Cultured Chronic Lymphocytic Leukemia Cells Research Articles

CLL Cells within the Proliferation Centers of the Patient Lymph Nodes Are Enriched for Notch Signaling, Thus NOTCH a Viable Target for CLL Therapy

CLL cells within the Proliferation Centers of the Patient Lymph Nodes are enriched for Notch signaling, thus a Target for CLL Therapy. Shantaram S. Joshi, Michael Pitner, Matt Kling, Furqan Mehdi, Mamta Shukla, Ashima Shukla, Vipul Shukla, Nagendra Chaturvedi, Hamid Band , Javeed Iqbal The tissue microenvironment, particularly the lymph node (LN), plays a major role in regulating the leukemic progression and clinical heterogeneity of chronic lymphocytic leukemia (CLL). CLL cells proliferate in the patient lymph nodes, resulting in the formation of proliferation centers (PCs). The molecular basis of the CLL cell proliferation within PCs is not well known. Several reports have implicated the activated Notch signaling pathway in the pathogenesis of CLL. Using the IRF4 -/-Vh11 mouse model of CLL, we have previously shown that Notch signaling is indispensable for CLL development (Vipul Shukla et al, Oncotarget, 2016). Based on these studies, we hypothesized that CLL cell proliferation in the lymph node PCs involves activated NOTCH signaling. To this end, we have examined the expression levels of Notch signaling associated molecules namely IRF4, Notch 1, Notch 2, Nedd4, Fbxw7 and Itch1 on CLL cells within the patient lymph node proliferation centers (n=12) using immunohistochemistry and western blotting. Expression levels were scored based on the percentage of cells with positive staining: 5+ (>80%), 4+ (60-80%), 3+ (40-60%), 2+ (20-40%), 1+ (<20%) and 0 (No staining). IHC analyses showed that the expression levels of IRF4 to be 2+, Notch 1 was 4+, Notch 2 was -3 to 4+, Nedd4 was 1+, Fbxw7 was 3+ and ITCH1 was 1+. Western blotting of CLL cells from the lymph node proliferation centers (n=6) confirmed a similar pattern of expression of the Notch-associated genes. Together, these results validate the association of Notch signaling in CLL cells of the patient lymph node PCs similar to CLL cells from the IRF4 -/-Vh11 mouse model. Therefore, as a next logical step we treated the MEC-1 and OSU-CLL cells in culture and primary CLL cell from patients with NOTCH inhibitor RO4929097 (RO), a γ-secretase inhibitor for 24, 48 and 72 hours in vitro. There was a significant decrease in the expression NOTCH 1, NOTCH 2 in MEC-1, OSU-CLL and primary CLL cells determined using western blot technique. Also, we observed a significant decrease in proliferation of CLL cells from MEC-1 and OSU-CLL cell lines, following treatment with NOTCH inhibitor RO. In addition, NOTCH inhibitor combined with Bruton's Tyrosine Kinase (BTK) a member of the B-Cell Receptor signaling, showed a further significant decrease in the proliferation of CLL cells in culture. Together these experiments demonstrated that NOTCH targeting strategy is a viable therapy for aggressive CLL warranting further preclinical and clinical studies. This work was partially supported by bridge funding from the American Association of Hematology awarded to Dr. Runqing Lu, who passed away during the pursuit of this study.

Wnt5a Induces ROR1-Dependent NFkB Activation to Upregulate MMP9 and Enhance Invasiveness in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) cells migrate to the lymphoid tissue compartments, where they receive growth/survival signals, and protect from spontaneous or drug induced apoptosis. Matrix metallopeptidases (MMP) proteins such as MMP9, a type IV collagenase and key MMP, expressed and released by CLL cells, is necessary to degrade the extracellular matrix (EM) to facilitate such trafficking process. Signaling pathways such as CXCR4/CXCL12 can regulate MMP9 expression in CLL cells, and ibrutinib, a BTK inhibitor can inhibit such upregulation of MMP9. However, we found that higher-level expression of ROR1 also had high-level expression of MMP9 compared to CLL cells with low-to-negligible levels of ROR1. ROR1 (receptor tyrosine kinase-like orphan receptor 1) is an evolutionarily conserved, type I membrane protein expressed on CLL cells. Wnt5a, a non-canonical Wnt factor and ligand for ROR1, was found expressed at significantly higher levels in the plasma of patients with CLL than in age-matched adults (Yu et al., J Clin Invest, 126:585, 2016). Culture of ROR1 expressing CLL cells overnight with serum free media resulted in reduction of expression of MMP9. Such serum starved CLL cells stimulated with exogenous Wnt5a can enhance MMP9 expression. CLL cells stimulated with Wnt5a also can enhance the release of MMP9 into the cultured media. Such effects of Wnt5a could be blocked by zilovertamab (also known as UC-961, or previously known as cirmtuzumab), a humanized mAb specific for ROR1 that can inhibit leukemia-cell ROR1-signaling in patients with CLL (Choi et al., Cell Stem Cell, 22:951, 2018). Next, we examined the NFkB signaling pathway which can regulate MMP9 expression in other cancers. We discovered that Wnt5a could induce NFkB activation via ROR1 dependent mechanism in CLL. Culture of CLL cells overnight with serum free media resulted in attrition of NFkB-p65 activation. Such serum starved CLL cells stimulated with exogenous Wnt5a can induce NFkB-p65 activation. siRNA directed silencing of NFkB-p65 inhibited the Wnt5a enhanced expression and release of MMP9. Moreover, the NFkB inhibitor (CAS 545380-34-5) blocked the capacity of Wnt5a to enhance the expression and release of MMP9. We also performed functional analysis such as invasion assay and found that Wnt5a enhanced the capacity of CLL cells to invade Matrigel in a Boyden-Chamber Assay; such effects could be inhibited by zilovertamab, but not by inhibitors of B-cell receptor/chemokine signaling (e.g., ibrutinib). Moreover, combined therapy of zilovertamab and BTK inhibitors (e.g., ibrutinib) provided additional efficacy to block invasive capability of CLL cells. siRNA directed reduced expression of NFkB-p65 inhibited the capacity of Wnt5a to enhance invasive capability of CLL cells. Wnt5a enhanced invasive capability of CLL cells also could be blocked by NFkB inhibitor. In addition, siRNA directed silencing of MMP9 inhibited the Wnt5a enhanced invasive capability of CLL cells. Moreover, the MMP9 inhibitor (CAS 1177749-58-4) blocked the capacity of Wnt5a to enhance the invasive capacity of CLL cells. These results revealed a better understanding in the regulation of MMP9 expression and invasive capacity of CLL cells, and support clinical evaluation of combined therapy of zilovertamab and BTK inhibitors (e.g., ibrutinib) in CLL or other ROR1 expressing cancers.

Enhanced IFN Sensing by Aggressive Chronic Lymphocytic Leukemia Cells.

Type I IFN is made by cells in response to stress. Cancer cells exist in a state of stress, but their IFN response is complex and not completely understood. This study investigated the role of autocrine IFN in human chronic lymphocytic leukemia (CLL) cells. CLL cells were found to make low amounts of IFN via TANK-binding kinase 1 pathways, but p-STAT1 and -STAT2 proteins along with IFN-stimulated genes that reflect IFN activation were variably downregulated in cultured CLL cells by the neutralizing IFNAR1 Ab anifrolumab. Patients with CLL were segregated into two groups based on the response of their leukemia cells to anifrolumab. Samples associated with more aggressive clinical behavior indicated by unmutated IGHV genes along with high CD38 and p-Bruton's tyrosine kinase expression exhibited responses to low amounts of IFN that were blocked by anifrolumab. Samples with more indolent behavior were unaffected by anifrolumab. Hypersensitivity to IFN was associated with higher expression of IFNAR1, MX1, STAT1, and STAT2 proteins and lower activity of negative regulatory tyrosine phosphatases. Autocrine IFN protected responsive CLL cells from stressful tissue culture environments and therapeutic drugs such as ibrutinib and venetoclax in vitro, in part by upregulating Mcl-1 expression. These findings suggest hypersensitivity to IFN may promote aggressive clinical behavior. Specific blockade of IFN signaling may improve outcomes for patients with CLL with higher-risk disease.

