Human Chronic Lymphocytic Leukemia Research Articles

Expressionof Sf3b1- K700Ein Murine B Cells Causes Pre-mRNA Splicing and Altered B Cell Differentiation and Function

Mutations in the RNA splicing factor SF3B1 have been identified by large-scale sequencing as putative drivers in chronic lymphocytic leukemia (CLL), but their precise roles in the pathogenesis of CLL remains unknown. Although prior transcriptomic studies using primary CLL samples have led to the appreciation of altered RNA splicing in association with these mutations, understanding of their impact on cellular function has been complicated by their variable mutant allele frequency across samples as well as their common co-occurrence with other heterogeneous gene mutations. We therefore generated a mouse line that conditionally expresses the commonly occurring Sf3b1-K700E mutation at its endogenous murine locus. We obtained B-cell lineage specific expression of the mutant allele by crossing heterozygous floxed Sf3b1-K700E mice with homozygous CD19-Cre knockin mice. We confirmed that expression of the mutant allele was uniquely present as a heterozygous mutation in B cells, but not in other cell lineages.We sought to characterize the impact of Sf3b1-K700E on RNA splicing, B cell function, and CLL in this in vivo model. By unbiased RNA-sequencing of splenic B cells from wildtype and mutant mice (n=3), we profiled the splice isoform changes that were associated with Sf3b1-K700E. Using the tool JuncBASE, we detected, classified and quantified 54 differentially spliced transcripts (P<0.05, absolute delta percent spliced in >10%). Consistent with the altered splicing pattern reported in human CLL samples, the splice variants in our mouse model were highly enriched with altered selection of 3' splice sites (49 of 54 events, P<0.001). Of these, we validated 3 selected splice variants in independent samples by qPCR. Our murine model of Sf3b1-K700E mutation thus recapitulates altered RNA splicing, as per in human disease.We therefore next investigated whether Sf3b1-K700E affects B cell development and function. Splenic B cell numbers were significantly lower in the mutant (n=37) compared to control (n=33, P=0.0027) mice while T cell numbers were equivalent. Flow cytometric analysis of various B cell subpopulations from bone marrow and spleen revealed a significant increase in marginal zone B cells in the mutant mice (n=6, P<0.01). Consistent with this finding, we observed evidence of enlarged marginal zone areas on sections of mutant mouse spleens by visual inspection, with a mean reduction by 25% of proliferating germinal centers (per Ki67+ staining, n=6, average, P<0.05). On average, in vitro culture of splenic B cells with LPS and IL4 revealed mutant B cells to undergo 10-15% less proliferation, with reduced survival upon stimulation. Moreover, serum analysis revealed a 30-45% reduction in production of IgG1 and IgG3 from mutant mice (n=8, P<0.05). Altogether, these results suggest that mutant Sf3b1 induces an intrinsic B cell defect leading preferentially to impaired cellular proliferation.Cellular senescence has been commonly detected in pre-malignant lesions and is related to impaired cell proliferation. Indeed, by quantitative PCR array of 84 genes associated with cellular senescence, we found comparable levels of expression of all genes in T cells from wildtype and mutant splenocytes, but an overall trend of upregulation (range: by 1.5- to 21-fold) in the entire set of 84 genes in mutant B splenocytes, with significant upregulation in 20 genes (n=5, P<0.05). These genes included the critical senescence regulators Cdkn2a (p16) and Cdkn1a (p21), whose elevated expression in Sf3b1 mutated B cells we confirmed at the protein level. Furthermore, we detected increased levels of the cellular senescence mediators Igfbp6 and Igfbp7 in the sera from mutant mice (n=31, P<0.05). Collectively, the data demonstrate that expression of Sf3b1-K700E in B cells leads to the cellular senescent phenotype.While we observed that expression of Sf3b1-K700E results in RNA splicing changes, B cell developmental dysregulation and cellular senescence, expression of this mutation alone did not lead to expansion of CD5+CD19+ cells in vivo over time, despite observations up to 18-months (n=50). Our ongoing studies are now focused on the combined effects of Sf3b1-K700E and other recurrent CLL mutations on evasion of senescence and CLL disease progression. DisclosuresNo relevant conflicts of interest to declare.

Immunoliposomal Delivery of Mir-29b By Targeting Tumor Antigen ROR1 Induces Epigenetic Reprograming in Human-ROR1-Expressed Mouse Model of Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL), characterized by accumulation of CD5+CD19+sIgM+ B lymphocytes in peripheral blood and lymphoid organs, is classified into indolent and aggressive forms. Patients with indolent CLL generally survive 5 to 10 years and do not require treatment until severe symptoms, while those with aggressive CLL show resistant to standard treatment and survive less than 24 months. While emerging B cell antigen receptor directed therapies are promising, resistance to such therapies pose problems warranting novel therapeutic approaches. MicroRNA (miR) profiling revealed lower expression of miR-29b in aggressive CLL associated with survival, drug resistance and poor prognosis via its up-regulation of anti-apoptotic proteins myeloid leukemia cell differentiation protein 1 (Mcl1) and oncogenic T-cell leukemia 1 (Tcl1). Thus, specific overexpression of miR-29b in B-CLL cells could be a potential therapy for aggressive CLL patients. Despite the promise, short circulation half-life, limited cellular uptake and off-target effects on non-desirable tissues pose a challenge for miR-based therapies. To promote efficiency and specificity of miR-29b delivery, we developed neutral immunonanoparticles with selectivity to CLL via targeting tumor antigen ROR1, which is expressed in over 95% of CLL but not normal B cells. We optimized a novel 2A2-immunoliposome (2A2-ILP) recognizing surface ROR1 on primary CLL cell to promote internalization and miR-29b uptake (n=6, p=0.042*). About 20-fold increased uptake of miR-29b was achieved with 2A2-ILP-miR-29b formulation compared to control. Further ROR1 targeted delivery of miR29b resulted in significant downregulation of DNMT1 and DNMT3a mRNA and protein (n=3, DNMT1: p= 0.0115*; DNMT3a: p=0.0231*, SP1; p=0.0031**) in primary CLL cells and a human CLL cell line OSU-CLL. Consistent with the downregulation of DNMTs, decreased global DNA methylation was observed in OSU-CLL cell line one week post- treatment with 2A2-ILP-miR-29b (n=3, p=0.0003***). To further study the in vivo ROR1-targeting efficiency of 2A2-ILP-miR-29b, we used our recently described Eµ-hROR1x Tcl1 CLL mouse model that develops CLL like disease with human ROR1 antigen in leukemic CD19+CD5+ B cells. Using hROR1+CD19+CD5+ leukemic cell engraftment model, we showed significant in-vivo efficacy of ROR1-ILP-miR-29b formulation associated with a) decreased number of circulating leukemic B220+CD5+ cells b) reduced splenomegaly (p=0.0461*, 2A2-29b: n=9; PBS: n=8) c) with extended survival (p=0.0075**, 2A2-29b: n=9; IgG-29b: n=7; 2A2-SC: n=7; PBS: n=8). In summary, 2A2-ILP effectively delivered functional miR-29b, resulting in downregulation of DNMT1 and DNMT3a, reduction of hypermethylation and anti-leukemic activity. Ongoing studies are aimed at understanding miR-29b mediated in-vivo methylome reprograming using our novel hROR1xTcl1 transgenic mouse model and ROR1-targeted miR-29b delivery formulation. [Display omitted] DisclosuresByrd:Acerta Pharma BV: Research Funding.

