Peripheral Blood Chronic Lymphocytic Leukemia Research Articles

Microenvironment-Induced Expression of PIM Kinases Supports Chronic Lymphocytic Leukemia Cells Survival and Promotes CXCR4-mTOR Pathway Dependent Migration

Lymph node microenvironment provides chronic lymphocytic leukemia (CLL) cells with pro-survival and protective signals, fostering resistance to conventional chemotherapeutics. CLL cells overexpress oncogenic PIM kinases, which modulate proteins engaged in transcription, translation, apoptosis, cell cycle and adhesion/motility (Mol Cancer Ther 2014, 13: 1231-45). Herein, we searched for the link between tumor microenvironment and PIMs expression, compared the clinical characteristics of CLL patients with high versus low expression of PIM kinases, and investigated the consequences of their inhibition with newly developed pan-PIM inhibitor, SEL24-B489 in primary CLL cells.We first evaluated the expression of PIM kinases in CD19+ cells derived from 88 newly diagnosed CLL cases. Patients with unmutated IGHV status exhibited significantly higher PIM1 transcript levels than patients with mutated IGHV genes. Subjects with advanced CLL (Binet C) exhibited higher PIM2 expression than patients in Binet A/B stage. Significantly higher PIM2 transcript abundance at the time of diagnosis was also observed in patients who relapsed after first line treatment (p=0.005). Expression of PIM2 and PIM3 kinases in lymph nodes was significantly higher than in peripheral blood, suggesting a relationship between PIM kinase expression/activity and CLL cell microenvironment. To further explore the role of microenvironment in the control of PIM expression, peripheral blood CLL cells were incubated with anti-IgM or CD40 ligand. Both stimuli induced PIM1 and PIM3 expression. Co-culture of CLL cells with stromal cell (HS5) monolayers promoted the expression of PIM3 isoform.We next assessed the consequences of PIM inhibition in CLL cells using novel pan-PIM inhibitor, SEL24-B489. Incubation with SEL24-B489 decreased phosphorylation of PIM substrates, p-FOXO1/3a(T24/T32) and p-4EBP1(S65), and induced dose-dependent apoptosis in 27 out of 28 analyzed cases, regardless of the IGHV mutation status and including relapsed patients. Of note, SEL24-B489 induced higher apoptotic response in primary CLL cells than referential pan-PIM inhibitor AZD1208. CLL cells with 17p13 deletion and obtained from chemo-refractory patients were also vulnerable to SEL24-B489, suggesting that functional p53 is not required for execution of SEL24-B489-mediated apoptosis. Importantly, SEL24-B489 was not toxic for cells derived from healthy donors.Since microenvironmental cues increase expression of PIM kinases, we hypothesized that interactions with stromal cells might hinder the in vitro activity of the PIM inhibitor. To explore this possibility, we compared apoptotic response to SEL24-B489 in CLL cells co-cultured on HS5 monolayers and CLL cells grown without the stromal support. In 6 out of 7 tested cases, SEL24-B489 overrode the protective signals from HS5 cells and induced apoptosis, although the cytotoxic effect of PIM inhibitor was stronger in the absence of stromal cells.PIM1 was shown to regulate CLL cells migration through CXCR4(S339) phosphorylation (Mol Cancer Ther 2014, 13: 1231-45). Accordingly, SEL24-B489 decreased phospho-CXCR4(S339), CXCR4 surface expression, and impaired CLL cells migration in the CXCL12 gradient. Surprisingly, decrease in the CXCR4 surface expression after SEL24-B489 was relatively modest when compared to the effect of this inhibitor on CXCL12-directed migration. We found that incubation of CLL cells with CXCL12 led to increase in the phosphorylation of mTOR(S2448) and Akt(S473). SEL24-B489 reduced the levels of p-mTOR(S2448), p-Akt(S473), p-4EBP1(T37/T46) and p-TSC2(S1798), revealing inhibitory effect on mTOR pathway. Pre-incubation of CLL cells with an mTOR inhibitor similarly restrained CXCL12-mediated mTOR activity and led to impaired CLL cells migration, uncovering the key role of mTOR axis in CXCR4-dependent migration. Thus, SEL24-B489 impairs the CLL cell migration by inhibiting CXCR4 surface expression and the CXCR4-triggered mTOR pathway.Taken together, we show that microenvironment signals increase expression of PIM kinases, supporting CLL cell survival and migration. Inhibition of PIM kinases impairs CXCR4-dependent migration and leads to CLL cells death, regardless of the p53 status. Targeting PIM kinases in CLL patients will likely release the cells from microenvironmental niches and might be a rational therapeutic strategy. DisclosuresWarzocha:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Czardybon:Selvita S.A.: Employment. Galezowski:Selvita S.A.: Employment. Windak:Selvita S.A.: Employment. Brzozka:Selvita S.A.: Employment. Juszczynski:Selvita S.A.: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Functional Differences between IgM and IgD Signaling in Chronic Lymphocytic Leukemia.

BCR signaling is a central pathogenetic pathway in chronic lymphocytic leukemia (CLL). Most CLL cells express BCRs of IgM and IgD isotypes, but the contribution of these isotypes to functional responses remains incompletely defined. We therefore investigated differences between IgM and IgD signaling in freshly isolated peripheral blood CLL cells and in CLL cells cultured with nurselike cells, a model that mimics the lymph node microenvironment. IgM signaling induced prolonged activation of ERK kinases and promoted CLL cell survival, CCL3 and CCL4 chemokine secretion, and downregulation of BCL6, the transcriptional repressor of CCL3 In contrast, IgD signaling induced activation of the cytoskeletal protein HS1, along with F-actin polymerization, which resulted in rapid receptor internalization and failure to support downstream responses, including CLL cell survival and chemokine secretion. IgM and IgD receptor downmodulation, HS1 and ERK activation, chemokine secretion, and BCL6 downregulation were also observed when CLL cells were cocultured with nurselike cells. The Bruton's tyrosine kinase inhibitor ibrutinib effectively inhibited both IgM and IgD isotype signaling. In conclusion, through a variety of functional readouts, we demonstrate very distinct outcomes of IgM and IgD isotype activation in CLL cells, providing novel insight into the regulation of BCR signaling in CLL.