Wnt5a Induces Interleukin-6 Via a ROR1-Dependent Pathway in Chronic Lymphocytic Leukemia, Leading to Leukemia-Cell Activation of STAT3

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic cell surface protein, which is expressed on the neoplastic B cells of patients with chronic lymphocytic leukemia (CLL), but not on virtually all normal post-partem tissues. ROR1 functions as a receptor for Wnt5a, which induces activation of Rho-GTPases, AKT, and HS-1 and promotes migration, proliferation, and survival of CLL B cells. We observed that freshly isolated CLL cells also had high levels of pSTAT3 (Y705), which attenuated over time in culture in serum-free media unless the CLL cells were stimulated with exogenous Wnt5a. This effect of Wnt5a could be blocked by cirmtuzumab, a humanized anti-ROR1 mAb that currently is undergoing clinical evaluation in patients with CLL. We examined the capacity of recombinant Wnt5a (rWnt5a) to induce activation of STAT3 in isolated CLL cells by performing a time-course study, evaluating for pSTAT3 in serum-starved CLL cells at time 0, 5', 15', 30' and 1, 2, 3, 4, and 24 hours after stimulation with exogenous Wnt5a. Surprisingly, activation of STAT3 in CLL cells was delayed, appearing first at 3 hours after addition of rWnt5a, and peaking at 24 hours, the last time point examined. In contrast, activation of CLL cells via treatment of CLL cells with interleukin (IL)-6, induced activation of STAT3 within 15' of the addition of this cytokine to the cultures. We hypothesized that the activation of STAT3 in CLL cells by Wnt5a was indirect, being caused by a factor(s) made by isolated CLL cells in response to stimulation with Wnt5a. To test this hypothesis, we examined CLL cells, and harvested supernatants of cultured CLL cells, at various times after stimulation with rWnt5a. RNA isolated from CLL cells was examined via real-time PCR for cytokine transcripts. This revealed a prominent, time-dependent increase in transcripts encoding IL-6 following stimulation of CLL cells with rWnt5a. Consistent with this observation we found Wnt5a induced CLL cells to secrete IL-6, which was detected via ELISA at increasing concentrations over time in culture-supernatants of CLL cells treated with rWnt5a, but not in the culture supernatants of unstimulated CLL cells. Culture supernatants collected from CLL cells stimulated for 24 hours with rWnt5a could induce activation of STAT3 within 15' in serum-starved CLL cells; this effect could be inhibited significantly by the anti-IL-6-receptor mAb, tocilizumab, but not by cirmtuzumab, even when used at concentrations that could block the capacity of concomitantly-added Wnt5a to induce latent STAT3 activation at 24 hours, suggesting that cirmtuzumab could inhibit Wnt5a-induced CLL-cell production of cytokines, such as IL-6. We examined for IL-6 in cultures of CLL cells treated for 24 hours with rWnt5a and cirmtuzumab, or rWnt5a and an antibody of irrelevant specificity. We found the concentration of IL-6 in the supernatants of CLL cells treated with rWnt5a and cirmtuzumab (110.4 ± 18.9 pg/ml) was significantly lower than that found in the supernatants of CLL cells treated with rWnt5a and control antibody (416.2 ± 53.7 pg/ml, p = 0.0007, Students t test). This study reveals that Wnt5a can induce CLL to secrete cytokines, such as IL-6, via a ROR1-dependent pathway, leading to latent, autocrine activation of STAT3. DisclosuresKipps:Gilead: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Research Funding; Oncternal: Research Funding; Roche: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria.

Development of a Novel Lymph Node-Based 3D Culture System Promoting Chronic Lymphocytic Leukemia Proliferation and Survival

INTRODUCTION. Primary chronic lymphocytic leukemia (CLL) cells, despite originating from a proliferative disease, rapidly undergo apoptosis in vitro in absence of microenvironmental survival signals1. Although co-culture with stromal cells or the addition of soluble factors can increase and extend CLL survival, no system permits the long-term expansion of CLL cells in vitro2. The difficulties of mimicking a physiologic microenvironment supporting CLL cells hinder in vitro studies of proliferation, drug screens and prevent propagation of rare subclones. For other cancers, various types of 3D cultures have been introduced utilizing scaffolds, gels, spheroid cultures and fluidic systems, representing a more accurate representation of the in vivo microenvironment3. Unlike solid tumors, secondary lymphoid tissues where CLL cells proliferate in vivo, do not derive from a single stem cell progenitor. Developing an appropriate 3D in vitro culture system for CLL is of obvious importance and may contribute pathophysiological relevance to study long-term CLL proliferation and more accurate drug screening4,5. Within the field of CLL, attempts have focused on bone marrow stroma, but it may be biologically and clinically more relevant to investigate the lymph node niche as this is the critical site of CLL proliferation6. METHODS. Primary CLL cells were cultured in various 3D systems including hydrogels, hanging drop cultures and ultra-low attachment plates (ULA) plates in parallel to an optimal 2D system, consisting of the culture of primary CLL cells on a monolayer of CD40L-presenting fibroblasts (3T40) or 3T3 negative control fibroblasts. CLL cells were either cultured as PBMCs alone, with or without T cells, or co-cultured with 3T40 or primary lymph node fibroblasts. CLL cells were either stimulated directly with IL-2, IL-15, IL-21 and CpG and/or indirectly via a T cell stimulation of anti-CD3/CD28. RESULTS. After testing and comparing multiple systems for the in vitro culture of CLL cells, we optimized a novel CLL culture system utilizing ULA plates creating spheroids of PBMCs isolated from peripheral blood. Without the addition of soluble factors or stroma, primary CLL cells in the ULA 3D model could be maintained in culture for 6 weeks as opposed to 1 week in the 2D system. Aside from significantly promoting CLL survival, cultures could be expanded approximately 3-4-fold over a course of 6 weeks using the ULA 3D model. 3D cultures showed a more consistent and significantly increased CLL proliferation compared to 2D cultures, independent of IGHV mutation status, increasing the average proliferation index of 2.87 to 3.90 (n=10). Additionally, co-culture with LN-derived stromal cells further increased CLL proliferation, reaching a maximum of 8 generations (n=6) (Figure 1). Lastly, when PBMCs were stimulated with IL-2, IL-15, IL-21 and CpG, spheroids developed proliferation center-like structures after 4 weeks of culture. CONCLUSIONS. We established a lymph node-based 3D in vitro culture system for CLL leading to increased CLL proliferation and survival compared to 2D systems. The set-up allows long-term expansion of CLL cells in vitro, as well as formation of proliferation center-like structures. We are currently optimizing drug resistance studies, expansion of specific CLL subclones and performing competition experiments.

Relation of Neutrophil Gelatinase-Associated Lipocalin Overexpression to the Resistance to Apoptosis of Tumor B Cells in Chronic Lymphocytic Leukemia.

