesion. The immunophenotypic changes identified may provide further insight into the mechanisms by which CLL cells interact with the tumor microenvironment and better define how ibrutinib and idelalisib release CLL cells from the lymph nodes and bone marrow.
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http://dx.doi.org/10.1080/10428194.2017.1403598DOI Listing
August 2018

Surface Profiling of Extracellular Vesicles from Plasma or Ascites Fluid Using DotScan Antibody Microarrays.

Methods Mol Biol 2017 ;1619:263-301

School of Life and Environmental Sciences, University of Sydney, Sydney, NSW, 2006, Australia.

DotScan antibody microarrays were initially developed for the extensive surface profiling of live leukemia and lymphoma cells. DotScan's diagnostic capability was validated with an extensive clinical trial using mononuclear cells from the blood or bone marrow of leukemia or lymphoma patients. DotScan has also been used for the profiling of surface proteins on peripheral blood mononuclear cells (PBMC) from patients with HIV, liver disease, and stable and progressive B-cell chronic lymphocytic leukemia (CLL). Fluorescence multiplexing allowed the simultaneous profiling of cancer cells and leukocytes from disaggregated colorectal and melanoma tumor biopsies after capture on DotScan. In this chapter, we have used DotScan for the surface profiling of extracellular vesicles (EV) recovered from conditioned growth medium of cancer cell lines and the blood of patients with CLL. The detection of captured EV was performed by enhanced chemiluminescence (ECL) using biotinylated antibodies that recognized antigens expressed on the surface of the EV subset of interest. DotScan was also used to profile EV from the blood of healthy individuals and the ascites fluid of ovarian cancer patients. DotScan binding patterns of EV from human plasma and other body fluids may yield diagnostic or prognostic signatures for monitoring the incidence, treatment, and progression of cancers.
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http://dx.doi.org/10.1007/978-1-4939-7057-5_20DOI Listing
March 2018

Lymphoma cell-of-origin assignment by gene expression profiling is clinically meaningful across broad laboratory contexts.

Br J Haematol 2018 04 15;181(2):272-275. Epub 2017 Feb 15.

Northern Blood Research Centre, Kolling Institute of Medical Research, University of Sydney, Sydney, New South Wales, Australia.

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http://dx.doi.org/10.1111/bjh.14556DOI Listing
April 2018

Modeling the chronic lymphocytic leukemia microenvironment in vitro.

Leuk Lymphoma 2017 02 18;58(2):266-279. Epub 2016 Oct 18.

a Northern Blood Research Centre , Kolling Institute of Medical Research, Royal North Shore Hospital , Sydney , Australia.

Microenvironments within the lymph node and bone marrow promote proliferation and drug resistance in chronic lymphocytic leukemia (CLL). Successful treatment of CLL must therefore target the leukemic cells within these compartments. A better understanding of the interaction between CLL cells and the tumor microenvironment has led to the development of in vitro models that mimic the mechanisms that support leukemic cell survival and proliferation in vivo. Employing these models as part of the pre-clinical evaluation of novel therapeutic agents enables a better approximation of their potential clinical efficacy. In this review we summarize the current literature describing how different aspects of the tumor microenvironment have been modeled in vitro and detail how these models have been employed to study the biology of the disease and potential efficacy of novel therapeutic agents.
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http://dx.doi.org/10.1080/10428194.2016.1204654DOI Listing
February 2017

Extensive surface protein profiles of extracellular vesicles from cancer cells may provide diagnostic signatures from blood samples.

J Extracell Vesicles 2016 15;5:25355. Epub 2016 Apr 15.

School of Life and Environmental Sciences, University of Sydney, Sydney, NSW, Australia.

