Survival Of B-CLL Cells Research Articles

A Non-Canonical Role for the Glycosyltransferase Enzyme UGT2B17 as a Novel Constituent of the B Cell Receptor Signalosome.

In chronic lymphocytic leukemia (CLL), an elevated glycosyltransferase UGT2B17 expression (UGT2B17HI) identifies a subgroup of patients with shorter survival and poor drug response. We uncovered a mechanism, possibly independent of its enzymatic function, characterized by an enhanced expression and signaling of the proximal effectors of the pro-survival B cell receptor (BCR) pathway and elevated Bruton tyrosine kinase (BTK) phosphorylation in B-CLL cells from UGT2B17HI patients. A prominent feature of B-CLL cells is the strong correlation of UGT2B17 expression with the adverse marker ZAP70 encoding a tyrosine kinase that promotes B-CLL cell survival. Their combined high expression levels in the treatment of naïve patients further defined a prognostic group with the highest risk of poor survival. In leukemic cells, UGT2B17 knockout and repression of ZAP70 reduced proliferation, suggesting that the function of UGT2B17 might involve ZAP70. Mechanistically, UGT2B17 interacted with several kinases of the BCR pathway, including ZAP70, SYK, and BTK, revealing a potential therapeutic vulnerability. The dual SYK and JAK/STAT6 inhibitor cerdulatinib most effectively compromised the proliferative advantage conferred by UGT2B17 compared to the selective BTK inhibitor ibrutinib. Findings point to an oncogenic role for UGT2B17 as a novel constituent of BCR signalosome also connected with microenvironmental signaling.

Identification of altered cell signaling pathways using proteomic profiling in stable and progressive chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is characterized by significant biologic and clinical heterogeneity. This study was designed to explore CLL B-cells' proteomic profile in order to identify biologic processes affected at an early stage and during disease evolution as stable or progressive. Purified B cells from 11 untreated CLL patients were tested at two time points by liquid chromatography-tandem mass spectrometry. Patients included in the study evolved to either progressive (n=6) or stable disease (n=5). First, at an early stage of the disease (Binet stage A), based on the relative abundance levels of 389 differentially expressed proteins (DEPs), samples were separated into stable and progressive clusters with the main differentiating factor being the RNA splicing pathway. Next, in order to test how the DEPs affect RNA splicing, a RNA-Seq study was conducted showing 4217 differentially spliced genes between the two clusters. Distinct longitudinal evolutions were observed with predominantly proteomic modifications in the stable CLL group and spliced genes in the progressive CLL group. Splicing events were shown to be six times more frequent in the progressive CLL group. The main aberrant biologic processes controlled by DEPs and spliced genes in the progressive group were cytoskeletal organization, Wnt/β-catenin signaling, and mitochondrial and inositol phosphate metabolism with a downstream impact on CLL B-cell survival and migration. This study suggests that proteomic profiles at the early stage of CLL can discriminate progressive from stable disease and that RNA splicing dysregulation underlies CLL evolution, which opens new perspectives in terms of biomarkers and therapy.

Immunostimulatory CpG Oligodeoxynucleotides Induce Sustained ERK2 Activation and Strong Proliferative Responses in CLL B-Cells from Patients with Progressive Disease.

Immunostimulatory DNA sequences that are present in microbial DNA, DNA-containing immune complexes and synthetic CpG-oligodeoxynucleotides (ODNs) can activate cells of the immune system via Toll-like receptor 9 (TLR9). Normal B-cells respond to such stimulation by upregulation of co-stimulatory molecules, proliferation and prolonged survival. The response of chronic lymphocytic leukemia B-cells to CpG ODN stimulation appears to be more variable, since both proliferation and induction of apoptosis have been reported. Because CLL B-cells with mutated and unmutated Ig VH genes show distinct clinical and biological behaviors, we compared their responsiveness to CpG ODN stimulation in terms of proliferation, survival, activation of TLR9 signaling pathways and expression of cell-cycle regulatory and anti-apoptotic proteins. A significant proliferative response was induced in most of the VH unmutated CLL samples (14/23), whereas 3H thymidine incorporation was low or absent in the majority of the VH mutated cases (22/27, P = 0.005). More strikingly, stratification of the CLL cases based on the clinical course showed strong proliferative responses in 14/21 cases with progressive disease and in only 5/29 cases with stable disease (P=0.001). The absence of a proliferative response to CpG ODN stimulation in the stable/VH mutated cases was not caused by different early signaling events, since phosphorylation of JNK, ERK1/2 and Akt, as well as degradation of IkB, was detected in almost all investigated samples (7/7 proliferating and 5/6 nonproliferating). However, a notable difference was observed in the duration of ERK2 activation, which was transient in the nonproliferating CLL B-cells, but lasted more than 24 hours in the proliferating cases. This difference was associated with dimished phosphorylation of the retinoblastoma protein (pRb) and less pronounced or absent induction of cyclin E and cyclin A in the nonproliferating cases, suggesting a block in the G1/S phase transition. On the other hand, cyclin D3 was induced and p27KIP1 was downregulated in all samples, indicating that CLL B-cells from both subgroups can enter the G1 phase of the cell cycle. Analysis of CLL B-cell survival by Annexin V/PI staining and PARP cleavage after 48 hours stimulation with CpG ODN showed significant levels of apoptosis in most of the nonproliferating cases. In contrast, proliferating CLL B-cells remained viable after CpG ODN stimulation, which was associated with upregulation of the anti-apoptotic proteins Mcl-1 and Bcl-xL. These data indicate that immunostimulatory CpG oligodeoxynucleotides can induce distinct responses in progressive/VH unmutated and stable/VH mutated CLL B-cells, which are characterized by proliferation and inhibition of spontaneous apoptosis in the former and induction of apoptosis in the latter group of cases. Although both subgroups show similar early activation of TLR9 signaling pathways and both enter into the G1 phase of the cell cycle, the absence of sustained ERK2 activation precludes cell cycle progression in most stable/VH mutated CLL B-cells. The greater capacity to traverse the cell cycle and proliferate in response to immunostimulatory CpG DNA may therefore be related to the more aggressive clinical course that is associated with CLL B-cells that express unmutated VH genes.

Abstract 3749: MER receptor tyrosine kinase overexpression potentiates survival signal in chronic lymphocytic leukemia cells