The resistance to apoptosis of chronic lymphocytic leukemia (CLL) cells partly results from the deregulated production of survival signals from leukemic cells. Despite the development of new therapies in CLL, drug resistance and disease relapse still occur. Recently, neutrophil gelatinase-associated lipocalin (NGAL), a secreted glycoprotein, has been suggested to have a critical role in the biology of tumors. Thus, we investigated the relevance of NGAL in CLL pathogenesis, analyzed the expression of its cellular receptor (NGAL-R) on malignant B cells and tested whether CLL cells are resistant to apoptosis through an autocrine process involving NGAL and NGAL-R. We observed that NGAL concentrations were elevated in the serum of CLL patients at diagnosis. After treatment (and regardless of the therapeutic regimen), serum NGAL levels normalized in CLL patients in remission but not in relapsed patients. In parallel, NGAL and NGAL-R were upregulated in leukemic cells from untreated CLL patients when compared to normal peripheral blood mononuclear cells (PBMCs), and returned to basal levels in PBMCs from patients in remission. Cultured CLL cells released endogenous NGAL. Anti-NGAL-R antibodies enhanced NGAL-R+ leukemia cell death. Conversely, recombinant NGAL protected NGAL-R+ CLL cells against apoptosis by activating a STAT3/Mcl-1 signaling pathway. Our results suggest that NGAL and NGAL-R, overexpressed in untreated CLL, participate in the deregulation of the apoptotic machinery in CLL cells, and may be potential therapeutic clues for CLL treatment.

Application of CpG Oligodeoxynucleotide Immunostimulation in Chromosome Study of Chronic Lymphoblastic Leukemia

To explore the value of CpG-oligonucleotide(CpG-ODN) immunostimulatory method in chromosome culture of chronic lymphocytic leukemia (CLL) cells and to compare the differences between related studies at home and abroad, so as to improve the success rate of CLL karyotype culture and the detection rate of abnormal karyo-types. Bone marrow samples from 82 CLL patients were collected and cultured with phytohemagglutinin (PHA), CpG-oligonucleotide plus interleukin-2 (CpG-ODN DSP30+IL-2) for 72 hours. Chromosomes were prepared and analyzed by conventional cytogenetics (CC). Meanwhile, D13S25, Rb1, ATM, p53 and CSP12 probes were used for interphase fluorescence in situ hybridization (iFISH) test. The differences of chromosome culture and iFISH test results between two cell stimulants were compared. The success rate of karyotype culture in PHA and CpG-ODN DSP30+IL-2 immunostimuli (analyzable mitotic t >20) was 90.2% (74 cases), 68.3% (56 cases) respectively, and the detection rate of abnormal karyotype was 13.5% (10 cases) and 46.4% (26 cases), respectively. The success rate of karyotype culture in PHA group was significantly higher than that in CpG-ODN DSP30+IL-2 group (P=0.01). The detection rate of abnormal karyotypes in CpG-ODN DSP30+IL-2 group was significantly higher than that in PHA group, and the difference was statistically significant (P=0.003). The detection rate of abnormal karyotypes in iFISH group was 74.4% (61 cases), which was significantly higher than that in CpG-ODN DSP30+IL-2 group (P=0.000). iFISH detection could verify the abnormalities detected by CC analysis. Application of CpG-ODN DSP30+IL-2 immunostimulation method in culture of CLL cells can enhance the detection rate of abnormal karyotypes, especially the detection of various translocations suggesting poor prognosis.

PB1872 IS THERE A ROLE OF VITAMIN D IN THE PATHOPHYSIOLOGY OF CHRONIC LYMPHOCYTIC LEUKEMIA?

Background:Calcitriol, the biologically active form of vitamin D, binds to the vitamin D receptor (VDR) which is translocated into the nucleus and regulates the transcription of target genes. Epidemiological studies have reported that low blood levels of vitamin D are associated with adverse disease outcome in chronic lymphocytic leukemia (CLL). However, very limited biological explanation is available for this observation.Aims:In this study, we investigated the potential pathophysiological implication of the VDR system in CLL.Methods:CD19+ primary CLL cells were negatively selected. VDR transcripts were detected by RT‐qPCR, VDR+ and CYP24A1+ cells were determined by Flow Cytometry (FC). CLL cell apoptosis was determined using FC (AnnexinV−/Pi− cells). CLL cells were cultured in vitro with calcitriol, anti‐IgM, CpG, and/or CD40 ligand, or co‐cultured with the HS‐5 mesenchymal cell line.Results:VDR mRNA expression was assessed in 75 CLL cases, revealing significantly higher (FD = 3.2, p < 0.01) levels in patients with unmutated IGHV genes (U‐CLL, n = 43) compared to patients with mutated IGHV genes (M‐CLL, n = 32). Interestingly, however, CLL cases showed remarkably reduced VDR mRNA levels (FD = 3.1, p < 0.0001) compared to tonsillar CD19+ B cells from healthy donors (n = 24). In order to examine whether the microenvironment of CLL cells affects VDR expression, we cultured U‐CLL cells (n = 8) for 14 hours under the following conditions (a) BcR stimulation with anti‐IgM; (b) TLR9 stimulation with CpG and (c) CD40 stimulation with CD40L. These treatments resulted in a statistically significant reduction of VDR mRNA expression compared to the unstimulated control (FD = 1.7, p < 0.01; FD = 2.9, p < 0.01; FD = 2, p < 0.05; for BcR, TLR9, CD40 respectively). Following, in order to activate the calcitriol/VDR system, we cultured CLL cells with calcitriol as well as a combination of anti‐IgM/CD40L/calcitriol for 24 hours and measured the protein expression of CYP24A1, a target gene of the activated VDR. The addition of calcitriol was found to increase the percentage of CYP24A1+ CLL cells compared to unstimulated control cells in both U‐CLL (n = 9;FD = 5.1, p < 0.01) and M‐CLL (n = 7; FD = 2.4, p < 0.05). Co‐stimulation with anti‐IgM/CD40L/calcitriol also induced CYP24A1 expression in U‐CLL (FD = 4.4, p < 0.01) and M‐CLL (FD = 2.1, p < 0.05) but to a lesser extent. Moreover, co‐culturing of CLL cells with the HS‐5 cells induced CYP24A1+ cells, while calcitriol showed additive effects. In contrast, no differences in VDR+ cells were observed under the conditions described above.Summary/Conclusion:We show that VDR is expressed at low levels in CLL cells compared to normal B cells, which can be further reduced by microenvironmental signals, indicating that the tumor microenvironment competes with VDR expression. However, calcitriol supplementation activates the VDR system suggesting that VDR is functional in CLL cells and can potentially induce the expression of target genes with antitumor activity.

NOTCH1 Stabilization By PEST Mutations Enhances IgM-Mediated Activity in Chronic Lymphocytic Leukemia