The Correlation of APOBEC Gene Family Member Expression with Worse CLL Patient Outcome Suggests a Role in CLL Mutational Evolution

A mutational signature consistent with APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide) activity has been identified in somatic mutations found in large-scale surveys of ultra-deep sequencing data from many human cancers including chronic lymphocytic leukemia (CLL). APOBEC is a cytidine deaminase family made up of eleven genes, including AID (activation-induced cytidine deaminase) and APOBEC3B, both of which have been implicated in somatic mutation in various cancers, including CLL. These observations have led to the hypothesis that APOBEC cytidine deaminases may be driving somatic mutations leading to the development of more aggressive cancers. Therefore, we examined APOBEC gene family member RNA expression levels in CLL to test for correlations with expression levels and patient outcome. We further examined if CLL cells generated de novo APOBEC family member mutational patterns in the immunoglobulin variable region gene (IGHV) after implantation in a mouse xenograft model of CLL.CLL peripheral blood mononuclear cells (PBMCs) and associated clinical data were collected from patients after informed consent as approved by the Institutional Review Board at the North Shore-Long Island Jewish Health System and in accordance with the Helsinki Declaration. CLL samples were chosen based on availability with no pre-established inclusion/exclusion criteria. CLL RNA expression levels were examined by microarray or quantitative real-time PCR (qPCR). For microarray studies, CLL B cells were purified prior to RNA isolation and acquisition of microarray expression data using Illumina Human WG6 and HT12 bead chips, followed by quantile normalization using GenomeStudio software (Illumina). For qPCR, RNA expression from CLL PBMCs was measured relative to glyceraldehyde 3-phosphate dehydrogenase gene expression by Taqman assay with Roche UPL probes and LightCycler 480. To examine de novo mutations in CLL, the IGHV region was ultra-deep sequenced (Roche 454 FLX system) from human CLL cells recovered from the NOD/Shi-scid,γcnull (NSG) xenograft mouse model of CLL as approved by the Institutional Animal Care and Use Committee at the North Shore-Long Island Jewish Health System.CLL patient (N = 65) RNA expression by microarray showed very low levels of APOBEC1, 2, 3A, 3B, 3D, 4, and AID, modest levels of APOBEC3C and 3H, and high levels of APOBEC3F and 3G. Higher AID expression levels significantly correlated (P <0.05) with shorter time to first treatment (TFT), which was anticipated based on previous reports. Interestingly APOBEC3B and APOBEC3F expression differences showed possible trends correlating with worse patient outcome. Therefore, we tested select APOBEC gene family members by qPCR. For qPCR, we utilized the CLL patient cohort (N= 83) previously found to indicate that AID expression was a risk factor for worse patient outcome in a multivariate analysis (Patten et al. 2012 Blood 120:4802). RNA expression by qPCR followed the same pattern as the microarray data: AID and APOBEC3B had very low levels, APOBEC3H had modest levels, and APOBEC3F and 3G had high levels. Similar to AID, patients could be grouped based on the presence or absence of detectable APOBEC3B, with its presence showing a significant correlation (P <0.05) with worse TFT and overall survival. Higher levels of APOBEC3F and 3H showed a trend towards a correlation with shorter TFT, while differences in APOBEC3G expression had no significant correlation with patient outcome. Thus, not only did we confirm the correlation of AID expression with worse patient outcome, but we also found APOBEC3B and potentially APOBEC3F and 3H correlate with worse patient outcome.To test if CLL cells can acquire de novo mutations indicative of APOBEC gene family member activity, human CLL cells were transferred into NSG mice. After CLL cells proliferated for 4-14 weeks in this xenograft model, the IGHV region was amplified, ultra-deep sequenced, and analyzed for specific mutational characteristics of various APOBEC gene family members. The results of these ongoing analyses will be presented.In conclusion, the expression levels of the APOBEC gene family members AID, APOBEC3B, and potentially APOBEC3F and 3H, correlate with worse patient outcome. These data are consistent with the hypothesis that APOBEC gene family member activity may promote new mutations at sites outside the IG gene loci leading to the evolution of aggressive CLL. DisclosuresBarrientos:Pharmacyclics, Celgene, and Genentech: Membership on an entity's Board of Directors or advisory committees; Gilead, Pharmacyclics, and AbbVie: Research Funding.