Extracellular vesicles (EV) are membranous particles (30-1,000 nm in diameter) secreted by cells. Important biological functions have been attributed to 2 subsets of EV, the exosomes (bud from endosomal membranes) and the microvesicles (MV; bud from plasma membranes). Since both types of particles contain surface proteins derived from their cell of origin, their detection in blood may enable diagnosis and prognosis of disease. We have used an antibody microarray (DotScan) to compare the surface protein profiles of live cancer cells with those of their EV, based on their binding patterns to immobilized antibodies. Initially, EV derived from the cancer cell lines, LIM1215 (colorectal cancer) and MEC1 (B-cell chronic lymphocytic leukaemia; CLL), were used for assay optimization. Biotinylated antibodies specific for EpCAM (CD326) and CD19, respectively, were used to detect captured particles by enhanced chemiluminescence. Subsequently, this approach was used to profile CD19(+) EV from the plasma of CLL patients. These EV expressed a subset (~40%) of the proteins detected on CLL cells from the same patients: moderate or high levels of CD5, CD19, CD31, CD44, CD55, CD62L, CD82, HLA-A,B,C, HLA-DR; low levels of CD21, CD49c, CD63. None of these proteins was detected on EV from the plasma of age- and gender-matched healthy individuals.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4834364PMC
http://dx.doi.org/10.3402/jev.v5.25355DOI Listing
April 2016

The Hsp90 inhibitor SNX-7081 is synergistic with fludarabine nucleoside via DNA damage and repair mechanisms in human, p53-negative chronic lymphocytic leukemia.

Oncotarget 2015 Dec;6(38):40981-97

School of Molecular Bioscience, University of Sydney, Darlington, NSW 2006, Australia.

Clinical trials of heat shock protein 90 (Hsp90) inhibitors have been limited by high toxicity. We previously showed that the Hsp90 inhibitor, SNX-7081, synergizes with and restores sensitivity to fludarabine nucleoside (2-FaraA) in human chronic lymphocytic leukemia (CLL) cells with lesions in the p53 pathway (Best OG, et al., Leukemia Lymphoma 53:1367-75, 2012). Here, we used label-free quantitative shotgun proteomics and comprehensive bioinformatic analysis to determine the mechanism of this synergy. We propose that 2-FaraA-induced DNA damage is compounded by SNX-7081-mediated inhibition of DNA repair, resulting in enhanced induction of apoptosis. DNA damage responses are impaired in part due to reductions in checkpoint regulators BRCA1 and cyclin D1, and cell death is triggered following reductions of MYC and nucleolin and an accumulation of apoptosis-inducing NFkB2 p100 subunit. Loss of nucleolin can activate Fas-mediated apoptosis, leading to the increase of pro-apoptotic proteins (BID, fas-associated factor-2) and subsequent apoptosis of p53-negative, 2-FaraA refractory CLL cells. A significant induction of DNA damage, indicated by increases in DNA damage marker γH2AX, was observed following the dual drug treatment of additional cell lines, indicating that a similar mechanism may operate in other p53-mutated human B-lymphoid cancers. These results provide valuable insight into the synergistic mechanism between SNX-7081 and 2-FaraA that may provide an alternative treatment for CLL patients with p53 mutations, for whom therapeutic options are currently limited. Moreover, this drug combination reduces the effective dose of the Hsp90 inhibitor and may therefore alleviate any toxicity encountered.
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http://dx.doi.org/10.18632/oncotarget.5715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4747384PMC
December 2015

Protein profiles distinguish stable and progressive chronic lymphocytic leukemia.

Leuk Lymphoma 2016 May 16;57(5):1033-43. Epub 2015 Nov 16.

a School of Molecular Bioscience, University of Sydney , Sydney , NSW , Australia ;

Patients with a stable chronic lymphocytic leukemia (CLL) double their blood lymphocyte count in >5 years, but may develop progressive disease with lymphocytes doubling in <12 months. To identify a protein signature for progressive CLL, whole cell extracts of peripheral blood mononuclear cells from patients with CLL (n=27) were screened using iTRAQ (isobaric tags for relative and absolute quantification) analysis. A total of 84 differentially abundant proteins were identified from patients with stable and progressive CLL. Subsequently, 32 of these proteins were quantified by SRM (selected reaction monitoring) using extracts of purified CD19+ CLL cells from patients (n=50). Hierarchical clustering of these protein profiles showed two clusters of patients that correlated with progressive and stable CLL, providing signatures that should be useful for triaging patients. Some of the proteins in the progressive cluster have not been linked with CLL, for example, glutamate dehydrogenase 1 and transcription intermediary factor 1-beta.
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http://dx.doi.org/10.3109/10428194.2015.1094692DOI Listing
May 2016

Targeting chronic lymphocytic leukemia cells in the tumor microenviroment: A review of the in vitro and clinical trials to date.