Phenotype and immune function of lymph node and peripheral blood CLL cells are linked to transendothelial migration

AbstractSeveral lines of evidence suggest that homing of tumor cells to lymphoid tissue contributes to disease progression in chronic lymphocytic leukemia (CLL). Here, we demonstrate that lymph node (LN)-derived CLL cells possess a distinct phenotype, and exhibit enhanced capacity for T-cell activation and superior immune synapse formation when compared with paired peripheral blood (PB) samples. LN-derived CLL cells manifest a proliferative, CXCR4dimCD5bright phenotype compared with those in the PB and higher expression of T-cell activation molecules including CD80, CD86, and HLA-D–related (DR). In addition, LN-CLL cells have higher expression of α4β1 (CD49d) which, as well as being a co-stimulatory molecule, is required for CLL cells to undergo transendothelial migration (TEM) and enter the proliferation centers of the LNs. Using an in vitro system that models circulation and TEM, we showed that the small population of CLL cells that migrate are CXCR4dimCD5bright with higher CD49d, CD80, CD86, and HLA-DR compared with those that remain circulating; a phenotype strikingly similar to LN-derived CLL cells. Furthermore, sorted CD49dhi CLL cells showed an enhanced capacity to activate T cells compared with CD49dlo subpopulations from the same patient. Thus, although PB-CLL cells have a reduced capacity to form immune synapses and activate CD4+ T cells, this was not the case for LN-CLL cells or those with the propensity to undergo TEM. Taken together, our study suggests that CLL cell immunologic function is not only modulated by microenvironmental interactions but is also a feature of a subpopulation of PB-CLL cells that are primed for lymphoid tissue homing and interaction with T cells.

BCR signaling inhibitors differ in their ability to overcome Mcl-1–mediated resistance of CLL B cells to ABT-199

AbstractThe Bcl-2 antagonist ABT-199 (venetoclax) has demonstrated promising clinical activity in patients with chronic lymphocytic leukemia (CLL). ABT-199 is strongly cytotoxic against unstimulated peripheral blood CLL cells in vitro but is much less effective against CLL cells that have received survival signals from the microenvironment. In particular, stimulation of CLL cells with CD40L results in substantial resistance mediated by induction of the antiapoptotic Bcl-2 family proteins Bcl-xL and Bfl-1. In this study, we investigated whether resistance to ABT-199 can be conferred by B-cell receptor (BCR) stimulation, which is another important survival signal from the leukemic microenvironment. We show that sustained BCR stimulation results in significant ABT-199 resistance, which correlates with induction of the antiapoptotic protein Mcl-1 and less consistently with downregulation of proapoptotic Bmf, Hrk, and BimEL. A major role for Mcl-1 in conferring ABT-199 resistance is additionally supported by knockdown and enforced expression experiments with primary CLL cells. We further show that SYK, BTK, and phosphatidylinositol 3-kinase δ (PI3Kδ) inhibitors significantly downregulate Mcl-1, but with different efficacy. Complete Mcl-1 downregulation was consistently achieved only with SYK inhibitors R406 and GS-9973 (entospletinib), whereas the BTK inhibitor ibrutinib and the PI3Kδ inhibitor idelalisib in more than half of the cases had only a partial effect. The greater ability of SYK inhibitors to downregulate Mcl-1 correlated with their greater capacity to block BCR-mediated inactivation of GSK-3, a major negative regulator of Mcl-1. The finding that BCR signaling inhibitors differ in their ability to target Mcl-1 is relevant for the design of clinical trials combining these agents with ABT-199.

A ribosome-related signature in peripheral blood CLL B cells is linked to reduced survival following treatment.

We have used polysome profiling coupled to microarray analysis to examine the translatome of a panel of peripheral blood (PB) B cells isolated from 34 chronic lymphocytic leukaemia (CLL) patients. We have identified a ‘ribosome-related' signature in CLL patients with mRNAs encoding for ribosomal proteins and factors that modify ribosomal RNA, e.g. DKC1 (which encodes dyskerin, a pseudouridine synthase), showing reduced polysomal association and decreased expression of the corresponding proteins. Our data suggest a general impact of dyskerin dysregulation on the translational apparatus in CLL and importantly patients with low dyskerin levels have a significantly shorter period of overall survival following treatment. Thus, translational dysregulation of dyskerin could constitute a mechanism by which the CLL PB B cells acquire an aggressive phenotype and thus have a major role in oncogenesis.

Ibrutinib Responsive Micro-RNAs and Upregulation of Tumor Suppressor Targets in Chronic Lymphocytic Leukemia

Background: MicroRNAs (miRNAs, miRs) are small (~22nt) noncoding RNAs that are involved in post-transcriptional regulation of specific mRNA targets. Aberrant miRNA expression has been shown to play a role in chronic lymphocytic leukemia (CLL) pathogenesis and to have prognostic implications. The lymph node (LN) has been demonstrated to be the site of CLL cell proliferation and activation. In particular, CLL cells in the LN display significantly increased BCR and NF-kB signaling in comparison to CLL cells in the peripheral blood (PB). Ibrutinib is a clinically approved BTK inhibitor that disrupts BCR and NF-kB signaling; and has been shown to be effective in relapsed or refractory CLL. We hypothesized that 1) miRNAs would be differentially expressed between activated CLL cells in the LN and CLL cells in the PB; and 2) ibrutinib would alter miRNA expression in CLL, which may affect therapeutic response by regulating the expression of miR target genes.Methods: RNA was isolated from CD19 positive selected cells from two separate cohorts of CLL patients. The first group included 17 treatment naïve patients donating paired LN and PB CD19 positive cells (10 IGHV un-mutated [59%], 4 IGHV mutated [23.5%] and 3 IGHV unknown [17.5%]); the second group contained 38 pairs of PB CD19 positive cells (22 IGHV un-mutated [58%] and 16 IGHV mutated [42%]) before and after ibrutinib treatment on day 28. A panel of 22 miRs previously reported to play a role in CLL pathogenesis, prognosis, or BCR signaling was profiled by qPCR. The expression level of a custom panel of genes encoding putative miR targets was quantified using the nanoString nCounter assay and verified by qPCR. Functional studies were performed in MEC-1 and SP-53 cell lines, which were treated separately with 1 μM ibrutinib or 1 μM fludarabine for 48 hours. Live cells were identified by trypan blue staining; miR and mRNA expression was quantified by qPCR.Results: Nineteen of 22 (86.4%) miRs were significantly increased (p < 0.05) in CLL cells from the LN in comparison to paired CLL cells from the PB, correlating with an activated B cell phenotype. Examination of mRNA transcripts of putative miR targets in 13 paired LN and PB samples showed that multiple tumor suppressor transcripts including CYLD, ARID1B and FOXP1 were significantly decreased in LN-derived CLL cells. Assay of 22 miRs in PB CLL cells from 38 patients before and after ibrutinib treatment indicated that a subset of miRs (miR-22, miR-34a, miR-146b and miR-181b) were significantly decreased (p < 0.05) in response to ibrutinib. Quantification of mRNA transcripts of putative targets of the ibrutinib responsive miRs by the nanoString nCounter assay showed that several tumor suppressor transcripts (ARID1B, ARID2, ATM, CYLD, FOXP1, HDAC1, IBTK, PTEN and SMAD4) were significantly increased. Functional studies in MEC-1 and SP-53 cell lines confirmed that miR-22, miR-34a, miR-146b and miR-181b were down-regulated and the tumor suppressor targets of these miRs (ARID1B, ARID2, ATM, CYLD, FOXP1, HDAC1, PTEN and SMAD4) were significantly increased after ibrutinib treatment. The alteration of miR and gene expression was specific to the effects of ibrutinib as similar changes were not observed post treatment with the chemotherapeutic agent fludarabine.Conclusions: miRNA expression patterns are significantly altered between CLL cells in the LN and those in the PB. An overall increase in the expression of miRs and down-regulation of mRNAs of tumor suppressor genes (ARID1B, CYLD and FOXP1) was observed in CLL cells in the LN, correlating with an activated B-cell phenotype. Ibrutinib treatment and inhibition of BCR signaling resulted in down-regulation of miR-22, miR-34a, miR-146b and miR-181b and concomitant up-regulation of multiple tumor suppressor targets including ARID1B, ARID2, ATM, CYLD, FOXP1, HDAC1, IBTK, PTEN and SMAD4 in PB CLL cells. Similar findings were also observed in ibrutinib treated MEC-1 and SP-53 cell lines. Additional studies are needed to address how much the down-regulation of miRs and consequent up-regulation of tumor suppressors in response to ibrutinib contributes to the overall disease response and whether this effect could sensitize CLL cells to treatment with cytotoxic agents. DisclosuresWiestner:Pharmacyclics: Research Funding.

In-Vivo Labelling Studies in Patients with Chronic Lymphocytic Leukemia Studies Demonstrate the Existence of Apparently Distinct Subpopulations That Differ in Phenotype and Proliferative Capacity

It is now generally believed that proliferation of the neoplastic clone in chronic lymphocytic leukemia (CLL) takes place in lymphoid tissues where interactions involving the B-cell receptor (BCR) and other microenvironmental elements take place. Previous studies using in-vivo labelling with deuterated water have shown that recently proliferated emigrants from lymphoid tissues express low levels of the chemokine receptor CXCR4 and high levels of CD5 (CXCR4loCD5hi). It has been proposed that, following entry into the peripheral blood (PB), these cells become quiescent, re-express CXCR4 and downregulate CD5 allowing re-entry into tissues and further rounds of proliferation. In the present study we used in-vivo labelling with deuterated glucose (6,6-2H2-glucose, D2G) to investigate the proliferation and release of CLL cells into PB. In contrast to deuterated water, this technique allows pulse labelling of a distinct cohort of cells, which can then be tracked over time in-vivo.Labelling studies were performed in 10 patients with previously untreated, non-progressive CLL. Patients underwent 10 hours of labelling with oral 2DG, after which peripheral blood and lymph node compartments were serially sampled. DNA Deuterium enrichment was measured in the entire CLL population and in flow-sorted subsets defined by CXCR4/CD5 and surface IgM (sIgM) expression.Maximum release of labelled cells into PB occurred after a median of 14 days (4-56 days). The disappearance rate was very slow, with labelled cells detectable after 56 days in half of the subjects. In one case we were able to track labelled cells in both lymph node (LN) and PB and demonstrated an increase in the fraction of labelled cells in the LN between days 7 and 28, consistent with re-entry into this compartment.Subpopulations of PB CLL cells, defined by CXCR4 and CD5 expression were studied over time to investigate the dynamic nature of these molecules in circulating cells. As previously reported, maximum rapid incorporation of deuterium was observed in the CXCR4loCD5hi fraction, equivalent to 1.15 ± 0.04 %/d fractional synthesis at 7 days. Conversely, CXCR4hiCD5lo cells remained largely unlabelled throughout the 8-week study, reaching a maximum of only 0.01 ± 0.003 %/d, suggesting that they represent a non-proliferating population, not derived from the CXCR4loCD5hi subset. In contrast, CXCR4/CD5 intermediate cells exhibited delayed and intermediate labelling, peaking at 0.1 ± 0.02 %/d at 28 days. This sequential labelling pattern suggests that they derive from the CXCR4loCD5hi subset.Since both CXCR4 and CD5 expression are modulated by BCR signalling, we went on to sort cells according to sIgM expression and found maximum labelling in the sIgM high subset with little or no deuterium incorporation in the sIgM low fraction at any time. Again, the sIgM intermediate subset showed delayed labelling, suggesting that they are derived from sIgM-high cells.These observations provide further evidence for clonal heterogeneity in CLL and suggest the existence of distinct but interdependent subpopulations. Recently-divided cells are CXCR4loCD5hi, but appear to transition to an intermediate phenotype by down-regulation of CD5 and sIgM and upregulation of CXCR4 over the ensuing weeks, but do not appear to transition to CXCR4hiCD5lo cells over the time-course of the experiment. Our findings have clinical relevance, since these functionally distinct subsets might also differ in their responsiveness to therapeutic agents, such as drugs that block BCR signalling. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Identification and characterization of distinct IL-17F expression patterns and signaling pathways in chronic lymphocytic leukemia and normal B lymphocytes.

Chronic lymphocytic leukemia (CLL) is characterized by a progressive accumulation of B lymphocytes. T cell abnormalities are a common feature of CLL and contribute to impaired immune function in these patients. T cells are ineffective in eliminating the leukemic clone and may actually promote tumor growth and survival. Previous work from our laboratory documented elevated circulating levels of IL-17A-producing Th17 cells in CLL patients as compared to healthy age-matched control subjects. These high circulating Th17 levels associated with better prognostic markers and significantly longer overall survival, even among patients whose clones used unmutated IGHVs (U-CLL). Recent studies suggest that Th17 cells are heterogeneous, expressing different profiles of cytokines, and that different subsets of Th17s mediate different biological functions. In the present study, we found significantly higher levels of IL-17F-expressing CD4+ T cells in CLL versus healthy peripheral blood mononuclear cells following in vitro stimulation in the presence of Th17-promoting cytokines. Furthermore, the differentiation of IL-17F-expressing Th17 cells was significantly enhanced when purified CD4+ T cells from CLL patients were cultured in the presence of autologous CLL B cells. Lastly, single-cell network profiling revealed that IL-17F triggers NFκB phosphorylation in T and B cells from patients with CLL, but not age-matched healthy controls. Taken together, our data suggest that the phenotype of Th17 cells in CLL patients is distinct from healthy individuals, expressing higher levels of IL-17F, and that B and T cells from CLL patients are particularly responsive to IL-17F, as compared to healthy age-matched control individuals.Electronic supplementary materialThe online version of this article (doi:10.1007/s12026-015-8722-5) contains supplementary material, which is available to authorized users.

Vincristine activates c-Jun N-terminal kinase in chronic lymphocytic leukaemia in vivo.

The authors' aim was to conduct a proof-of-principle study to test whether c-Jun N-terminal kinase (JNK) phosphorylation and Noxa induction occur in peripheral blood chronic lymphocytic leukaemia (CLL) cells in patients receiving a vincristine infusion. Patients with CLL received 2 mg vincristine by a 5-min intravenous infusion. Blood samples were collected at baseline and up to 6 h after the vincristine infusion, and assayed for JNK activation, Noxa induction and vincristine plasma concentrations. Ex vivo treated peripheral CLL cells activated JNK in response to 10-100 nM vincristine in 6 h. Noxa protein expression, while variable, was also observed over this time frame. In CLL patients, vincristine infusion led to rapid (<1 h) JNK phosphorylation in peripheral blood CLL cells which was sustained for at least 4-6 h after the vincristine infusion. Noxa protein expression was not observed in response to vincristine infusion. This study confirmed that vincristine can activate JNK but not induce Noxa in CLL cells in vivo. The results suggest that novel JNK-dependent drug combinations with vincristine warrant further investigation.

Targeting Nedd8 Activating Enzyme Induces DNA Damage and Cell Cycle Arrest and Sensitizes Chronic Lymphocytic Leukemia (CLL) B-Cells to Alkylating Agents

Microenvironment-mediated upregulation of the B-cell receptor (BCR) and nuclear factor-kappaB (NFκB) signaling in the CLL cells resident in the lymph node and bone marrow promotes apoptosis evasion and clonal expansion. We recently reported that stromal-mediated NFκB pathway activity and CLL cell survival may be successfully abrogated in vitrousing MLN4924. MLN4924 is an investigational agent that inhibits the NEDD8-activating enzyme (NAE) and thereby prevents neddylation of Cullin-RING ubiquitin ligases (CRL), resulting in stabilization of their protein substrates, including inhibitor of NFκB. However, targeting NAE also slows degradation of other proteins and thus full biological consequences are tissue-dependent. In adherent solid tumor cell lines, MLN4924 induced expression of Cdt1, a DNA replication licensing factor, followed by DNA damage and cell cycle arrest, but the importance of this mechanism in primary neoplastic B-cells has not been determined. Here we show that, under the growth conditions, targeting NAE induces DNA damage and cell cycle arrest in primary CLL B-cells.B-cells obtained from patients with CLL were co-cultured in vitro with CD40L-expressing stroma to mimic the pro-survival conditions present in lymphoid tissue. CLL cells were incubated in the presence or absence of 25 ng/mL IL-21. MLN4924 was provided by Millennium Pharmaceuticals, Inc.CD40L-expressing stromal co-cultures rapidly induced anti-apoptotic BCL2 family proteins MCL1 and BCLX in peripheral blood CLL cells, leading to protection from spontaneous apoptosis and chemoresistance. Prolonged (72 h) co-cultures induced cell cycle progression in ~5% of the CLL cells. IL-21 further enhanced proliferation and sensitized CLL cells to MLN4924. Thus, treatment with 1 μM MLN4924 induced apoptosis in 44.6±5.4% of the CD40L-stimulated, and in 63.7±2.6% of the CD40L/IL-21-stimulated CLL cells at 24 h (p=0.00004). CD40-stimulated CLL cells treated with MLN4924 showed Cdt1 accumulation, DNA re-replication, activation of DNA damage response pathways as evidenced by accumulation of pRPA, pChk1, pChk2 and gH2AX which preceded PARP cleavage, and G2 arrest. DNA damage and G2 arrest were more prominent in IL-21-treated cells. Targeting NAE also resulted in early accumulation of the endogenous cyclin-dependent kinase inhibitors p21 and p27, both CRL targets. By contrast, retinoblastoma protein hypophosphorylation occurred late and did not precede apoptosis. siRNA-mediated knockdown of Cdt1, but not p21 or p27 prevented MLN4924-mediated DNA damage response and CLL cell apoptosis. Finally, MLN4924 did not lead to accumulation of Cdt1, p21 or p27 in CLL cells cultured off stroma.Since alkylating agents target primarily cycling cells, we further determined whether they would cooperate with MLN4924 to induce DNA damage in CLL. While either chlorambucil or bendamustine exhibited little toxicity against the CD40L-stimulated CLL cells, MLN4924 sensitized CLL cells to those agents. MLN4924 further augmented bendamustine toxicity in the presence of IL-21. MLN4924-mediated DNA damage response was augmented in the presence of bendamustine. Interestingly, this combination proved ineffective in the CLL samples with del(17p), suggesting that bendamustine effect depends on the intact p53 pathway.Here we characterize DNA damage response pathway in CLL, where it remains understudied. Targeting NAE in cycling CLL cells induces Cdt1 accumulation, DNA damage and cell cycle arrest. This data provide additional insights into the biological consequences of targeting NAE in CLL B-cells as well as preclinical rationale to study the potential clinical activity of MLN4924 in CLL, alone or in combination with bendamustine or chlorambucil. DisclosuresBerger:Takeda: Employment.

STAT3-Driven, Aberrantly Expressed Lipoprotein Lipase Mediates Free Fatty Acid Metabolism in Chronic Lymphocytic Leukemia Cells

The metabolic profile of mammalian cells is determined primarily by the cells’ proliferation rate. Unlike circulating memory B cells, which are typically quiescent and proliferate only in response to external stimuli, approximately 1% of chronic lymphocytic leukemia (CLL) cells proliferate daily. We sought to determine how CLL cells adjust their metabolism to meet increased energy demands imposed by their proliferation rate. Muscle cells, which proliferate at rates similar to those of CLL cells, preferentially use intracellular stored triglycerides as an available energy source. Similar to muscle cells, CLL cells express lipoprotein lipase (LPL), an enzyme that catalyzes the hydrolysis of tryglycerides into free fatty acids (FFA). We wondered whether CLL cells use a similar pathway. In reviewing bone marrow biopsies of patients with CLL, we identified clear-appearing oil red O-positive vacuoles in the cytoplasm of CLL cells. Using electron microscopy, we confirmed that these lipid vacuoles were present in 95% of CLL peripheral blood cells but not in normal B cells. To determine whether CLL cells metabolize FFA, we incubated CLL cells with or without FFA (palmitate or oleate) in a sealed flask and measured the dissolved O2 (dO2) content in the medium of the cultured cells after 48 h. Compared with CLL cells incubated in the absence of FFA, dO2 levels were significantly reduced when FFA was added. In contrast, dO2 levels were not reduced after FFA was added to cultures of normal B lymphocytes, suggesting that unlike normal B cells, CLL cells acquired the capacity to metabolize FFA. Transfecting CLL cells with LPL small interfering RNA abrogated the capacity of CLL cells to metabolize FFA, suggesting that FFA metabolism in CLL cells is LPL dependent. We and other groups found that LPL is abundantly expressed in CLL cells. Because STAT3 is constitutively activated in CLL cells and because we identified putative STAT3 binding sites in the LPL promoter, we hypothesized that STAT3 induces aberrant expression of LPL in CLL cells. By transfecting a luciferase reporter gene driven by LPL promoter fragments into MM1 cells, we found that STAT3 activates the LPL promoter, and by using chromatin immunoprecipitation and electrophoretic mobility shift assays, we confirmed that STAT3 binds to the LPL promoter in MM1 and in CLL cells. To confirm these data, we transfected CLL cells with a lentiviral STAT3 short hairpin RNA. Unlike the empty lentiviral vector, STAT3–small interfering RNA downregulated mRNA levels of LPL and several STAT3 target genes and downregulated LPL protein levels. Taken together, our data suggest that CLL cells store lipids in cytoplasmic vacuoles, produce LPL, and adapt their metabolism to utilize intracellular stored lipids for energy production, a process that is driven by constitutively activated STAT3. DisclosuresO’Brien:Amgen, Celgene, GSK: Consultancy; CLL Global Research Foundation: Membership on an entity’s Board of Directors or advisory committees; Emergent, Genentech, Gilead, Infinity, Pharmacyclics, Spectrum: Consultancy, Research Funding; MorphoSys, Acerta, TG Therapeutics: Research Funding.

A novel method using blinatumomab for efficient, clinical-grade expansion of polyclonal T cells for adoptive immunotherapy.

Current treatment of chronic lymphocytic leukemia (CLL) patients often results in life-threatening immunosuppression. Furthermore, CLL is still an incurable disease due to the persistence of residual leukemic cells. These patients may therefore benefit from immunotherapy approaches aimed at immunoreconstitution and/or the elimination of residual disease following chemotherapy. For these purposes, we designed a simple GMP-compliant protocol for ex vivo expansion of normal T cells from CLL patients' peripheral blood for adoptive therapy, using bispecific Ab blinatumomab (CD3 × CD19), acting both as T cell stimulator and CLL depletion agent, and human rIL-2. Starting from only 10 ml CLL peripheral blood, a mean 515 × 10(6) CD3(+) T cells were expanded in 3 wk. The resulting blinatumomab-expanded T cells (BET) were polyclonal CD4(+) and CD8(+) and mostly effector and central memory cells. The Th1 subset was slightly prevalent over Th2, whereas Th17 and T regulatory cells were <1%. CMV-specific clones were detected in equivalent proportion before and after expansion. Interestingly, BET cells had normalized expression of the synapse inhibitors CD272 and CD279 compared with starting T cells and were cytotoxic against CD19(+) targets in presence of blinatumomab in vitro. In support of their functional capacity, we observed that BET, in combination with blinatumomab, had significant therapeutic activity in a systemic human diffuse large B lymphoma model in NOD-SCID mice. We propose BET as a therapeutic tool for immunoreconstitution of heavily immunosuppressed CLL patients and, in combination with bispecific Ab, as antitumor immunotherapy.

Tiam1/Rac1 signals contribute to the proliferation and chemoresistance, but not motility, of chronic lymphocytic leukemia cells

AbstractSignals from the tumor microenvironment promote the migration, survival, and proliferation of chronic lymphocytic leukemia (CLL) cells. Rho GTPases control various signaling pathways downstream of microenvironmental cues. Here, we analyze the function of Rac1 in the motility and proliferation of CLL cells. We found decreased transcription of the Rac guanine nucleotide exchange factors Tiam1 and Vav1 in unstimulated peripheral blood CLL cells with almost complete loss of Tiam1 but increased transcription of the potential Rac antagonist RhoH. Consistently, stimulation of CLL cells with the chemokine CXCL12 induced RhoA but not Rac1 activation, whereas chemokine-induced CLL cell motility was Rac1-independent. Coculture of CLL cells with activated T cells induced their activation and subsequent proliferation. Here, Tiam1 expression was induced in the malignant cells in line with increased Ki-67 and c-Myc expression. Rac1 or Tiam1 knockdown using siRNA or treatment with the Tiam1/Rac inhibitor NSC-23766 attenuated c-Myc transcription. Furthermore, treatment of CLL cells with NSC-23766 reduced their proliferation. Rac inhibition also antagonized the chemoresistance of activated CLL cells toward fludarabine. Collectively, our data suggest a dynamic regulation of Rac1 function in the CLL microenvironment. Rac inhibition could be of clinical use by selectively interfering with CLL cell proliferation and chemoresistance.

Functional Effects Of NOTCH1 Mutations In Chronic Lymphocytic Leukemia Patients

Chronic lymphocytic leukemia (CLL), is characterized by the expansion of mature B lymphocytes present in blood, bone marrow and lymphoid organs. Clinical behavior is highly heterogeneous, thus requiring timely identification of high-risk patients.NOTCH1 encodes a trans-membrane receptor acting as a ligand-activated transcription factor. NOTCH signaling initiates when the ligand, from either the Jagged or Delta families, binds to the receptor and induces successive proteolytic cleavages, resulting in the release and nuclear translocation of the NOTCH intra-cellular domain (NICD). Signaling is terminated by phosphorylation of the PEST domain of NOTCH1, triggering its ubiquitination and proteasomal degradation.Whole exome sequencing approaches have revealed NOTCH1 mutations in 5-10% newly diagnosed CLL cases, with their prevalence increasing to 15-20% in progressive or relapsed patients. The most frequent mutation is 7544-7545delCT frameshift deletion in exon 34, resulting in disruption of the C-terminal PEST domain. Truncation of the PEST domain is predicted to result in NOTCH1 impaired degradation, stabilization of the active NOTCH1, and deregulated signaling.The present study was undertaken with the aim to compare NOTCH1 expression and functional role in CLL patients harboring wild type (WT) or mutated (M) NOTCH1 gene.NOCTH1 mRNA and surface protein were expressed at comparable high levels in peripheral blood (PB) CLL cells obtained from NOTCH1 M and WT patients, consistent with a more general requirement of NOTCH1 signaling in this leukemia. However, at a variance of NOTCH1 WT cases, NOCTH1 M patients displayed remarkable accumulation of both the intermediate molecular species of the activated NOCTH1 receptor, as well as of the active NICD. Consistently, by gene expression profiling NOCTH1 M patients displayed significantly higher levels of HES1 and DTX1, the main NOTCH1 target genes. Overall, these data suggest a more active signaling pathway in NOTCH1 M CLL than in NOTCH1 WT cases.Expression of NOTCH1 and of its target gene (DTX1) varied across disease compartments, being higher in CLL cells obtained from the lymph nodes (LN), as compared to paired samples derived from the PB or the bone marrow (BM). By immunohistochemical analyses of primary LN tissue samples, NOTCH1 M CLL showed an intense nuclear staining as opposed to the more cytoplasmic distribution observed in NOTCH1 WT samples. These data suggest a more active NOTCH1 signaling in CLL residing in the LN microenvironment and confirm the functional effect of NOTCH1 mutations in vivo.When PB CLL cells were cultured in vitro in the absence of any supporting layer or stimulation, they showed a rapid down regulation of the NOTCH pathway, with complete loss of NICD after 24 hours paralleled by a sharp decrease in HES1 and DTX1 transcription. Consistently, levels of presenilin-1 (PSEN1), the catalytic subunit of the g-secretase complex, were also down-regulated offering a partial mechanistic explanation for the NICD loss. NOTCH1 mRNA levels remained unchanged, with accumulation of the receptor at the plasma membrane. These effects were independent of NOTCH1 mutation status and suggested the dependence of NOTCH1 signaling activation upon in vivo microenvironmental interaction, even in NOTCH1 M CLL.Within primary LN biopsies from CLL patients, the NOTCH1 ligand, was highly expressed on CD68+ elements of myeloid origin. This observation prompted the in vitro recreation of a lymphoid niche by co-culturing Jagged1+ nurse-like cells (NLC) with autologous CLL cells. Under these conditions, NOTCH1 activity in CLL cells was sustained over time, as shown by Q-PCR analyses of DTX1 and PSEN1. Moreover, NLCs protect NOTCH1 M CLL cells from fludarabine-induced apoptosis. This microenvironment-induced chemoresistance was prevented by pre-treatment of NOTCH1 M CLL cells with specific g-secretase inhibitors, to block NOTCH1 activation.Taken together, these results show that the 7544-7545delCT mutation in the PEST domain of NOTCH1 has a stabilizing effect on NOTCH1 signaling pathway. They also show that micro-environmental interactions are critical in activating NOTCH1 pathway both in the M and WT patients. Lastly, these results show that NOTCH1 signals micht create local conditions that favour drug resistance, thus making NOTCH1 a potential molecular target in CLL. Disclosures:No relevant conflicts of interest to declare.

Clinical Impact Of Small TP53 Mutated Subclones In Chronic Lymphocytic Leukemia

▪ IntroductionTP53 mutations are strong predictors of poor survival and refractoriness in chronic lymphocytic leukemia (CLL) and have direct implications for disease management. Clinical information on TP53 mutations are currently limited to lesions that are represented in the majority of CLL cells. Next generation sequencing (NGS) allows sensitive detection of mutations harbored by a small fraction of the tumor cell population. Here we aim at assessing the frequency, evolution during disease course, and prognostic impact of small TP53 mutated subclones in newly diagnosed CLL. MethodsThe study was based on a consecutive series of 309 newly diagnosed and previously untreated CLL (median age: 71 years; Binet A/B/C: 79/12/9%; unmutated IGHV genes: 35%; clonal TP53/NOTCH1/SF3B1/BIRC3 lesions: 11/11/7/5%; median follow-up: 8.1 years). TP53 mutations (exons 4-8) were screened on peripheral blood (PB) samples (tumor representation 70-98%) by amplicon-based deep-NGS (GSJ, 454 Life Sciences) (average depth: 2660). A bioinformatic algorithm was developed to call TP53 variants out of background noise. By dilution experiments, deep-NGS allowed to detect mutant allele fractions of 0.3%. TP53 variants were considered subclonal if missed by Sanger sequencing, which was performed in parallel. Subclonal TP53 variants were confirmed by duplicate deep-NGS and independently validated by allele specific PCR (AS-PCR). TP53 variant allele frequency (VAF) was corrected for tumor representation. ResultsDeep-NGS identified 50 subclonal TP53 mutations (VAF 0.3%-11%) in 28/309 (9%) CLL (Fig 1A). All subclonal mutations were non-silent, were missed by Sanger sequencing, and were validated by AS-PCR. The molecular spectrum of subclonal TP53 mutations (i.e. missense/truncating ratio, transition/transversion ratio, distribution across hot spot codons; p>.05; Fig. 1B-D), as well as the residual transactivational activity of mutants toward the p21 promoter (p=.872) were highly consistent with that of fully clonal TP53 mutations reported in CLL (Zenz T, Leukemia 2010). Subclonal TP53 mutations were the sole TP53 genetic event in 15/309 (4.8%) CLL, while in 13/309 (4.2%) cases subclonal TP53 mutations co-existed in the same leukemic population along with a clonal TP53 mutation or with 17p deletion. In cases (n=12) harboring more than one TP53 mutation, the variants mapped on distinct sequencing reads from the same amplicon suggesting that they belonged to different CLL subclones. By combining subclonal TP53 mutations, clonal TP53 mutations and 17p deletion, 50/309 (16%) CLL harbored at least one TP53 defect at diagnosis. Subclonal TP53 mutations were significantly enriched among cases presenting with advanced stage (Binet C: 26%; p=.005) and clonal TP53 abnormalities (37%; p<001). Cases harboring solely subclonal TP53 mutation showed a median overall survival (3.4 years) significantly shorter than TP53 wild type cases (10.8 years; p=.028), and similar to that of cases with clonal TP53 genetic defects (3.1 years; p=.375) (Fig. 1E). By multivariate analysis, cases harboring subclonal TP53 mutations had a significantly increased hazard of death (HR: 2.0; p=.023) after adjusting for age, disease stage, IGHV mutation status, clonal TP53 genetic defects, and lesions of NOTCH1, SF3B1 and BIRC3. Subclonal TP53 mutations showed a similar allele fraction in paired PB and lymph-node CLL cells in 3/5 assessable cases, suggesting a systemic spread of mutated subclones across disease compartments. Among cases harboring solely subclonal TP53 mutations, longitudinal deep-NGS of sequential samples documented the outgrowth of the TP53 variant to a fully clonal level in 57% (4/7) of cases. In all these cases, clonal selection was strongly associated with treatment exposure and development of a chemorefractory phenotype. Conversely, in cases managed by watch-and-wait only, the load of TP53 mutations did not increase during follow-up. [Display omitted] ConclusionsSmall TP53 mutated subclones detected by deep-NGS occur in a significant fraction of newly diagnosed CLL, have the same unfavorable prognostic impact as clonal TP53 defects, and anticipate the development of a chemorefractory phenotype among CLL requiring treatment. Search of minor subclones by deep-NGS should be considered for a comprehensive assessment of TP53 disruption in CLL. D.R. and H.K equally contributed; R.F., R.R. and G.G equally contributed. Disclosures:No relevant conflicts of interest to declare.

Combined ROR1 and CD160 Detection For Improved Minimal Residual Disease In Patients With Chronic Lymphocytic Leukemia (CLL)

IntroductionOver the past decade, treatments for patients with chronic lymphocytic leukemia (CLL) have produced complete remissions (CR) without evidence for minimal residual disease (MRD), particularly for younger and/or fitter patients. In this setting, achieving an MRD-negative CR has prognostic implications, yielding longer progression free survival (PFS) and overall survival (OS) than for patients who achieve a CR with persistent MRD. Complicating efforts to incorporate testing for MRD in clinical practice has been the lack of a defined consensus on the methods of MRD detection. In this study, we report on a novel combination of mAbs for MRD detection by flow cytometry based on two antigens: the NK-cell receptor and tumor specific antigen, CD160; and the tumor associated antigen, receptor tyrosine kinase-like orphan receptor 1 (ROR-1). ObjectiveTo compare a novel single-tube, tumor-specific (CD160+ROR1) targeted approach to MRD detection against the previously published CD160 flow cytometric assay (CD160FCA) (Farren et al, 2011) and the new, single-tube 8-color ERIC assay. MethodsBetween October 2012 and July 2013, prospective assessment of MRD was performed on peripheral blood in 56 patients (86 samples). We developed a flow cytometric assay using mAbs specific for CD160 or ROR1 (Fukuda et al, 2008). For this we used the following mAb from BD Biosciences: CD2 FITC, CD5 Pe-Cy7, CD19 PerCP5.5, CD45V500, CD160PE, ROR-1 AF647 (“ROR-160FCA”) and a sequential gating strategy. This was compared with CD160FCA and the 8-color ERIC consortium protocol (unpublished). Light chain analysis (LCR) was performed in all cases and reported where detectable. A proof of concept spiking experiment simulating MRD was prepared by mixing CLL and normal peripheral blood leukocytes in a serial dilution to a level of 10-5(n=3). Statistical analysis was performed using Spearman Rank correlation coefficients, Mann-Whitney t-test, and Bland-Altman method comparison. Significance was set at <0.05%. ResultsTo establish the proof of principle, MRD levels ranging from 0.001% to 100% (Neat CLL) were prepared by serial dilutions, in which MRD levels could be established by the ROR-160FCA to 10-5 (n=3). Assessment of the observed incidence against expected incidence of CLL MRD demonstrated a highly significant correlation (R2=0.96, p=0.01). In the study, the range of detectable disease went from <0.01% to 38.59%, of which 37% of samples had levels below <0.01%. Analyzing all flow cytometric methods, a highly significant correlation was observed between all three: CD160FCA vs ERIC: Spearman R=0.96 (95%CI: 0.93 - 0.97, p<0.001); CD160FCA vs ROR-160FCA: Spearman R=0.97 (95%CI: 0.96 – 0.99, p<0.001); ROR-160FCA vsERIC: Spearman R=0.97 (95%CI: 0.94 – 0.98, p<0.001).54 samples had levels of disease <1%. Bland-Altman assay comparison in these patients again demonstrated significant associations between the assays (CD160FCA vs ERIC: mean 0.08 ±0.15; CD160FCA vs ROR-160FCA: mean 0.04 ±0.19; ROR-160FCA vs ERIC: mean 0.03 ±0.25). Light chain restriction was detectable in 24 patients (size of the restricted population ranged from 0.2% to 47% of all cellular events). This sub-group of patients also demonstrated excellent correlation between level of LCR and detectable disease by CD160FCA (Spearman R=0.96, 95%CI: 0.92-0.98, p<0.001), ERIC (R=0.95, 95%CI: 0.92-0.98, p<0.01) and ROR-160FCA (R=0.97, 95%CI 0.93-0.98, p<0.001). ConclusionMonitoring minimal residual disease in CLL is a key focus for clinical trials, as MRD is an important prognostic marker in CLL in terms of PFS and OS. Here we provide a single tube assay, ROR-160FCA, which is unique by targeting two antigens restricted to malignant B-cells, CD160 and ROR1. ROR-160FCA is equivalent in MRD detection compared to both CD160FCA and the current ERIC assay under development. The two tumor-specific antigens give ROR-160FCA the potential for improved sensitivity, particularly where limited sample is available. Furthermore, it only requires a simple sequential gating strategy, is rapid, and appears more cost effective than other Methods.ReferencesFarren TW, Giustiniani J, Liu FT et al. Blood. 2011;118 (8):2174-2183.Fukuda T, Chen L, Endo T et al. Proc Natl Acad Sci U S A. 2008;105 (8):3047-3052. Disclosures:Farren:BD Biosciences: Research Funding. Warner:BD Biosciences: Employment, Research Funding. Agrawal:BD Biosciences: Research Funding.

CD62L as a Therapeutic Target in Chronic Lymphocytic Leukemia

Despite advances in the treatment of chronic lymphocytic leukemia (CLL), the disease remains incurable with standard therapies and relapse is inevitable. A growing body of evidence indicates that alterations in the adhesion properties of neoplastic cells play a pivotal role in the development and progression of CLL. The expression of 71 cell surface molecules was examined on CLL peripheral blood mononuclear cells (PBMCs) over 3 weeks in culture. The most highly upregulated marker, CD62L, was examined further for expression on CD5(+)/CD19(+) CLL cells in vitro and in lymph node and bone marrow biopsies. The prosurvival role of CD62L was examined using a functional blocking antibody and therapeutic potential evaluated by comparison with current chemotherapy agents. Blocking CD62L resulted in apoptosis of CLL cells but not PBMCs from healthy donors suggesting a novel role for CD62L in CLL cell survival. The beneficial effect of coculturing CLL cells with bone marrow stromal cells or endothelial cells does not protect CLL cells from anti-CD62L-related toxicity. Moreover, combining fludarabine or mafosfamide with the anti-CD62L in vitro produced an additive effect both with and without stromal cells. This is the first reported data showing that blocking the activation and homing marker, CD62L, regulates CLL cell survival in vitro. These data also suggest that therapeutic antibodies against CD62L may provide additional clinical benefit to patients with CLL receiving current standard chemotherapy protocols.

CLL cells are resistant to smac mimetics because of an inability to form a ripoptosome complex

In the lymph node (LN) environment, chronic lymphocytic leukemia (CLL) cells display increased NF-κB activity compared with peripheral blood CLL cells, which contributes to chemoresistance. Antagonists of cellular inhibitor of apoptosis proteins (cIAPs) can induce apoptosis in various cancer cells in a tumor necrosis factor-α (TNFα)-dependent manner and are in preclinical development. Smac-mimetics promote degradation of cIAP1 and cIAP2, which results in TNFR-mediated apoptosis via formation of a ripoptosome complex, comprising RIPK1, Fas-associated protein with death domain, FLICE-like inhibitory protein and caspase-8. CD40 stimulation of CLL cells in vitro is used as a model to mimic the LN microenvironment and results in NF-κB activation and TNFα production. In this study, we investigated the response of CLL cells to smac-mimetics in the context of CD40 stimulation. We found that treatment with smac-mimetics results in cIAP1 and cIAP2 degradation, yet although TNFα is produced, this did not induce apoptosis. Despite the presence of all components, the ripoptosome complex did not form upon smac-mimetic treatment in CLL cells. Thus, CLL cells seem to possess aberrant upstream NF-κB regulation that prevents ripoptosome formation upon IAP degradation. Unraveling the exact molecular mechanisms of disturbed ripoptosome formation may offer novel targets for treatment in CLL.

Abstract LB-141: Potent single agent activity of Ibrutinib (PCI-32765) in patients with chronic lymphocytic leukemia (CLL): clinical and translational results from an ongoing phase II study.

Abstract Background: B-cell receptor (BCR) activation plays a pivotal role in the pathogenesis and for progression of CLL. Ibrutinib (PCI-32765), an irreversible inhibitor of Bruton's tyrosine kinase (BTK), can disrupt BCR signaling and has durable antitumor activity in CLL. We initiated a phase II single center trial to extend this experience and assess the impact of ibrutinib on tumor cells in vivo. Methods: Treatment naïve (TN) and relapsed/refractory (R/R) pts with CLL are treated with 420 mg ibrutinib daily. Pts are enrolled in 2 cohorts: 1) deletion (del) of chromosome 17p and 2) &amp;gt;65yo and treated until disease progression. Response is evaluated at 6 months (mo) and every 6 mo thereafter. 17p del was assessed by FISH cytogenetics. Spleen volume on computed tomography (CT) scans was computed using a General Electric Advanced Workstation Server. Results: With a median follow-up of 9 mo we report on the first 53 patients: for del 17p n=29; 15 TN, 14 R/R; and for &amp;gt;65yo n=24; 8 TN, 16 R/R. Estimated event free survival at 12 mo is 94%. Most adverse events were mild with non hematologic toxicities (regardless of causality) grade ≥3 in &amp;lt;13% of pts. Two deaths on study were not treatment related. At 6 mo 95% of pts (n=44 evaluable) had a nodal response (median reduction in lymph node size of 73%) and 52% of pts had a partial response (PR) by IWCLL criteria. One pt had progressive disease (presumed Richter's transformation). All pts had a decrease in splenomegaly. The median reduction of splenic volume computed from CTs was 55% (30-1716 ml). Tumor infiltration in bone marrow biopsies (n=26), decreased by a median 82% as assessed by immunohistochemistry for CD79a and the median ALC decreased from 79 k/µl (0.5-402) to 32 k/µl (0.8-167) at 6 mo for a median reduction of 62%. We evaluated the in vivo effects of ibrutinib using blood and tissue samples collected pre and during ibrutinib treatment. On treatment expression of genes known to be induced by B-cell receptor (BCR) activation (Herishanu et al 2011) was significantly reduced in CLL cells in the peripheral blood (PB) in all pts (n=15; median reduction 57%; P&amp;lt;.001). Consistent with inhibition of BCR signaling, we found concurrent reduction in PLCγ2 phosphorylation; a direct target of BTK. Patients also donated matched lymph node (LN) core biopsies pre-treatment and within the first 4 days on treatment. Ibrutinib inhibited BCR signaling in the LN (n=8, P=.004) to a similar degree as observed in the PB. Similarly, ibrutinib decreased expression of NF-κB target genes in PB and LN. In keeping with inhibition of BCR and NF-κB signaling surface markers of cellular activation such as CD38, CD69, and CD86 were significantly reduced by ibrutinib as measured by flow cytometry (P&amp;lt;.02). Finally, ibrutinib significantly reduced tumor proliferation as determined by the percentage of PB CLL cells expressing Ki67 (median reduction of 80%; P&amp;lt;.001). Conclusions: Ibrutinib as a single agent is well tolerated and highly effective in both TN and R/R pts achieving rapid disease control in blood, lymph nodes, spleen and bone marrow that is durable. Correlative studies on patient samples during ibrutinib treatment provide direct evidence for on target effects of ibrutinib in vivo demonstrating effective and sustained inhibition of BCR and NF-κB signaling in both PB and LNs. Ibrutinib deserves further investigation as targeted therapy for CLL. We thank our patients for participation and donation of blood and tissue samples. Supported by the Intramural Research Program of NIH. Citation Format: Adrian Wiestner, Sarah Herman, Rashida Mustafa, Janet Valdez, Jade Jones, Nakhle Saba, Andrew Lipsky, Diane C. Arthur, Gerald Marti, Francine Thomas, Irina Maric, Stefania Pittaluga, Xin Tian, Susan Soto, Georg Aue, Mohammed Z. Farooqui. Potent single agent activity of Ibrutinib (PCI-32765) in patients with chronic lymphocytic leukemia (CLL): clinical and translational results from an ongoing phase II study. [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 LB-141. doi:10.1158/1538-7445.AM2013-LB-141

CLL Cells Respond to B-Cell Receptor Stimulation with a MicroRNA/mRNA Signature Associated with MYC Activation and Cell Cycle Progression

Chronic lymphocytic leukemia (CLL) is a disease with variable clinical outcome. Several prognostic factors such as the immunoglobulin heavy chain variable genes (IGHV) mutation status are linked to the B-cell receptor (BCR) complex, supporting a role for triggering the BCR in vivo in the pathogenesis. The miRNA profile upon stimulation and correlation with IGHV mutation status is however unknown. To evaluate the transcriptional response of peripheral blood CLL cells upon BCR stimulation in vitro, miRNA and mRNA expression was measured using hybridization arrays and qPCR. We found both IGHV mutated and unmutated CLL cells to respond with increased expression of MYC and other genes associated with BCR activation, and a phenotype of cell cycle progression. Genome-wide expression studies showed hsa-miR-132-3p/hsa-miR-212 miRNA cluster induction associated with a set of downregulated genes, enriched for genes modulated by BCR activation and amplified by Myc. We conclude that BCR triggering of CLL cells induces a transcriptional response of genes associated with BCR activation, enhanced cell cycle entry and progression and suggest that part of the transcriptional profiles linked to IGHV mutation status observed in isolated peripheral blood are not cell intrinsic but rather secondary to in vivo BCR stimulation.