Primary Chronic Lymphocytic Leukemia Samples Research Articles

Anergy in CLL: Moving Towards the Clinic

B-Cell Receptor (BCR) triggering and responsiveness play a crucial role in the survival and expansion of Chronic Lymphocytic Leukemia (CLL) clones. In the recent past, several groups including ours have investigated the activation status of the signaling pathways originating from the leukemic BCR. Specifically we found that around 50% of CLL patients display a biochemical signature characterized by constitutive phosphorylation of ERK1/2 (pERK(+)) and constitutive nuclear translocation of NF-ATc1. These cases are unable to respond in vitro to BcR stimulation and are resistant to spontaneous apoptosis, thus resembling B lymphocytes previously anergized in vivo. Similar biochemical and functional features have been recently demonstrated in B leukemic cells persisting in the blood in patients treated with the BTK inhibitor, Ibrutinib, thereby making anergy an attractive target on the way to obtain eradication of the disease.CLL-associated B cell anergy can be specifically targeted by using different MAPK-inhibitors that have been shown to induce apoptosis selectively in the group of pERK(+) CLL. These data suggested that MAPK signalling can be efficiently inhibited in CLL for therapeutic purpose and that the phosphorylation status of ERK1/2 may represent a reliable biomarker to predict and monitor treatment response. However, even if the tested compounds were shown to be extremely efficient in inhibiting ERK1/2 phosphorylation in vitro, a lack of clinical activity was reported for many of them when tested in patients, mostly with solid tumors.In the present work, we used Trametinib, a specific MEK1/2 inhibitor, recently approved as a single-agent for the treatment of V600E mutated metastatic melanoma, and we investigated, at preclinical level, its activity in both primary CLL samples and a xenograft leukemic mouse model. Trametinib treatment completely inhibited constitutive ERK1/2 phosphorylation in 10 pERK1/2(+) samples at 3uM after 30 minutes treatment. Additionally, in 23 patients Trametinib treatment for 48 hours reduced cell viability in the cells from all 12 pERK1/2(+) patients (28,2% ± 3,5 mean survival) tested as compared to those from the pERK(-) group (11 cases, 58,1% ± 3,8 mean survival, p< 0,0001).To strengthen our in vitro data, we evaluated the effect of Trametinib administration in the xenograft Rag2-/-gc-/- mouse model subcutaneously transplanted with the CLL cell line MEC1, characterized by specific features of anergy.Mice were subcutaneously injected with 10x106 cells and then challenged with Trametinib (oral gavage with 1mg/kg or with vehicle alone) starting from day 21 after tumour injection for 14 days. The effect of the inhibitor was monitored by tumour volume growth.Trametinb administration delayed tumour growth (p<0.05 starting at days 27) and inhibited leukemic cell dissemination in the peripheral blood, peritoneal cavity and bone marrow.In summary, our data further support the idea that blocking anergic pathways may be highly effective not only in vitro but also in vivo with potential clinical implications at least in the subset of patients whose cells are characterized by anergic features, including those with persistent lymphocytosis when treated with Ibrutinib. The preclinical efficacy shown by Trametinib, a drug already approved for clinical use, warrants the implementation of controlled studies in CLL patients. DisclosuresNo relevant conflicts of interest to declare.

The Dual PI3K/mTOR Inhibitor PF-04691502 Induces Substantial Apoptosis in Chronic Lymphocytic Leukemia Cells in Vitro and Prolongs Survival in the Eµ-TCL1 Mouse Model

Pharmacological inhibition of specific B cell receptor signalling pathways within chronic lymphocytic leukemia (CLL) cells offers the potential for improved therapeutic options with reduced off target toxicity. Idelalisib, the PI3Kδ selective inhibitor, has been approved for CLL and significantly improved overall survival among patients with relapsed CLL in combination with rituximab compared to rituximab alone. In addition to PI3Kδ however, there are three other Class I PI3K isoforms, PI3Kα, PI3Kβ and PI3Kγ, with PI3Kα known to have a role in CLL survival and chemotaxis. In neutrophils, functional redundancy between PI3K isoforms is evident, with inhibition of at least three PI3K isoforms required for maximal apoptosis. Inhibition of mTOR is known to induce cell cycle arrest and apoptosis in CLL cells, however prolonged inhibition of mTOR results in activation of a positive feedback loop resulting in PI3K\\Akt reactivation. To overcome these caveats, pan PI3K inhibition alongside mTOR inhibition may achieve superior cytotoxicity against CLL cells compared to PI3Kδ or mTOR inhibition alone. We therefore sought to investigate the effect of a dual pan PI3K and mTOR inhibitor, PF-04691502, on primary CLL cells and in the Eµ-TCL1 mouse model of CLL.Twenty five primary CLL samples were treated with PF-04691502 and downstream signalling and subsequent apoptosis assessed by immunoblotting and Annexin V/PI staining respectively. In primary CLL cells, PF-04691502 induced 80% apoptosis after 24 hours at 10µM concentration. PF-04691502 had an IC50 value of 1µM as assessed by Annexin V/PI staining, with minimal toxicity to normal human B or T cells and a trend towards more specific killing in unmutated CLL (p=0.09) compared to mutated CLL. PF-04691502 inhibited both soluble and immobilised anti-IgM induced signalling and overcame anti-IgM induced survival signals. PF-04691502 abrogated CXCL12 induced signalling and impaired subsequent CLL migration towards the chemokine in a transwell chemotaxis assay. Finally, PF-04691502 was able to overcome protection from co-culture with stroma inducing significant apoptosis of CLL cells when added continuously and in wash out experiments.To assess the effect of PF-04691502 in vivo, twenty Eµ-TCL1 mice were inoculated with tumour cells and after day 21 dosed once daily with vehicle, 5mg/kg or 10mg/kg PF-04691502 for a further 14 days. Mice were assessed for leukemic cell counts, spleen size and white blood cell count throughout the duration of the experiment. The effect of PF-04691502 on B cell receptor and chemokine receptor induced signalling in ex vivo Eµ-TCL1 cells was assessed by immunoblotting.Near identical results to the CLL data above were observed. Using Eµ-TCL1 cells isolated from the spleen, PF-04691502 was shown to profoundly inhibit anti-IgM and CXCL12 induced signalling and chemotaxis as well as induce substantial apoptosis as measured by Annexin V/PI staining. Therefore, we subsequently assessed the effect of PF-04691502 in vivo using this model. Mice treated with PF-04691502 displayed a transient increase in leukemic cell numbers in the blood persisting for 1-4 days, followed by a reduction to levels significantly below that of the vehicle control. The whole white blood cell count remained stable in the PF-04691502 treated mice during the course of treatment, whilst the vehicle treated mice showed significant increases in tumour load up to 40×106 cells/ml 33 days post inoculation. Spleen sizes increased steadily over time in the control mice, whilst PF-04691502 treatment prevented this. After 33 days, PF-04691502 treated mice had spleen sizes comparable to non tumor-recipient control animals. In addition, CD5+ B220+ Eµ-TCL1 cells were significantly reduced in the bone marrow, spleen and lymph nodes following PF-04691502 treatment compared to control mice (p=0.0198; p< 0.0001; p=0.0151 respectively). These results demonstrate that PF-04691502 induces substantial apoptosis of primary CLL cells in vitro and significantly prolongs survival in an in vivo murine model of CLL. Such data indicate that dual pan PI3K/mTOR inhibitors may prove efficacious in the treatment of CLL patients. DisclosuresNo relevant conflicts of interest to declare.