The Eµ-Myc/TCL1 Transgenic Mouse As a New Aggressive B-Cell Malignancy Model Suitable for Preclinical Therapeutics Testing

Background: Aggressive B-cell lymphomas occurring in the setting of Chronic Lymphocytic Leukemia (CLL) as a large cell transformation are an important clinical problem, and improved mouse models to test novel and targeted therapeutics are needed. The Eµ-Myc mouse overexpresses c-Myc gene which is placed under control of the Myc promoter and lymphoid-specific IgH enhancer (Eµ), resulting in c-Myc overexpression and spontaneous B-cell lymphoma development. The Eµ-Myc mice have been used in drug development, however malignancy develops at variable ages and with differing genetics and response to therapeutic agents, making drug studies difficult. The Eµ-TCL1 transgenic mouse overexpresses the human TCL1 oncogene, under the control of the B-cell specific IgVH promoter and Eµ enhancer. Mice develop a spontaneous mature B-cell leukemia after a long latency period and represent a well-established model of human CLL. We crossed the Eµ-Myc and TCL1 mice to create a new model of aggressive B-cell lymphoma to test novel therapeutics that would be more homogeneous than the Eµ-Myc model.Methods: Eµ-Myc and TCL1 mice on C57BL/6 background strain were crossed and resulting genotypes were verified by PCR. All mice were kept in standard pathogen-free housing until death or removal from study. All mice born within a 24 month time period were followed for survival. At the time of death, spleen, lymph nodes, bone marrow, and liver were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 4uM, and stained with hematoxylin and eosin for histologic evaluation. For engraftment studies, adoptive cell transfer was done by injecting fresh splenic white blood cells (1e7 cells, >75% CD5+/CD19+) into the tail vein of C57BL/6 wild type recipient mice. In the drug treatment experiments ibrutinib (~30mg/kg/day) was administered in drinking water and KPT-8602 (15mg/kg) by daily oral gavage. Survival was shown using Kaplan-Meier curves and survival curves compared statistically using a log-rank test.Results: The Eµ-Myc/TCL1 mice had significantly worse survival (median 45.5, range 29-74 days) compared to Eµ-Myc (median 118, range 42-520 days) or TCL1 (median 359.5, range 188-531 days) mice (p=<0.0001). Survival curves are show in Figure 1. Pathologic examination revealed aggressive B-cell histology similar to Burkitt lymphoma in both the Eµ-Myc mice and Eµ-Myc/TCL1 mice, whereas the TCL1 mice showed mature B-cell morphology. Infiltration of the bone marrow of Eµ-Myc and Eµ-Myc/TCL1 mice was extensive in comparison with TCL1 mice. In all three mouse genotypes the normal architecture in the spleen was effaced by neoplastic cells. Adoptive transfer of Eµ-Myc/TCL1 splenic lymphocytes produced similar pathologic findings to the Eµ-Myc/TCL1 transgenic mice with less severe splenic enlargement in the engrafted mice. Median survival of the engrafted mice was short (38 days). There was a differential response to targeted therapeutics between the groups. Ibrutinib, an irreversible inhibitor of Bruton tyrosine kinase (BTK) previously demonstrated to improve survival in TCL1 mice, had no survival advantage over vehicle treatment in either Eµ-Myc or Eµ-Myc/TCL1 mice (p=0.876 and P=0.83 respectively). KPT-8602, a second generation selective inhibitor of exportin 1 (XPO1) was tested in Eµ-Myc/TCL1 engrafted mice, and demonstrated a significant improvement in survival compared to vehicle alone (p=<0.0001). A first generation agent (selinexor) targeting XPO1 is currently in human clinical trials for aggressive lymphomas, including large cell transformation of CLL.Conclusions: Mice with both c-Myc and TCL1 transgenes develop an aggressive B-cell malignancy and have decreased survival compared to Eµ-Myc or TCL1 mice. The histology of the disease is similar to that of Eµ-Myc mice, but with shorter survival with less variability. The resulting mouse model has differential responses to targeted therapeutics, with a poor response to ibrutinib more similar to aggressive lymphoma than CLL. The malignant B cells can be adoptively transferred into immunocompetent mice for experimental drug treatment studies. These qualities make Eµ-Myc/TCL1 mice a useful tool to test new therapies for aggressive lymphoma where there is an unmet clinical need. Work is currently underway to further define the genetic characteristics of this mouse in comparison to human lymphoma sub-types and responsiveness to targeted agents. [Display omitted] DisclosuresByrd:Acerta Pharma BV: Research Funding.

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.

Cd1d regulates B cell development but not B cell accumulation and IL10 production in mice with pathologic CD5(+) B cell expansion.

BackgroundCD1d is a widely expressed lipid antigen presenting molecule required for CD1d-restricted invariant natural killer T (iNKT) cell development. Elevated CD1d expression is detected in CD5+ IL10-producing B cells, called B10 B cells, and is correlated with poorer prognosis in chronic lymphocytic leukemia (CLL), a CD5+ B cell malignancy with B10-like functional properties. Whether CD1d expression regulates CD5+ B cell accumulation, IL10 competence, and antibody production in naïve mice with pathologic CD5+ B cell expansion remains untested.ResultsUsing three different transgenic mouse models of benign or leukemic CD5+ B cell expansion, we found that CD1d was differentially expressed on CD5+ B cells between the three models, but loss of CD1d expression had no effect on CD5+ B cell abundance or inducible IL10 expression in any of the models. Interestingly, in the CLL-prone Eμ-TCL1 model, loss of CD1d expression suppressed spontaneous IgG (but not IgM) production, whereas in the dnRAG1xEμ-TCL1 (DTG) model of accelerated CLL, loss of CD1d expression was associated with elevated numbers of splenic CD4+ and CD8+ T cells and an inverted CD4+:CD8+ T cell ratio. Unexpectedly, before leukemia onset, all three transgenic CD1d-deficient mouse strains had fewer splenic transitional B cells than their CD1d-proficient counterparts.ConclusionsThe results show that CD1d expression and iNKT cells are dispensable for the development, accumulation, or IL10 competence of CD5+ B cells in mice prone to benign or leukemic CLL-like B cell expansion, but reveal a novel role for iNKT cells in supporting B cell progression through the transitional stage of development in these animals. These results suggest CD1d-directed therapies to target CLL could be evaded by downregulating CD1d expression with little effect on continued leukemic CD5+ B cell survival. The data also imply that iNKT cells help restrain pro-leukemic CD8+ T cell expansion in CLL, potentially explaining a reported correlation in human CLL between disease progression, the loss of NKT cells, and a paradoxical increase in CD8+ T cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-015-0130-z) contains supplementary material, which is available to authorized users.