BACKGROUND. Mutations in NOTCH1 PEST domain (NOTCH1-M) are present in ~10% of Chronic Lymphocytic Leukemia (CLL) patients, result in accumulation of more stable NOTCH1 protein, and associate with poorer prognosis. NOTCH1-M are enriched in unmutated (U) immunoglobulin gene heavy-chain variable region (IGHV) CLL, which show high surface IgM (sIgM) expression and signaling capacity. mRNA translation is a prominent response to B cell receptor (BCR) engagement, increased in U-CLL, and for which therapeutic inhibitors are under active development. In CLL, c-MYC is an essential mediator of BCR-driven translation and direct target of NOTCH1, suggesting the impact of NOTCH1 on anti-IgM-mediated cell growth via MYC.AIMS AND METHODS. Our aim was to investigate the functional role of NOTCH1-M on anti-IgM-mediated signaling, compared to wild-type (WT) NOTCH1. The impact on global mRNA translation was studied using a flow cytometry-based O-propargyl-puromycin (OPP) incorporation assay and polysome fractionation assays. The effects of stabilized vs WT NOTCH1 were measured after 24-hour cultures of CLL cells, when data demonstrate differences in the expression of the two forms. Two cohorts of U-CLLs were compared: i) a subset of samples carrying NOTCH1-M [variant allele frequency (VAF) ≥30%, n=21] and ii) a cohort of samples with WT NOTCH1 (VAF<1%, n=23). In both subsets no additional cytogenetic lesions other than 13q deletion were present.RESULTS. sIgM levels and signaling capacity (measured by anti-IgM mediated iCa2+ mobilization) were higher in NOTCH1-M than in -WT samples, consistent with previous observations (1). Conceivably, anti-IgM-mediated phosphorylation of PLCg2 and ERK1/2 was stronger in M than in WT CLLs. In keeping with these results, expression of downstream targets as MYC and CCL3 was also induced at higher levels in M samples. Interestingly, inhibition of NOTCH1 with g-secretase inhibitor (DAPT) significantly decreased BCR target genes induction in M cells, reducing the differences with WT samples, and further enhanced the effects of ibrutinib when used in combination. In order to investigate the impact of NOTCH1 on IgM-mediated CLL cell growth, anti-IgM-induced global mRNA translation was compared in the two cohorts. Consistent with the higher MYC mRNA and protein levels, anti-IgM led to higher global mRNA translation in NOTCH1-M than in -WT cells. DAPT inhibited it in both CLL subsets, while ibrutinib led to complete inhibition of mRNA translation only in the -WT subset, suggesting a major contribution of NOTCH1 to the process. Consistently, the combination of DAPT+ibrutinib abrogated the difference between M and WT CLL cells.Importantly, MYC (but not translation initiation factors eIF4G, eIF4A or eIF3b) was already induced at 6 hours following anti-IgM stimulation and was maintained at high levels at 24 hours, while up-regulation of eIF4G, eIF4A and eIF3b was evident only at 24 hours, supporting the hypothesis of a direct MYC-dependent regulation of the translation machinery (2).NOTCH1 itself was post-transcriptionally regulated upon BCR ligation, as we observed increased NOTCH1 mRNA in polysome-enriched actively translated fractions and increased protein levels on the surface of anti-IgM stimulated cells, specifically inhibited by ibrutinib. Consequently, NOTCH1 pathway was significantly more activated upon anti-IgM stimulation in M than WT cells, as determined by qPCR of NOTCH1 target genes. Both Ibrutinib and DAPT significantly prevented NOTCH1 activation upon BCR triggering, with the drug combination being the most effective treatment. Moreover, in line with data showing NOTCH1-dependent regulation of a B cell gene signature, expression of BTK, LYN and BLNK was significantly increased in anti-IgM activated NOTCH1-M samples, an effect prevented by DAPT.CONCLUSIONS. These data indicate that NOTCH1 stabilization associates with stronger IgM signaling capacity and suggest an interplay between BCR and NOTCH1 pathway, with the former promoting NOTCH1 expression and activation. The evidence that NOTCH1 pathway inhibition reverts this difference suggests a direct effect of NOTCH1 on IgM signaling. In this scenario, stabilizing NOTCH1 mutations may enhance BCR signaling by boosting translation through MYC induction and by directly regulating expression of BCR cascade elements.NOTES. SD and FF share senior authorshipD'Avola, Blood 2016Ruggero, Cancer Res 2009 DisclosuresCoscia:Abbvie, Gilead, Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen, Karyopharm: Research Funding. Gaidano:Janssen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Morphosys: Honoraria; Amgen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Roche: Consultancy, Honoraria. Allan:Genentech: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Sunesis: Membership on an entity's Board of Directors or advisory committees; Acerta: Consultancy; Verastem: Membership on an entity's Board of Directors or advisory committees. Furman:Gilead: Consultancy; AbbVie: Consultancy; Verastem: Consultancy; Janssen: Consultancy; Genentech: Consultancy; Incyte: Consultancy, Other: DSMB; Loxo Oncology: Consultancy; TG Therapeutics: Consultancy; Sunesis: Consultancy; Acerta: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy. Packham:Aquinox: Research Funding. Deaglio:iTeos therapeutics: Research Funding; VelosBio inc: Research Funding; Verastem: Research Funding. Forconi:Abbvie: Consultancy; Janssen-Cilag: Consultancy.

In Vitro Co-Culture of CLL-B Cells Reveals Long-Term Survival, Proliferation, and Maintenance of Telomere Length

Telomere length is a prognostic factor in Chronic Lymphocytic Leukemia (CLL) with short telomere length a powerful predictor of early time to first treatment and reduced overall survival. However, little is known about telomere dynamics through the course of an individual patient's disease. Our recent longitudinal analysis of CLL B-cell telomere length revealed very little dynamic change within individual patients with a mean erosion rate of -52bp/year (p=0.05). In marked contrast, T-cells derived from the same patients showed a significantly higher mean erosion rate of -119bp/year (p=0.02) with a median follow up time of 69 months. Here we present data derived from long-term in-vitro co-culture of peripheral blood from CLL patients coupled with temporal analysis of their telomere length dynamics.We utilized a multi-cellular co-culture system, comprised of autologous T-cells and CD40L-expressing mouse fibroblasts, to maintain CLL cells in long-term culture. Patient-derived peripheral blood mononuclear cells (n=16) were maintained for a median of 70 days (range 54-154); samples were analyzed every two weeks for tumor cell telomere length and evidence of proliferation. We used fluorescence-activated cell sorting (FACS) to sort populations of CD19+CD5+ CLL B-cells and CD3+ T-cells from each of the cultures. We then performed high-resolution single telomere length analysis (STELA) on these sorted subsets of cells and analyzed their telomere dynamics over this extended time course. Analysis of CLL B-cells from these cultures revealed significantly increased Ki-67+ at day 14 when compared to day 0 (p<0.001) and this was evident for the duration of the cultures. Despite sustained tumor cell proliferation, we observed no significant difference in the CLL B-cell telomere length with a mean TL at the start of 4.5kb vs 4.3kb at the end (p=0.14).The presence of T-cells was shown to be critical for the maintenance of the long-term cultures in two ways. Firstly, cultures that were treated with 4μM fludarabine showed a catastrophic reduction in T-cells (p=0.01), which was associated with a significantly shorter duration of survival of CLL B-cells when compared to untreated controls (median 17.5 days (range 7-70); p<0.001). Secondly, it proved impossible to maintain T-cell depleted, purified CLL B-cells, in long-term culture. T-cells isolated from the long-term cultures showed evidence of proliferation with Ki-67+ again being increased at day 14 in comparison to baseline (p=0.003). Furthermore, T-cells derived from these cultures showed a significant alteration in subset composition over time with a decrease in the numbers of naive CD4+ (p=0.05) and CD8+ (p=0.02) T-cells and a corresponding increase in effector memory (p=0.2) and terminally differentiated effector memory (EMRA) subsets (p=0.07).In conclusion, this study demonstrates that we have developed a robust, long-term culture method for the maintenance of CLL cells. Despite evidence of sustained CLL proliferation, CLL B-cells showed little telomere length erosion during long-term co-culture and this is compatible with our recent ex-vivo analysis, which showed that the telomere length of CLL B-cells are remarkably stable with a mean erosion rate of only -52bp/year. In both ex-vivo and in-vitro analysis, telomere erosion correlated with starting telomere length (r2=0.14, p=0.04 and r2=0.3 p=0.03 respectively). Taken together, our in-vitro and ex-vivo data imply that the radically short telomeres observed in some CLL patients are not the result of increased proliferation of the malignant B-cell, but rather the mutagenic event occurs in a B-cell which already has short telomeres. Furthermore, our novel long-term culture model has reinforced the vital role of T-cells in sustaining CLL B-cells viability and proliferation in-vitro. Given the consistent skewing of the T-cell pool towards a memory phenotype it seems unlikely that this is driven in-vitro by cognate TCR antigen recognition but rather a cytokine-mediated response. DisclosuresFegan:Gilead Sciences: Honoraria; Roche: Honoraria; AbbVie: Honoraria.