Abstract Chronic lymphocytic leukemia (CLL) is the most common form of adult B-cell leukemia in the United States. It is predominantly characterized by the accumulation of CD19+/CD5+/CD23+ monoclonal B-cells in peripheral blood, bone marrow and lymphoid tissues. BCR signaling pathway has emerged as a key driver for the expansion of neoplastic B-cell clones and pathogenesis in several B-cell malignancies including CLL. Stimulation of BCR induces activation of a cascade of downstream signal mediators including Bruton's tyrosine kinase (BTK), PI3K/AKT and ERK1/2. While the use of BCR-targeted agents, ibrutinib (BTK-inhibitor) or idelalisib (PI3Kδ-inhibitor), has been shown to be effective in relapsed/refractory CLL patients, the responses are limited to partial remissions. Therefore, insights into drugs with unique mechanism(s) of action that are nontoxic to the bone marrow/immune system and feasible in the older patient population (average age: 65 - 70 years) are urgently needed. To achieve such a goal it is critical to identify other targets that are constitutively active and may crosstalk with the BCR signal or other receptor tyrosine kinases (RTKs). MER RTK of the TAM (Tyro3, AXL, MER) family offers such an alternative approach. Previously, we detected that CLL B-cells express highly active AXL that acts as a docking site of multiple kinases/lipase including LYN, SYK/ZAP70, PI3K and PLCγ2 that, when subject to specific inhibition induces apoptosis. Most recently, we found that CLL B-cells from 50 - 60% of previously untreated CLL patients (n=30) also overexpressed MER at variable levels while, normal B-cells from health subjects did not show any such expression. Interestingly, MER in CLL B-cells remains constitutively phosphorylated (active). In addition, we also detected overexpression of its ligand, growth arrest specific gene 6 (Gas6) in CLL B-cells as compared to normal B-cells. Of note, leukemic B-cells from the Eμ-TCL1 transgenic mice which recapitulate aggressive human CLL also showed overexpression of MER. To this end, we found that exogenous addition of recombinant Gas6 to primary CLL B-cell culture or MEC1 (a CLL cell line) cells activated MER in a time-dependent manner. Interestingly, we also detected co-activation of BTK of the BCR pathway, following MER activation in CLL cells while, in vitro stimulation of BCR did not activate MER. Together, these findings suggest that MER activation may regulate the BCR signal in CLL B-cells in order to potentiate the survival signal. In conclusion, we detected overexpression of constitutively activated MER in CLL B-cells from some CLL patients which may represent high-risk CLL clones for clinical progression. Importantly, activation of MER may positively regulate the BCR signal inducing increased survival and apoptotic resistance of the leukemic B-cells to BCR-targeted agents. Further studies are in progress to elucidate the impact of targeting MER in CLL B-cell survival and resistance to therapy. Citation Format: Mariana T. Mendez, Hasan Mahmud, Sara K. Vesely, Jennifer Holter-Chakrabarty, Asish K. Ghosh. MER receptor tyrosine kinase overexpression potentiates survival signal in chronic lymphocytic leukemia cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3749.

Tumor-stroma interaction increases CD147 expression in neoplastic B lymphocytes in chronic lymphocytic leukemia

BackgroundChronic lymphocytic leukemia (CLL) microenvironment plays a critical role in disease pathogenesis. Matrix metalloproteinases (MMPs) are involved in CLL-B cell migration and survival. CD147 is associated with MMPs production by tumor and stromal cells. AimTo analyze CD147, MMP2 and MMP9 expression in CLL-B cells and its modulation by fibroblasts (Fb)-CLL-B cell interaction. MethodsCLL-B cells were co-cultured with Fb, as a simulation of CLL microenvironment. CD147 was evaluated in healthy donor (HD)-B cells and CLL-B cells by flow cytometry. MMP2 and MMP9 activity in CLL-plasma samples and conditioned media (CMs) was studied by zymography. ResultsMMP9/MMP2 plasma levels were significantly higher in CLL patients than in HD. CD147 expression (median fluorescence intensity) in CLL patients characterized 3 groups: low- (19.1 ± 3.2; n=3), middle- (42.7 ± 12.8; n=18) and high- (76.5 ± 9.6; n=5) related to CD147 expression in HD-B cells. CD147 expression significantly increased in CLL-B cells after Fb-CLL-B cell co-culture. A significant increase in proMMP2 activity was observed in CMs obtained from Fb-CLL-B cell co-cultures in comparison with isolated CLL-B cells. ConclusionsCD147 expression in CLL-B cells and MMPs secretion was induced by Fb-CLL-B cell contact, suggesting CD147 participation in the CLL pathophysiology.

Β-Catenin and Axl Receptor Tyrosine Kinase Modulation in CLL B-Cells with Co-Culture on Marrow Stromal Cells: Implications for Drug Resistance

Introduction: Bone marrow stromal cells (BMSCs), a major component of CLL microenvironment influences CLL B-cell survival. We have utilized an in vitro BMSC model system and found unique alterations in CLL B-cells with short term BMSC co-culture that point to previously unidentified biologic changes in the CLL B-cells that may influence CLL B-cell signaling and their drug resistance. Methods: Purified primary CLL B-cells (n=41) from previously untreated CLL patients were cultured alone or co-cultured with primary BMSCs from either normal individuals (n=24) or CLL patients (n=18) at a 50:1 ratio in AIMV medium. After 48 hours, separated CLL B-cells or BMSCs were examined by immunoprecipitation/Western blot analyses to assess the presence of intracellular signal proteins and real time PCR to determine the RNA level expression. In separate experiments to assess CLL B-cell response in co-culture with BMSC, purified CLL B-cells were treated with the following agents at sub lethal doses; fludarabine, chlorambucil, ibrutinib and venetoclax as single agents. Results: Consistent and significant increase in expression of Axl receptor tyrosine kinase for both mRNA and protein levels was observed in CLL B-cells co-cultured with BMSCs compared to CLL B-cells cultured alone. We also detected significantly increased expression of β-catenin at the protein level in CLL B-cells co-cultured with BMSCs. In contrast there was no significant change in β-catenin or Axl protein expression in the co-cultured BMSCs. Co-culturing of CLL B-cells with BMSCs using transwells confirmed that the upregulation of both Axl and β-catenin is dependent on the direct contact of CLL B-cells with BMSCs. Experiments showed that the increased Axl level in co-cultured CLL B-cells was independent of Axl kinase activity. Furthermore, the CLL B-cells from co-culture also had clear upregulation of downstream P-ERK-1/2 but no change in P-AKT(Ser473). Next we reasoned that if increases in Axl and β-catenin are related to drug resistance then CLL B-cells should have increases in those proteins when cultured with drugs and in presence of BMSCs. Treatment with chemotherapeutic or targeted therapy drugs, (i.e. fludarabine, chlorambucil, ibrutinib, venetoclax) with sublethal doses was found to lead to increase in expression levels of both β-catenin and Axl in co-cultured CLL B-cells. However these increases were significantly over that seen with simple co-culture of CLL B-cells with BMSCs. Since CLL B-cells were less sensitive to these latter chemotherapy drugs in presence of BMSCs, this suggests to us a role for both Axl and β-catenin in stromal mediated CLL B-cell drug resistance to these agents. Moreover, high nuclear β-catenin and P-ERK-1/2 levels were also detected in co-cultured CLL B-cells. It is also known that ERK associates with and inactivates GSK-3β resulting in the up-regulation of β-catenin. We found upregulation in P-GSK-3β (Ser9) an inactive molecule which can result in increasing accumulation of β-catenin. Inhibition of P-ERK-1/2 using the ERK inhibitor PD98059 in co-cultured CLL B-cells inhibited β-catenin as well as Axl expression levels. Furthermore, it has been shown that Axl expression can be regulated by c-Jun activity. In that regard we have observed upregulation of P-c-Jun(Ser73) which can enhance Axl level in co-cultured CLL B-cells and inhibition of c-Jun activity using SP600125 (c-jun upstream JNK inhibitor), as well inhibited the Axl expression. Finally, we studied 5 CLL patients before and while being treated with ibrutinib/chemo-immuno therapy for the expression of CLL B-cell Axl, β-catenin, P-ERK-1/2, P-c-Jun(Ser73) levels. We found that 2 patients had increase in Axl expression, 4 patients had increased β-catenin and P-ERK-1/2 levels and 3 patients showed increase in P-c-Jun(S73) level after the therapy. Conclusion: Here we show that marrow stromal cell contact with CLL B-cells consistently mediate increased expression in both β-catenin and Axl in CLL B-cells (Figure1). The mechanism for this may, in part, via activated ERK and c-Jun levels (Figure1). We believe that these changes in both molecules are associated with leukemic B-cell survival and drug resistance (Figure1). These studies suggest that a further understanding of the roles of Axl and β-catenin in the CLL B-cells mediated by contact with BMSC will help to develop potential strategies for the management of CLL disease resistant to various drugs. Disclosures Warner: Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment. Kay:Agios: Other: DSMB; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; MorphoSys: Other: Data Safety Monitoring Board.