Standardized Extract of the Persian Gulf Sponge, Axinella Sinoxea Selectively Induces Apoptosis through Mitochondria in Human Chronic Lymphocytic Leukemia Cells

Sponges are important components of the Persian Gulf animal communities. The marine sponges of the genus Axinella sinoxea is are a genus of sponges in the family Axinellidae. Species of Axinella sinoxea occur in the India, Pacific Oceans and also Persian Gulf. Chronic lymphocytic leukemia (CLL) is a disease characterized by the relentless accumulation of CD5+ B lymphocytes. CLL is the most common leukemia in adults, about 25-30% of all leukemias. In this study B lymphocytes mitochondria (both cancerous and non-cancerous) were isolated using differential centrifugation from peripheral blood samples and succinate dehydrogenase activity, mitochondrial reactive oxygen species (ROS) production, collapse of mitochondrial membrane potential (MMP), mitochondrial swelling and finally release of cytochrome C were examined following the addition of methanolic extract of Axinella sinoxea. Our results showed that only in mitochondria isolated from cancerous BUT NOT normal lymphocytes a significant (P &lt; 0.05) increase in mitochondrial ROS formation, MMP collapse, mitochondrial swelling and cytochrome c release. These results showed that Axinella sinoxea extract has a selective toxicity on chronic lymphocytic leukemia lymphocytes and their mitochondria and hence may be considered as a promising anti CLL candidate for further studies needed as a supplement for cancer patients in the future.

ID: 63: Defective IFN-signaling through endoplasmic reticulum causes loss of Autophagy resulting in human chronic lymphocytic leukemia progression

The molecular and genetic bases for the progression of chronic lymphocytic leukemia (CLL), an indolent disease, are poorly understood. The Death associated protein kinase 1 (DAPK1), an IFN-inducible calcium-calmodulin-dependent serine/threonine kinase, is a critical regulator of autophagy and apoptosis. Its expression is frequently suppressed in a wide variety of tumors including CLL. Recently, we have identified a novel non-STAT signaling pathway, where in an endoplasmic resident transcription factor ATF6, in association with the transcription factor C/EBP- β regulates the IFN- γ -induced expression of DAPK1. IFN- γ -induced proteolytic processing of ATF6 and phosphorylation of C/EBP- β are obligatory for the formation of ATF6/C/EBP- β transcriptional complex that regulates DAPK1. Our studies show that in B-CLL, failure of IFN- γ activated functional collaboration between C/EBP- β and/or ATF6 contributes to a loss of growth control via autophagy. We also show a correlation between loss of DAPK1 levels and a failure of ATF6 and/or C/EBP- β activation primary CLL tumors. Restoration of ATF6 or C/EBP- β into respective defective-primary tumor cells, re-established DAPK1 expression. Furthermore, IFN- γ and chemotherapeutics failed to activate autophagy in CLL patient samples lacking ATF6 or C/EBP- β .Together, these results identify a mechanistic basis for the loss of DAPK1 expression, drug resistance and progression of CLL.

NOD/SCID IL2Rγ-null mouse xenograft model of human p53-mutated chronic lymphocytic leukemia and ATM-mutated mantle cell lymphoma using permanent cell lines

Xenograft models represent a promising tool to study the pathogenesis of hematological malignancies. To establish a reliable and appropriate in vivo model of aggressive human B-cell leukemia and lymphoma we xenotransplanted four p53-mutated cell lines and one ATM-mutated cell line into immunodeficient NOD/SCID IL2Rγ-null mice. The cell lines MEC-1, SU-DHL-4, JEKO-1, REC-1, and GRANTA-519 were transplanted intraperitoneally or subcutaneously and the engraftment was investigated using immunohistochemistry and flow cytometry. We found significant differences in engraftment efficiency. MEC-1, JEKO-1 and GRANTA-519 cell lines engrafted most efficiently, while SU-DHL-4 cells did not engraft at all. MEC-1 and GRANTA-519 massively infiltrated organs and the whole intraperitoneal cavity showing very aggressive growth. In addition, GRANTA-519 cells massively migrated to the bone marrow regardless of the transplantation route. The MEC-1 and GRANTA-519 cells can be especially recommended for in vivo study of p53-mutated chronic lymphocytic leukemia and ATM-mutated mantle cell lymphoma, respectively.

Targeting deposited complement component C3d to potentiate monoclonal antibody cancer therapy (VAC3P.1057)

Abstract Treatment-induced loss of targeted cell surface antigens through trogocytosis or internalization reduces efficacy of monoclonal antibody (mAb) therapy of cancer. However, cells that escape therapy mediated by complement-fixing mAbs carry covalently deposited complement activation fragments on their cell surfaces, in particular C3d. We hypothesized that cell-associated C3d constitutes a neoantigen that could be exploited to selectively retarget cells escaping from therapeutic mAbs. We generated an anti-C3d IgG1 human/mouse chimeric mAb specific for human C3d that is not competed by C3 in human serum. For proof of concept, we used cells from chronic lymphocytic leukemia (CLL) patients that had substantially reduced CD20 levels due to treatment with the anti-CD20 mAb ofatumumab (OFA). The chimeric anti-C3d mAb bound cell surface C3d on these CLL cells ex vivo (KD = 6.7nM), and mediated complement-dependent, and antibody-dependent cellular cytotoxicity and phagocytosis in vitro. CLL cells opsonized by C3d in vivo and reacted with the anti-C3d mAb in vitro were further C3d opsonized, resulting in an amplification loop that enhanced anti-C3d mAb binding capacity and killing of target cells. In vivo, the anti-C3d mAb was effective in reducing tumor burden in blood and spleens of NSG mice xenografted with human CLL cells. Our results identify anti-C3d mAbs as a means to specifically and potently augment the therapeutic efficacy of complement-fixing mAbs.