Combined Inhibition of mTOR and DNA-PK Blocks Survival, Adhesion, Proliferation and Chemoresistance in Primary Chronic Lymphocytic Leukemia (CLL) Cells

CLL progression and chemoresistance can result from signals from the lymph node (LN) microenvironment and from acquired aberrations in the DNA damage repair (DDR) pathway. Clinical targeting of kinases upstream in the B cell receptor (BCR) activation pathway, such as Btk or PI3Kδ results in egress of cells from the LN microenvironment [1,2]. Such prolonged lymphocytosis during kinase-inhibitor treatment appears to pose no clinical disadvantage [3]. However, it enhances the chance of accumulating resistance-inducing mutations, and therefore drugs that combine LN egress with direct cytoxicity could provide an improved therapeutic strategy for CLL.The mTOR complex, consisting of mTOR1 and 2, is the main downstream kinase of the PI3K/Akt pathway and contributes to proliferation and survival. DNA-PK is a kinase required for non-homologous end joining (NHEJ) of the DNA repair pathway. Inhibitors of crucial components of the DDR pathway might be active in CLL, especially in patients harboring mutations in DNA repair molecules such as Ataxia telangiectasia mutated (ATM). In this study the potency of a novel dual mTOR1,2 and DNA-PK inhibitor (CC-115) was studied in primary CLL samples of different prognostic subgroups with respect to induction of cytotoxicity, and inhibition of adhesion, CD40-mediated chemoresistance and proliferation.In vitro, CC-115 inhibited mTOR1 and 2 and also affected the DDR reflected by inhibition of irradiation-induced γH2AX, not only in ATM-mutated but also in ATM-wild type CLL cells. CC-115 showed induction of caspase-dependent cell killing (IC50=0.625µM) which was more robust than selective kinase inhibitors (table 1), irrespective of p53 or ATM status. This cytotoxic effect was not observed in the T cells from CLL patients. BCR-mediated adhesion to fibronectin [4] was inhibited by CC-115 to a similar extent as PI3Kδ inhibitor (idelalisib) (table 1). CD40-mediated chemoresistance [5] could be reverted completely by CC-115 while more specific inhibitors had only modest effects. CLL proliferation induced by CD40L+IL-21 treatment [6] was completely blocked by both CC-115 and a dual mTOR1,2 inhibitor but not by inhibitor of the more upstream kinase PI3Kδ (table 1).In conclusion, these data show that CC-115 induces direct cytotoxicity and inhibits several clinically relevant biological features of CLL, and provide a rationale for clinical trials with CC-115 in CLL patients.[1] Hoellenriegel J, Meadows SA, Sivina M et al. The phospoinositide 3’-kinase delta inhibitor, CAL-101, inhibits B-cell receptor signaling and chemokine networks in chronic lymphocytic leukemia. Blood 2011;(118):3603-3612[2] Burger JA. Bruton’s Tyrosine Kinase (BTK) Inhibitors in Clinical Trials. Curr Hematol Malig Rep (2014) 9:44–49[3] Herman SE, et al. Ibrutinib-induced lymphocytosis in patients with chronic lymphocytic leukemia: correlative analyses from a phase II study. Leukemia. 2014 Apr 4. doi: 10.1038/leu.2014.122. [Epub ahead of print][4] de Rooij MF, Kuil A, Geest CR et al. The clinically active BTK inhibitor PCI-32765 targets B-cell receptor- and chemokine-controlled adhesion and migration in chronic lymphocytic leukemia. Blood 2012;(119):2590-2594.[5] Differential Noxa/Mcl-1 balance in peripheral versus lymph node chronic lymphocitic leukemia cells correlates with survival capacity. Blood 2007;(109):1660-1668.[6] Pascutti MF, Jak M, Tromp JM et al. IL-21 and CD40L signals from autologous T cells can induce antigen-independent proliferation of CLL cells. Blood 2013;(122):3010-3019.Smit LA, Hallaert DY, Spijker R et al.Table 1The effect of the mTOR1,2 + DNA-PK inhibitor (CC-115), mTOR1,2 inhibitor (CC-214), DNA-PK inhibitor (NU7441) and PI3Kδ inhibitor (idelalisib) on CLL cells in functional assays.CC-115CC-214NU7441Idelalisib (CAL-101)TargetmTOR1,2DNA-PKmTOR1,2DNA-PKPI3KδCytotoxicity (IC50)0.625 µ M>10µM>10µM>10µMInhibition of adhesion40%**0%22%48%**ActivationInhibition of CD40-mediated resistance to fludarabine (6.25 µ M)92%**42%*n.d.21%Inhibition of CD40L+IL21 induced proliferation98%**98%*28%51%**n.d. = not done The one sample T test was used to determine the significance of differences between means of treated samples and normalized values of untreated samples (100%). * p <0,05;** p<0,01 DisclosuresKersten:Celgene: Research Funding. Kater:Celgene: Research Funding.

The Oxazolidinone Derivative Locostatin Induces Apoptosis in CLL Cells through Inhibition of AKT and MAPK-ERK1/2 Signaling Under Conditions That Mimic the Tumor Microenvironment

BACKGROUND The Raf-1/MEK/MAPK-ERK1/2 pathway plays an important role in the pathogenesis and progression of multiple forms of cancer, including Chronic Lymphocytic Leukemia (CLL). Recent pre-clinical trials of the Raf-1 inhibitor Sorafenib demonstrate that targeting this pathway may represent a therapeutic option for CLL.Raf-1 activity is negatively regulated by the Raf-1 kinase inhibitory protein (RKIP). However, in CLL cells evidence suggests that phosphorylation of RKIP by protein kinase C (PKC) may restrict RKIP binding to Raf-1 and promote its binding and inhibition of G-coupled protein receptor 2 (GRK2). Consequently, GRK2, which would normally bind AKT and MEK1/2, would no longer suppress activity of these proteins. It is conceivable, therefore, that deregulation of RKIP may contribute to the over activity of the Raf-1 and AKT-mediated pathways that has been described in freshly isolated CLL cells.METHODS We sought to determine the effects of inhibiting RKIP on the survival and migratory capacity of CLL cells in vitro by using Locostatin, a specific inhibitor of RKIP binding to Raf-1 and GRK2. The proportion of apoptotic primary CLL cells following Locostatin treatment in either media alone or in the presence of a supportive stroma was assessed by flow cytometry. The mechanisms of action of Locostatin were investigated by western blotting for the phosphorylated forms of AKT and ERK1/2.RESULTS Locostatin induced a dose dependent decrease in the proportion of viable cells from CLL patients (n = 6) and a panel of haematological lines (n = 8). The IC50 for Locostatin was significantly (P < 0.001) lower against primary CLL samples analysed (11.14 +/- 4.86 µM) than in peripheral blood mononuclear cells from healthy volunteers (72.71 +/- 9.85 µM). Locostatin was also effective against CLL cells cultured in the presence of a CD40L-expressing fibroblast layer.Locostatin treatment reduced the expression of the chemokine receptor CXCR4 on the CLL cell surface with a concomitant decrease in their migration along a stroma-derived factor 1α (SDF-1α) gradient and significantly reduced the proportion of cycling CLL cells following induction by CpG oligonucleotide/IL2 (Dsp30/IL-2).We demonstrate by western blotting that RKIP is phosphorylated on S153 in CLL patient samples (Figure 1), supporting our hypothesis that RKIP may not be able to bind Raf-1 in these cells. Furthermore, we show that Locostatin treatment inhibits phosphorylation of AKT (S473) and ERK1/2 in CLL cells cultured in media alone or on CD40L fibroblasts (Figure 2).CONCLUSIONS These data suggest that deregulation of RKIP in CLL cells may contribute to their survival and proliferation by increasing the activities of AKT and ERK1/2. We show that Locostatin, a specific RKIP inhibitor, down-regulates the activity of both AKT and ERK1/2 in CLL cells cultured in media or a model of the tumor microenvironment, decreases cell viability and reduces the migratory and proliferative capacities of the leukemic cells. Our data suggest that RKIP may represent a novel target for therapy in CLL, which can be effectively inhibited by Locostatin. [Display omitted] [Display omitted] DisclosuresMulligan:Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi Aventis: Research Funding; Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria.