Overexpression of HMGB1 Receptor RAGE Is Associated with Worse Clinical Outcome in Patients with Chronic Lymphocytic Leukemia

Background: Receptor for advanced glycation end-products (RAGE) is the major receptor for inflammatory molecule HMGB1. Increased levels of RAGE are associated with poor prognosis in many types of tumours. Following engagement with extracellular HMGB1, RAGE internalises and promotes the survival and migration of cancer cells through activation of NF-kB. We have previously determined significantly increased HMGB1 levels in the plasma of CLL patients compared to healthy controls, associated with poorer clinical outcomes. HMGB1 induces differentiation of nurse like cells via RAGE and/or Toll-like receptor 9 (TLR9) harboring CLL progression and maintenance. However, it is unknown whether HMGB1/RAGE/TLR9 signaling axis can directly promote CLL B-cell survival and contribute to disease progression.We hypothesise that HMGB1 could contribute to CLL progression via direct activation of RAGE/TLR9 pathways. The aim of this study is to characterise whether activation of HMGB1-mediated pathways is associated with poorer clinical outcome in patients with CLL.Methods: HMGB1-mediated colocalisation of TLR9 and RAGE proteins was assessed in fresh primary CLL cells using the ImageStream X Mark II flow cytometer and fluorescent microscopy. Expression of RAGE and TLR9 were determined by immunohistochemistry on tissue microarrays (TMAs), containing CLL lymph node samples (n=91). The correlation between RAGE and TLR9 with other established CLL markers were analysed by both Spearman's multiple correlation and chi-square analyses. The association between protein expression and overall survival (OS) was analysed by uni- and multivariate cox regression analysis on both continuous and categorical variables to increase statistical robustness. The test/validation set-defined data cut points were determined by X-tile software.Results: HMGB1-mediated internalisation of RAGE and its colocalisation with TLR9 in primary CLL-B-cells were detected qualitatively by fluorescent microscopy and quantitatively by ImageStream X Mark II flow cytometry, demonstrating that HMGB1 directly activates both RAGE and TLR9 in CLL B-cells.RAGE and TLR9 proteins are heterogeneously expressed in CLL lymph nodes. Multiple correlation analysis identified a significant correlation between RAGE and TLR9 receptor expression (p<0.0001) suggesting they have co-linked functions. RAGE expression positively correlates with levels of Ki67 and positivity of ZAP70 (p<0.0001; p<0.05), and negatively correlates with the median OS rates, suggesting that higher expression of RAGE is associated with CLL cell proliferation and poor OS. However, RAGE expression is not correlated with CD68, a marker of tumor-associated macrophages, suggesting that increased RAGE protein may be mainly expressed by CLL B-cells.We analysed the prognostic effect of RAGE, TLR9, Ki67 and age individually by continuous univariate cox regression analysis and found that only the age of CLL patients is an independent prognostic marker for this cohort of CLL patients. Expression cut points were statistically generated using the X-tile software to define RAGE expression as high/medium/low. Increased RAGE expression (n=13) confers inferior OS (median 3 years) compared to patients with medium/low RAGE expression (n=29 and 48 respectively; median OS 7.5 years; log rank test p<0.05). There is a significant prognostic interaction between high RAGE, high Ki67 and ZAP70 categorical variables (p<0.0001), highlighting the inferior effect of overexpression of RAGE on disease outcome.Conclusions: Our data demonstrates for the first time that overexpression of HMGB1 receptor RAGE in CLL lymph nodes is associated with a worse clinical outcome in patients with CLL and positively correlated with higher expression of Ki67 and positivity of ZAP70. HMGB1 can directly activate both RAGE and TLR9 in primary CLL B-cells. We therefore propose that HMGB1 may play an important role in promoting CLL cell proliferation and survival and could be a potential target for the treatment of CLL. However, we need to further confirm our findings by combining other independent validation sets and intensive functional studies on the role of HMGB1 in CLL. DisclosuresGribben:Pharmacyclics: Honoraria; Roche/Genentech: Honoraria; Celgene: Consultancy, Honoraria; Janssen: Honoraria; Gilead: Honoraria.

Targeting PI3Kδ: emerging therapy for chronic lymphocytic leukemia and beyond.

Chronic lymphocytic leukemia (CLL) remains the most incurable leukemia. Early chemotherapeutic treatments, including alkylating agents, purine nucleoside derivatives, and immunotherapeutic antibodies, only show limited benefits for patients but severe off-target related side effects. Recent advances in understanding of the critical molecular pathways of regulating proliferation and survival of B-CLL cells have spurred a new therapeutical strategy by selectively targeting phosphoinositide 3-kinase delta (PI3Kδ). Idelalisib, a first-in-class PI3Kδ-selective small molecule has received the FDA's fast-track approval in July of 2014 as a new treatment of CLL, indolent B-cell non-Hodgkin's lymphoma, and relapsed small lymphocytic lymphoma. Undoubtedly, the success of idelalisib has provided a solid support in the development of PI3Kδ-specific inhibitors and reformed the concept of treating CLL. However, the number of reported selective inhibitors of PI3Kδ is very limited and very few have advanced into clinical trials. The mechanism of their actions remains elusive. More profound understanding on the modes of action of new PI3Kδ inhibitors will further validate the PI3Kδ-targeting strategy, and help to identify biomarkers capable of stratifying patients who will most likely benefit from the therapy.

PKC Inhibitor AEB071 Demonstrates Pre-Clinical Activity In Chronic Lymphocytic Leukemia