World J Clin Cases 2015 Aug;3(8):694-704

Kyle Crassini, Stephen P Mulligan, O Giles Best, Northern Blood Research Centre and CLL Australian Research Consortium, Kolling Institute of Medical Research, Royal North Shore Hospital, Sydney NSW 2065, Australia.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. Despite significant advances in therapy over the last decade CLL remains incurable. Current front-line therapy often consists of chemoimmunotherapy-based regimens, most commonly the fludarabine, cyclophosphamide plus rituximab combination, but rates of relapse and refractory disease are high among these patients. Several key signaling pathways are now known to mediate the survival and proliferation of CLL cells in vivo, the most notable of which are the pathways mediated by the B-cell receptor (BCR) and cytokine receptors. A better understanding of the pathogenesis of the disease, the underlying biology of the CLL-cell and the roles of the tumour microenvironment has provided the rationale for trials of a range of novel, more targeted therapeutic agents. In particular, clinical trials of ibrutinib and idelalisib, which target the Brutons tyrosine kinase and the delta isoform of phosphoinositol-3 kinase components of the BCR signaling pathway respectively, have shown extremely promising results. Here we review the current literature on the key signaling pathways and interactions of CLL cells that mediate the survival and proliferation of the leukemic cells. For each we describe the results of the recent clinical trials and in vitro studies of novel therapeutic agents.
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http://dx.doi.org/10.12998/wjcc.v3.i8.694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4539409PMC
August 2015

The MEK1/2 inhibitor, MEKi-1, induces cell death in chronic lymphocytic leukemia cells under conditions that mimic the tumor microenvironment and is synergistic with fludarabine.

Leuk Lymphoma 2015 26;56(12):3407-17. Epub 2015 Aug 26.

a Northern Blood Research Centre, Kolling Institute of Medical Research, Royal North Shore Hospital , St Leonards, Sydney , NSW , Australia.

The Raf-1/MEK/ERK1/2 pathway has become a focus for novel cancer therapies. This study sought to investigate whether targeting MEK1/2 may represent a therapeutic option for chronic lymphocytic leukemia (CLL). The MEK1/2 inhibitor, MEKi-1, induced apoptosis of CLL cells and was synergistic with fludarabine under conditions that mimic the tumor microenvironment, irrespective of poor-risk characteristics. MEKi-1 down-regulated the activities of AKT and ERK1/2 and was synergistic with fludarabine through a mechanism that involved potentiation of DNA damage and attenuation of the activity of ERK1/2 and expression of Mcl-1. This study highlights the significant role of the mitogen-activated protein kinase (MAPK)-ERK1/2 pathway in mediating the effects of the CLL tumor microenvironment and suggests that targeting MEK1/2 in CLL cells may impact upon the activity of both ERK1/2 and AKT. Inhibitors of MEK1/2 as single agents or in combination with DNA-damaging agents may represent a novel therapeutic strategy for CLL.
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http://dx.doi.org/10.3109/10428194.2015.1032963DOI Listing
September 2016

Chronic lymphocytic leukaemia, monoclonal B-lymphocytosis and pregnancy: five cases, a literature review and discussion of management.

Br J Haematol 2015 Feb 25;168(3):350-60. Epub 2014 Sep 25.

Department of Haematology, Royal North Shore Hospital, St Leonards, Sydney, NSW, Australia; Kolling Institute, University of Sydney, St Leonards, Sydney, NSW, Australia; Chronic Lymphocytic Leukaemia Australian Research Consortium (CLLARC), Sydney, NSW, Australia.

Chronic lymphocytic leukaemia (CLL) occurs rarely with pregnancy and monoclonal B-Lymphocytosis (MBL) has not previously been described in this setting. CLL is predominantly a disease of the elderly and affects men twice as often as women and hence only an estimated 2% of patients are females of childbearing age. We identified only five reported cases of CLL in pregnancy in the literature. We describe two additional cases, plus three other women with CLL dealing with pregnancy-related decisions. We review the literature and discuss proposals for management and issues that arise in this relatively uncommon occurrence. In contrast to many other haematological malignancies where longer remissions are typically associated with a lower risk of relapse, most patients with CLL who require treatment will ultimately relapse with current therapy. This complex setting requires careful consideration and well informed patients to assist with decisions related to pregnancy.
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http://dx.doi.org/10.1111/bjh.13134DOI Listing
February 2015

Mechanisms of action of fludarabine nucleoside against human Raji lymphoma cells.