Novel Putative Driver Gene Mutations in Chronic Lymphocytic Leukemia (CLL): Results from a Combined Analysis of Whole-Exome Sequencing of 262 Primary CLL Samples

Unbiased high-throughput massively parallel sequencing methods have transformed the process of discovery of novel putative driver gene mutations in cancer. In chronic lymphocytic leukemia (CLL), these methods have yielded several unexpected findings, including the driver genes SF3B1, NOTCH1 and POT1. Recent analysis, utilizing down-sampling of existing datasets, has shown that the discovery process of putative drivers is far from complete across cancer. In CLL, while driver gene mutations affecting >10% of patients were efficiently discovered with previously published CLL cohorts of up to 160 samples subjected to whole exome sequencing (WES), this sample size has only 0.78 power to detect drivers affecting 5% of patients, and only 0.12 power for drivers affecting 2% of patients. These calculations emphasize the need to apply unbiased WES to larger patient cohorts.To this end, we performed a combined analysis of CLL WES data joining together our previously published cohort of 159 CLLs with data from 103 CLLs collected by the International Cancer Genome Consortium (ICGC). The raw sequencing reads from these 262 primary tumor samples (102 CLL with unmutated IGHV, 147 with mutated IGHV, 13 with unknown IGHV status) were processed together and aligned to the hg19 reference genome. Somatic single nucleotide variations (sSNVs) and indels were detected using MuTect. Subsequently, inference of recurrently mutated genes was performed using the MutSig algorithm. This method combined several characteristics such as the overall mutation rate per sample, the gene specific background mutation rate, non-synonymous/synonymous ratio and mutation clustering to detect genes that are affected by mutations more than expected by chance.This analysis identified 40 recurrently mutated genes in this cohort. This included 22 of 25 previously identified recurrently mutated genes in CLL. In addition, 18 novel candidate CLL drivers were identified, mostly affecting 1-2% of patients. The novel candidates included two histone proteins HIST1H1D and HIST1H1C, in addition to the previously identified HIST1H1E. Another was IKZF3, affected by a recurrent sSNV resulting in a p.L162R change in its DNA binding domain, in close proximity to a region recently identified as critical for lenalidomide resistance in multiple myeloma (MM). An additional recurrently mutated gene was nuclear RNA export factor 1 (NXF1), which along with previously known recurrently mutated genes (SF3B1, XPO1, DDX3X), highlights the importance of RNA processing to CLL biology. Finally, this search for putative CLL driver genes also identified ASXL1 and TRAF3, already characterized as drivers in acute myeloid leukemia and MM, respectively. Of the 59 of 262 samples for which RNA-seq data were available, 76% of the identified driver mutations were detected and thereby validated. Validation using RNAseq detection of driver mutations and targeted sequencing within the entire cohort are ongoing.The larger size of our cohort enabled the separate application of the somatic mutation discovery process to samples with mutated or unmutated IGHV. Among the 147 samples with mutated IGHV, only 5 driver genes (TP53, SF3B1, MYD88, CHD2, RANBP2) retained significance. In contrast, analysis of the 102 IGHV unmutated samples revealed a distinct and more diverse pattern of recurrently mutated genes (lacking MYD88 and CHD2, and including NOTCH1, RPS15, POT1, NRAS, EGR2, BRAF, MED12, XPO1, BCOR, IKZF3, MAP2K1, FBXW7 and KRAS). This extended cohort also allowed for better resolution of the clinical impact of those genetic variants with greater than 4% prevalence in the cohort. For example, samples with POT1 mutations were found to be associated with shorter time from sample to therapy compared with those with wild-type POT1 (P= 0.02).Our study demonstrates that with larger cohort size, we can effectively detect putative driver genes with lower prevalence, but which may nonetheless have important biological and clinical impact. Moreover, our interrogation shows that subset analysis can reveal distinct driver patterns in different disease subsets. In particular, the marked clinical difference between CLLs with mutated and unmutated IGHV may reflect the higher likelihood of the latter group to harbor a broader spectrum of driver mutations with a more complex pattern of co-occurrence. DisclosuresBrown:Sanofi, Onyx, Vertex, Novartis, Boehringer, GSK, Roche/Genentech, Emergent, Morphosys, Celgene, Janssen, Pharmacyclics, Gilead: Consultancy.

Abstract 4205: Mass-spectroscopy phosphorylation profiling identified differential activation of signaling molecules via spleen tyrosine kinase in B-cell receptor signaling and stromal cell stimulation in DLBCL and CLL cells that are inhibited by GS-9973

Abstract Dysregulation of protein kinase activity leading to aberrant or excessive downstream signaling is widely believed to underlay the pathogenesis of multiple cancers. The purpose of this study was to identify the spleen tyrosine kinase (SYK)-dependent phosphoproteins that were activated in a diffuse large B-cell lymphoma (DLBCL) cell line, DHL4, and primary chronic lymphocytic leukemia (CLL) cells, upon either B-cell receptor (BCR) activation, stromal cell contact, or in a resting state and demonstrate inhibition with a selective SYK inhibitor, GS-9973. A commercial PTMScan Direct procedure from Cell Signaling Technology was utilized in this assessment. Cell lysates generated from resting, αIgG treatment or HS-5 human stromal cell line contact were digested with trypsin and the resultant peptides were enriched by immunoprecipitation with either an anti-phosphotyrosine or anti-phosphoserine/threonine antibody cocktail, and subjected to mass spectrometric analysis to identify and quantify phosphorylated peptides. The phosphorylated peptide profile was then compared to a similarly treated lysate after incubation with 1 μM GS-9973. Protein phosphorylations that were significantly changed by &amp;lt; or &amp;gt; 2.5-fold with GS-9973 treatment were identified and confirmed, where possible, by western blot of DHL-4 lysates or in primary CLL samples with antibodies specific for the identified sites. PTMScan Direct analysis showed overlapping, but not equivalent, patterns of phosphorylated peptides upon αIgG or stromal cell activation. A shared set of GS-9973 inhibited phosphoproteins were identified including pSyk, pAkt, pMek, pErk, pJnk2, pPKCδ, and pS6 kinase. Western blot analysis validated 13 out of 14 proteins as being modulated by 1 μM GS-9973 treatment. Furthermore, dose-response inhibition was demonstrated for pAkt-T473, pBCR-Y177, pBlnk-Y84, pMek-S217/221, and pErk-T202/Y204 with EC50s ranging from 23 to 50 nM. These data support the concept that Syk inhibition can block multiple signaling pathways known to be important in malignant B-cells function. Furthermore, identification of these sites elucidates possible pharmacodynamic signatures of GS-9973 inhibition that may be followed in clinical investigations of GS-9973 in oncology. Citation Format: Julie A. Di Paolo, Astrid Clarke, Hong Rong, Emma Rousseau. Mass-spectroscopy phosphorylation profiling identified differential activation of signaling molecules via spleen tyrosine kinase in B-cell receptor signaling and stromal cell stimulation in DLBCL and CLL cells that are inhibited by GS-9973. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4205. doi:10.1158/1538-7445.AM2014-4205

