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 effect of HYPE knock-out on the AMPylome of human OSU-CLL leukemia cells.

In humans, AMPylation of cellular proteins is carried out by Huntingtin yeast-interacting protein E (HYPE), activated under conditions of endoplasmic reticulum stress, such as in cancer cells. Extracts of the human chronic lymphocytic leukemia cell line, OSU-CLL, were fractionated using immuno-precipitation with antibodies against adenosine-phosphate and then AMP-Tyr. The proteins isolated were modified with AMP, the 'AMPylome.' AMP-labelled peptides isolated from HYPE wild-type (WT) and HYPE knock-out (KO) cells were identified using tandem mass spectrometry. A total of 213 proteins were identified from WT cell extracts, while only 23 of these were pulled down from KO cells, consistent with the presence of another AMPylator, besides HYPE. The KO cells were more sensitive to fludarabine nucleoside (2-FaraA) than WT cells. Ingenuity Pathway Analysis of the AMPylated proteins identified in WT cells clustered actin binding proteins of the cytoskeleton, and proteins of the RHO GTPase pathway that would jointly stimulate cell proliferation.

Fludarabine nucleoside induces major changes in the p53 interactome in human B-lymphoid cancer cell lines.

Triple combination FCR (fludarabine, cyclophosphamide and rituximab) is often used as front-line treatment for chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma. Results from our laboratory indicate that 2-FaraAMP (fludarabine) has multiple mechanisms of cytotoxicity that include accumulation of isoforms and phosphorylated derivatives of p53, and induction of the unfolded protein response (UPR). Using protein pull-downs with Dynabeads coated with p53 antibody, we have found that 2-FaraA (fludarabine nucleoside) induces major changes in the p53 interactome in human Raji lymphoma and IM9 multiple myeloma cells. These changes are likely driven by DNA strand breaks induced by 2-FaraA that activate protein kinases such as ATM, ATR and Chk1.

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.

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

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.

Molecular pathogenesis of chronic lymphocytic leukaemia.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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.

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.

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

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.

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

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.

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

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.