Hedgehog/GLI and PI3K signaling in the initiation and maintenance of chronic lymphocytic leukemia.

The initiation and maintenance of a malignant phenotype requires complex and synergistic interactions of multiple oncogenic signals. The Hedgehog (HH)/GLI pathway has been implicated in a variety of cancer entities and targeted pathway inhibition is of therapeutic relevance. Signal cross-talk with other cancer pathways including PI3K/AKT modulates HH/GLI signal strength and its oncogenicity. In this study, we addressed the role of HH/GLI and its putative interaction with the PI3K/AKT cascade in the initiation and maintenance of chronic lymphocytic leukemia (CLL). Using transgenic mouse models, we show that B-cell-specific constitutive activation of HH/GLI signaling either at the level of the HH effector and drug target Smoothened or at the level of the GLI transcription factors does not suffice to initiate a CLL-like phenotype characterized by the accumulation of CD5+ B cells in the lymphatic system and peripheral blood. Furthermore, Hh/Gli activation in Pten-deficient B cells with activated Pi3K/Akt signaling failed to enhance the expansion of leukemic CD5+ B cells, suggesting that genetic or epigenetic alterations leading to aberrant HH/GLI signaling in B cells do not suffice to elicit a CLL-like phenotype in mice. By contrast, we identify a critical role of GLI and PI3K signaling for the survival of human primary CLL cells. We show that combined targeting of GLI and PI3K/AKT/mTOR signaling can have a synergistic therapeutic effect in cells from a subgroup of CLL patients, thereby providing a basis for the evaluation of future combination therapies targeting HH/GLI and PI3K signaling in this common hematopoietic malignancy.

Generation of a poor prognostic chronic lymphocytic leukemia-like disease model: PKCα subversion induces up-regulation of PKCβII expression in B lymphocytes.

Overwhelming evidence identifies the microenvironment as a critical factor in the development and progression of chronic lymphocytic leukemia, underlining the importance of developing suitable translational models to study the pathogenesis of the disease. We previously established that stable expression of kinase dead protein kinase C alpha in hematopoietic progenitor cells resulted in the development of a chronic lymphocytic leukemia-like disease in mice. Here we demonstrate that this chronic lymphocytic leukemia model resembles the more aggressive subset of chronic lymphocytic leukemia, expressing predominantly unmutated immunoglobulin heavy chain genes, with upregulated tyrosine kinase ZAP-70 expression and elevated ERK-MAPK-mTor signaling, resulting in enhanced proliferation and increased tumor load in lymphoid organs. Reduced function of PKCα leads to an up-regulation of PKCβII expression, which is also associated with a poor prognostic subset of human chronic lymphocytic leukemia samples. Treatment of chronic lymphocytic leukemia-like cells with the selective PKCβ inhibitor enzastaurin caused cell cycle arrest and apoptosis both in vitro and in vivo, and a reduction in the leukemic burden in vivo. These results demonstrate the importance of PKCβII in chronic lymphocytic leukemia-like disease progression and suggest a role for PKCα subversion in creating permissive conditions for leukemogenesis.

Chrysin Induces Apoptosis in Peripheral Blood Lymphocytes Isolated from Human Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) develops due to an imbalance between apoptosis and proliferation of B lymphocytes. Chrysin induced apoptosis in leukemia cell lines such as U937, MO7e, THP-1 and HL-60, but there has not yet been data demonstrating the apoptotic effect of chrysin on CLL cells. Therefore, in our investigation we examined the cytotoxicity of chrysin against two leukemia cell lines, MOLT-4 and JVM-13, peripheral blood lymphocytes isolated from B-CLL patients and peripheral blood mononuclear cells (PBMC) from healthy individuals in vitro. The effect of chrysin on viability of MOLT-4 and JVM-13 cell lines, B-CLL cells derived from 28 patients and PBMC from 16 healthy subjects was determined by MTT assay. The type of cell death induced by chrysin was verified by Annexin V/7AAD assay and acridine orange and ethidium bromide (AO/EB) staining assay. Intracellular localisation and endogenic expression of apoptotic proteins including Bax, Bcl-2, cytochrome c and caspase-3 were determined by flow cytometry and fluorescent microscopy. Our results demonstrated that exposure of MOLT-4, JVM-13 cell lines and B-CLL cells to the concentration of chrysin of 10μM and higher selectively decreased viability of cells in this cell population, but not in the PBMC derived from healthy subjects; LC50 values of chrysin for B-CLL cells were 51μM for 24 hours and 32μM for 48 hours of incubation, respectively. Our findings demonstrated that chrysin induces the activation of proapoptotic Bax and decreases the expression of antiapoptotic Bcl-2 protein, releases cytochrome c from mitochondria into cytosol and cleavages/activates caspase-3, subsequently leading to the activation of apoptosis of B-CLL cells. Together, these findings suggest that chrysin selectively induces apoptosis of peripheral blood lymphocytes isolated from human chronic lymphocytic leukemia patients via mitochondrial pathway in vitro and that it might have a promising role as a potential future antileukemic remedy.

Effects of TNF secreting HEK cells on B lymphocytes' apoptosis in human chronic lymphocytic leukemias.