Proteomics-based strategies to identify proteins relevant to chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL), a malignant B-cell disorder, is characterized by a heterogeneous clinical course. Two-dimensional nano liquid chromatography (2D-nano-LC) coupled with matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS) (LC-MALDI) was used to perform qualitative and quantitative analysis on cellular extracts from 12 primary CLL samples. We identified 728 proteins and quantified 655 proteins using isobaric tag-labeled extracts. Four strategies were used to identify disease-related proteins. First, we integrated our CLL proteome with published gene expression data of normal B-cells and CLL cells to highlight proteins with preferential expression in the transcriptome of CLL. Second, as CLL's outcome is heterogeneous, our quantitative proteomic data were used to indicate heterogeneously expressed proteins. Third, we used the quantitative data to identify proteins with differential abundance in poor prognosis CLL samples. Fourth, hierarchical cluster analysis was applied to identify hidden patterns of protein expression. These strategies identified 63 proteins, and 4 were investigated in a CLL cohort (39 patients). Thyroid hormone receptor-associated protein 3, T-cell leukemia/lymphoma protein 1A, and S100A8 were associated with high-risk CLL. Myosin-9 exhibited reduced expression in CLL samples from high-risk patients. This study shows the usefulness of proteomic approaches, combined with transcriptomics, to identify disease-related proteins.

Epigenetic inactivation of mir-34b/c in addition to mir-34a and DAPK1 in chronic lymphocytic leukemia

BackgroundTP53 mutation/deletion is uncommon in chronic lymphocytic leukemia (CLL). We postulated that components of TP53-centered tumor suppressor network, miR-34b/c, in addition to DAPK1 and miR-34a might be inactivated by DNA hypermethylation. Moreover, we tested if miR-34b/c methylation might correlate with miR-203 or miR-124-1 methylation in CLL.MethodsmiR-34b/c, miR-34a and DAPK1 methylation was studied in 11 normal controls, 7 CLL cell lines, and 78 diagnostic CLL samples by methylation-specific polymerase chain reaction. MEC-1 cells were treated with 5-Aza-2’-deoxycytidine for reversal of methylation-associated miRNA silencing. Tumor suppressor properties of miR-34b were demonstrated by over-expression of precursor miR-34b in MEC-1 cells.ResultsmiR-34b/c promoter was unmethylated in normal controls, but completely methylated in 4 CLL cell lines. miR-34b/c expression was inversely correlated with miR-34b/c methylation. Different MSP statuses of miR-34b/c, including complete methylation and complete unmethylation, were verified by quantitative bisulfite pyrosequencing. 5-Aza-2’-deoxycytidine treatment resulted in promoter demethylation and miR-34b re-expression in MEC1 cells. Moreover, over-expression of miR-34b resulted in inhibition of cellular proliferation and increased cell death. In primary CLL samples, miR-34a, miR-34b/c and DAPK1 methylation was detected in 2.6%, 17.9% and 34.6% of patients at diagnosis respectively. Furthermore, 39.7%, 3.8% and 2.6% patients had methylation of one, two or all three genes respectively. Overall, 46.2% patients had methylation of at least one of these three genes. Besides, miR-34b/c methylation was associated with methylation of miR-34a (P = 0.03) and miR-203 (P = 0.012) in CLL.ConclusionsTaken together, miR-34b/c is a tumor suppressor miRNA frequently methylated, and hence silenced in CLL. Together with DAPK1 methylation, miR-34b/c methylation is implicated in the disruption of the TP53-centered tumor suppressor network. Moreover, the association of miRNA methylation warrants further study.

Epigenetic inactivation of miR-9 family microRNAs in chronic lymphocytic leukemia - implications on constitutive activation of NFκB pathway

BackgroundThe miR-9 family microRNAs have been identified as a tumor suppressor miRNA in cancers. We postulated that miR-9-1, miR-9-2 and miR-9-3 might be inactivated by DNA hypermethylation in chronic lymphocytic leukemia (CLL).MethodsMethylation of miR-9-1, miR-9-2 and miR-9-3 was studied in eight normal controls including normal bone marrow, buffy coat, and CD19-sorted peripheral blood B-cells from healthy individuals, seven CLL cell lines, and seventy-eight diagnostic CLL samples by methylation-specific polymerase chain reaction.ResultsThe promoters of miR-9-3 and miR-9-1 were both unmethylated in normal controls, but methylated in five (71.4%) and one of seven CLL cell lines respectively. However, miR-9-2 promoter was methylated in normal controls including CD19 + ve B-cells, hence suggestive of a tissue-specific but not tumor-specific methylation, and thus not further studied. Different MSP statuses of miR-9-3, including complete methylation, partial methylation, and complete unmethylation, were verified by quantitative bisulfite methylation analysis. 5-Aza-2′-deoxycytidine treatment resulted in miR-9-3 promoter demethylation and re-expression of pri-miR-9-3 in I83-E95 and WAC3CD5+ cells, which were homozygously methylated for miR-9-3. Moreover, overexpression of miR-9 led to suppressed cell proliferation and enhanced apoptosis together with downregulation of NFκB1 in I83-E95 cells, supporting a tumor suppressor role of miR-9-3 in CLL. In primary CLL samples, miR-9-3 was detected in 17% and miR-9-1 methylation in none of the patients at diagnosis. Moreover, miR-9-3 methylation was associated with advanced Rai stage (≥ stage 2) (P = 0.04).ConclusionsOf the miR-9 family, miR-9-3 is a tumor suppressor miRNA relatively frequently methylated, and hence silenced in CLL; whereas miR-9-1 methylation is rare in CLL. The role of miR-9-3 methylation in the constitutive activation of NFκB signaling pathway in CLL warrants further study.