Targeting B-cell receptor (BCR) downstream pathways is of therapeutic importance in eradicating chronic lymphocytic leukemia (CLL) cells. Members of the protein kinase C (PKC) family play an important role in B-cell activation. PKC-β has recently been shown to be over-expressed in CLL and essential to CLL development in the TCL1 mouse model. Mice deficient in PKC-β exhibit a survival defect in response to BCR stimulation, correlating with an inability to induce the NF-κB-dependent anti-apoptotic proteins as Bcl-xL and A1. Moreover, PKC-β-dependent activation of NF-κB in stromal cells is pivotal for the survival of B-CLL cells in vivo; wherein PKC-β inhibition was shown to prevent microenvironment protection of CLL. Additionally, PKC-β lies downstream of PLC-γ2 where activating mutations have been noted in BTK (ibrutinib) resistant patients, which conveys a potential mechanism to target resistance related to mutations in this target protein. Therefore exploration of a PCK-β inhibitor in CLL is highly justified and innovative. Sotrastaurin (AEB071) is an orally administered potent inhibitor of classical and novel PKC isotypes; with strong and specific activity on PKC-α, PKC-β and PKC-θ and lesser activity on PKC-δ, PKC-ε, and PKC-η. Pre-clinically, AEB071 has demonstrated in vivo pre-clinical activity in activated B-cell diffuse large B-cell lymphoma (DLBCL) models and is currently being tested for efficacy in CD79b mutated DLBCL. Since PKC-β is indispensable for BCR-induced NF-κB activation and B-cell survival, herein we evaluate the impact of AEB071 on CLL cell survival as a promising therapeutic to target this pathway.Our preliminary work demonstrated that AEB071 was markedly cytotoxic to CLL cells in a dose-dependent (≤6.25uM, p<0.001) and time-dependent manner (p=0.011) as measured by MTS analysis. In a whole blood assay, AEB071 exhibits a retained selective cytotoxicity against tumor cells with a modest reduction in B-CLL cells whereas no effect on T-cells or natural killer cells was detected in CLL patient samples. Notably, upon treatment of blood from healthy subjects, AEB071 showed no toxic effects on normal B-cells, T-cells and natural killer cells. AEB071 inhibits CPG-induced survival of CLL cells in vitro (p<0.01), and effectively blocks the protection induced by soluble factors such as CD40L, IL-4, and TNF (p<0.01), which are known to reduce the spontaneous apoptosis associated with CLL cells. Similar effects were observed with stromal cell contact; wherein AEB071 showed enhanced cytotoxic potency on CLL cells under co-culture conditions with stromal cells compared to CLL alone (p<0.05). Additionally, AEB071 attenuated anti-IgM-induced survival of CLL cells with a modest induction of apoptosis (p<0.001). Furthermore, treatment of PMA- or BCR-activated CLL cells with AEB071 could effectively abrogate downstream survival pathways including ERK1/2, p38MAPK, AKT, GSK3β, and NF-κB as revealed by immunoblot analysis. Collectively, this data indicate that therapeutic strategies to inhibit PKC-β have the potential to disrupt signaling from the microenvironment that lead to in vivo CLL cell survival and potentially drug resistance. Current studies are ongoing to evaluate the in vivo tolerability and therapeutic efficacy of AEB071 in the Eμ-TCL1 transgenic mouse model of CLL.In conclusion, PKC-β represents an innovative target for CLL and therefore, future efforts targeting PKC with the PKC inhibitor AEB071 as monotherapy in clinical trials of relapsed and refractory CLL patients may be warranted. Disclosures:No relevant conflicts of interest to declare.

Abstract 932: Predictive markers of the in vitro anticancer effect of the pan class I PI3K inhibitor BKM120 in primary B-CLL lymphocytes.

Abstract B-chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD5+ B-lymphocytes that are long-lived in-vivo but die quickly by apoptosis when cultured in-vitro. The phosphatidylinositol-3 kinase (PI3K) cascade which is a critical component of survival signaling has increased activity in CLL lymphocytes as compared to normal B lymphocytes. There are three classes of PI3Ks of which class I is the most clearly implicated in human cancer. The PI3K-δ inhibitor, CAL-101, promotes apoptosis and abrogated protection from spontaneous apoptosis induced by CD40 in primary CLL lymphocytes in-vitro. Thus the PI3K pathway appears to play a critical role in B-CLL cell survival. BKM120 is a pan class I PI3K inhibitor developed by Novartis. Phase I trials demonstrated that plasma concentrations (Cmax) of 5 μM can be obtained. In view of the critical role of PI3K in CLL homeostasis, the activity of BKM120 was examined in CLL lymphocytes in-vitro. BKM120 was cytotoxic in the 3 CLL cell lines and in 78% of the primary B-CLL lymphocytes samples isolated from 65 B-CLL patients, including patients with del11 and del17. Interestingly, 60% of the B-CLL samples tested in our study have an IC50 below the Cmax. Furthermore, BKM120 cytotoxicity correlated with the basal expression of proteins involved in the PI3K/Akt pathway (Akt, rictor, raptor, p70S6K and 4E-BP1) but not with PTEN, mTor, IgVH or CD38 expression in these primary B-CLL lymphocytes. Further analysis of these different markers demonstrated that only patients very sensitive to BKM120 (IC50 ≤3 μM) expressed low basal levels of mTor, raptor and p70S6K simultaneously. Inhibition of PI3K was demonstrated by detection of phosphorylated Akt (S473) after BKM120 treatment in the cell lines. Molecular biomarkers such as p70S6K and 4E-BP1 have been used as indicators of PI3K pathway inhibition in-vivo. We also have demonstrated that BKM120 decreased the phosphorylation status of 4E-BP1 and p70S6K in the primary B-CLL lymphocytes tested. As detected by AnnexinV/propidium iodine staining and analysis of caspase-3 cleavage, BKM120 induced apoptosis in primary B-CLL cells culture in the presence and absence of stromal cell support. Taken together, our results demonstrated that the class I PI3K inhibitor BKM120 may be useful as a single agent in CLL patients independently of their IgVh mutational status, CD38 expression or genomic deletions (del11 and del17). Furthermore, a combination of different biomarkers (expression of mTor, raptor, and p70S6K) may predict the response to BKM120 treatment. Also, BKM120 abolished the protection against apoptosis and drug resistance conferred by the microenvironment to the primary B-CLL lymphocytes in-vitro. Our preclinical study demonstrated that BKM120, a molecule with acceptable clinical toxicity, may be useful in the treatment for CLL. Citation Format: Lilian Amrein, May Shawi, Jeremy Grenier, Raquel Aloys, Lawrence Panasci. Predictive markers of the in vitro anticancer effect of the pan class I PI3K inhibitor BKM120 in primary B-CLL lymphocytes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 932. doi:10.1158/1538-7445.AM2013-932

Somatic Mutation As a Mechanism of Wnt/β-Catenin Pathway Activation in CLL

Abstract 559One goal of cancer genome sequencing is to identify key genes and pathways that drive tumor pathogenesis. While many studies identify driver genes based on recurrence of mutations in individual genes, subsets of genes with non-recurrent mutations may also be defined as drivers if they affect components of a single biological pathway. By large-scale DNA sequencing, we recently identified the Wnt pathway as a significantly mutated pathway in chronic lymphocytic leukemia (CLL) (Wang et al NEJM 2011). This pathway is known to be critical for hematopoietic stem cell development and differentiation, and is highly dysregulated in CLL. However, the functional alterations generated by specific mutations in members of this pathway and their contributions to CLL pathogenesis have not been previously characterized.In our previously reported DNA sequencing of 91 CLL samples, we identified a broad spectrum of mutated Wnt pathway members. Fifteen non-synonymous coding mutations were identified in 13 CLL samples (1–2 mutations per sample) within core Wnt pathway members. These nonsilent coding mutations involved components of the entire Wnt pathway–ranging from extracellular factors (DKK2, WNT1, WNT10A, RSPO4, CSNK1E), to cell surface receptors (FZD5, RYK), cytoplasmic factors (CSNK1E, PRICKLE1) and nuclear transcriptional factors (CHD8, BRD7, CREBBP, BCL9). All of the identified Wnt pathway mutations localized to evolutionarily conserved sites, supporting a role for these mutations in perturbing Wnt pathway function.To assess the contribution of the Wnt pathway to CLL-B cell survival, we optimized a novel miniaturized transfection platform, silicon nanowires (SiNWs), to deliver siRNAs targeting core Wnt pathway members into normal and CLL-B cells. By exploring various nanowire sizes, lengths and densities, we achieved conditions to consistently deliver siRNAs into B cells (90%) without impacting cellular viability (>95%). Using SiNW-mediated delivery of gene-specific siRNAs, we established that silencing of the Wnt pathway members LEF1, CTTNB1, and DVL1 in CLL samples (n=3) resulted in greater loss of cell viability (by∼20–35%) compared to delivery of non-targeting controls (P=0.06, 0.01, and 0.005, respectively). These results demonstrate that perturbation of key nodes in the Wnt pathway reduce CLL-B cell survival, and support the importance of this pathway in CLL.We then explored whether mutated Wnt pathway members could functionally alter Wnt pathway signaling. We focused our studies on two mutated Wnt pathway members, DKK2-R197H and BCL9-G548S. We generated plasmids expressing either wildtype or mutant alleles and measured Wnt pathway activation in 293T cells using a TCF/LEF-dependent luciferase reporter system following coexpression of WNT1 with the wildtype or mutant gene or both. For both DKK2 and BCL9, mutated forms abolished their normal repressive effects on Wnt activation. Elimination of the repressive effects of the wildtype protein was also observed when the wildtype and mutant alleles were expressed together, suggesting a dominant activating effect of the mutated allele. We hypothesized that chronic pathway activation by these mutations could lead to dependence of patient CLL cells on Wnt signaling for their survival. We directly addressed this question by examining the survival of normal B cells (n=4) and patient CLL-B cells with or without (n=7) mutated DKK2 or BCL9 following SiNW-mediated delivery of gene-specific siRNAs. This revealed that a BCL9-mutated CLL sample was more dependent on BCL9 expression for survival than normal B cells (p=0.02) or CLL samples lacking Wnt pathway mutations (p=0.07). Similarly, silencing of DKK2 in a DKK2- mutated CLL sample led to greater cell death than gene silencing in normal B cells, or in CLL samples without Wnt pathway mutations (p=0.003). These results demonstrate that CLL samples harboring mutations in DKK2 or BCL9 exhibit greater dependency on Wnt pathway signaling than samples without Wnt pathway mutations, and support a driving role for these mutations in CLL.Taken together, our results demonstrate that somatic mutations in Wnt pathway components in CLL-B cells alter the activity of this pathway and increase its sensitivity to pathway inhibition. Our findings further support the notion that non-recurrent mutations at different nodes of the Wnt pathway contribute to CLL pathogenesis. Disclosures:Brown:Calistoga, Celgene, Genentech, Pharmacyclics, Novartis, Avila: Consultancy; Genzyme, Celgene: Research Funding.