Global and MYC-Specific Translation Is Enhanced in Activated Chronic Lymphocytic Leukemia Cells Carrying NOTCH1 C.7541_7542delct Mutations

Introduction: mRNA translation is increased in activated tumor cells of the aggressive form of Chronic Lymphocytic Leukemia (CLL), typically unmutated (U) immunoglobulin gene heavy-chain variable region (IGHV) with a strong sIgM signaling capacity (Yeomans et al, Blood 2016). C-MYC protein is a master regulator of cell performance and its expression is controlled at both transcriptional and translational levels. C-MYC protein is over-expressed in the proliferation centers of CLL and high c-MYC mRNA expression is associated with poor prognosis. In leukemic cell lines, c-MYC is an essential mediator and direct target of NOTCH1. Pro-activating c.7541_7542delCTmutations in NOTCH1 PEST domain of chromosome 9 exon 34 (NOTCH1ΔCT) are enriched in U-CLL with high sIgM levels/signaling capacity and associate with poorer prognosis in CLL (D’Avola et al, Blood, 2016), likely due to accumulation of more stable NOTCH1 protein and enhanced signaling in tissue activated CLL cells (Arruga et al, Leukemia, 2014). Aims and Methods: We investigated the consequences of NOTCH1ΔCT on global mRNA and c-MYC translation using a novel flow cytometry-based O-propargyl-puromycin (OPP) incorporation assay (’Click-iT’ assay) and by c-MYC-specific immunoblotting in U-CLL. Since prolonged culture of CLL cells in vitro in the absence of stimuli led to spontaneous inactivation of NOTCH1 pathway, CpG-mediated TLR9 induction was used as a tool for activation of CLL cells in vitro. Cycloheximide (CHX) was used as a negative control for mRNA translation. For this study, 2 cohorts were investigated: i) a test "CLLΔCT cohort" of U-CLL with NOTCH1ΔCT (variant allele frequency [VAF] by droplet digital PCR, range 42.6-48.9%, median 47% of the CD19+CD5+ CLL cell population), but no additional genetic lesion other than 13q deletion, and ii) a control "CLLWT cohort" of U-CLL with no NOTCH1ΔCT (VAF<1% in all cases) or additional genetic lesion other than 13q deletion. CLL cells were incubated with 7.5 μg/ml CpG-ODN 2006 for 24 hours and assays were performed at baseline, 3 and 24 hours. NOTCH1 pathway γ-secretase inhibition was performed with DAPT GSi. Results: The CLLΔCT cohort had higher sIgM levels (range 31-372 MFI, median 81 MFI) and signaling capacity (Fab’2 anti-IgM induced intracellular calcium mobilization sIgM [iCa2+] range 47-54%, median 51) than the CLLWT cohort (sIgM levels range 19-288 MFI, median 47 MFI; IgM iCa2+ range 2-78%, median 25%). Following TLR9-mediated cell activation, the CLLΔCT cohort had sustained NICD (NOTCH1-intracellular cleaved domain) protein accumulation for up to 24 hours and expressed higher NOTCH1 target gene HES1 (hairy enhancer of split) transcript levels than in the CLLWT cohort. These data indicated NOTCH1 canonical pathway sustainment in the CLLΔCT upon activation. Global mRNA translation after 24 hours in the presence of CpG was 11.5 fold higher than that without CpG in the CLLΔCT cohort and only 4 fold higher in the CLLWT cohort, revealing significantly higher levels of translation in CLLΔCT than in CLLWT (p=0.03). CpG-induced global mRNA translation in the CLLWT cohort was similar to that in the CLLΔCT cohort treated with CHX. By using CpG-induced global mRNA translation in the presence of CHX inhibitor as background levels for each group, DAPT GSiat 2.5 to 10 μM showed from 47% to 63% inhibition of the residual CpG-induced global translation in CLLΔCT (p<0.05), but no effect in CLLWT. Remarkably, c-MYC mRNA translation after 3 hour culture with CpG was higher in CLLΔCT than in CLLWT (p= 0.02), and a similar trend was maintained in the cases investigated at 24 hour. Treatment of CLLΔCT cells with DAPT GSi decreased expression of c-MYC in a dose-dependent manner. Conclusion: NOTCH1ΔCT mutations associate with a very aggressive clinical behavior in CLL. These results now indicate that pro-activating mutations of NOTCH1 pathway associate with increased global mRNA translation and c-MYC expression. They highlight a mechanism by which NOTCH1 pathway may induce c-MYC overexpression in CLL, likely leading to increased proliferation and survival. The association of increased NOTCH1 variant allele frequency with sIgM levels and signaling capacity indicate that these mechanisms are predominant in the less anergic subgroup of U-CLL and make NOTCH1 mediated c-MYC translation an attractive target for therapeutic inhibition. DisclosuresSteele:Portola Pharmaceuticals: Honoraria. Packham:Karus Therapeutics: Other: Share Holder & Founder; Aquinox Pharmaceuticals: Research Funding.

The Syk\\Jak Inhibitor Cerdulatinib (PRT062070) Shows Promising Preclinical Activity in Chronic Lymphocytic Leukemia By Antagonising B Cell Receptor and Microenvironmental Signalling

The emergence of B cell receptor (BCR) kinase inhibitors has proved effective for the treatment of a number of B-cell malignancies including chronic lymphocytic leukemia (CLL). BTK and PI3K inhibitors have clear efficacy in suppressing tumor progression but have not been curative. A number of patients have developed resistance to these drugs following mutation of the BTK or PLCγ2 gene. Whilst, other patients are unable to tolerate these drugs due to adverse events or progress whilst on therapy for unknown reasons. Thus the development of novel drugs which are still effective once other BCR-kinases inhibitors become ineffective is of paramount importance. Spleen tyrosine kinase (Syk) is essential for B cell receptor signalling pathways as well as a variety of other surface receptors such as MHCII, FC receptors and integrins, all of which have been shown to play a role in CLL biology. Importantly, Syk inhibition has been shown to overcome resistance to ibrutinib, identifying Syk inhibition as a promising strategy to treat these patients. Furthermore, we have previously shown that IL-4 is found in CLL lymph nodes and can promote resistance to ibrutinib and idelalisib by restoring αIgM induced calcium flux and phosphorylated ERK (ASH 2014, abstract #3299). IL-4 signalling is mediated through the JAK/STAT signalling pathways via JAK1 and JAK3, therefore simultaneous inhibition of both Syk and JAK1/3 may be therapeutically beneficial over BCR kinase inhibitors alone. Cerdulatinib (PRT062070) is a dual JAK/Syk inhibitor in a phase I open label dose escalation study and is currently demonstrating clinical activity in patients with relapsed/refractory B cell malignancies including CLL. Our group has now demonstrated in vitro that cerdulatinib, at plasma concentrations achievable in patients, can induce apoptosis of CLL cells in a concentration and time dependent manner with a mean IC50 of 3µM and 1µM at 48 and 72h respectively, defined by annexin V/PI and cleavage of caspase 3 and poly ADP ribose polymerase (PARP). Apoptosis was caspase dependent since treatment with the pan caspase inhibitor ZVAD.fmk significantly inhibited cerdulatinib induced cell death at 24h. Cerdulatinib induced apoptosis coincided with an increase in pro-apoptotic proteins Noxa and Puma and a decrease in the anti-apoptotic protein Mcl-1. Cerdulatinib significantly inhibited IL-4 induced phosphorylation of STAT6 at 300nM (p=.005), BCR induced phosphorylation of AKTS473 with soluble (p=.008) and bead immobilised (BI) (p=.025) αIgM at 30nM and phosphorylation of AKTT308 with BI αIgM at 300nM (p=.008). Furthermore, in patients with CLL, it is thought that CD40L and IL-4 are key factors, which promote survival of CLL cells in proliferation centres within the lymph node microenvironment. Therefore, we cultured CLL cells with a vehicle control or IL-4\\CD40L, prior to treatment with cerdulatinib. Cerdulatinib alone induced similar levels of apoptosis irrespective of IL-4/CD40L treatment, suggesting cerdulatinib may be able to overcome microenvironmental signals and target cells within the lymph node. Next we explored the possibility of augmenting cerdulatinib induced apoptosis by simultaneous inhibition with the Bcl-2\\Bcl-XL inhibitor ABT-199. In vitro in the presence of IL-4/CD40L, ABT-199 synergised with cerdulatinib to induce significantly greater cell death than with either agent alone. Therefore these data provide in vitro evidence for the use of cerdulatinib in clinical trials for the treatment of CLL as either a single agent or in combination with other therapies such as ABT-199. DisclosuresStrefford:Roche: Research Funding. Davies:Seattle Genetics: Research Funding; Takeda: Honoraria. Coffey:Portola Pharmaceuticals Inc: Employment, Equity Ownership, Research Funding. Steele:Portola Pharmaceuticals: Other: Travel bursary to ASH 2015; Janssen: Other: Travel bursary to EHA 2015.