Nucleosides Nucleotides Nucleic Acids 2014 ;33(4-6):375-83

a School of Molecular Bioscience , University of Sydney , Sydney , NSW , Australia.

Fludarabine (2-FaraAMP) is a purine analog that is effective against chronic lymphocytic leukemia (CLL) and non-Hodgkins lymphoma (NHL). For some cases of CLL, 2-FaraAMP as a single agent can clear the blood of leukemia cells, but leukemia stem cells usually remain protected in sanctuary sites. It is clear that 2-FaraAMP has multiple mechanisms of action that may collectively result in strand breaks in DNA, accumulation of phosphorylated p53 and apoptosis. We have demonstrated using the human Burkitt's lymphoma B-cell line, Raji, that p53, p63 and p73 all accumulate in the nucleus, following treatment of cells with fludarabine nucleoside (2-FaraA). In addition, phosphorylated p53 accumulates in the cytosol and at mitochondria. Using sophisticated methods of proteomic analysis with mass spectrometry, proteins that become differentially abundant after treatment of cells with 2-FaraA have been identified, providing considerable additional information about the cellular responses of B-lymphoid cancers to this purine analog. The levels of proteins involved in the unfolded protein response increase, indicating that endoplasmic reticulum stress is likely to be one mechanism for induction of apoptosis. The levels of a number of proteins found on the outer plasma membrane change on cells treated with 2-FaraA, suggesting that signaling from the B-cell antigen receptor (BCR) is stimulated, resulting in induction of apoptosis through the intrinsic pathway. Increased levels of the cell surface proteins, CD50, CD100 and ECE-1, would promote survival of these cells; the balance between these survival and death responses would determine the fate of the cell.
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http://dx.doi.org/10.1080/15257770.2013.863334DOI Listing
February 2015

Profiles of surface mosaics on chronic lymphocytic leukemias distinguish stable and progressive subtypes.

J Pharm Pharm Sci 2013 ;16(2):231-7

School of Molecular Bioscience, University of Sydney, Sydney, NSW 2006, Australia.

Purpose: Chronic lymphocytic leukemia (CLL) is a heterogeneous disease, some patients may survive for many years, while 20-30% of patients progress and may die within several years. Currently, there is not a single procedure that enables accurate prognosis and triaging of those patients who need immediate and aggressive treatment. All CLL cells are characterised by the expression of the B-cell antigens CD19, CD20, CD21, CD22 and CD23, with aberrant expression of the T-cell antigen CD5.

Methods: We have developed a CD antibody microarray (DotScan) containing 182 immobilised CD antibodies that has been used to obtain extensive surface profiles of CLL cells obtained from 96 patients.

Results: Of these 182 antigens, 27 were significantly differentially expressed between stable, stable-progressive and progressive CLL. Some of these antigens are not expressed on normal B-cells and may be targets for therapeutic antibodies against CLL. Unsupervised hierarchical clustering of the surface profiles from 96 patients showed that those with progressive CLL could be distinguished based solely upon this 'disease signature'. The sensitivity (proportion of actual positives correctly identified) was 67.9%, the specificity (proportion of negatives correctly identified) was 77.5%, and the accuracy was 71.9%.

Conclusions: Considerable effort by a number of research groups has resulted in identification of individual markers for progressive CLL, but their collective use is yet to provide a test that identifies CLL patients at risk. Data presented here provide a basis for development of a simple test using an antibody microarray.
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http://dx.doi.org/10.18433/j3f01cDOI Listing
March 2014

Protein kinase C isoform expression in chronic lymphocytic leukemia: a potential target for therapy?

Leuk Lymphoma 2013 Oct 19;54(10):2098-9. Epub 2013 Apr 19.

Northern Blood Research Centre, Kolling Institute of Medical Research, Royal North Shore Hospital , NSW , Australia.