The Kinin-Kallikre System in Chronic Lymphocytic Leukemia - A Potential Target for Therapy?

AbstractAbstract 3904CLL remains an incurable disease with standard therapy regimens. The hyper reactivity of the B cell Receptor (BCR) to unknown antigen ligation plays a pivotal role in B-cell survival. Recent clinical trials of BCR-targeted therapies prompted the need for a better understanding of the biology of BCR signaling. We previously employed proteomics to study protein expression changes associated with BCR ligation. Using 2-dimensional gel electrophoresis (2DE) with MALDI-TOF mass spectrometry (MS) we identified that Kininogen (KNG) was upregulated in 3/3 “poor prognosis” clinical samples upon BCR stimulation. KNG (a critical protein of Kinin-Kallikrein System) is the precursor for Kinins which act via the B1 and B2 receptors (B1R and B2R, respectively) and is known to play a critical role in cell migration, proliferation, vascular permeability, inflammation and intracellular Ca2+ influx. Consequently we hypothesized that there is a functional role for B1R and B2R, Kinins and their precursor KNG in CLL B-cell survival which may offer a potential therapeutic target.Following ethical approval, blood samples were collected from CLL patients. Time to first treatment, clinical stage, IgVh status, CD38 and ZAP-70 data were available. “Poor prognosis” samples were defined as unmutated IgVh status, high WBC and hyper-responsive BCR (p-ERK expression ≥ 2.0- fold increase upon stimulation). Immunoblotting was employed to confirm KNG upregulation and Tissue Kallikrein expression in CLL B-cells. A series of 59 CLL samples was screened for constituent KNG expression using immunoblotting. The presence of mRNA transcripts for KNG was assessed by PCR. An Automated One-stage Factor Assay on the Instrumentation Laboratory ACL-TOP analyser was used to determine the level of Plasma Kallikrein in CLL patients vs healthy controls. Fluorescence Activated Cell Sorting (FACS) was utilized for CLL B-cell enrichment according to CD20 status. Isolated CLL B-cells were subjected to immunodetection and flow cytometry for B1R and B2R cell surface expression identification.Proteomic analysis by 2DE/MALDI-TOF MS previously revealed upregulation of KNG after 5.5 hours of in vitro BCR stimulation in 3/3 clinical samples. Upregulation of KNG after stimulation was confirmed by immunoblotting in 5 samples, including all 3 which were previously analyzed using proteomics. There are 2 forms of KNG: HMWK and LMWK. The analysis of constitutive KNG by immunoblotting revealed positive expression in 72.9% (43/59) and 80% (28/35) for LMWK and HMWK, respectively. No statistical significance was found with preliminary clinical correlations, however, there was a trend towards shorter median survival in LMWK positive cases (152 months vs. 264 months for LMWK negative cases) (p=0.161). PCR confirmed the presence of KNG transcript in CLL samples. An automated one-stage factor assay revealed that 47.2% (26/55) CLL patients expressed elevated Plasma Prekallikrein (the zymogen form of Plasma Kallikrein) level. Tissue Kallikrein overexpression was confirmed in 14 CLL samples, using an anti-KLK 6 antibody. We investigated the presence of inducible B1R and constitutive B2R on CD20+ CLL B-cells and demonstrated that B1R and B2R are expressed by CLL B-cells, however, B1R expression was highly upregulated.We demonstrate for the first time that CLL B-cells express differing levels of KNG protein and that the expression can be significantly increased after BCR ligation. Consequently, high concentration of KNG is achieved in near proximity to B1R and B2R, identified on the surface of CLL B-cells. Therefore, we show that CLL B-cells comprise the complete system for the synthesis and liberation of Kinins which in turn mediate the activation of B1R and B2R via an autocrine loop. We suggest, that simultaneous and sustained activation of B1R and B2R, which is known to be advantageous for immediate triggering of ERK1/2 global phosphorylation and PLCγ2-dependent intracellular Ca2+ mobilization, play a functional role in the induction of pathologic signal transduction leading to B-cells survival and that Ca2+ influx-dependent transcriptional activation and MEK1/ERK1/2 pathway signaling may be dependent upon simultaneous complimentary stimuli input from BCR, B1R and B2R and possibly other disease-associated stimuli. The inhibition of Kinin receptor function may be a potential target for combined therapy in CLL. Disclosures:No relevant conflicts of interest to declare.

Increased Survival and Migration of CLL B-Cells in the Presence of Marrow Mesenchymal Stromal Cells: Novel Findings for Microenvironment-Targeted Therapies