Tumor necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL) is an antitumor candidate in cancer therapy. This study focused on effects of TRAIL, as a proapototic ligand that causes apoptosis, in B-CELL chronic lymphocytic leukemia cells (B-CLL) . A population of HEK 293 cells was transducted by lentivirus that these achieved ability for producing the TRAIL protein and then HEK 293 cells transducted were placed in the vicinity of CLL cells. After 24 hours of co-culture, apoptosis of CLL cells was assessed by annexin V staining. The amount of Apoptosis was examined separately in four groups: 293 HEK TRAIL (16.17±1.04%); 293 HEK GFP (2.7±0.57%); WT 293 HEK (2±2.6%); and CLL cells (0.01±0.01%). Among the groups studied, the maximum amount of apoptosis was in the group that the vector encoding TRAIL was transducted. In this group, the mean level of soluble TRAIL in the culture medium was 253 pg/ml; also flow cytometry analyzes showed that proapotosis in this group was 32.8±1.6%, which was higher than the other groups. In this study, we have demonstrated that TNF secreted from HEK 293 cells are effective in death of CLL cells.

Harnessing the fcμ receptor for potent and selective cytotoxic therapy of chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a B-cell malignancy in need of new, effective, and safe therapies. The recently identified IgM receptor FcμR is overexpressed on malignant B cells in CLL and mediates the rapid internalization and lysosomal shuttling of IgM via its Fc fragment (Fcμ). To exploit this internalization and trafficking pathway for targeted drug delivery, we engineered an IgM-derived protein scaffold (Fcμ) and linked it with the cytotoxic agent monomethylauristatin F. This Fcμ-drug conjugate was selectively toxic for FcμR-expressing cell lines in vitro and for CLL cells but not autologous normal T cells ex vivo. Notably, the cytotoxic activity of the Fcμ-drug conjugate was maintained in CLL cells carrying a 17p deletion, which predicts resistance to standard chemotherapy. Next, we tested the possible therapeutic application of the Fcμ-drug conjugate in immunodeficient NOD/SCID/IL-2Rγ(null) (NSG) mice engrafted with peripheral blood cells from patients with leukemia. Three intravenous injections of the Fcμ-drug conjugate over a 10-day period were well tolerated and selectively killed the human CLL cells but not the coengrafted autologous human T cells. In summary, we developed a novel strategy for targeted cytotoxic therapy of CLL based on the unique properties of FcμR. FcμR-targeted drug delivery showed potent and specific therapeutic activity in CLL, thus providing proof of concept for FcμR as a valuable therapeutic target in CLL and for IgM-based antibody-drug conjugates as a new targeting platform.

Stereospecific ligands and their complexes. XXII. Synthesis and antitumor activity of palladium(II) complexes with some esters of (S,S)-ethylenediamine-N,N′-di-(2,2′-di(4-hydroxy-benzyl))-acetic acid

Four new ligands and their palladium(II) complexes of general formula R2-S,S-eddtyr (L1–L4) and [PdCl2(R2-S,S-eddtyr)] (C1–C4) (R=ethyl, n-propyl, n-butyl and n-pentyl; S,S-eddtyr·2HCl=ethylenediamine-N,N′-di-(2,2′-di(4-hydroxy-benzyl))-acetic acid dihydrochloride have been synthesized and characterized by microanalysis, infrared, 1H and 13C NMR spectroscopy. Cytotoxicity for ligands and complexes on two different cell lines (human breast cancer, MDA-MB-231 and human lung cancer, A549 cell lines) and human chronic lymphocytic leukemia cells (CLL) was investigated using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay.

Pharmacologic Inhibition with Duvelisib (IPI-145) and Genetic Inhibition of PI3K p110δ Antagonizes Intrinsic and Extrinsic Survival Signals in Chronic Lymphocytic Leukemia (CLL)

Background: Chronic lymphocytic leukemia (CLL), the most common adult leukemia in the western world, remains an incurable B-cell malignancy. It is characterized by the accumulation of malignant mature B cells in the blood, lymph nodes, spleen, and bone marrow. CLL cells display up-regulated B-cell receptor (BCR) activation, which maintains B cell survival and proliferation through transmitting microenvironmental stimuli. Due to aberrant regulation of the BCR, CLL cells display constitutively activated survival and proliferation pathways, such as phosphoinositide-3 kinase (PI3K) and Bruton’s tyrosine kinase (BTK) pathways. Small molecules that target such kinases in the BCR pathway have shown significant clinical activity in CLL patients. Both the PI3K p110δ inhibitor, idelalisib, and the BTK inhibitor, ibrutinib, have received approval by FDA for treatment of relapsed CLL. However, patients still relapse on these therapies.Methods/Results: Here we use pharmacologic and genetic approaches to further characterize the role of PI3K signaling in the leukemia pathogenesis in the CLL cell and in the microenvironment. We describe that a PI3K p110δ and p110γ inhibitor, duvelisib (IPI-145), which is in late stage clinical development, attenuates pro-survival signals in the OSU-CLL cell line and primary human and murine CLL cells and promotes apoptosis and downstream pathway inactivation in primary human and murine CLL cells in a dose- and time-dependent fashion. To examine the cytotoxicity of duvelisib in normal immune cells, we incubated whole blood from CLL patients with 0.25-5 μM duvelisib for 48 hours and analyzed by flow cytometry for absolute count of live CD3+ T cells, CD56+ NK cells and CD19+ B cells. T cells and NK cells were sensitive to duvelisib, displaying about 20% decrease in viability at concentrations greater than 0.5μM; however, the B cell population showed about 50% decrease in viability. To specifically examine normal B cells, we isolated CD19+ B cells from healthy volunteer blood and incubated with 1 μM duvelisib for 48 hours and observed no cytotoxicity, despite observing a significant decrease in CLL cells viability under the same conditions. Additionally, duvelisib is highly effective at reducing downstream PI3K signaling in a B cell line with the ibrutinib resistance conferring BTK C481S mutation. Genetically we show that the PI3K p110δ-inactivating and the TCL1 leukemia murine models can be utilized to further explore the differential role of PI3K p110δ in the leukemic cell and microenvironment. Our study indicates that systemic disruption of PI3K p110δ function in the TCL1 mouse significantly prevents spontaneous leukemia development, indicating that PI3K p110δ is a critical kinase for CLL disease initiation and expansion. Moreover, inactivation of PI3K p110δ in the microenvironment showed a dose-dependent effect in delaying leukemia engraftment. This suggests that PI3K p110δ activity is also critical in the non-B cell compartment for leukemia progression. While our group has focused on the role of PI3K p110δ, we continue to examine the role of PI3K p110γ using the PI3K p110δ and p110γ inhibitor, duvelisib. DisclosuresDubovsky:Principia Inc.: Research Funding. Kutok:Infinity Pharmaceuticals, Inc.: Employment, Equity Ownership. Byrd:Pharmacyclics: Research Funding.