Adenosine Signaling Mediates Hypoxic Responses in the Chronic Lymphocytic Leukemia Microenvironment

Abstract Extracellular adenosine generated from ATP/ADP through the concerted action of the ectoenzymes CD39 and CD73 elicits potent cytoprotective and immunosuppressive effects mediated by type-1 purinergic receptors. Chronic lymphocytic leukemia (CLL) cells expressing the ectoenzymes CD39 and CD73 can actively produce adenosine, activating an autocrine adenosinergic axis that supports engraftment of leukemic cells in a growth-favorable environment. These effects are mediated by the A2A adenosine receptor, which inhibits chemotaxis and limits spontaneous and drug-induced apoptosis of CLL cells. Following the reported cross-talk between hypoxia and adenosine, we tested the hypothesis of a functional interplay between the adenosinergic axis and hypoxic signals in the CLL microenvironment. Results indicate that culture of CLL cells under hypoxic conditions, such as those observed in lymph nodes from CLL patients, boosts adenosine production, mainly because of the significant increase in the mRNA and protein levels of CD73, the rate-limiting enzyme in adenosine synthesis. CLL also underwent a robust up-regulation of CD26, which functions as an adenosine-deaminase scaffold protein, in keeping with the hypothesis that extracellular nucleotides enter a scavenging pathway, with conversion to inosine and re-uptake by the leukemic cells. Confirmation was obtained using HPLC assays, which showed increased inosine generation under hypoxia. Consistently, expression of membrane nucleoside transporters was also significantly up-regulated. However, hypoxic CLL cells also expressed high levels of the A2A adenosine receptor, which delivered cytoprotective signals and which supported CLL proliferation in response to TLR signaling. Attention was then focused on the stromal and T cell compartments, which are critical to the formation and maintenance of the leukemic niche. Hypoxia enhanced differentiation of circulating monocytes into nurse-like cells, macrophages of the M2 type playing an essential role in nurturing leukemic cells. The enhancement of NLC differentiation under hypoxic conditions relied, at least in part, on the activation of A2A: its engagement by a pharmacological agonist favored NLC generation, with overexpression of indoleamine 2,3-dioxygenase (IDO) and of the M2 macrophage markers CD163 and CD206. Moreover, activation of A2A induced secretion of immunomodulatory cytokines, such as IL-6, IL-10 and CCL18, while pharmacological blockade of A2A under hypoxia prevented NLC differentiation, expansion, expression of immunosuppressive molecules and secretion of cytokines and chemokines. In the T cell compartment, hypoxic cultures were followed by the sharp up-regulation of A2A, without significantly affecting the enzymes that generate adenosine, which were anyway restricted to the regulatory T cell (Treg) compartment. Co-cultures of T lymphocytes and CLL cells under hypoxia resulted in a dramatic decrease of T cell proliferation, partially rescued by A2A receptor antagonists. Furthermore, hypoxic T cells underwent a metabolic switch, with increased expression of nucleoside transporters and enzymes involved in glucose metabolism, suggesting a Warburg effect. This was accompanied by the differentiation of a population of Tr1 cells, characterized by the expression of LAG3 and CD49b and by the secretion of high levels of IL-10 and VEGF. Expression of the PD-1 immuno-inhibitory receptor was enhanced in hypoxic T cells, suggesting that multiple inhibitory mechanisms are activated. We also observed expansion of classical Tregs, defined on the basis of a CD4+/CD25high/CD127low/foxp3+ phenotype. Blockade of the A2A receptor prevented this phenotype, partially restoring T cell proliferation and immune competence. Together, these findings indicate that the adenosinergic and hypoxic axes synergize in shaping the CLL niche, suggesting that pharmacological inhibition of the adenosinergic signals may counteract some of the effects mediated by an hypoxic environment, contributing to disrupt the leukemic niche and to restore the immune system. Disclosures Gaidano: Celgene: Research Funding; MorphoSys; Roche; Novartis; GlaxoSmithKline; Amgen; Janssen; Karyopharm: Honoraria, Other: Advisory boards.

Dual Inhibition of PIM and PI3-Kinase By Ibl-202 Is Highly Synergistic Compared to Mono-Molecular Inhibition and Represents a Novel Treatment Strategy for Chronic Lymphocytic Leukemia