Targeting Chronic Lymphocytic Leukemia With DNA Nanoparticles

A significant challenge in many areas of cancer research and treatment is the lack of cancer cell-specific binding agents. In Chronic Lymphocytic Leukemia (CLL), numerous genetic and molecular markers are used to divide patients into prognostic categories but clearly distinguished etiological or biological subtypes remain elusive. The current targeted treatments for CLL include monoclonal antibodies: alemtuzumab, ofatumumab, and rituximab, which target CD52 and CD20. While these markers are present on CLL cells, they do not distinguish the leukemia from other lymphocytes.We have developed a technology that uses library bio-panning and molecular evolution approaches to create hyper-avid DNA nanoparticles that recognize cells through multiple interactions. This technology utilizes rolling circle amplification (RCA) of circular oligonucleotide templates containing random nucleotides to produce libraries of single-stranded DNA concatamers, each of which forms a distinct nanoparticle. Each library contains 1010 unique particles and each particle contains 50-200 copies of its template. To select cancer-specific DNA nanoparticles, we employ random library selection methods, in which a library of particles is incubated with cells, stringently washed to remove non-specific particles, and the retained particles are reamplified to their oligonucleotide template (Figure 1). Templates are recircularized and the entire process is repeated 5-7 times to identify specific particles. Oligonucleotide templates from a pool of specific particles are then cloned, sequenced, and synthesized to identify individual clones. We are agnostic as to the molecular target(s) of the particles, thus circumventing the need to identify a unique molecular identifier on the cancer cell surface, as is the case for developing monoclonal antibodies. Our DNA nanoparticles have an intrinsic multivalent display of modules, allowing for recognition of a diverse landscape on the cell through many low, monovalent affinity interactions, which translate into high overall avidity. [Display omitted] In our current study, we have performed selections on primary CLL samples and identified CLL-binding particles. Different patient samples were used in each round of selection (one patient sample/round), and several particles were identified that bound to multiple CLL patient samples after 5 rounds of selection. CLL-binding particles can be identified by flow cytometry, in a method akin to cell staining with fluorescently-labeled antibodies. To label the DNA nanoparticles, a fluorescently labeled short oligonucleotide with complementary sequence to the particle is annealed at a low molar ratio. We identified one particle that bound to 6/6 CLL samples tested, including both fresh and frozen samples. Four of those samples were not part of the original sequential selection process. The mean fluorescence intensity was 1.4-2.6-fold above background staining with control particles. These particles were also found to bind to normal B cells, as no counter-selections were performed to remove general binders from the pool. In ongoing experiments a depletion step with counter screening against PBMCs is introduced prior to CLL selection in order to enrich the CLL-specific proportion of binders. Furthermore, a nextgen sequencing approach has been developed that will allow quantitative assessment of the populations of particles present in each round of selection as well as pre and post counterscreening, thus enabling bioinformatic monitoring of the population dynamics as well as pointing towards the most specific candidate particles for subsequent experimental validation. CLL specific particles could be used in diagnostic testing, in vivo targeting, and drug delivery. Disclosures:No relevant conflicts of interest to declare.

Systematic Mapping Of Drug and Pathway Sensitivity In Chronic Lymphocytic Leukemia Identifies Synthetic Lethal Interactions Of Mutant p53

IntroductionCritical pathway alterations contribute to heterogeneous biology and clinical course in chronic lymphocytic leukemia (CLL). The role of particular mutations for drug sensitivity has been demonstrated for CLL and other entities. Here we systematically investigated heterogeneity of drug response and genetic lesions in primary CLL. The comprehensive identification of pathway dependencies will lead to a better understanding of hematologic malignancies and this may be exploited by development of rational-driven treatment approaches. MethodsPrimary CLL cells were characterized through an ex vivo high-throughput drug screening platform in 384-well format. The drug library consisted of 67 compounds (inhibitors targeting critical pathways in CLL and other cancers as well as compounds with known activity against CLL cells (including ABT-263, CAL-101, dasatinib, fludarabine, ibrutinib)) at different concentrations. Heat inactivated human serum was supplemented to mimic micro-environmental conditions. CLL samples represented untreated CLL, sensitive or refractory disease as well as major genetic subgroups. Cell viability was assessed by quantification of ATP (CellTiterGlo®) 48 and 72 hours after drug application. To understand heterogeneous pathway dependencies, drug sensitivity was regressed on genetic profiles. Genetic characterization was performed by FISH, targeted sequencing of recurrent aberrations (BRAF, MYD88, NOTCH1, SF3B1, TP53) as well as whole exome sequencing. ResultsWe report results from 97 primary CLL samples screened ex vivo. Validity of the approach was demonstrated by the response profiles of p53-interfering compounds: Nutlin-3a and fludarabine induced cell death in cells with wild-type (n=80) but considerably lessin CLL with mutated p53(n=17; Nutlin-3a 10µM: 51±18vs80±18;fludarabine 10µM: 40±36vs 67±23 [% viability of untreated control], both p<0.001). Furthermore inhibitors with related targets (e.g. drugs targeting the B-cell receptor (BCR) pathway: CAL-101, dasatinib, ibrutinib, R406) showed similar response profiles across patients, suggesting that prediction of drug effects can be based on functional and genetic properties of tumors. Patients can be divided into two major subgroups according to their sensitivity towards BCR and related kinase inhibition (e.g. Akt, Btk, Lyn, PI3K, Syk). A number of genotype-specific screening hits for single genetic aberrations were obtained. We identified compounds with preferential activity in groups with high risk aberrations (e.g. TP53 mutation, NOTCH1 mutation, 11q deletion; Fig. 1A) suggesting synthetic lethal interactions. Validation of the top hit with increased response of TP53-mutated CLL (compound A 10µM wt vs mutTP53: 58±31 vs 37±18 [% viability of untreated control]; Fig. 1A) confirmed genotype-specific cytotoxicity across concentration ranges (0.1-10µM; Fig 1B). Other compounds inhibiting the same target support our observation (compounds A2 and A3; Fig. 1B). The effect was reproducible in co-culture of CLL cells with HS-5 stroma cells (66±16 vs 32±13 [% viability of untreated control], p<0.05; Fig 1C). [Display omitted] ConclusionOur data provide a first comprehensive map of genetically determined drug sensitivity in CLL. The work offers a novel functional classification of CLL based on drug sensitivity and identifies synthetic lethal interaction for TP53 mutant CLL. Disclosures:No relevant conflicts of interest to declare.

Ibrutinib Differentially Interferes With Surface IgM and IgD BCR Signaling Kinetics In Chronic Lymphocytic Leukemia