AbstractAbstract 4571 Background.The accumulation of CD19+/CD5+/CD23+ B cells with a prolonged lifespan in peripheral blood, secondary lymphoid organs and bone marrow (BM) is a peculiar feature of B-cell chronic lymphocytic leukemia (B-CLL). Since CLL cells removed from the in vivo microenvironment and in vitro cultured rapidly undergo spontaneous apoptosis, bidirectional interactions between malignant and by-stander cells may lead to an abnormal microenvironment that confers growth advantages to neoplastic clone. Mesenchymal Stromal Cells (MSCs) are the dominant marrow stromal population in indolent subtype of CLL/small lymphocytic leukemia (SLL) and follicular lymphoma (FL), rather than other aggressive B-cell lymphomas, and are involved in B-CLL cell survival. Despite the phenotypic and cytologic homogeneity, CLL is characterized by extremely variable clinical courses, suggesting that malignant B-cells hold variable degrees of dependency on pro-survival signals coming from the microenvironment. The aim of this study was to assess the role of MSCs in CLL B-cell localization and survival, defining the degree of dependency of leukemic B-cells from external pro-survival signals, with the ultimate goal of identifying patients that mostly benefit microenvironment-targeted therapies. Methods.MSCs isolated from the BM of 47 B-CLL patients were expanded ex vivo and characterized through flow cytometry analysis and differentiation cultures. Fresh isolated CLL peripheral blood mononuclear cells were co-cultured with CLL-MSCs or stromal cells and apoptosis were measured by Annexin V test and western blotting analysis (PARP-1 detection). Chemotactic assays were performed. Results.The survival of neoplastic cells ranged from 13.3% (±13.2) in leukemic cells cultured in medium alone to 58.5% (±17.2) when leukemic cells were cultured in presence of CLL-MSCs (p<0.01). Transwell experiments showed that the anti-apoptotic effect is mediated by soluble factors produced by MSCs. We investigated whether different CLL clones show a different susceptibility to spontaneous apoptosis when co-cultured in presence of MSCs recovered from B-CLL patients. The detection of the 85KDa cleaved PARP fragment in all CLL B-cells cultured in medium alone confirmed that they underwent spontaneous apoptosis. At the same time, the presence or the lacking of the cleaved fragment of PARP-1 on CLL B-cells after 7 day-co-cultures with MSCs discriminated patients into two groups: non-responder (89 kDa Parp fragment detectable) and responder (89 kDa Parp fragment not detectable) to microenvironment pro-survival signals. Finally, chemotaxis tests showed the ability of MSCs to produce and release molecules promoting the migration and the localization of neoplastic B-cells in bone marrow (Migration Index of leukemic cells: 5.1; Migration Index of normal B cells: 1.9; p<0.01). Conclusions.MSCs derived from patients with B-CLL provide survival signals to neoplastic cells, extending their lifespan and producing chemotactic factors that favour their accumulation into BM. On the other hand, CLL cells display heterogeneous responses to environmental pro-survival signals, suggesting that each CLL clone could differently react to the microenvironment protection. The blocking of the cross-talk between malignant clone and accessory cells within the microenvironment might represent an attractive novel strategy for CLL therapy. Our data provide the rationale for tailored therapies which powerfully target the cross-talk with marrow cells, particularly on patients carrying a clone more sensitive to anti-apoptotic signals coming from the microenvironment. Disclosures:No relevant conflicts of interest to declare.

Altered BCR and CD40 signalling are associated with clinical outcome in small lymphocytic lymphoma/chronic lymphocytic leukaemia and marginal zone lymphoma patients

Altered <scp>BCR</scp> and <scp>CD</scp>40 signalling are associated with clinical outcome in small lymphocytic lymphoma/chronic lymphocytic leukaemia and marginal zone lymphoma patients

Axl Receptor Tyrosine Kinase Signaling Pathway and the p53 Tumor Suppressor Protein Exist In A Novel Regulatory Loop In B-Cell Chronic Lymphocytic Leukemia Cells

Abstract 799 Background:Axl is a constitutively active receptor tyrosine kinase (RTK) in CLL B-cells. We have shown that Axl is physically associated with multiple non-receptor cellular kinases and regulates their activities in CLL B-cells including Lyn kinase (a member of Src-family kinases reported to be overexpressed in CLL), PI3K, Syk and phospholipase γ2 (PLCγ2). These aspects likely relate to drug resistance and survival. Importantly, inhibition of Axl by pharmacological inhibitor (R428) induces massive cell death suggesting its critical role in CLL B-cell survival. We hypothesized that in addition to its role in regulating cellular kinases, Axl might modulate the tumor suppressor p53 to make CLL B-cells more resistant to conventional therapeutic agents. This is based on our unpublished findings that CLL B-cells from patients who have undergone treatment without any reduction in tumor burden expressed increased levels of Axl vs. their pre-therapy levels. In addition, we have detected higher levels of Axl in 17p-deleted CLL patients. Thus to understand the mechanism of Axl RTK-mediated regulation of CLL B-cell growth and survival, we interrogated the functional association between Axl and the HDM2 (human homolog of mouse MDM2)/p53 axis. Methods:We cloned the entire (∼2 kb) 3’-untranslated region (UTR) of the Axl transcript into pMIR-Report vector downstream of the fire-fly luciferase gene. Human embryonic kidney cells (HEK293) were transfected with a full length Axl gene containing the first 500 nucleotides of its 3’-UTR or the pMIR-Report plasmid with or without miR-34a. Cell lysates were examined for the expression of Axl by Western blot or performed luciferase assay. Purified CLL B-cells were also treated with various kinase inhibitors including, Wortmannin (pan-PI3K-inhibitor), PP1 (Src-inhibitor) and SKI-606 (targets both Src and Axl) to detect signaling pathway components downstream of Axl involved in regulating HDM2 function. Results:CLL B-cells from majority of CLL patients (n=15) showed constitutive levels of Axl phosphorylation which we found was associated with the phosphorylation level of HDM2 at Ser-166. HDM2 has an important function in controlling p53 stability in response to genotoxic stress. Recent studies have shown that HDM2 can be phosphorylated on Ser-166 by AKT, MAPKs, and other kinases and this phosphorylation regulates HDM2 nuclearcytoplasmic shuttling under certain conditions. However, we found that HDM2 phosphorylation in CLL is independent of PI3K/AKT, p70S6K, and Erk1/2 activation. Interestingly, we found that inhibition of Axl and/or Src kinases (using SKI-606 and PP1) inhibited HDM2 phosphorylation suggesting that Axl and Src kinases are critically involved in regulating HDM2 affinity towards p53 in CLL B-cells. We also identified a complementary binding sequence of miR-34a microRNA on the first 100 nucleotides of Axl 3’-UTR, suggesting a possible post-translational regulation of Axl. Interestingly, miR-34 family transcription was reported to be regulated by p53, and miR-34a was found differentially expressed in CLL versus normal CD5+ B-cells and is linked to response to therapy in CLL. Indeed, exogenous expression of the Axl gene containing the first 500 nucleotides of the Axl 3’-UTR in HEK293 cells exhibited a dose-dependent down regulation of the Axl protein upon co-transfection with miR-34a using Western blot analysis. Furthermore, activation of p53 in CLL B-cells upon treatment with the DNA-damage agent doxorubicin showed a 4–5 fold decrease of Axl protein by flow cytometry. Activation of p53 by doxorubicin was verified by detecting increased expression of p53 and its acetylation at lysine-382 by Western blot. In addition, luciferase assay data using the Axl-3’-UTR reporter plasmid DNA and miR-34a revealed a dose-dependent inhibition of the luciferase activity. Further studies are underway to fully dissect these novel regulatory signaling pathways. Conclusions: Together, these findings suggest that the two functionally distinct cell growth regulatory pathways can modulate CLL B-cell survival in response to various stimuli. This study has established a link between Axl RTK cell-survival signaling pathway and the tumor suppressor p53-mediated apoptosis-signaling pathway in CLL. These first of a kind observations also underscore that the relative balance of p53 status and Axl levels in tumor cells could relate to CLL therapeutic outcome. Disclosures:Kay:Biothera: Research Funding; Clegene: Research Funding; Cephalon: Research Funding; Genentech: Research Funding; Glaxo Smith Kline: Research Funding; Hospira: Research Funding; Novartis: Research Funding; Supergen: Research Funding; Calistoga: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Emergent Biosolutions (Formerly Trubion): Membership on an entity's Board of Directors or advisory committees.