BACKGROUND Trials of ibrutinib and idelalisib for Chronic Lymphocytic Leukemia (CLL) targeting Btk and PI3-kinase respectively, illustrate the potential of targeting components of the B-cell receptor (BCR) signalling pathway. Emerging evidence suggests that subgroups of CLL patients develop resistance to these agents. In particular late relapses on ibrutinib appear to be associated with a high frequency of acquired mutations in Btk and PLCg2. Identification of novel combination therapies or agents that target multiple molecules in key signaling pathways represents a rational approach in the development of novel treatment strategies. Pim (provirus integration site for Moloney murine leukemia virus) family proteins are proto-oncogenic and involved in B-cell development and lymphoid malignancies. They are highly conserved serine/threonine kinases and are overexpressed in CLL. Given the clinical efficacy of idelalisib and results of preclinical studies of the PIM kinase SGI-1776 [Chen et al., 2009], we sought to investigate the potential of simultaneous inhibition of PIM and PI3-kinase for CLL therapy.METHODS The effects of the novel inhibitor of PIM kinase, pPIMi, and the dual inhibitor of PIM/PI3-kinase, IBL-202 (both from Inflection Bioscience Ltd, Ireland), were investigated against panels of primary CLL patient samples and haematological lines. The in vitro efficacy of both agents was compared to that of SGI-1776 and idelalisib and studied under conditions involving CLL cell co-culture using CD40L-expressing fibroblasts that mimic the support offered by the CLL tumor microenvironment.RESULTS Inhibition of PIM kinase by pPIMi was as effective as SGI-1776, inducing a dose dependent decrease in CLL-cell viability. The rationale for dual inhibition of PIM and PI3-kinase was investigated by combining pPIMi with idelalisib and by calculation of combination indices (CI), where CI values of < 1 are indicative of synergy. In primary CLL patient samples (Figure 1) and 3 B-cell lines (Mec1, Ramos and Raji) the combination proved highly synergistic, with CI values of 0.07 + /- 0.03 and 0.11 +/- 0.07 respectively, for fractional cell killing effects of between 0.1 and 0.9.Consistent with our assessment of synergy between pPIMi and idelalisib, dual inhibition of PIM and PI3-kinase by IBL-202 was significantly more effective at inducing cell death than either pPIMi or idelalisib as single agents (Figure 2), evidenced by the significantly lower IC50 against CLL patient samples (7.47 µM, 43.12 µM, 1.23 µM for pPIMi, idelalisib and IBL-202 respectively).The rationale for targeting PIM and PI3-kinase using IBL-202 was further highlighted by culturing CLL cells in the presence of a CD40L-expressing fibroblast monolayer, which represents a model of the tumor microenvironment. CD40L fibroblast co-culture significantly increased CLL-cell survival under control conditions but also conferred resistance to the PIM inhibitor pPIMi. In contrast CLL cells cultured under these conditions were sensitive to IBL-202 in a dose dependent manner.CONCLUSION Inhibition of PIM and PI3-kinase is potently synergistic against CLL cells and is particularly effective under conditions that mimic the tumor microenvironment. Dual targeting of PIM and PI3-kinase by IBL-202 may represent a novel therapeutic strategy for CLL that minimizes the risk of acquired mutations associated with mono-molecular inhibition of BCR-signaling components. [Display omitted] [Display omitted] DisclosuresO’Neill:Inflection Biosciences: Employment, Equity Ownership. O’Dwyer:Inflection Biosciences Ltd: Membership on an entity’s Board of Directors or advisory committees. Mulligan:Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi Aventis: Research Funding; Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria.

Inhibition of the Notch1 Pathway in Chronic Lymphocytic Leukemia Promotes Apoptosis and Inhibits Proliferation

In chronic lymphocytic leukemia (CLL), mutations in the NOTCH1 receptor occur in 4-10% of newly diagnosed patients and 15–20% of multiply relapsed patients. Using next-generation sequencing, our group previously reported NOTCH1 p.P2514fs mutations in 15 CLL patients (9.4%) in an initial cohort of 160 CLL patients in which NOTCH1 mutations were associated with IGHV unmutated (UM) CLL (p=0.0001). Further analysis using a three-group comparison (NOTCH1 mut, IGHV UM vs. NOTCH1 wild-type [wt] IGHV UM vs. NOTCH1 wt IGHV mut) showed that NOTCH1 mutations associated with both trisomy 12 (p=0.049) and 17p deletion (p=0.0008) and poor overall survival (HR 2.99, p=0.008).Given that targeting activating mutations has proven an effective therapeutic strategy in many cancers, we explored the therapeutic potential of a Notch1 inhibitor, PF-03084014, in CLL. Previous studies in T-cell acute lymphoblastic leukemia cells harboring NOTCH1 mutations have shown that gamma secretase inhibitors can induce apoptosis by blocking Notch1 receptor activation. When we tested the gamma secretase inhibitor (GSI) PF-03084014 in 18 CLL samples with NOTCH1 mutations, it consistently induced apoptosis of all CLLs after 48 hours in culture across all cytogenetic groups tested (13.3-47.2% death with 5 μM GSI, p<0.0001)(Figure A). The induction of apoptosis was similar (GSI vs. ibrutinib, p=ns) to that of ibrutinib (n=11,11.9-74.4% death with 5 μM ibrutinib, p<0.0001). In contrast, GSI treatment only induced apoptosis in some (n=10), but not all NOTCH1 wt CLLs (n=6) (p=0.0137). We next tested the effect of GSI PF-03084014 in the context of a stromal environment. Co culture of CLL cells with CD40L-expressing fibroblasts partially mimics the lymph node and bone marrow microenvironments, which are known sites of drug-resistance and proliferation of CLL in vivo. We tested whether the interaction with stromal cells protects CLL cells from Notch1 inhibitor-induced apoptosis, as seen with other drugs including ibrutinib. We found that co culture with CD40L-expressing 3T3s decreased GSI-induced apoptosis in NOTCH1 mutant CLLs (p=0.0006) and in the majority of the NOTCH1 wt CLLs that responded to the GSI (Figure A).Since NOTCH1 mutations have been reported to be an independent marker of aggressive disease in CLL, we tested whether CLL cells with NOTCH1 mutations were more proliferative in vitro compared to NOTCH1 wt CLL cells. We showed that CLL cells upregulate Ki67 expression in co culture with 3T3-CD40L cells and in a cohort of 10 NOTCH1 mutants and 11 NOTCH1 wt, we found the NOTCH1 mutants to be more proliferative than the NOTCH1 wt (median of 7.6% vs. 2.3%, p=0.015). To then address whether blocking of the Notch1 pathway decreases proliferation, we treated CLL cells in co culture with 3T3-CD40L cells with 5 μM GSI for 7 days. GSI treatment decreased the percentage of Ki67+ CLL cells in all but one NOTCH1 mutant (median decrease 28.3%, p=0.044) as wells as in the majority of NOTCH1 wt samples (median decrease 38.7%, p=0.037).Having established that inhibition of Notch1 can reduce proliferation and induce apoptosis in CLL cells in vitro, we were interested in determining the downstream genes that may be the effectors of this activity. We therefore compared the gene expression profiles (GEP) of NOTCH1 mut vs. NOTCH1 wt CLLs, and found upregulation of genes involved in the Notch1 pathway, in apoptosis and in chemokine signaling in the NOTCH1 mutants. Furthermore, comparing GEP of high Ki67 vs. low Ki67 expressing CLL cells revealed higher expression of a range of both upstream and downstream Notch1 pathway genes in high Ki67 expressing CLL cells.In conclusion, we show that PF-03084014 induces apoptosis and decreases proliferation in both NOTCH1 mutant and wt CLL cells. We find NOTCH1 mutant CLL cells to be more proliferative than NOTCH1 wt and show upregulation of Notch1 pathway genes in NOTCH1 mutants compared to wt CLL cells and in high Ki67 expressing compared to low Ki67 expressing CLL cells. Taken together, these results emphasize the important role of Notch1 signaling in CLL in general, perhaps particularly in proliferative compartments like lymph nodes, and demonstrate that Notch1 pathway inhibitors are worthy of therapeutic investigation in CLL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

MiR-26a and miR-214 down-regulate expression of the PTEN gene in chronic lymphocytic leukemia, but not PTEN mutation or promoter methylation.

We previous found the expression level of PTEN was low in the chronic lymphocytic leukemia (CLL) patients. To assess the pathogenic contribution of the low expression of PTEN, we determined PTEN-regulating miRNA interference, PTEN promoter methylation and PTEN gene mutation condition in CLL. One hundred and fifty-four previously untreated CLL patients and 200 cases of healthy controls were sequenced in exons 5-9 of PTEN. None of single nucleotide polymorphism site or mutation was detected in the coding sequences of those exons. Methylation of PTEN promoter was found in one (1.33%) of the 75 patients with CLL, but none of the 25 age-matched control subjects. We found that PTEN was a potential target of miR-26a and miR-214, which had been confirmed following dual-luciferase reporter assays, reverse transcription polymerase chain reaction and Western blotting. High expression of miR-26a was associated with advanced Binet stage (P=0.012), p53 aberrations (P=0.014) and inferior time to first treatment (P=0.038), and high expression of miR-214 was only associated with p53 aberrations (P=0.041). Inhibition of miR-26a or miR-214 could induce more apoptosis in primary cultured CLL cells. These findings support miR-26a and miR-214 down-regulate expression of PTEN in CLL, but not PTEN mutation or promoter methylation.