B cell receptor (BCR) signaling is a driving event in Chronic Lymphocytic Leukemia (CLL) progression. Most CLL cells express both surface IgM (sIgM) and sIgD molecules, and previous reports showed heterogeneous responses to anti-IgM stimulation: CLL cells carrying IGHV unmutated-BCRs are highly responsive in terms of intracellular signaling activation, while cells with IGHV-mutated receptors are usually anergic to BCR cross-linking. In contrast, it has been described that almost all CLL cells retain responsiveness to sIgD, suggesting that sIgD signaling may not be the most relevant event in disease progression and heterogeneity. As the clinical significance of these mechanisms still needs to be unraveled, in the present study we analyzed the kinetics of BCR signaling activation following either sIgM or sIgD stimulation in CLL cells. The activation of HS1 protein, along with BCR proximal kinases (i.e. SRC/LYN and BTK) and the downstream effector ERK, was tested by Western Blot and flow-cytometry analysis up to 60 minutes following receptor engagement. HS1 protein was chosen as an early marker of BCR signaling and cytoskeletal activation (ten Hacken E et al., Blood 2013), also in the light of its prognostic relevance, as HS1 hyperphosphorylation correlates with a dismal clinical outcome of patients.Purified B cells from 16 primary CLL samples were stimulated with either soluble anti-IgM or anti-IgD and the level of HS1 activating tyrosine (Y)397 phosphorylation was analyzed at different time points (2, 5, 15, 30, 60 minutes) by densitometric analysis of Western Blot results or Mean Fluorescent Intensity analysis of flow cytometry data. HS1 activation peaked after 2-5 minutes following stimulation, and then returned to baseline at later time points with different patterns. In 13/16 samples, we observed a protracted HS1 phosphorylation after anti-IgM stimulation. In contrast, 6/16 samples showed protracted HS1 phosphorylation after anti-IgD stimulation, while 10/16 samples showed a very transient HS1 activation that rapidly returned to baseline. The activation kinetics of the other signaling molecules (i.e. SRC/LYN, BTK and ERK) was in line with that of HS1. As protracted BCR signaling activation is linked to cell cycle entry and proliferation, the protracted IgD signaling observed in some patients suggests that not only IgMs, but also IgDs may have pathogenetic relevance in CLL progression and heterogeneity.We then analyzed how the BTK inhibitor ibrutinib affects BCR signaling activation kinetics and analyzed phosphoprotein activation patterns in 10 CLL samples after 1 hour pretreatment with ibrutinib and up to 60 minutes following anti-IgM and anti-IgD stimulation. Both IgM and IgD-induced phosphorylation of HS1, SRC/LYN, BTK and ERK was reduced by ibrutinib treatment, demonstrating that ibrutinib is effective in blocking signaling pathways downstream not only of IgM receptors, as previously demonstrated, but also downstream of IgDs. Interestingly, in 7/10 samples, HS1 partially retained the ability to be phosphorylated after BCR triggering following 1-hour ibrutinib treatment and 5-minute anti-IgM or anti-IgD stimulation, suggesting that HS1 activation can bypass BTK kinase inhibition at least at early time points following receptor stimulation. A broad analysis on a large cohort of patients will further allow the correlation between BCR signaling molecule activation and cell viability with molecular/cytogenetic prognostic factors.Overall, these results show that sIgM stimulation of CLL cells promotes protracted activation of BCR signaling, which can only be observed in a restricted set of patients after sIgD stimulation. These observations support the notion that sIgM signaling may be more relevant for CLL progression, but also suggest that sIgD signaling may contribute to CLL pathogenesis in selected patients. Both signaling pathways can be inhibited by ibrutinib, further supporting the effectiveness of ibrutinib in inhibiting BCR signaling in CLL. Disclosures:O'Brien:Pharmacyclics: Research Funding. Burger:Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Pharmacological targeting of eIF4E in primary CLL lymphocytes

Pharmacological targeting of eIF4E in primary CLL lymphocytes

The BH3-only protein Puma plays an essential role in p53-mediated apoptosis of chronic lymphocytic leukemia cells

The purpose of this study was to explore the characteristics and functions of BH3-only proteins Puma, Noxa and Bim in the prognosis, therapy and drug resistance of chronic lymphocytic leukemia (CLL). Puma, Noxa and Bim mRNAs were evaluated by real-time quantitative reverse transcriptase-polymerase chain reaction, and correlations between their expression levels and CLL prognostic markers were analyzed. Primary CLL samples were treated in vitro with fludarabine to investigate the role of Puma, Noxa and Bim in the response to chemotherapeutic drugs which act through activation of the p53 pathway. We found that a low expression level of Puma was associated with some markers of poor prognosis. However, the level of Noxa or Bim was not different in patients with CLL with variant clinical features and prognostic factors. Puma expression was up-regulated after fludarabine treatment in primary CLL cells, but there was no significant difference for Noxa and Bim. Up-regulation of Puma occurred only in CLL cells with functional p53. CLL cells with p53 abnormalities were deficient in the activation of Puma by chemotherapeutics. These results suggest that a lack of Puma induction may contribute to the development of resistance to anticancer agents in CLL.

Overexpression of uridine diphospho glucuronosyltransferase 2B17 in high-risk chronic lymphocytic leukemia

AbstractUridine diphospho glucuronosyltransferase 2B17 (UGT2B17) glucuronidates androgens and xenobiotics including certain drugs. The UGT2B17 gene shows a remarkable copy number variation (CNV), which predisposes for solid tumors and influences drug response. Here, we identify a yet undescribed UGT2B17 mRNA overexpression in poor-risk chronic lymphocytic leukemia (CLL). In total, 320 CLL patients and 449 healthy donors were analyzed. High (above median) UGT2B17 expression was associated with established CLL poor prognostic factors and resulted in shorter treatment-free and overall survival (hazard ratio ([death] 2.18; 95% CI 1.18-4.01; P = .013). The prognostic impact of mRNA expression was more significant than that of UGT2B17 CNV. UGT2B17 mRNA levels in primary CLL samples directly correlated with functional glucuronidation activity toward androgens and the anticancer drug vorinostat (R > 0.9, P < .001). After treatment with fludarabine containing regimens UGT2B17 was up-regulated particularly in poor responders (P = .030). We observed an exclusive involvement of the 2B17 isoform within the UGT protein family. Gene expression profiling of a stable UGT2B17 knockdown in the CLL cell line MEC-1 demonstrated a significant involvement in key cellular processes. These findings establish a relevant role of UGT2B17 in CLL with functional consequences and potential therapeutic implications.

Expression level of DEK in chronic lymphocytic leukemia is regulated by fludarabine and Nutlin-3 depending on p53 status

Human oncogene DEK has been shown to be upregulated in a number of neoplasms. The purpose of this study was to investigate DEK expression level in chronic lymphocytic leukemia (CLL), analyze the correlation between DEK expression and CLL prognostic markers, and characterize the role of DEK in the response to either chemotherapeutic drugs or nongenotoxic activators of the p53 pathway. DEK mRNA was evaluated by real-time quantitative reverse transcriptase-polymerase chain reaction (qPCR), and primary CLL samples were treated in vitro with either fludarabine or Nutlin-3 to explore the interaction of p53 status and DEK mRNA expression. The median expression levels of DEK mRNA were 6.792 × 10−2 (1.438 × 10−2−3.201 × 10−1) in 65 patients with CLL. A marked increase of DEK mRNA expression was observed in the CLL patients with unmutated immunoglobulin heavy chain variable (IGHV) gene (p = 0.025), CD38-positive (p = 0.047), del(17p13) (p = 0.006). Both fludarabine and Nutlin-3 significantly downregulated DEK in the primary CLL cells which were with normal function of p53, or without deletion or mutation of p53 (p = 0.042, p = 0.038; p = 0.021, p = 0.017; p = 0.037, p = 0.017). However, the downregulation of DEK was not observed in the primary CLL cells which were with dysfunction of p53, or with deletion or mutation of p53 (p = 0.834, p = 0.477; p = 0.111, p = 0.378; p = 0.263, p = 0.378). These data show that DEK might be applied for the assessment of prognosis in patients with CLL, and fludarabine and Nutlin-3 regulate DEK expression depended on p53 status.