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http://dx.doi.org/10.3109/10428194.2013.779692DOI Listing
October 2013

Hsp90 Inhibitor SNX-7081 dysregulates proteins involved with DNA repair and replication and the cell cycle in human chronic lymphocytic leukemia (CLL) cells.

J Proteome Res 2013 Apr 22;12(4):1710-22. Epub 2013 Mar 22.

Cancer Proteomics Laboratory, School of Molecular Bioscience, University of Sydney , Sydney, NSW 2006, Australia.

The proteomic effects of the Hsp90 inhibitor, SNX-7081, have been determined on the p53-mutated B-cell chronic lymphocytic leukemia (CLL) cell line, MEC1. Following SNX-7081 treatment (500 nM, 24 h), 51 proteins changed abundance by more than 2-fold (p < 0.05); 7 proteins increased while 44 proteins decreased. Proteins identified as differentially abundant by LC-MS/MS were validated by Western blotting (DDB1, PCNA, MCM2, Hsp90, Hsp70, GRP78, PDIA6, HLA-DR). RT-PCR showed that SNX-7081 unexpectedly modulates a number of these proteins in MEC1 cells at the mRNA level (PCNA, MCM2, Nup155, Hsp70, GRP78, PDIA6, and HLA-DR). Pathway analysis determined that 3 of the differentially abundant proteins (cyclin D1, c-Myc and pRb) were functionally related. p53 levels did not change upon SNX-7081 treatment of p53 wild-type Raji cells or p53-mutated MEC1 and U266 cells, indicating that SNX-7081 has a p53-independent mechanism. The decreases in DDB1, MCM2, c-Myc, and PCNA and increases of pRb and cyclin D1 were confirmed in MEC1, U266, Raji, and p53 null HL60 cells by Western blotting. These data suggest that SNX-7081 arrests the cell cycle and inhibits DNA replication and r epair and provides evidence for the mechanism of the observed synergy between Hsp90 inhibitors and drugs that induce DNA strand breaks.
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http://dx.doi.org/10.1021/pr301055yDOI Listing
April 2013

The phosphoinositide 3-kinase pathway in chronic lymphocytic leukemia: evidence for phosphatase and tensin homolog deletion on chromosome 10 deregulation.

Leuk Lymphoma 2013 Jun 5;54(6):1123-4. Epub 2012 Dec 5.

Northern Blood Research Centre, Kolling Institute, Royal North Shore Hospital, Sydney, Australia.

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http://dx.doi.org/10.3109/10428194.2012.746685DOI Listing
June 2013

Immunoglobulin G subclass deficiency and infection risk in 150 patients with chronic lymphocytic leukemia.

Leuk Lymphoma 2013 Jan 8;54(1):99-104. Epub 2012 Sep 8.

Department of Haematology, Royal North Shore Hospital, Kolling Institute, University of Sydney, St Leonards, Sydney, Australia.

Hypogammaglobulinemia is a common complication of chronic lymphocytic leukemia (CLL), but the significance of immunoglobulin G (IgG) subclass deficiency is unknown. We analyzed the prevalence of immunoglobulins G, A and M, IgG subclass deficiency and infection in 150 patients with CLL. Low IgG, IgA and IgM levels were observed in 27.3%, 30.7% and 56.7% of patients, respectively. IgG subclass deficiency was frequent, with reduced IgG1, IgG2, IgG3 and IgG4 in 28%, 19.3%, 52% and 22.7% of patients, respectively. IgG subclass deficiency (total 64.6%) and hypogammaglobulinemia (27.3%) were more prevalent than clinically significant infection (16%). Recurrent or significant infections were seen in 24 patients (16%), of whom 50% had hypogammaglobulinemia but 100% had at least one IgG subclass deficiency, indicating that half the patients with infection had IgG subclass deficiency but normal total IgG level. Deficiencies of IgG3 and IgG4 were statistically associated with infection risk. Normal immunoglobulin and IgG subclass levels were seen in 26 patients (17%) and none had infections. IgG subclass deficiency is commonly observed in patients with CLL with both normal and reduced total IgG levels, and is associated with infection. Screening patients with CLL for IgG subclass deficiency may be a useful adjunct in stratifying their infection risk.
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http://dx.doi.org/10.3109/10428194.2012.706285DOI Listing
January 2013

Heat shock protein-90 inhibitor, NVP-AUY922, is effective in combination with fludarabine against chronic lymphocytic leukemia cells cultured on CD40L-stromal layer and inhibits their activated/proliferative phenotype.