The Involvement of NF-κB Subunits in the Interaction Between CLL-B Cells and Bone Marrow Stromal Cells

Abstract 4590 Objective:To investigate the function of NF-κB signaling pathway in the interaction between chronic lymphocytic leukemia B cells and bone marrow stromal cells. Method:The expression of NF-κB family members at mRNA and protein levels were examined by quantitative RT-PCR and Western bloting analyses individualy, and the differences between CLL-human bone marrow stromal cell (hBMSC) and non-CLL-hBMSC were investigated with the methods above. Cell death was measured by flow cytometry analysis after B-CLL cells were co-cultured with hBMSC and treated with proteasome inhibitors. The changes of NF-κB expression at the protein levels were examined by Western bloting after co-culture experiment. Result:The expression of NF-κB family members turned out to be heterogeneous at both mRNA and protein levels in B-CLL cells, and the members demonstrated a different κB-DNA binding activities. The mRNA expression of NF-κB family members in B-CLL cells was shown at a remarkable higher level than that of the controls. The relative mRNA expression of relA was 0.0214±0.012, whereas it was 0.0130±0.012 for the controls of CD19 positive cells. The relative mRNA expression of p50 and p52 were 66.0860±21.649 and 0.0208±0.011 respectively, and the corresponding expression of CD19 positive cells were 24.8440±9.749 and 0.0065±0.002. The differences were statistically significant. There was no notable difference between CLL-hBMSC and Non-CLL-hBMSC. hBMSC protected B-CLL cells against the proteasome inhibitors and facilitated the survival of B-CLL cells. Conclusion:NF-κB expression is heterogeneous in bone marrow B-CLL cells. There is no significant difference between CLL-hBMSC and non-CLL-hBMSC. hBMSC can protect the survival of B-CLL cells dependending on the endogenous NF-κB activity. hBMSC can increase the drug resistance of B-CLL cells to proteasome inhibitors. Disclosures:No relevant conflicts of interest to declare.

CCL19 Induced Responses Are Differentially Regulated by Atypical Chemokine Receptor CRAM and Its Classical Chemokine Receptor CCR7 in B-CLL Cells

Abstract 4896 Introduction:Chemokines are known to play an important role in the migration and survival of B-CLL cells. The non-signalling chemokine receptors, including DARC, D6 and CCX-CKR, have recently been shown to be involved in chemokine clearance and activity regulation. The human chemokine receptor CRAM is the most recently identified member of this atypical group. CRAM is expressed on B cells in a maturation-stage dependent manner, and to variable degrees on B-CLL cells. We have recently shown that it competitively binds CCL19 and that this binding is not followed by classical chemokine responses. CCL19 and its signalling receptor CCR7 are centrally involved in B cell localisation and maturation within the secondary lymphoid tissues. CCR7 is also highly expressed on B cells from CLL patients and mediates migration towards its ligands CCL19 and CCL21 which have been shown to be present at higher concentrations in serum of patients with lymphadenopathia compared to patients without. In this study we investigate the influence of CRAM on the CCL19 dependent responses of B-CLL cells and potential correlations to clinical data with a specific focus on lymphadenopathia. Results:We demonstrate that B cells from patients with B-CLL present high, but variable degrees of CCR7 and CRAM expression. Patients with compared to patients without lymphadenopathia show a higher CRAM expression level whereas the CCR7 expression is not significantly different. In single samples showing extremly high CRAM expression the migration towards CCL19 is reduced compared to patients with lower CRAM expression. These observations confirm results in the B-CLL cell line MEC-1 showing increased migration toward CCL19 when CRAM expression is reduced using CRAM-siRNA. On the other hand, CRAM seems to be a chemokine presenter as we can show that it does not degrade its chemokine ligand but presents it on the surface of polarised cell layers. Thus, we assume that CRAM plays a role for cell migration, possibly transmigration and cell localisation within lymph nodes of B-CLL cells. Conclusions:We show that CRAM can act as an integrator of different recruitment and activation factors. It is associated to CCR7 driven recruitment of B cells by regulating CCL19 availability. Expression of CRAM differs in B cell malignancies for which CCL19 and CCL21 have already been shown to be implicated in lymphadenopathia. We therefore suggest that CRAM is an additional player in the localisation and differentiation/maturation processes of malignant B cells of B-CLL patients. Disclosures:No relevant conflicts of interest to declare.

P53 Homolog TAp63 elicits apoptosis in Chronic Lymphocytic Leukemia (CLL) B-Cells,

Abstract 3894p63 is a p53 homolog whose function depends on the cellular context. The full-length TAp63 variant may carry an anti-oncogenic potential in solid tumor models, where it mediates Ras-induced cellular senescence, antagonizes tumorigenesis and suppresses the development of metastases. In hepatoma cells TAp63 is involved in activation of both extrinsic and intrinsic apoptosis pathways. By contrast, ΔNp63, an amino-terminally truncated p63 variant, is oncogenic in tumors of squamous cell origin. TAp63 is the predominantly expressed p63 isoform in lymphoid malignancies. Increased expression of TAp63 in diffuse large B-cell lymphoma confers an unfavorable prognosis. In CLL TAp63 was shown to mediate B-cell homing to the bone marrow, thus possibly contributing to apoptosis evasion. We studied expression of p63 in CLL and determined whether p63 plays a role in CLL B-cell survival and sensitivity to chemotherapy.We enrolled 25 previously untreated subjects with B-CLL at the Norris Cotton Cancer Center (Lebanon, NH). CLL B-cells were isolated from peripheral blood with standard Ficoll-Hypaque technique and purified using a B-cell (CLL) Isolation Kit. Small interfering RNA against p63 were delivered using Lonza Nucleofector with transfection efficiency of 20–50% and viability of 60–80% at 24 h. Viability was enhanced when cells were cocultured with “feeder cells”. To test sensitivity to chemotherapy, cells were treated with 10 μM chlorambucil and 5 μM fludarabine for 48–72 h. For apoptosis analysis cells were stained with Annexin V and 7-AAD and assayed by flow cytometry. Five lymph node tissues were analyzed for p63 expression by immunohistochemistry using a mouse monoclonal p63 antibody (clone 4A4).Of 25 CLL patients 15 were males (60%). Median age was 61 years. Median follow up was 3 years. Most patients presented in Rai stage 0–1 (92%). TAp63 mRNA transcripts were expressed in all CLL samples, while ΔNp63 variant was not detected. TAp63α was the predominantly expressed splicing variant. A C-terminally truncated TAp63γ variant was detected at a low level. TAp63 mRNA transcript levels were higher in CLL B-cells than in normal B-cells, but 10- to 100-fold lower than in high-grade lymphoma cell lines. Of five lymph nodes analyzed, weak nuclear p63 staining was detected in one. In this small cohort, p63 expression in the peripheral blood CLL B-cells did not correlate with ZAP-70 expression. CD38-positive (>30%) CLL samples expressed higher TAp63 mRNA transcript levels (p<0.05). siRNA knockdown of p63 in CLL cells resulted in protection from spontaneous apoptosis at 24–48 h, which persisted when cells were cultured on a “feeder layer”. Furthermore, p63 knockdown conferred protection against chlorambucil and fludarabine. B-cell receptor crosslinking with IgM resulted in a 2.5-fold repression of TAp63 transcription, consistent with the pro-survival role of a B-cell receptor in CLL.Thus, TAp63α is the predominantly expressed p63 variant in the peripheral blood CLL cells. TAp63a contributes to the intrinsic apoptosis program in CLL and may play a role in sensitizing CLL B-cells to standard chemotherapy drugs. Disclosures:No relevant conflicts of interest to declare.