T Cell Exhaustion Contributes to Immune Evasion in Chronic Lymphocytic Leukaemia

Abstract 1773T cell exhaustion is a state of T cell dysfunction in response to chronic antigen exposure, marked by impaired effector function and the continued expression of inhibitory receptors such as Programmed Death 1 (PD-1) (Wherry EJ. Nat Immunol. 2011 Jun;12(6):492–9.). Because tumour growth in chronic lymphocytic leukaemia (CLL) occurs over a long period of time, we hypothesized that the continued exposure of T cells to a CLL-derived antigen could also lead to a state of T cell exhaustion. We therefore investigated whether T cell exhaustion is induced in CLL by using the Eμ-TCL1 transgenic (tcl1tg) tumour transfer mouse model for this disease (Hofbauer JP, et al. Leukemia. 2011 Sep;25(9):1452-8) and by analyzing primary samples from CLL patients. We found that the number of PD-1+ T cells was increased in both CD4+ and CD8+ populations and in all lymphoid compartments examined of the Eμ-TCL1 transgenic (tcl1tg) tumour recipient mice, but not in recipient mice receiving wildtype (WT) splenocytes showing that leukemic mice have an increased number of T cells displaying an exhausted phenotype that is induced by the presence of CLL cells. We next assessed the expression of the ligands for PD-1 on the surface of murine CLL cells. Peripheral CLL tumour cells showed only a modest increase in PD-L1 expression as compared to WT B cells. However, lymph node and spleen residing tumour cells showed a marked increase in PD-L1 expression, which suggests a microenvironment-induced upregulation of PD-L1 on tumour cells, e.g. by their close contact to accessory cells.To validate our results on primary human CLL samples, we collected peripheral blood from 89 unselected CLL patients and 18 healthy donors and observed an increase in surface expression of PD-1 on the CD4+ and CD8+ T cell populations. While the percentage of PD-1+ CD4+ T cells in chemonaive patients was comparable to healthy donors, chemotherapy drastically increased the number of PD-1-expressing CD4+ T cells (63.81% ±19.75 vs 35.70% ±19.22; p<.001). In contrast, treatment apparently had little impact on the PD-1+ CD8+ population, indicating that PD1 induction on CD8+ T cells might be a more general CLL specific phenomenon. PD-1 expression in chemonaive patients did not correlate with the prognostic markers CD38, Zap-70 and IgVH mutations, however, it significantly correlated with Rai stage (CD4+: p<.001; CD8+: p=.003; spearman correlation). Our immune phenotyping analyses also included markers to distinguish between naïve, memory, and effector T cell subsets. We observed that PD-1 expressing T cells belong primarily to the memory compartment, characterized by the absence of CD45RA expression. PD-1 expression levels in T cells was independent from chronic human cytomegalovirus (HCMV) infection, indicating that increased PD-1+ T cell numbers in CLL do not simply reflect a higher incidence of HCMV infections, which has been described in this patient population.As we speculated that PD1/PDL1 pathway might be exploited by CLL to evade a T cell dependent cytotoxic attack, we next wanted to know whether blocking this pathway by recombinant PD1 (rPD1) or PD-L1 specific Fab fragments in vivo would lead to reinvigoration of CLL specific T cells reflected in CLL clearance. We found that inhibition of PD1/PD-L1 binding in vivo in the tcl1tg tumour transfer model led to a decrease in CLL load in the peripheral blood and in all analysed organs (lymph node, spleen, bone marrow, liver, lung) while in vitro treatment with rPD-1 or PD-L1 Fab fragment did not affect the viability of mouse tumour cells. Our results imply that in tumour bearing mice, a vast number of tumour-specific exhausted CTLs exist that can be immediately reactivated by PD1/PD-L1 blockage leading to cytolysis of the tumour, thereby representing a promising target for therapeutic intervention in CLL patients. Disclosures:No relevant conflicts of interest to declare.

A Potential Therapeutic Strategy for Chronic Lymphocytic Leukemia by Combining GS-1101, a PI3 kinase Delta (PI3Kδ) Inhibitor and a Novel Highly Selective Spleen Tyrosine Kinase (Syk) Inhibitor, GS-9973

AbstractAbstract 3876 Background:Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western World and remains incurable with standard therapies. Despite significant advances, novel treatments are essential to improve outcomes. A number of therapeutic agents have recently been developed including some that have shown significant activity and tolerability in clinical trials. Among these drugs, are small molecule kinase inhibitors that inhibit B-cell receptor (BCR)-mediated signaling pathways and disrupt essential CLL cell-microenvironment interactions. Specifically, when primary CLL cells are treated with GS-1101, a PI3 kinase delta-specific isoform (PI3Kδ) inhibitor, Bruton’s tyrosine kinase (Btk) inhibitors or spleen tyrosine kinase (Syk) inhibitors, inhibition of signaling pathways downstream of the BCR, cell killing, and disruption of chemokine-mediated CLL cell migration are observed. Although significant clinical activity has been observed in patients treated with single agents that target these critical pathways, very little is known about the effects of inhibiting multiple nodes in the BCR pathway. Simultaneous inhibition of multiple pathways downstream of the BCR has the potential to result in a synergistic response that may overcome the resistance observed with single compound use. These considerations prompted us to assess the effects of PI3Kd and Syk inhibition and to determine if dual inhibition might enhance antitumor effects in CLL. Methods and Results:CLL patient samples were assessed for growth inhibition, chemokine release, and pathway activation status using a tetrazolium-based MTS assay, ELISA, and flow cytometry, respectively. PBMCs were isolated from primary patient samples using a Ficoll-Paque gradient. GS-1101, a PI3Kδ inhibitor, and GS-9973, a novel Syk inhibitor, were plated either alone in concentrations known to span their IC50 (.6nM to 10uM), or in combination using equimolar concentrations of each drug (.6nM to 10uM) in 96-well plates. 5×105 primary cells were cultured in triplicate in HS-5 stromal cell conditioned media and growth inhibition was determined after 72 hours.To explore potential additive, synergistic, or antagonistic interactions between GS-1101 and GS-9973, dose response and interaction indices were calculated using R (Lee et al. 2007). Given the importance of microenvironmental signals in the ability of malignant cells to survive, proliferate, and resist standard therapies, we investigated the effect of combining PI3Kd and Syk inhibitors for use on CLL cells in the presence of stroma-conditioned media. Immunoblotting and flow cytometry were used to measure the inhibitory effects of each drug alone or in combination on downstream targets known to be associated with BCR activation, including AKT, ERK, and S6 in Ramos cells, a Burkitt’s lymphoma cell line, and in primary CLL cells. Of the 14 primary CLL samples treated with the single agent GS-9973, the median cell viability IC50 for all samples was 3.7 μM and an IC50 of ≤ 2μM was obtained in 7 samples (50%). Interaction indices were calculated from combination studies using GS-1101 and GS-9973 in 7 samples. At least one significantly synergistic concentration of the combination of GS-1101 and GS-9973 was found in five of the seven samples. The two remaining CLL samples showed additive responses when treated with the combination. Patient samples cultured in the presence of conditioned media resulted in increased CCL2, CCL3, and CCL4 levels. Production of these chemokines by CLL cells was reduced by both GS-1101 and GS-9973, alone and in combination. Furthermore, treatment with individual inhibitors decreased S6 and ERK, phosphorylation, an effect further enhanced by the combination of PI3Kd and Syk inhibition. Conclusion:Our findings indicate that both PI3Kd and Syk inhibition reduces CLL survival. Dual targeting can also induce synergistic growth inhibition and further disrupt chemokine signaling. Given the complexity of BCR signaling pathways, simultaneous targeting of multiple kinases has the potential to significantly increase clinical activity. Since inhibition of BCR mediated kinases has demonstrated good patient tolerability, combination therapy targeting both PI3Kd and Syk may provide a novel treatment approach, especially in patients with poor risk disease that may not respond optimally to single agents. Disclosures:Clarke:Gilead Sciences Inc: Employment. Meadows:Gilead sciences: Employment. Loriaux:Gilead Sciences Inc: Research Funding. Maciejewski:Gilead Sciences Inc: Employment. Di Paolo:Gilead Sciences Inc: Employment. Lannutti:Gilead Sciences Inc: Employment. Druker:Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cylene: Consultancy, Membership on an entity's Board of Directors or advisory committees. Spurgeon:Gilead: Research Funding.