Histone Deacetylase 6 (HDAC6) As a Regulator of Immune Check-Point Molecules in Chronic Lymphocytic Leukemia (CLL)

Co-inhibitory receptors and ligands such as PD-1, PD-L1, TIM3 and LAG3 are commonly over expressed in malignancies resulting in immune-surveillance evasion and generation of a tumorigenic microenvironment. Targeting of these molecules has become a new therapeutic strategy demonstrating promising and favorable clinical outcomes in a variety of human cancers. Although, inhibitory antibody therapy has been successful for many patients, other subsets of patients receive no clinical benefit. This is potentially due to the activation of redundant untargeted co-inhibitory pathways. In CLL, an immunosuppressive phenotype enables the malignant B-cell to evade immune detection and obtain a vast array of immune regulatory mechanisms, causing T-cell dysfunction and ultimately immunosuppression. In fact CD8+ effector and terminal memory subsets are skewed and over-express PD-1, LAG3 and TIM3 in a Eu-TCL1 CLL mice model. Epigenetic changes triggered by proteins such as histone deacetylases are gaining special attention particularly because of their active role in the regulation of pathogenesis and immune-related pathways in CLL; however the exact mechanism of action is yet to be elucidated. Recently, we have identified one member of the HDAC family, HDAC6 which plays a role in the regulation of immunogenicity in CLL. Utilizing CLL murine models, CLL patient samples, and CLL cell lines we have found the following: First, HDAC6 is overexpressed in human CLL Second, using selective HDAC6 inhibitor (ricolinostat) we have been able to modulate the expression of co-inhibitory molecules in CLL. Indeed, treatment of MEC1 cells with varying doses of ricolinostat for 48hrs resulted in decreased expression of PD-L1 Interestingly in primary B-cells isolated from CLL patient samples; PD-1 expression was decreased when exposed to various concentrations of ricolinostat for 48hrs. Third, silencing of HDAC6 in the Eu-TCL1 mice (Eu-TCL1/HDAC6KO mouse model) results in 1) significantly lower levels of PD-L1 expression and 2) a restoration of the T-cell CD4:CD8 ratio, from 8:10 (Eu-TCL1) to 1:1 (Eu-TCL1/HDAC6KO). Although the overall absolute count of T-cells was observed to be higher than the wild-type, the memory compartment was returned to mostly naive T-cells, doubling in numbers from 4% of total lymphocytes to 9%. Consequently, a total reduction in surface expression of co-inhibitory molecules was observed on all T-cell memory subsets, but with substantial reduction in the CD8+ effector and terminal memory phenotype. In conclusion, selective inhibition of HDAC6 in both B and T-cell compartment of CLL results in the reduction of co-inhibitory molecules, which in combination with subsequent therapeutic regimen could provide a successful immunotherapeutic strategy for this disease. DisclosuresQuayle:Acetylon Pharmaceuticals: Employment, Equity Ownership. Jones:Acetylon Pharmaceuticals Inc.: Employment.