TRIS (DIBENZYLIDENEACETONE) Dipalladium a Small-Molecule Palladium Complex Is Effective in the Induction of Apoptosis for B-Chronic Lymphocytic Leukemia B-Cells

Abstract 2851 Background:In an attempt to better treat B-Chronic Lymphocytic Leukemia (CLL) patients, we have examined a unique organometallic complex called Tris (dibenzylideneacetone) dipalladium (Tris DBA). This novel agent is a small-molecule palladium complex shown to have antiproliferative activity against melanoma cells and possible antitumor activity. Tris DBA is known to inhibit activation of signaling molecules such as PI3K and STAT-3, critical factors in CLL B-cell survival and resistance to therapeutic agents. Because B-CLL remains incurable and treatment strategies challenging, our findings may be useful in pursing new therapies to improve patient care. Therefore, this information prompted us to investigate whether Tris DBA could induce apoptosis in CLL B-cells.Method of Approach: Primary leukemic B-cells from CLL patients were cultured in serum free media and treated at 2 × 106 cells per ml with vehicle or various doses of Tris DBA (1 – 10 μg/ml) for 24 – 72 hours. After treatment, cells were analyzed for apoptosis by a FITC-labeled annexin-V/propidium iodide assay using the FACScan. To assess changes in anti-apoptotic proteins, CLL-B cells were cultured for 24 hours with either vehicle or 2.5μg/mL of Tris DBA. Total protein extracts were used for immunoblot methodology with specific antibodies. In some experiments we developed primary marrow stromal cells (MSC) that when nearly confluent were used for co-culture experiments in the presence of freshly isolated CLL B-cells with various chemotherapeutic drugs and/or Tris DBA. Finally, we compared CLL patients with known immunoglobulin heavy chain variable region (IgVH) status because of the clear differences in clinical outcome for these two prognostic subgroups. Results:CLL B-cells were shown to be sensitive to Tris-DBA with a steep dose response curve that significantly increased apoptosis over control levels as early as 24 hours of culture. Thus the LD50 of Tris DBA was between 2.5 and 5.0 μg/ml for the CLL B-cells who were IgVH mutated while between 5 and 7.5μg/ml for the unmutated status CLL B cells (n=10). Normal B or T lymphocytes were found to be equally sensitive to Tris DBA when compared to CLL B-cells however mesenchymal stromal cells (MSC) from CLL marrow (CLL MSC) were far less sensitive to the Tris DBA drug. Thus the LD50 for normal T cells, B cells or NK cells was between 2.5 and 5.0 μg/ml while the LD50 for the MSC was not reached over the dose range of 1–10 μg/ml. When CLL B-cells were added to CLL derived primary MSC in the presence of increasing concentrations of Tris DBA (2.5–10 μg/ml), the cytotoxic impact of Tris DBA on CLL B-cells could be overcome in the presence of MSC. To delineate the mechanism of apoptosis induction of CLL B-cells, we treated CLL B-cells with sub-lethal dose of Tris DBA and prepared cell lysates for Western blot analyses. We found that the anti-apoptotic proteins XIAP and Bcl-2, but not Mcl-1, were down regulated in Tris- DBA-treated leukemic B-cells. In addition, phosphorylation levels of Akt and STAT-3 were inhibited with exposure to Tris DBA as measured by Western blot using specific antibodies. Finally, we studied if Tris DBA could be synergistic or additive with fludarabine in induction of apoptosis of CLL B- cells. Interestingly, we found that in 2 of 3 assays there was synergy between Tris DBA and fludarabine in the enhancement of CLL B-cell apoptosis. Conclusions:We have established that a unique compound, Tris dipalladium, is effective at inducing apoptosis in CLL B-cells whether these leukemic cells are IGVH mutated (low risk) or unmutated (high risk). In addition this compound can modify the levels of XIAP and Bcl-2, Akt and STAT3 activation in the leukemic B-cells. Importantly, by itself the Tris DBA is effective even in the presence of stromal cells albeit losing some effectiveness at high doses in the co-culture and can generate synergy when added to a purine nucleoside for induction of B-cell apoptosis. In total these findings support future clinical applications of this compound in treating CLL patients. Disclosures:Kay:Biothera: Research Funding; Clegene: Research Funding; Cephalon: Research Funding; Genentech: Research Funding; Glaxo Smith Kline: Research Funding; Hospira: Research Funding; Novartis: Research Funding; Supergen: Research Funding; Calistoga: Membership on an entity’s Board of Directors or advisory committees; Celgene: Membership on an entity’s Board of Directors or advisory committees; Emergent Biosolutions (Formerly Trubion): Membership on an entity’s Board of Directors or advisory committees. Arbiser:Natuderm: Patents & Royalties.

Cysteinyl Leukotrienes Signal Via Overexpressed CysLT1, but Not CysLT2 Receptor In Chronic Lymphocytic Leukemia

AbstractAbstract 3612The G Protein-coupled receptors (GPCRs) CysLT1 and CysLT2 are both expressed in peripheral blood mononuclear cells (PBMNC). Their ligands are inflammatory mediators of the cysteinyl leukotriene (cysLT) family and contribute, together with ligands of other GPCRs such as chemokines, to migration and proliferation of a variety of cell types. In the present study, we demonstrate by real-time RT-PCR that CysLT1 mRNA is overexpressed in B-CLL cells compared to both normal PBMNC and normal CD19+ B cells, whereas only low levels of CysLT2 mRNA are present. GPCR-typical cellular responses such as intracellular calcium fluxes and actin polymerization, which were induced by the cysLT LTD4 in CLL cells in a dose-dependent manner, were suppressed by the CysLT1 antagonist MK571, the prototype of the lukast family of drugs used in asthma treatment (e.g., montelukast). However, MK571 and other lukasts also block the leukotriene transporter MRP. The fact that the CysLT1 antagonist LY171883, which has no effect on MRP, also abrogated the responses of CLL cells indicates that the effects of cysLTs are mediated solely by CysLT1. Moreover, also chemotaxis was induced by the cysLT LTD4 in B-CLL cells (optimum at low nanomolar concentrations) and could be blocked by MK571. We further observed inhibiting effects of MK571 on cysLT-induced Erk/MAP kinase phosphorylation in B-CLL cells, which suggests an involvement of CysLT1 in cell proliferation. Indeed, MK571 significantly induced apoptosis of CLL cells in vitro and reduced survival of cultured B-CLL cells. However, the concentrations for a significant reduction of cell viability were higher (1-10 μM) than the concentration required for efficient CysLT1 receptor inhibition (0.1 μM). Thus, the influence of MK571 on B-CLL cell survival may mainly be due to direct effects on apoptosis occuring at the higher dose level and/or blocking of the leukotriene transporter MRP. Accordingly, LY171883 and the combined CysLT1/2 antagonist Bay-u9773 did not induce CLL apoptosis. Our results suggest that CysLT1 is overexpressed in B-CLL cells and involved in cell trafficking, similar to the chemokine receptor CXCR4. Considering the redundancy among GPCRs and the limited effect of MK571 on cell survival, CysLT1 for its own represents a less attractive therapeutic target using the currently available CysLT1 antagonists of the lukast family. However, combined inhibition of several GPCRs highly expressed in CLL cells including cysLT1 and CXCR4 may constitute a promising therapeutic approach and should be further evaluated. Disclosures:No relevant conflicts of interest to declare.