Leuk Lymphoma 2012 Nov 9;53(11):2314-20. Epub 2012 Jul 9.

Royal North Shore Hospital, St Leonards, Sydney, NSW, Australia.

Chronic lymphocytic leukemia (CLL) involves disease infiltration into active proliferation centers within the lymph nodes and marrow. Successful treatment of CLL must involve targeting the leukemic cells in these supportive microenvironments. Our recent data suggest that inhibition of heat shock protein-90 (Hsp90) may be an effective treatment for CLL. We sought to further these data to determine whether the Hsp90 inhibitor, AUY922 (Novartis), is effective against CLL cells in a supportive in vitro environment. AUY922 significantly attenuated changes in immunophenotype and signal transducer and activator of transcription 3 (STAT3) signaling induced by CD40L-fibroblast co-culture but had no effect on the viability of CLL cells in this model. However, AUY922 in combination with fludarabine was significantly more effective at inducing apoptosis in cells in co-culture than either drug alone, an effect that was irrespective of ATM/TP53 dysfunction. In conclusion, our data suggest that further studies and clinical trials of AUY922 in combination with fludarabine may be warranted.
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http://dx.doi.org/10.3109/10428194.2012.698278DOI Listing
November 2012

Fludarabine nucleoside induces accumulations of p53, p63 and p73 in the nuclei of human B-lymphoid cell lines, with cytosolic and mitochondrial increases in p53.

Proteomics Clin Appl 2012 Jun;6(5-6):279-90

School of Molecular Bioscience, University of Sydney, Sydney, NSW, Australia.

Purpose: Human Raji cells treated with fludarabine nucleoside (2-FaraA, 3 μM) undergo apoptosis with accumulation of p53 in the nuclei as multiple phosphorylated isoforms and C-terminal truncated derivatives. Changes induced by 2-FaraA in the levels of p53, p63 and p73 in the nuclear, cytosolic and mitochondrial fractions have been determined in four human B-lymphoid cell lines that are TP53-functional (Raji and IM9) and TP53-mutated (MEC1 and U266).

Experimental Design: The B-lymphoid cell lines were treated with 2-FaraA (3 μM, 24 h, 48 h) and viability determined. Protein extracts of subcellular fractions from 2-FaraA-treated cells were analysed by 1D and 2D electrophoresis; multiple phosphorylated isoforms and truncated derivatives were identified by Western blots for p53, p63 and p73.

Results: p53 and p63 were present in all three fractions, while p73 was only detected in nuclei. After treatment with 2-FaraA, nuclear p53, p63 and p73 accumulated as multiple phosphorylated isoforms and truncated derivatives. The association of p63 with mitochondria in human cells is novel.

Conclusions And Clinical Relevance: Comprehensive information on the subcellular distributions and responses of p53, p63 and p73 to 2-FaraA provides additional insight into mechanisms for induction of apoptosis in the treatment of B-lymphoproliferative disorders with fludarabine.
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http://dx.doi.org/10.1002/prca.201200003DOI Listing
June 2012

Diagnostic techniques and therapeutic challenges in patients with TP53 dysfunctional chronic lymphocytic leukemia.

Leuk Lymphoma 2012 Nov 31;53(11):2105-15. Epub 2012 May 31.

Royal North Shore Hospital, St Leonards, NSW, Australia.

Abstract Aberrations of the TP53 pathway, whether by deletion or mutation, are increasingly recognized as one of the most important biological risk factors in chronic lymphocytic leukemia. Yet, there is little consensus on how to assess for TP53 defects in the clinic, and very few clinical studies to guide optimal management of such patients. In this review, we discuss the state-of-the-art in the assessment of the TP53 pathway, and review the evidence-base for therapeutic recommendations.
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http://dx.doi.org/10.3109/10428194.2012.692088DOI Listing
November 2012