Notch2 Is Critical for CLL Development in the IRF4-/-Vh11 Mice

Chronic Lymphocytic Leukemia (CLL) is the most common adult leukemia in the western world. Despite considerable progress in our current understanding of CLL, the molecular events underlying the complex pathogenesis and clinical behavior of CLL have not been fully elucidated. IRF4 belongs to the IRF superfamily of transcription factors and plays critical roles at multiple stages of B cell development. Genome Wide Association Studies had linked Single Nucleotide Polymorphism (SNP) mediated IRF4 down regulation to the pathogenesis of human CLL (Di Bernardo et al. Nature Genetics, 2008). Moreover, low levels of IRF4 were found to correlate with poorer disease outcomes in human CLL (Chang et al. Blood, 2002). More recently, the studies from our group have established a causal relationship between low levels of IRF4 and development of CLL. We used Vh11 knock-in (Vh11) mice that express a prearranged immunoglobulin variable heavy chain (Vh11) leading to preferential generation of B1 cells (CLL precursors). Interestingly, we have shown that mice expressing the Vh11 transgene in the IRF4 deficient background (IRF4-/-Vh11) spontaneously develop CLL at complete penetrance (Shukla et al. Blood, 2013). Additionally, we have also shown that low levels of IRF4 dramatically accelerate CLL development in the New Zealand Black (NZB) mice (Ma et al. JBC, 2013). Although our studies have demonstrated a critical role for IRF4 in the pathogenesis of CLL, the molecular mechanism through which IRF4 controls CLL development remains unclear.In an attempt to identify the molecular networks regulated by IRF4 in CLL cells we performed gene expression profiling of CLL cells isolated from IRF4-/-Vh11 mice. Intriguingly, our analysis revealed that Notch signaling was hyperactive in the IRF4-/-Vh11 CLL cells compared to the IRF4+/+Vh11 B1 cells. This is particularly important because recent high depth sequencing studies have identified mutations in Notch1 protein leading to its stabilization and activation in ~10% of all CLL patients (Puente et al. Nature, 2011, Fabbri et al. JEM, 2011). Furthermore, the individuals carrying these mutations have an aggressive disease course and poor overall survival (Fabbri et al. JEM, 2011). Recently, studies by Simonetti et al have shown that the IRF4 deficient B cells express high levels of Notch receptors and display increased Notch activity (Simonetti et al. JEM, 2013). We also detected high levels of Notch2 but not Notch1 on the cell surface of IRF4-/-Vh11 CLL cells. To examine the contribution of Notch signaling in the pathogenesis of CLL, we crossed IRF4-/-Vh11 mice with Notch2 conditional mutant mice and with mice expressing cre recombinase in B cells (CD19cre). This approach allowed us to genetically delete Notch2 receptor specifically in B cells of IRF4-/-Vh11 mice (CD19creNotch2fl/flIRF4-/-Vh11). Surprisingly, CLL cells could still emerge from the majority of CD19creNotch2fl/flIRF4-/-Vh11 mice. However, further analysis revealed that those CLL cells expressed high levels of Notch2 on their surface and were apparently derived from IRF4-/-Vh11 B1 cells that escaped cre-mediated deletion. Importantly, our analysis also showed that the majority of B1 and B2 cells in the CD19creNotch2fl/flIRF4-/-Vh11 mice had lost the expression of Notch2, indicating that Notch2 is dispensable for their generation.We also performed a series of analysis to determine how Notch signaling affects B1 and CLL cells in vitro. We used a co-culture system to engage Notch receptors to their ligand (Delta like 1 or DL1) expressed on S17 stromal cells (S17-DL1). Co-culture of wild type B1 cells with S17-DL1 stromal cells led to enhanced proliferation and survival of B1 cells when compared with cells co-cultured on control vector transfected S17 stromal cells (S17-R1). Importantly, Notch2 receptor deficient B1 cells failed to respond to DL1 expressing S17 stromal cells. Similarly, IRF4-/-Vh11 CLL cells also showed enhanced survival on S17-DL1 stromal cells compared to S17-R1 stromal cells.In summary, our studies show that Notch2 is absolutely required for CLL development in the IRF4-/-Vh11 mice, thereby supporting a critical role for Notch signaling in the pathogenesis of CLL. DisclosuresNo relevant conflicts of interest to declare.

CD200 May be a Potential Target for Therapy in Standard Risk Childhood ALL

Multiagent therapies for children with acute lymphoblastic leukaemia (ALL) have resulted in impressive remission rates of around 90%. Unfortunately, relapse rates remain high and most children who relapse will not survive. Innovative antibody and chimeric antigen receptor (CAR) modified T-cell therapies have shown promising results in ALL. However, this is a heterogeneous disease and several groups have shown ALL evolves in a branching fashion. Moreover, some leukaemia stem cell (LSC) populations do not express antigens that are the targets of developing therapies such as CD19, CD20, and CD22. Consequently, these cells may be largely unaffected and could provide a reservoir of resistant cells, from which relapse may develop. The diversity of reported LSC populations may be a reason for treatment failure and improvements in therapy will require targeting of all cells that have the potential to initiate and maintain this disease. In an attempt to identify more appropriate therapeutic targets we conducted an extensive microarray screen of several LSC subpopulations and bulk leukaemia cells from a group of paediatric patients with favourable cytogenetic abnormalities. This screen identified a number of genes that were up-regulated in all LSC subpopulations compared to levels in normal haemopoietic cells. The up-regulated genes encode for cell surface antigens, including the type-1 membrane protein, CD200 and signalling pathways. Expression of CD200 was 12-fold higher in bulk ALL cells compared to normal BM cells (p=0.001). CD200 is a key immunosuppressive molecule that has been implicated as a poor prognostic factor in multiple myeloma and acute myeloid leukaemia. To validate the microarray results, we assessed expression of CD200 in 20 BCP-ALL cases (11 pre B, 9 c-ALL) by flow cytometry and in functional assays. Fifteen of these cases were classified as MRD low risk and had favourable cytogenetic abnormalities and 5 were classified as high risk by MRD and cytogenetic abnormalities. ALL cells were stained with antibodies against CD34, CD19 and CD200 and cells were sorted on the basis of expression/lack of expression of CD34/CD200. The functional ability of the sorted populations was assessed in NSG mice and serial transplantation experiments were performed to assess self-renewal capacity. Overall, the flow cytometric analyses confirmed the microarray data in that expression of CD200 was high in this patient cohort (60±6.6%) compared to normal BM (0.1%±0.04, p ≤ 0.001), regardless of cytogenetic abnormality or MRD risk status. In standard risk cases, BM engraftment was achieved with the CD34+/CD200+ (2-66% human leukaemia) and CD34-/CD200+ (10-26%) subpopulations only, using inocula of 4x104-107 cells. No engraftment was attained using similar numbers of CD200- cells, regardless of expression of CD34 or CD19 in this group. Interestingly, when cells from the 5 patients defined as high risk were inoculated, engraftment was achieved using all four sorted subpopulations. The levels of leukaemia engraftment in these cases ranged from 2-95% using 1x103-5x106 cells and levels were similar across all subpopulations in individual patient samples. Results from serial transplantation studies to date demonstrate the sorted subpopulations were capable of self-renewal. As with the primary transplants, all four subpopulations from high risk samples engrafted while this ability was restricted to CD200+ subpopulations in standard risk cases. FISH analyses have confirmed all the engrafting cells had an abnormal karyotype. These data provide further evidence that surface antigen immunophenotype is not a good predictor of function in paediatric ALL, particularly in high risk cases. However, in standard risk cases it may be possible to eliminate LSC by targeting CD200. Treating human chronic lymphocytic leukaemia cells with anti-CD200 antibodies has been shown to prevent engraftment in NSG mice. Our data suggests that investigation of the effects of anti-CD200 in standard risk childhood ALL are warranted. DisclosuresNo relevant conflicts of interest to declare.