The gene expression signature associated with TP53 mutation/deletion in chronic lymphocytic leukaemia is dominated by the under‐expression of TP53 and other genes on chromosome 17p

In chronic lymphocytic leukaemia (CLL), TP53 mutation and deletion are strongly associated with one another and with adverse clinical outcome. Mutant TP53 protein typically accumulates to high levels and has been reported to have transcriptional regulatory activity distinct from that of wild-type TP53. To investigate whether such an effect is relevant to CLL, carefully balanced primary CLL samples with or without TP53 mutation/deletion were compared for their gene expression profiles using high-density DNA microarrays. Ninety-six and eight differentially expressed genes were identified, respectively, using two alternative statistical approaches with different stringencies. None of the differentially expressed genes were known to be regulated by mutant TP53, and only four of the 67 under-expressed genes were known transcriptional targets of wild-type TP53. Significantly, both approaches showed that gene under-expression was the dominant feature of TP53-mutant CLL samples. Furthermore, a disproportionate number of the under-expressed genes were located on chromosome 17p, the most significant being TP53 itself. Together, these results indicate that any transcriptional regulatory effects of mutant TP53 in CLL cells are overshadowed by the under-expression of co-deleted TP53 and other genes on chromosome 17p. Our findings have implications for emerging therapeutic strategies that target mutant TP53.

LNA Anti-MicroRNA-155: A Novel Therapeutic Strategy in Waldenstrom Macroglobulinemia and Chronic Lymphocytic Leukemia

Abstract 2728 Background.We and others have previously demonstrated that primary Waldenstrom's Macroglobulinemia (WM) and Chronic lymphocytic leukemia (CLL) cells show increased expression of microRNA-155 (miR-155), suggesting a role in regulating pathogenesis and tumor progression of these diseases. However, developing therapeutic agents that specifically target miRNAs has been hampered by the lack of appropriate delivery of small RNA inhibitors into tumor cells. We tested the effect of a novel LNA (locked nucleic acid)-modified anti-miR-155 in WM and CLL. Methods.WM and CLL cells, both cell lines (BCWM.1; MEC.1) and primary tumor cells; BCWM.1 Luc+ cells; and primary WM bone marrow (BM) stromal cells were used. WM and CLL cells were treated with antisense LNA anti-miR-155 or LNA scramble oligonucleotide. Efficiency of delivering FAM-labeled LNA into cells was determined by flow cytometry. Survival and cell proliferation were assessed by MTT and thymidine uptake assay, respectively. Synergistic effects of LNA with bortezomib were detected on BCWM.1 or MEC1 cells. Co-culture of BCWM.1 or MEC1 cells with WM bone marrow stromal cells was performed to better define the effect of the LNA-anti-miR155 in the context of the bone marrow microenvironment. miR-155 levels were detected in stromal cells from WM patients by qPCR. Co-culture of BCWM.1 or MEC1 cells with either wild-type or miR155−/− mice BM stromal cells was examined after LNA treatment. Gene expression profiling analysis was performed on BCWM.1 cells treated with either LNA anti-miR-155 or scramble control. miR-155 target gene candidates were predicted by TargetScan software. mRNA levels of miR-155, and its known target genes or gene candidates were detected by qRT-PCR. A microRNA luciferase reporter assay was used to determine whether miR-155 target candidates could be directly regulated by miR-155. mRNA levels of miR-155 targets were detected by qRT-PCR from primary WM or CLL cells treated with LNA. The activity of the LNA-anti-miR-155 was also detected in vivo using bioluminescence imaging and mRNA levels of miR-155 targets were detected by qRT-PCR ex vivo. Efficiency of introducing the FAM-labeled LNA into mice BM cells was determined by flow cytometry 1 week or 2 weeks after intravenous injection. Results.The efficiency of delivering LNA oligos into both WM and CLL-derived cell lines and primary samples was higher than 90%. LNA antimiR-155 reduced proliferation of WM and CLL-derived cell lines by 30–50%, as compared to LNA scramble control. In contrast, LNA antimiR-155 didn’t exert significant cytotoxicity in BCWM.1 or MEC.1. LNA synergistically decreased BCWM.1 or MEC1 cell growth co-treated with bortezomib and decreased BCWM.1 or MEC1 cell growth co-cultured with WM BM stromal cells in vitro. A higher level of miR-155 was found in WM BM stromal cells compared to normal ones. LNA decreased BCWM.1 or MEC1 cell growth when co-cultured with BM stromal cells from miR155−/− mice compared with wild-type. We demonstrated increased expression of miR-155-known targeted genes, including CEBPβ, SOCS1, SMAD5, and several novel target candidates including MAFB, SH3PXD2A, and SHANK2, in WM cells upon LNA anti-miR-155 treatment. These target candidates were confirmed to be directly regulated by miR-155 using a luciferase reporter assay. mRNA levels of miR-155 targets were upregulated by 1.5–2 fold at 48 hr after direct incubation of the LNA with primary WM or CLL samples, indicating efficient delivery and biologic effect of the LNA in cells. Moreover, this LNA showed significant in vivo activity by inhibiting WM cell proliferation in a disseminated xenograft mouse model. Upregulation of miR-155 targeted genes were confirmed ex vivo, in WM cells isolated from the BM of treated mice compared to control. Mice BM cells were FAM positive 1 or 2 weeks after injection indicating efficient delivery of FAM-labeled LNA into cells in vivo. Summary.A novel LNA (locked nucleic acid)-modified anti-miR against miR-155 could be highly efficiently delivered into tumor cells in vivo in the bone marrow microenvironment. Anti-WM activity of LNA anti-miR-155 was confirmed both in vitro and in vivo and anti-CLL activity was confirmed in vitro. Novel miR-155 direct target genes including MAFB, SH3PXD2A, and SHANK2 were identified. These findings will help to design individualized clinical trials for WM and CLL patients with elevated levels of miR-155 in their tumor cells. Disclosures:Roccaro:Roche:. Obad:Santaris Pharma: Employment. Broom:Electroporation: Employment. Kauppinen:Santaris Pharma: Employment. Brown:Calistoga: Consultancy, Research Funding; Celgene: Honoraria, Research Funding; Genzyme: Research Funding; GSK: Research Funding. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium: Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees.