Chronic Lymphocytic Leukemia Cell Lines Research Articles

STAT3 mediates C6-ceramide-induced cell death in chronic lymphocytic leukemia

The pathogenesis of chronic lymphocytic leukemia (CLL) is poorly understood and it remains incurable with current therapies. We have previously shown that nanoliposomal C6-ceramide (CNL) is an effective therapy in an in vivo murine model of CLL. However, the key signaling pathways mediating CNL-induced cell death in CLL remains unknown. We hypothesized that CNL targets STAT3, a critical regulator of hematopoietic biology. We observed that CNL treatment reduced phosphorylated STAT3 at both Y705 and S727 residues in CLL cell lines and patient cells. This, in turn, reduced STAT3 transcriptional activity and expression of critical STAT3-dependent survival factors like Mcl-1 and survivin. The effect of CNL on STAT3 was further confirmed ex vivo as shown by reduced STAT3 phosphorylation in xenograft tumors obtained from mice treated with CNL. CNL suppressed STAT3 phosphorylation at Y705 and S727 through reduction in BTK activity and MEK1/2 kinase/PKC activities, respectively. Moreover, a synergistic reduction in CLL cell viability was observed on co-treatment with CNL and the BTK inhibitor, ibrutinib. Expression of an oncogenic form of STAT3 conferred partial resistance to CNL, providing confirmation that STAT3 mediates CNL-induced cell death. Taken together, these findings provide the first body of evidence demonstrating ceramide regulation of STAT3 phosphorylation. These results are also the first to demonstrate an effect of ceramide on BTK, a critical kinase mediating the B-cell receptor signaling in CLL cells and suggest a novel and synergistic combination of CNL and BTK inhibitors for CLL treatment.

Abstract 154: The phosphatidylinositol-3-kinase (PI3K) inhibitor (i) copanlisib is active in preclinical models of B-cell lymphomas as single agent and in combination with conventional and targeted agents including venetoclax and palbociclib

Abstract Introduction Copanlisib (BAY 80 6946) is a highly selective pan class I PI3K-i with predominant inhibitory activity against PI3Kδ and PI3Kα, in clinical development as single agent and in combination for lymphoma patients. To address single agent antitumor activity in different lymphomas and to understand the molecular basis of resistance mechanisms for rational combination, we performed a screening of copanlisib as single agent and in combination with 15 other anticancer agents in 17 cell lines derived from mantle cell lymphoma (MCL), marginal zone lymphoma (MZL) and chronic lymphocytic leukemia (CLL). Methods MCL (Jeko1, Rec1, JVM2, Granta519, Maver1, Mino1, SP-49, SP-53, UPN1, Z138), MZL (Karpas1718, VL51, SSK41, ESKOL, HAIR-M, HC-1) and CLL (MEC1) cell lines were exposed to increasing doses of copanlisib alone and in combination with other compounds using the fixed ratio set-up. Tested compounds included approved and experimental inhibitors of key regulatory pathways. Synergy was assessed via Chou-Talalay combination index (CI). Gene expression profiling (GEP) was done using Illumina Human HT12Expression BeadChips and GSEA (FDR<0.25). Results Copanlisib showed antitumor activity in most cell lines (median IC50=22nM; 95%C.I.: 15-98). The other most active drugs were bortezomib (5nM; 5-7), romidepsin (34nM; 2-94), roniciclib (23nM; 18-29), panobinostat (161nM; 11-1263), MI2 (490nM; 224-1000). The remaining had median IC50s >500nM. Copanlisib-containing combinations often gave synergy/additive effects: copanlisib with venetoclax was beneficial in 16/17; with MI2 in 15; with palbociclib or ibrutinib in 14; with BAY 1125976 or panobinostat in 13; with lenalidomide or BAY 1238097 in 12; with rituximab in 11; with romidepsin in 10; with roniciclib in 9; with bortezomib in 8; with BAY 1143572 in 7; with bendamustine in 6; with ruxolitinib in 2. Combinations with venetoclax and with palbociclib were the most promising, achieving CI values <0.5 in 7 and 6 cell lines, respectively. GEP before treatment identified genesets associated with different sensitivity to these 2 combinations. High expression of genes involved in IFN signaling, oxidative phosphorylation, fatty acid metabolism, apoptosis, PI3K/AKT/mTOR and IL6/JAK/STAT signaling and low expression of cell cycle genes were associated with synergism to copanlisib/venetoclax. Largely the opposite was observed for the palbociclib combination, more active with high expression of E2F/MYC targets and cell cycle genes and low expression of genes involved in IFN PI3K/AKT/mTOR and IL6/JAK/STAT signaling. Conclusion Copanlisib was active in MCL, MZL and CLL models. Combinations with BCL2-i venetoclax and CDK4/CDK6-i palbociclib were the most synergistic. Specific GEP features might predict lymphomas that could benefit from these regimens. Citation Format: Eugenio Gaudio, Ivo Kwee, Filippo Spriano, Chiara Tarantelli, Andrea Rinaldi, Thibaud Jourdan, Melanie Berthold, Alberto Arriibas, Anastasion Stathis, Davide Rossi, Ningshu Liu, Martin Lange, Oliver Politz, Emanuele Zucca, Francesco Bertoni. The phosphatidylinositol-3-kinase (PI3K) inhibitor (i) copanlisib is active in preclinical models of B-cell lymphomas as single agent and in combination with conventional and targeted agents including venetoclax and palbociclib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 154. doi:10.1158/1538-7445.AM2017-154

Abstract 5583: Pegylated adenosine deaminase 2 (PEG-ADA2) abrogates the cytoprotective effects of adenosine against chronic lymphocytic leukemia cells

Abstract Extracellular adenosine can be generated from ATP and/or ADP through the concerted action of the ectoenzymes CD39 and CD73. Adenosine can bind different type-1 purinergic receptors eliciting potent cytoprotective and immunosuppressive effects. We previously focused on chronic lymphocytic leukemia (CLL), a disease characterized by the progressive expansion of a mature population of B lymphocytes, showing that a subset of patients with a CD73+ clone possess a poor prognosis. This patient subset can actively generate adenosine, which favors leukemic cell survival, synthesis of immunosuppressive cytokines, while inhibiting T lymphocyte proliferation. ADA2 is a human enzyme that catalyzes the conversion of adenosine into inosine. We hypothesized that depletion of high adenosine levels from the tumor microenvironment through the administration of exogenous ADA2 could be therapeutically relevant to limit tumor protection and immunosuppression. Several engineered PEG-ADA2 variants possessed improved enzyme activity and pharmacokinetics compared to wild-type ADA2. Inhibition of adenosine receptor activation was evaluated by measuring intracellular cAMP concentrations in T lymphocytes purified from healthy donors. Adenosine depletion was observed with increasing concentrations of PEG-ADA2-R222Q/S265N and significantly reduced cAMP levels. PEG-ADA2 variants were then tested on CD73+ CLL lymphocytes to determine their effects on extracellular adenosine and inosine. Treatment of leukemic B cells with PEG-ADA2-R222Q/S265N and PEG-ADA2-R222Q/S265N/K374D resulted in complete removal of high levels of adenosine, as measured by HPLC. Conversely, PEG-ADA2-E182T, which possesses significantly attenuated catalytic activity, was less effective in reducing adenosine. The functional effects of adenosine depletion on CLL cell survival were analyzed following treatment with the chemotherapeutic, DNA-damaging agent etoposide that robustly induces apoptosis within 16 hours. Addition of exogenous adenosine to cultures of purified CD73+ CLL cells significantly rescued cells from etoposide-induced apoptosis. However, when these primary leukemic cells were pretreated with PEG-ADA2-R222Q/S265N or PEG-ADA2-R222Q/S265N/K374D, the cell viability rate was significantly decreased, abrogating the cytoprotective effects of adenosine. On the contrary, PEG-ADA2-E182T had a minimal effect, suggesting enzymatic depletion of adenosine is critical to observe these effects. Similar effects were observed by the PEG-ADA2 variants on a CLL cell line expressing CD73+. These preliminary results suggest that enzymatic depletion of extracellular adenosine following treatment with PEG-ADA2 is a relevant approach to counteract the cytoprotective effects of adenosine, warranting further development of PEG-ADA2 as a possible approach to treat CLL. Citation Format: Sara Serra, Cinzia Bologna, Luz Londono, Lin Wang, Michael Shepard, Sanna Rosengren, Christopher Thanos, Silvia Deaglio. Pegylated adenosine deaminase 2 (PEG-ADA2) abrogates the cytoprotective effects of adenosine against chronic lymphocytic leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5583. doi:10.1158/1538-7445.AM2017-5583

The HDAC inhibitor valproate induces a bivalent status of the CD20 promoter in CLL patients suggesting distinct epigenetic regulation of CD20 expression in CLL in vivo.

Treatment with anti-CD20 antibodies is only moderately efficient in chronic lymphocytic leukemia (CLL), a feature which has been explained by the inherently low CD20 expression in CLL. It has been shown that CD20 is epigenetically regulated and that histone deacetylase inhibitors (HDACis) can increase CD20 expression in vitro in CLL. To assess whether HDACis can upregulate CD20 also in vivo in CLL, the HDACi valproate was given to three del13q/NOTCH1wt CLL patients and CD20 levels were analysed (the PREVAIL study). Valproate treatment resulted in expected global activating histone modifications suggesting HDAC inhibitory effects. However, although valproate induced expression of CD20 mRNA and protein in the del13q/NOTCH1wt I83-E95 CLL cell line, no such effects were observed in the patients studied. In contrast to the cell line, in patients valproate treatment resulted in transient recruitment of the transcriptional repressor EZH2 to the CD20 promoter, correlating to an increase of the repressive histone mark H3K27me3. This suggests that valproate-mediated induction of CD20 may be hampered by EZH2 mediated H3K27me3 in vivo in CLL. Moreover, valproate treatment resulted in induction of EZH2 and global H3K27me3 in patient cells, suggesting transcriptionally repressive effects of valproate in CLL. Our results suggest new in vivo mechanisms of HDACis which may have implications on the design of future clinical trials in B-cell malignancies.

Therapeutic inhibition of USP7-PTEN network in chronic lymphocytic leukemia: a strategy to overcome TP53 mutated/deleted clones.

Chronic Lymphocytic Leukemia (CLL) is a lymphoproliferative disorder with either indolent or aggressive clinical course. Current treatment regiments have significantly improved the overall outcomes even if higher risk subgroups - those harboring TP53 mutations or deletions of the short arm of chromosome 17 (del17p) - remain highly challenging. In the present work, we identified USP7, a known de-ubiquitinase with multiple roles in cellular homeostasis, as a potential therapeutic target in CLL. We demonstrated that in primary CLL samples and in CLL cell lines USP7 is: i) over-expressed through a mechanism involving miR-338-3p and miR-181b deregulation; ii) functionally activated by Casein Kinase 2 (CK2), an upstream interactor known to be deregulated in CLL; iii) effectively targeted by the USP7 inhibitor P5091. Treatment of primary CLL samples and cell lines with P5091 induces cell growth arrest and apoptosis, through the restoration of PTEN nuclear pool, both in TP53-wild type and -null environment. Importantly, PTEN acts as the main tumor suppressive mediator along the USP7-PTEN axis in a p53 dispensable manner. In conclusion, we propose USP7 as a new druggable target in CLL.

The Human CD38 Monoclonal Antibody Daratumumab Shows Antitumor Activity and Hampers Leukemia-Microenvironment Interactions in Chronic Lymphocytic Leukemia.

Purpose: To establish a proof-of-concept for the efficacy of the anti-CD38 antibody daratumumab in the poor prognosis CD38+ chronic lymphocytic leukemia (CLL) subtype.Experimental Design: The mechanism of action of daratumumab was assessed in CLL primary cells and cell lines using peripheral blood mononuclear cells to analyze antibody-dependent cell cytotoxicity (ADCC), murine and human macrophages to study antibody-dependent cell phagocytosis (ADCP), or human serum to analyze complement-dependent cytotoxicity (CDC). The effect of daratumumab on CLL cell migration and adhesion to extracellular matrix was characterized. Daratumumab activity was validated in two in vivo models.Results: Daratumumab demonstrated efficient lysis of patient-derived CLL cells and cell lines by ADCC in vitro and ADCP both in vitro and in vivo whereas exhibited negligible CDC in these cells. To demonstrate the therapeutic effect of daratumumab in CLL, we generated a disseminated CLL mouse model with the CD38+ MEC2 cell line and CLL patient-derived xenografts (CLL-PDX). Daratumumab significantly prolonged overall survival of MEC2 mice, completely eliminated cells from the infiltrated organs, and significantly reduced disease burden in the spleen of CLL-PDX. The effect of daratumumab on patient-derived CLL cell dissemination was demonstrated in vitro by its effect on CXCL12-induced migration and in vivo by interfering with CLL cell homing to spleen in NSG mice. Daratumumab also reduced adhesion of CLL cells to VCAM-1, accompanied by downregulation of the matrix metalloproteinase MMP9.Conclusions: These unique and substantial effects of daratumumab on CLL viability and dissemination support the investigation of its use in a clinical setting of CLL. Clin Cancer Res; 23(6); 1493-505. ©2016 AACR.

Phosphatidylinositol 3-kinase δ blockade increases genomic instability in B cells.

Activation-induced cytidine deaminase (AID) is a B-cell specific enzyme that targets immunoglobulin (Ig) genes to initiate class switch recombination (CSR) and somatic hypermutation (SHM)1. Through off-target activity, however, AID has a much broader impact on genomic instability by initiating oncogenic chromosomal translocations and mutations involved in lymphoma development and progression2. AID expression is tightly regulated in B cells and its overexpression leads to enhanced genomic instability and lymphoma formation3. The phosphatidylinositol 3-kinase (PI3K) δ pathway plays a key role in AID regulation by suppressing its expression in B cells4. Novel drugs for leukemia or lymphoma therapy such as idelalisib, duvelisib or ibrutinib block PI3Kδ activity directly or indirectly5–8, potentially affecting AID expression and, consequently, genomic stability in B cells. Here we show that treatment of primary mouse B cells with idelalisib or duvelisib, and to a lesser extent ibrutinib, enhanced the expression of AID and increased somatic hypermutation (SHM) and chromosomal translocation frequency to the Igh locus and to several AID off-target sites. Both these effects were completely abrogated in AID deficient B cells. PI3Kδ inhibitors or ibrutinib increased the formation of AID-dependent tumors in pristane-treated mice. Consistently, PI3Kδ inhibitors enhanced AID expression and translocation frequency to IgH and AID off-target sites in human chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) cell lines, and patients treated with idelalisib, but not ibrutinib, showed increased SHM in AID off-targets. In summary, we show that PI3Kδ or BTK inhibitors increase genomic instability in normal and neoplastic B cells by an AID-dependent mechanism, an effect that should be carefully considered as such inhibitors are administered for years to patients.

Transposon Mutagenesis Reveals Fludarabine Resistance Mechanisms in Chronic Lymphocytic Leukemia

To identify resistance mechanisms for the chemotherapeutic drug fludarabine in chronic lymphocytic leukemia (CLL), as innate and acquired resistance to fludarabine-based chemotherapy represents a major challenge for long-term disease control. We used piggyBac transposon-mediated mutagenesis, combined with next-generation sequencing, to identify genes that confer resistance to fludarabine in a human CLL cell line. In total, this screen identified 782 genes with transposon integrations in fludarabine-resistant pools of cells. One of the identified genes is a known resistance mediator DCK (deoxycytidine kinase), which encodes an enzyme that is essential for the phosphorylation of the prodrug to the active metabolite. BMP2K, a gene not previously linked to CLL, was also identified as a modulator of response to fludarabine. In addition, 10 of 782 transposon-targeted genes had previously been implicated in treatment resistance based on somatic mutations seen in patients refractory to fludarabine-based therapy. Functional characterization of these genes supported a significant role for ARID5B and BRAF in fludarabine sensitivity. Finally, pathway analysis of transposon-targeted genes and RNA-seq profiling of fludarabine-resistant cells suggested deregulated MAPK signaling as involved in mediating drug resistance in CLL. To our knowledge, this is the first forward genetic screen for chemotherapy resistance in CLL. The screen pinpointed novel genes and pathways involved in fludarabine resistance along with previously known resistance mechanisms. Transposon screens can therefore aid interpretation of cancer genome sequencing data in the identification of genes modifying sensitivity to chemotherapy. Clin Cancer Res; 22(24); 6217-27. ©2016 AACR.

DNA Hypomethylation within B-Cell Enhancers and Super Enhancers Reveal a Dependency on Immune and Metabolic Mechanisms in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL), characterized by the progressive and uncontrolled accumulation of CD19+ B cells, currently remains as an incurable malignancy. The difficulties of eliciting curative measures in CLL are partly driven by the adaptability of the transcriptional response mediated by epigenetic mechanisms. In this study, we sought to better characterize the complexities of the CLL transcriptional profile by conducting an integrative analysis between the B cell enhancer and super enhancer signatures defined from 3 B cell H3K27Ac ChIPseq samples (CD19+ B cell, GM12878, and MEC1), the DNA methylation signatures defined from reduced-representation bisulfite sequencing (RRBS) of 42 CLL patient and 8 healthy donor samples, and the mRNA expression signatures defined from RNA sequencing of 47 CLL patient and 5 healthy donor samples. From our analysis, we identified super enhancers (SEs) in each of the ChIPseq profiles (approximately 4% of called enhancers) and discovered 741 SEs in GM12878, 374 SEs in MEC1, and 523 SEs in the CD19+ B cell profiles, respectively. Based on MSigDB gene ontology analysis, many of the genes corresponding with SEs were involved in pathways regulating immune signaling activation (e.g. TNFA_SIGNALING_VIA_NFKB, INFLAMMATORY RESPONSE) or metabolic homeostasis (e.g. MTORC1_SIGNALING, FATTY_ACID_METABOLISM). By further analyzing the corresponding expression level of SE-associated genes in CLL patients, we identified 190 transcripts associated with SEs that were significantly overexpressed in CLL patient B cells (Student’s t-test p<0.05), and this overexpressed subset of SE-associated transcripts was enriched in genes involved in either immune signaling (e.g. LCK, FCER2) or metabolic regulation (e.g. LSR, ENO2). Based on the differential expression of genes associated with enhancers occurring between CLL patient and healthy donor B cells, we then wanted to determine whether differential DNA methylation within enhancers corresponded with upregulation of CLL transcripts. Based on differential DNA methylation (DM) analysis (methylation difference +/- .25; Student’s t-test p-value<0.05) from our RRBS samples, we discovered 744 DM CpG sites that overlapped within our identified B cell enhancers, and most of the DM CpG sites in CLL were significantly hypomethylated (avg. DM GpG difference: enhancer = -0.40; non-enhancer = -0.08). Examples of hypomethylated enhancers included super enhancers corresponding with overexpressed transcripts ENO2, SEPT9, RXRA, and CCR7 as well as a typical enhancer that corresponded with the overexpressed transcript PDCD1. Based on the derived information from our integrative analysis of B cell enhancers, we then compared the effects of preferentially targeting enhancer-mediated expression with either the BET bromodomain inhibitor JQ1 or the cyclin dependent kinase-7 (CDK7) inhibitor THZ1. Based on in vitro assays and RNAseq expression analysis comparing THZ1 and JQ1-treated CLL cell lines MEC1 and MEC2, we saw that JQ1 could inhibit CLL cell line proliferation, suppress IgM-mediated primary CLL proliferation, and differentially disrupt transcription of genes involved in immune signaling cascades. Contrastingly, we saw that THZ1 elicited a different response in CLL cell lines and primary cells by disrupting cell viability, inducing apoptosis, and differentially downregulating genes involved in metabolic homeostasis. The specific enhancer-associated genes disrupted by the respective treatments further highlight the dichotomy of JQ1 and THZ1-mediated effects, as JQ1 selectively suppressed the B cell activation marker gene FCER2 and the PD-1 receptor gene PDCD1, whereas THZ1 selectively suppressed the glycolytic enolase gene ENO2 and the proto-oncogene FGR. Collectively, these results reveal how CLL DNA hypomethylation within B cell enhancers can mediate immune signaling and metabolic expression signatures in CLL and can differentially be disrupted by BET bromodomain or CDK7 inhibition. DisclosuresAwan:Innate Pharma: Research Funding; Pharmacyclics: Consultancy; Novartis Oncology: Consultancy.

The Bcl-2 BH4 Antagonist Bda-366 Induces Apoptosis in CLL and DLBCL Cells through a Bcl-2 Independent Mechanism

Overexpression of the antiapoptotic protein Bcl-2 is considered primarily responsible for the increased apoptosis resistance and prolonged survival of the malignant B cells in chronic lymphocytic leukemia (CLL), follicular lymphoma and subsets of diffuse large B cell lymphoma (DLBCL). The importance of Bcl-2 overexpression is corroborated by the significant clinical activity of the recently approved Bcl-2 BH3 antagonist venetoclax. Venetoclax induces apoptosis by sequestering Bcl-2 from the pro-apoptotic Bcl-2 family members Bim, Bax and Bak, resulting in consequent Bax/Bak activation, mitochondrial outer membrane permeabilization and cytochrome c release. Although venetoclax is extremely toxic against CLL cells in vitro, resistance can arise in vivo because of overexpression of other anti-apoptotic Bcl-2 family members, such as Bcl-xL and Mcl-1.In the present study we investigated the activity of BDA-366, another small molecule Bcl-2 inhibitor that was recently reported to selectively induce apoptosis in Bcl-2-overexpressing lung cancer cells through a distinct mechanism (Han B et al, Cancer Cell. 2015;27:852-63). This drug was designed to selectively bind the BH4 domain of Bcl-2 and was reported to trigger apoptosis by inducing a conformational change in Bcl-2 that converts it into a pro-apoptotic protein. To investigate whether BDA-366 is also selectively toxic for CLL cells, we analyzed the viability of CLL (n=31) and normal peripheral blood mononuclear cells (PBMCs)(n=6) after 48 hours in culture with increasing concentrations of BDA-366. Annexin V/PI analysis showed that BDA-366 is significantly more toxic against CLL (LD50=1.11±0.46 mM) than normal PBMCs (LD50=2.03±0.31 mM, P<0.001). However, considerable variability was noted in the sensitivity of the different CLL samples, with approximatelly 50% of the cases demonstrating LD50 values in the nanomolar range and the remaining cases displaying sensitity similar to normal PBMC. To investigate the reasons for this variability, we correlated LD50 values with IGHV mutation status, ZAP-70 expression, cytogenetic abnormalities, and expression of various Bcl-2 family members, including Bcl-2, Bcl-xL, Mcl-1, BimEL, BimL, Bax, Bak and Hrk. The only positive finding from this analysis was the inverse correlation between LD50 and expression of BimL (n=23, r=-0.446, p=0.033), whereas no correlation was observed with Bcl-2 expression.Variable sensitivity to BDA-366 and absence of correlation with Bcl-2 expression was also observed in the analysis of a series of DLBCL and CLL cell lines. These cell lines showed LD50 values ranging from nanomolar (Pfeiffer, 0.19 µM; OCI-LY-18, 0.32 µM; OCI-LY-1, 0.33 µM; TOLEDO, 0.41 µM; OCI-Ly10, 0.50 µM; HT, 0.56 µM; Ri-1, 0.58 µM; and SU-DHL-6, 0.81 µM) to micromolar range (MEC1, 1.5 µM; HBL-1, 4.0 µM; Karpas, 4.34 µM; BJAB, 4.8 µM; and SU-DHL-4, 6.31 µM). Although most of the sensitive lines expressed Bcl-2, some had very low or even undetectable Bcl-2 expression (e.g., Pfeiffer and HT), suggesting that BDA-366 can induce apoptosis independently of Bcl-2. In support of this possibility, we observed that BDA-366 was equally effective against Bcl-2-negative HT cells and HT cells stably transfected with Bcl-2. In addition, transient overexpression of Bcl-2 in the DLBCL cell line BJAB and the CLL cell line MEC1 resulted in greater resistance to BDA-366, further suggesting that BDA-366 does not induce apoptosis by converting Bcl-2 into a proapoptotic protein.To determine whether mechanisms that induce resistance to venetoclax can also induce resistance to BDA-366, we investigated viability of BDA-366-treated CLL cells following stimulation with immobilized anti-IgM. Pre-treatment of CLL cells with immobilized anti-IgM resulted in increased resistance to both BDA-366 and venetoclax. Interestingly, the combination of BDA-366 and venetoclax was significantly more toxic than each agent alone (Figure 1), suggesting that combining these drugs could be a potential approach to overcome resistance induced by BCR signals.In conclusion, these data show that BDA-366 is selectively toxic at low nanomolar concentrations for a large proportion of primary CLL samples and DLBCL cell lines. The primary mechanism of action is still unclear, but the lack of a correlation with Bcl-2 expression suggests that it differs from the one initially proposed. [Display omitted] DisclosuresEfremov:Gilead: Honoraria.

Exploring the Role of the Recurrent Exportin 1 (XPO1/CRM1) Mutations E571G and E571K in Chronic Lymphocytic Leukemia

BackgroundExportin 1 (XPO1/CRM1) is a critical component of the nuclear to cytoplasmic export machinery. As such, XPO1 shuttles tumor suppressor proteins (e.g. p53, IkB) out of the nucleus thereby preventing their anti-apoptotic function and contributing to enhanced proliferation. XPO1 also transports a subset of mRNAs (e.g. c-fos, IFN-alpha) by virtue of 3'-UTR AU-rich elements. XPO1 is upregulated in most hematologic malignancies, including CLL, where elevated XPO1 expression correlates with poor prognosis and resistance to therapy. To date, the role of nuclear export in CLL is poorly understood. Interestingly, recurrent mutations at a highly conserved residue (E571G; E571K) in the cargo binding pocket of XPO1 have been described in chronic lymphocytic leukemia (CLL) and are associated with worse overall survival. We hypothesized that the E571G/K mutation confers novel cargo-binding abilities to XPO1 and may contribute to the pathogenesis of CLL.MethodsTo interrogate this question in an unbiased, transcriptome-wide manner we developed CLL cell lines expressing mutant XPO1 and a novel RNA immunoprecipitation procedure using RNase I digestion followed by RNA-seq to capture direct interaction sites or 'footprints'. Reads were mapped to the transcriptome and peaks (regions where RNA footprints are enriched) were identified. We examined footprints for enriched sequence motifs using the MEME suite. Mass-array screening identified a cohort of CLL patients bearing the E571G/K mutation for validation purposes. The XPO1 transgenic mice were generated via pronuclear injection of linearized plasmid containing human cDNA corresponding to XPO1WT, XPO1E571G, or XPO1E571K under the heavy chain promoter/enhancer.ResultsWe identified a transcriptome-wide pool of >100 XPO1-interacting RNAs in CLL cells and identified RNA footprints of wild-type XPO1 contributing to the first XPO1 RNA profile of a cell line model of CLL. The majority of bound RNA species associated with XPO1 are ribosomal, as expected; however, we identified multiple additional classes of RNAs such as miRs, mRNA, long-non coding RNAs, and many un-annotated RNAs. These data are consistent with additional preliminary data we have generated indicating that XPO1 may regulate mRNAs important in CLL development. We are currently searching for structural motifs. Validation experiments using patient-derived CLL cells with and without XPO1 mutations are ongoing.To further assess the independent transforming role of XPO1 dysfunction in B-cells, we generated transgenic mice overexpressing XPO1WT, XPO1E571G, or XPO1E571K under the heavy chain promoter/enhancer in the B cell compartment of C57BL/6 mice. Overexpression of XPO1 at the protein level in B cells was confirmed in multiple transgenic founder lines. Preliminary studies were performed to characterize the XPO1 transgenic animals for defects or alterations in major stages of B cell maturation. We analyzed the pro-B (IgM-CD43+), pre-B (IgM-CD43-), and immature (IgM+CD43-) B cell stages of development from the bone marrow of XPO1 transgenic animals and littermate controls. We also examined immature (IgM+IgD-) and mature (IgM+IgD+) B cells isolated from the spleen of these animals. In this preliminary analysis no significant differences in the B cell compartment were observed between transgenic and non-transgenic animals. Functional studies of these B-cells are ongoing. A cohort of animals from each founder line is being followed for evidence of B-cell expansion and overall survival analysis. In addition, the XPO1 transgenic mice were crossed with the Eµ-TCL1 mice to establish the XPO1WT/E571K/G/TCL1 double transgenic mouse model whose disease is currently being monitored.ConclusionsXPO1 is a recurring, high variant allele mutation that occurs in a small subset of CLL patients. XPO1 transports select mRNA and also novel RNA species whose function in CLL have yet to be characterized, but points to a novel role of this oncoprotein in the pathogenesis of this disease. DisclosuresNo relevant conflicts of interest to declare.

Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) Is a New Biomarker for Mantle Cell Lymphoma: Expression, Localization, and Phosphorylation Study

Mantle cell lymphoma (MCL) is a relatively distinct B-cell non-Hodgkin lymphoma subtype with aggressive and often recurrent clinical course. At diagnosis, MCL often manifests with leukemization, a feature more common to chronic lymphocytic leukemia (CLL). Common features and differences between MCL and CLL were not yet explored by comprehensive global approaches, despite such understanding potentially being very neat for deciphering pathogenesis and tailoring therapies of these clinically distinct diseases.In our study, we have compared MCL(n=10), CLL(n=10) and normal control(n=8) B-cell samples using the Affymetrix Human Genome HG-U133 Plus 2.0 Array. We studied different mRNA levels of ~47.000 transcripts represented on the array. The comparative analyses identified a set of 892 differentially expressed genes between MCL and NBC; and 774 differentially expressed genes between CLL and NBC. In order to find MCL/CLL-specific biomarkers we focused on the intersection of differently expressed genes in both groups (CLL vs NBC and MCL vs NBC). There were 222 mRNAs in the intersection, 216 of them were deregulated in the same direction in both groups while 6 mRNAs were deregulated in the opposite direction. This set of 6 disease-specific mRNAs contained previously reported biomarkers (CD200, LEF1), and also the Myristoylated alanine-rich C-kinase substrate (MARCKS) that has not yet been studied in MCL. Thus we utilized the validation patient groups (NMCL=6, NCLL=8) and confirmed differential expression of MARCKS on protein levels by flow cytometry and immunofluorescence. As MARCKS was previously shown to either bound to the cell membrane, to reside in the cytosol, or alternatively become transmitted to nuclei, we investigated subcellular localization of MARCKS using immunofluorescence (IF). The cytoplasmic MARCKS signal in MCL was significantly higher than in CLL while the opposite was observed for the nuclear IF signal. The ratio between cytoplasmic and nuclear signal was 2.5 for MCL and 0.8 for CLL (p < 0,0001). The active forms of MARCKS were shown to become phosphorylated on serineresidues and this prompted us to study the phosphorylation forms of MARCKS in MCL. Indeed, one of the residues, Ser159/163, was hyperphosphorylated in the MCL cytoplasm and its level and distribution markedly differed from CLL or NBC. We next searched for regulatory mechanisms upstream of the MARCKS expression in MCL vs CLL. MARCKS is a predicted target of several microRNAs (according to DIANA-TarBase v7.0), among them also of miR-155 (that is differentially expressed between MCL and CLL). To further investigate the regulatory relationship between mir-155 and MARCKS we utilized a CLL cell line MEC-1 and using the CRISPR/Cas9 technology we prepared individual cell clones that were mutated within the mature miR-155 sequence that recognizes MARCKS mRNA. As expected, the miR-155-MEC-1 mutants expressed markedly higher level of MARCKS compared to the control MEC-1 cells.In conclusion, our work identified a set of six differentially expressed mRNAs when comparing MCL and CLL, among them, MARCKS. We further showed that MARCKS is differentially expressed, localized, and phosphorylated between MCL and CLL, and that MARCKS is partly controlled by oncogenic microRNA miR-155. MARCKS may play an important role in MCL pathogenesis and can serve as useful MCL biomarker.Grant support: GAČR 16-05649S & P305/12/1033, AZV: 16-27790A and 16-31586A. Institutional support: CZ.1.05/1.1.00/02.0109, UNCE 204021, LH15170, PRVOUK P24, LQ1604. DisclosuresNo relevant conflicts of interest to declare.

Differing Mutational Profiles of IgHV+ and IgHV- Patients Revealed By Whole Genome Sequencing in Chronic Lymphocytic Leukaemia (CLL)

BackgroundDuring B-cell development, somatic mutations are introduced into the variable region (V) of Immunoglobulin Heavy (IGH) genes by activation-induced cytidine deaminase (AID). In CLL, the degree of mutation in these regions is tied to clinical outcome, with IgHV hypermutated status (IgHV+, <98% homology to germline) strongly predicting increased survival rates over unmutated patients (IgHV-) (Gardiner et al., Blood, 1999). In addition to AID, APOBEC signatures have been found in many human cancers (Gordenin at al., Nature Genetics 2013). So far, WGS efforts have focused primarily on IgHV+ patients (Puente et al, Nature 2015; Kasar et al, Nature Com 2015). Here, we perform comparative analyses between IgHV+/- patients using Whole Genome Sequencing (WGS) to explore this link.MethodsWhole genome sequencing was performed on matched tumour and germline DNA from a cohort of 46 CLL patients, divided into two groups; 16 IgHV+ and 30 IgHV-. Sequence data was generated using the Illumina HiSeq 2500 platform, and somatic variants were generated by Strelka 2.4.7.SNVs were annotated using ANNOVAR (version 2015 Dec 14) and supplemented with information from primary CLL cell lines and B-cell ENCODE databases for the non-coding regions. Kataegis was identified based on the methods of Lawrence et al. (Nature, 2013) and Alexandrov et al. (Nature, 2013). Mutation signatures were analysed according to Alexandrov et al. (Nature, 2013).ResultsWe identified a total of 64,420 high confidence somatic SNVs from 46 samples (mean=1400), of which 44% were from the IgHV+ cohort (mean=1680) and 56% from IgHV- (mean=1237). Of these; SNVs in coding regions (exons, introns, UTRs) occurred at significantly higher proportions in IgHV- patients (P=0.0004, Fishers Exact test). Mutations in predicted active DNAse hypersensitivity regions and H3k27 acetylated regions, however, were significantly more likely to occur in IgHV+samples (P<0.0001).Mutational signature analysis revealed three distinct signatures shared between the two cohorts. Two of these (Tsig1 and Tsig2) clustered with Alexandrov signature 1A, and the third to signature 1B (Tsig3), both of which were designated as ageing signatures. Despite this, our signatures significantly correlated with the proportion of mutated AID (P<0.03; P<0.03; Tsig1 and Tsig3 respectively), and APOBEC sites (P<0.001; P<0.001; Tsig1 and Tsig3 respectively), and not with age. These signatures were found to differ significantly between cohorts (P < 0.001), regardless of treatment. Tsig2 was not found to correlate with either patient age, AID signature or APOBEC signature, suggesting that it may be a novel signature.A total of 53 kataegis regions were identified across all patients, of which three were found on chromosomes 2, 14 and 22, corresponding to the IG loci. Coding mutation hotspots were located in known CLL driver genes, including TP53, ATM, IKZF3 and SF3B1. Non-coding recurrent hotspots caused by AID were found to predominately affect promoter and enhancer regions of key B-cell pathways, including BCL6, BCL2, BTG2, IGLL5, and PAX5. This observation is closely linked to the IgHV status; the IgHV+ cases frequently harboured mutated non-coding variants in genes involved in B cell signalling, whilst IgHV- cases were more likely to contain exonic driver mutations. Kataegis analysis also revealed novel non-coding mutations in recurrently mutated genes that were common in IgHV- cases, including CDK6 and BIRC3, and a non-coding RNA region on chromosome 9 that was hypermutated only in IgHV-cases.ConclusionHere, we present a whole genome sequencing study on 46 patients divided into two cohorts of IgHV+ and IgHV-. WGS revealed distinct changes in mutation distribution and signatures between these cohorts; differences that are mirrored in both the recurrently mutated gene profiles, and the regions of somatic hypermutation. We demonstrate that mutational differences in IgHV+ and IgHV- patients extend far beyond the IgHV regions and the 1% of the coding genome. This study paves the way for future work into understanding the genomic differences between these cohorts and thus, contribute to increasing our understanding of the molecular mechanisms underlying the different clinical outcomes. DisclosuresHillmen:Pharmacyclics: Research Funding; Janssen: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Research Funding.

CLL Exosome-Derived Y RNA hY4 Induces TLR7/8-Mediated Inflammation and PD-L1 Expression in Monocytes

Chronic lymphocytic leukemia (CLL) is a B-cell malignancy associated with an inflammatory milieu and impaired anti-tumor immunity. Both in human CLL samples and the Eµ-TCL1 mouse model of CLL, monocytes and macrophages were identified as key players in the involved processes, as they secrete immune regulatory cytokines and show enhanced expression of the immune checkpoint protein PD-L1 which is known to be involved in T-cell suppression. We recently showed that reactivation of T-cell activity using anti-PD-L1 antibodies controls leukemia development in mice and is associated with a normalization of CLL-associated immune defects.The current study aimed at unraveling the molecular mechanisms of CLL-induced changes in monocytes and macrophages and focused on CLL-derived exosomes and their role in the tumor microenvironment.Exosomes were isolated from blood plasma of CLL patients as well as culture supernatant of the CLL cell line MEC-1 by a serial centrifugation protocol. Characterization of isolated exosomes by electron microscopy, Nanoparticle Tracking Analysis (NTA), and Western blot analysis revealed vesicles, 30 to 350 nm in size, which were positive for various exosome marker proteins. Quantification of exosomes by NTA and ELISA indicated an enrichment of B-cell derived exosomes in plasma of CLL patients compared to healthy controls, although absolute exosome counts were not different. RNA sequencing and proteome analysis of CLL exosomes revealed a disease-specific composition and identified non-coding Y RNA hY4 as the most abundant exosomal RNA species. Transfer of CLL exosomes or hY4 RNA alone to monocytes triggered release of cytokines like CCL2, CCL3, CCL4, and IL-6, and increased expression of PD-L1. As these are key features associated with CLL, a novel role for exosomal Y RNAs in the tumor microenvironment of CLL is suggested. Of interest, exosome or hY4-induced responses in monocytes were significantly reduced by chloroquine treatment and completely abolished in TLR7-deficient monocytes. Therefore, exosomal hY4 was identified as novel ligand that activates Toll-like receptor 7/8 signaling pathway in monocytes.Tumor-derived exosomes as well as exosomal Y RNAs were detected in a number of malignancies, suggesting their more general contribution to cancer-related sterile inflammation and the formation of a tumor-supportive myeloid microenvironment. Ongoing studies will show whether these novel findings can be exploited in treatment approaches for CLL and other malignancies. DisclosuresStilgenbauer:Hoffmann-La Roche: Consultancy, Honoraria, Other: Travel grants , Research Funding; Sanofi: Consultancy, Honoraria, Other: Travel grants , Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: Travel grants , Research Funding; Boehringer Ingelheim: Consultancy, Honoraria, Other: Travel grants , Research Funding; AbbVie: Consultancy, Honoraria, Other: Travel grants, Research Funding; Novartis: Consultancy, Honoraria, Other: Travel grants , Research Funding; Janssen: Consultancy, Honoraria, Other: Travel grants , Research Funding; Genentech: Consultancy, Honoraria, Other: Travel grants , Research Funding; Amgen: Consultancy, Honoraria, Other: Travel grants, Research Funding; GSK: Consultancy, Honoraria, Other: Travel grants , Research Funding; Mundipharma: Consultancy, Honoraria, Other: Travel grants , Research Funding; Genzyme: Consultancy, Honoraria, Other: Travel grants , Research Funding; Gilead: Consultancy, Honoraria, Other: Travel grants , Research Funding; Celgene: Consultancy, Honoraria, Other: Travel grants , Research Funding.

BRAFV600E Accelerates Disease Progression and Increases Immune Suppression in a Mouse Model of B-Cell Leukemia

Background: Mitogen-activated protein kinase (MAPK) pathway components are frequently mutated in cancer, and abnormal MAPK signaling can drive malignancy by promoting tumor survival and proliferation. Furthermore, work in solid tumors shows that the presence of BRAFV600E further enhances tumor-induced immune suppression, but if and how this occurs in a disseminated disease such as leukemia is unknown. MAPK mutations occur in nearly 100% of classic hairy cell leukemia (HCL) and approximately 10% of chronic lymphocytic leukemia (CLL). Furthermore, up to 30% of B cell lymphomas exhibit activated MAPK signaling via expression of a BRAF pseudogene. However, the pathomechanisms of these abnormalities and the clinical utility of MAPK inhibitors in hematologic malignancies are unclear, and few models are available to evaluate this. To study these aspects, we developed in vitro and in vivo models of BRAF-mutated B cell malignancy.Methods: To mechanistically interrogate the role of BRAFV600E in malignant B cells, we transfected the CLL cell line OSUCLL with doxycycline-inducible constructs containing normal BRAF or BRAFV600E and incubated the cells -/+ doxycycline. These cells were also co-cultured with healthy donor T cells pre-incubated with anti-CD3/anti-CD28, and T cell proliferation was measured by flow cytometry. Cytokines and surface proteins were assessed by flow cytometry. To mimic human HCL, we developed a transgenic mouse model of BRAFV600E B cell leukemia. Mice with Cre-activatable BRAFV600E expression were crossed with mice carrying Cre under the control of the CD19 promoter. CD19-Cre x BRAFV600E or CD19-Cre only mice were then crossed with the well-characterized Eµ-TCL1 model of CLL to generate mice with spontaneous B cell leukemia expressing either wild-type BRAF (CD19-Cre x TCL1) or BRAFV600E (BRAFVE x CD19-Cre x TCL1) under the native BRAF promoter. B cell restricted expression of BRAFV600E was confirmed by immunoblots of purified B and T cells using a BRAFV600E specific antibody. Blood and spleen cells were examined by flow cytometry. For adoptive transfer experiments, leukemia cells (2x10^7) from transgenic mice were introduced intravenously into syngeneic healthy adult animals.Results: OSUCLL cells expressing BRAFV600E showed no significant changes in growth vs. wild-type BRAF expressing cells, but more strongly inhibited anti-CD3/CD28-induced proliferation of normal donor T cells. Transwell assays showed this effect was due both to soluble and contact-dependent factors. Expression of PD-L1 was not different between cells expressing normal and mutated BRAF, indicating this common checkpoint molecule is not the reason for greater immune suppression in this context. TNF levels were higher in BRAFV600E expressing cells and reduced by vemurafenib, but a TNF neutralizing antibody did not alter the inhibitory effect of BRAFV600E expressing cells on T cell proliferation. BRAFV600E x CD19-Cre x TCL1 mice developed B cell leukemia significantly earlier (median 4.9 vs. 8.1 months; P<0.001) and had significantly shorter lifespan (median 7.3 vs. 12.1 months; P<0.001) than their wild-type BRAF counterparts. In contrast to effects of BRAFV600E described in some solid tumors, BRAFV600E expression in B cells had no impact on the rate of B cell proliferation in vivo and only modestly reduced spontaneous apoptosis. To study the effects of this activating mutation on tumor-mediated immune suppression in vivo, leukemia cells from BRAFV600E x CD19-Cre x TCL1 or CD19-Cre x TCL1 mice were adoptively transferred into syngeneic mice. In 3 separate studies, tumor cells from BRAFV600E mice produced leukemia (>10% CD5+/CD19+ cells in blood) sooner than CD19-Cre x TCL1 tumor cells. In mice matched for disease load, BRAFV600E B cells produced a greater negative impact on T cells as evidenced by lower overall percentage of T cells, increased expression of T cell exhaustion markers PD-1, CD244, and CD160, and higher percentage of CD44+ memory T cells. Current studies are investigating the mechanism of these effects in vivo as well as the potential for pharmacologic reversal.Conclusions: Together, these results demonstrate the immune-suppressive impact of BRAFV600E in B-cell leukemias and introduce a novel mouse model to develop rational combination strategies to both directly target the tumor cell and overcome tumor-mediated immune evasion. DisclosuresLozanski:Genentech: Research Funding; Stemline Therapeutics Inc.: Research Funding; Beckman Coulter: Research Funding; Boehringer Ingelheim: Research Funding.

Establishment of Human Chronic Lymphocytic Leukemia Model in the BALB/c Nude Mice by Using MEC-1 and HG3 Cell Lines

To explore the the optimal condition for establishing immune deficiency mouse(BALB/c) model with CLL via subcutaneous inoculation of human chronic lymphocytic leukemia (CLL) cells at different inoculative locations and different cell concentrations. Firstly, Two CLL cell lines (MEC-1-GFP and HG3-GFP)with the green fluorescent protein (GFP) were established by lentivirus system respectively, and then the MEC-1-GFP cells (5×107/ml) were inoculated into forelimb, hindlimb and abdomen to observe the tumorigenesis. Secondly, the MEC-1-GFP and HG3-GFP cells with same density (5×107/ml) were inoculated into forelimb to compare the time and rate of tumor formation. Thirdly, the MEC-1-GFP cells (1×107/ml) and HG3-GFP cells (5×107/ml) were inoculated into forelimb to compare the time and rate of tumor formation at different inoculative density. After observation for 5 weeks, the peripheral blood was collected and treated with EDTA and erythrocytolysin, then the of GFP positive cells were detected by flow cytomety. Meanwhile, the tumor-bearing mice were killed, and the tumors were isolated and cut into slices for histopathological examination. MEC-1-GFP and HG3-GFP cell lines were successfully established, and after inocutation of MEC-1-GFP cells with 5×107/ml the xenograt tumors were formated in forelimb, hindlimb and abdomen of mice, especially in the forelimb with a higher tumorigenic rate. In addition, the inoculation of same density of MEC-1-GFP and HG3-GFP cells (5×107/ml) also resulted in xenograft in forelimb, and the tumorigenic rate reached to 80% after 5 weeks. Moreover, the inocutation of MEC-1-GFP and HG3-GFP cells with 1×107 and 5×107/ml respectively also effectively resulted to xenograft tumor in forelimb. The flow cytometry showed that there was no MEC-1-GFP and HG3-GFP cells in peripheral blood, while histopathological examination demonstrated CLL cell metastasis towards peritoneal cavity. The BALB/c nude mouse model is successfully established by subcutaneous injection of MEC-1-GFP and HG3-GFP cells. This model is a useful tool to explore the pathogenic mechanism.

Targeting transcription-coupled nucleotide excision repair overcomes resistance in chronic lymphocytic leukemia.

Treatment resistance becomes a challenge at some point in the course of most patients with chronic lymphocytic leukemia (CLL). This applies to fludarabine-based regimens, and is also an increasing concern in the era of more targeted therapies. As cells with low-replicative activity rely on repair that triggers checkpoint-independent noncanonical pathways, we reasoned that targeting the nucleotide excision repair (NER) reaction addresses a vulnerability of CLL and might even synergize with fludarabine, which blocks the NER gap-filling step. We interrogated here especially the replication-independent transcription-coupled-NER ((TC)-NER) in prospective trial patients, primary CLL cultures, cell lines and mice. We screen selected (TC)-NER-targeting compounds as experimental (illudins) or clinically approved (trabectedin) drugs. They inflict transcription-stalling DNA lesions requiring TC-NER either for their removal (illudins) or for generation of lethal strand breaks (trabectedin). Genetically defined systems of NER deficiency confirmed their specificity. They selectively and efficiently induced cell death in CLL, irrespective of high-risk cytogenetics, IGHV status or clinical treatment history, including resistance. The substances induced ATM/p53-independent apoptosis and showed marked synergisms with fludarabine. Trabectedin additionally perturbed stromal-cell protection and showed encouraging antileukemic profiles even in aggressive and transforming murine CLL. This proof-of-principle study established (TC)-NER as a mechanism to be further exploited to resensitize CLL cells.

Epigenetic silencing of tumor suppressor long non-coding RNA BM742401 in chronic lymphocytic leukemia.

BM742401 is a tumor suppressor lncRNA downregulated in gastric cancer. As the promoter region and the entire transcript are embedded in a CpG island, we postulated that BM742401 is a tumor suppressor lncRNA inactivated by DNA methylation in chronic lymphocytic leukemia (CLL). The promoter of BM742401 was unmethylated in normal controls including three each of normal bone marrow, peripheral blood buffy coats, and CD19-sorted peripheral B-cells, but methylated in four (57.1%) CLL cell lines. Methylation of BM742401 correlated inversely with expression. In the completely methylated WAC3CD5+ CLL cells, 5-Aza-2′-deoxycytidine treatment led to promoter demethylation and re-expression of BM742401 transcript. Functionally, stable overexpression of BM742401 resulted in inhibition of cellular proliferation and enhanced apoptosis through caspase-9-dependent intrinsic but not caspase-8-dependent extrinsic apoptosis pathway, suggesting a tumor suppressor role of BM742401 in CLL. In primary CLL samples, methylation of BM742401 was detected in 43/98 (43.9%) of patients. Moreover, among CLL patients with standard-risk cytogenetic aberrations, methylation of BM742401 correlated with advanced Rai stage (≥ stage 2)(P = 0.002). Furthermore, BM742401 methylation was associated with miR-129-2 methylation (P = 0.05). BM742401 is a tumor suppressor lncRNA frequently methylated in CLL. The mechanism of BM742401 as a tumor suppressor warrants further studies.

Carfilzomib Triggers Cell Death in Chronic Lymphocytic Leukemia by Inducing Proapoptotic and Endoplasmic Reticulum Stress Responses.

Carfilzomib, while active in B-cell neoplasms, displayed heterogeneous response in chronic lymphocytic leukemia (CLL) samples from patients and showed interpatient variability to carfilzomib-induced cell death. To understand this variability and predict patients who would respond to carfilzomib, we investigated the mechanism by which carfilzomib induces CLL cell death. Using CLL patient samples and cell lines, complementary knockdown and knockout cells, and carfilzomib-resistant cell lines, we evaluated changes in intracellular networks to identify molecules responsible for carfilzomib's cytotoxic activity. Lysates from carfilzomib-treated cells were immunoblotted for molecules involved in ubiquitin, apoptotic, and endoplasmic reticulum (ER) stress response pathways and results correlated with carfilzomib cytotoxic activity. Coimmunoprecipitation and pull-down assays were performed to identify complex interactions among MCL-1, Noxa, and Bak. Carfilzomib triggered ER stress and activation of both the intrinsic and extrinsic apoptotic pathways through alteration of the ubiquitin proteasome pathway. Consequently, the transcription factor CCAAT/enhancer-binding protein homology protein (CHOP) accumulated in response to carfilzomib, and CHOP depletion conferred protection against cytotoxicity. Carfilzomib also induced accumulation of MCL-1 and Noxa, whereby MCL-1 preferentially formed a complex with Noxa and consequently relieved MCL-1's protective effect on sequestering Bak. Accordingly, depletion of Noxa or both Bak and Bax conferred protection against carfilzomib-induced cell death. Collectively, carfilzomib induced ER stress culminating in activation of intrinsic and extrinsic caspase pathways, and we identified the CHOP protein level as a biomarker that could predict sensitivity to carfilzomib in CLL. Clin Cancer Res; 22(18); 4712-26. ©2016 AACR.

Abstract A16: Establishment and characterization of CCGL007, a novel human chronic lymphocytic leukemia cell line

Abstract Chronic lymphocytic leukemia (CLL) is a monoclonal disorder characterized by a progressive accumulation of functionally incompetent lymphocytes. However, lack of immortalized cell line representative of the CLL disease has hampered a full understanding of disease pathogenesis and development of novel therapeutic agents for treatment. There were very few representative CLL cell lines are available now. To this end, we have tested different culture condition with addition of growth factor supplements to generate CLL cell lines from highly purified peripheral blood B cells of CLL patients. Recently, we have successfully established a new CLL cell line, named CCGL007. CCGL007 cells grow as suspension in liquid culture. Surface marker analysis with a panel of monoclonal antibodies revealed that CCGL007 cell line expresses B cell markers. The chromosomal, immunophenotypic, molecular biologic characteristics, and in vivo tumorigenesis potential of CCGL007 is under investigation. We have used this cell model to test the efficacy of selected standard chemotherapy drugs and new therapeutic agents, such as ibrutinib and several novel in-house BTK inhibitors, against CLL cell growth, and CCGL007 represents as a suitable preclinical model for testing pharmacological agents. Citation Format: Ching-Chuan Kuo, Chih-Cheng Chen, Chih-Hsiang Huang, Lin Li-Mei. Establishment and characterization of CCGL007, a novel human chronic lymphocytic leukemia cell line. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A16.