Matrix metalloproteinase-9 is involved in chronic lymphocytic leukemia cell response to fludarabine and arsenic trioxide.

BackgroundMatrix metalloproteinase-9 (MMP-9) contributes to chronic lymphocytic leukemia (CLL) pathology by regulating cell migration and preventing spontaneous apoptosis. It is not known if MMP-9 is involved in CLL cell response to chemotherapy and we address this in the present study, using arsenic trioxide (ATO) and fludarabine as examples of cytotoxic drugs.MethodsWe used primary cells from the peripheral blood of CLL patients and MEC-1 cells stably transfected with an empty vector or a vector containing MMP-9. The effect of ATO and fludarabine was determined by flow cytometry and by the MTT assay. Expression of mRNA was measured by RT-PCR and qPCR. Secreted and cell-bound MMP-9 was analyzed by gelatin zymography and flow cytometry, respectively. Protein expression was analyzed by Western blotting and immunoprecipitation. Statistical analyses were performed using the two-tailed Student's t-test.ResultsIn response to ATO or fludarabine, CLL cells transcriptionally upregulated MMP-9, preceding the onset of apoptosis. Upregulated MMP-9 primarily localized to the membrane of early apoptotic cells and blocking apoptosis with Z-VAD prevented MMP-9 upregulation, thus linking MMP-9 to the apoptotic process. Culturing CLL cells on MMP-9 or stromal cells induced drug resistance, which was overcome by anti-MMP-9 antibodies. Accordingly, MMP-9-MEC-1 transfectants showed higher viability upon drug treatment than Mock-MEC-1 cells, and this effect was blocked by silencing MMP-9 with specific siRNAs. Following drug exposure, expression of anti-apoptotic proteins (Mcl-1, Bcl-xL, Bcl-2) and the Mcl-1/Bim, Mcl-1/Noxa, Bcl-2/Bax ratios were higher in MMP-9-cells than in Mock-cells. Similar results were obtained upon culturing primary CLL cells on MMP-9.ConclusionsOur study describes for the first time that MMP-9 induces drug resistance by modulating proteins of the Bcl-2 family and upregulating the corresponding anti-apoptotic/pro-apoptotic ratios. This is a novel role for MMP-9 contributing to CLL progression. Targeting MMP-9 in combined therapies may thus improve CLL response to treatment.

Gossypol Increases Expression of the Pro-apoptotic BH3-only Protein NOXA through a Novel Mechanism Involving Phospholipase A2, Cytoplasmic Calcium, and Endoplasmic Reticulum Stress

Gossypol is a putative BH3 mimetic proposed to inhibit BCL2 and BCLXL based on cell-free assays. We demonstrated previously that gossypol failed to directly inhibit BCL2 in cells or induce apoptosis in chronic lymphocytic leukemia (CLL) cells or platelets, which require BCL2 or BCLXL, respectively, for survival. Here, we demonstrate that gossypol rapidly increased activity of phospholipase A2 (PLA2), which led to an increase in cytoplasmic calcium, endoplasmic reticulum (ER) stress, and up-regulation of the BH3-only protein NOXA. Pretreatment with the PLA2 inhibitor, aristolochic acid, abrogated the increase in calcium, ER stress, and NOXA. Calcium chelation also abrogated the gossypol-induced increase in calcium, ER stress, and NOXA, but not the increase in PLA2 activity, indicating that PLA2 is upstream of these events. In addition, incubating cells with the two products of PLA2 (lysophosphatidic acid and arachidonic acid) mimicked treatment with gossypol. NOXA is a pro-apoptotic protein that functions by binding the BCL2 family proteins MCL1 and BFL1. The BCL2 inhibitor ABT-199 is currently in clinical trials for CLL. Resistance to ABT-199 can occur from up-regulation of other BCL2 family proteins, and this resistance can be mimicked by culturing CLL cells on CD154(+) stroma cells. We report here that AT-101, a derivative of gossypol in clinical trials, overcomes stroma-mediated resistance to ABT-199 in primary CLL cells, suggesting that a combination of these drugs may be efficacious in the clinic.

Loss of cooperativity of secreted CD40L and increased dose-response to IL4 on CLL cell viability correlates with enhanced activation of NF-kB and STAT6.

Chronic lymphocytic leukemia (CLL) cells fail to enter apoptosis in vivo as opposed to their non-malignant B-lymphocyte counterparts. The ability of CLL cells to escape apoptosis is highly dependent on their microenvironment. Compared to non-malignant B cells, CLL cells are more responsive to complex stimuli that can be reproduced in vitro by the addition of cytokines. To understand the molecular mechanism of the environment-dependent anti-apoptotic signaling circuitry of CLL cells, we quantified the effect of the SDF-1, BAFF, APRIL, anti-IgM, interleukin-4 (IL4) and secreted CD40L (sCD40L) on the survival of in vitro cultured CLL cells and found IL4 and sCD40L to be most efficient in rescuing CLL cells from apoptosis. In quantitative dose-response experiments using cell survival as readout, the binding affinity of IL4 to its receptor was similar between malignant and non-malignant cells. However, the downstream signaling in terms of the amount of STAT6 and its degree of phosphorylation was highly stimulated in CLL cells. In contrast, the response to sCD40L showed a loss of cooperative binding in CLL cells but displayed a largely increased ligand binding affinity. Although a high-throughput microscopy analysis did not reveal a significant difference in the spatial CD40 receptor organization, the downstream signaling showed an enhanced activation of the NF-kB pathway in the malignant cells. Thus, we propose that the anti-apoptotic phenotype of CLL involves a sensitized response for IL4 dependent STAT6 phosphorylation, and an activation of NF-kB signaling due to an increased affinity of sCD40L to its receptor.

The Co-Culture Of Primary CLL Cells With Bone Marrow Mesenchymal Cells, CD40 Ligand and CpG ODN Promotes The Proliferation Of Chemoresistant CLL Cells Phenotypically Comparable To Those From The Proliferative Centers

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation and proliferation of monoclonal CD5+ mature B-cells in peripheral blood (PB), lymph nodes (LN), and bone marrow (BM). The microenvironment found in BM and LN, where CLL cells interact with different accessory cells, induces active proliferation of CLL cells and protects them from spontaneous and chemotherapy-induced apoptosis. This may explain why the BM and secondary lymphoid organs are preferential sites for minimal residual disease persistence and for relapses observed in all patients after treatment. Therefore, further characterization of primary CLL cells found in the proliferative niches can facilitate the discovery and study of new targets specifically expressed by this proliferative and resistant subset of primary CLL cells.In order to further characterize the proliferative subset of primary CLL cells we analyzed the characteristics of proliferating subclones of CLL cells found in PB and compared it to the phenotype of primary CLL cells co-cultured ex vivo in conditions mimicking the microenvironment of the proliferative niches. Firstly, we compared actively proliferating CLL cells from 40 patients diagnosed with CLL with their quiescent counterpart. For this, we analyzed by flow cytometry (FC) the differential expression of ZAP-70, CD38, and the chemokine receptors CXCR4, CXCR5 and CCR7 in Ki-67 positive vs. negative CLL cells. Ki-67 positive CLL cells had higher expression levels of ZAP-70 and CD38, while the expression levels of all the chemokine receptors analyzed were significantly lower (Figure 1). This phenotype indicates that these cells may have an increased capability to respond to different survival and migration signals provided by the cellular microenvironment, since high expression of ZAP-70 and CD38 has been related to increased response to this kind of signaling. Moreover, downregulation of chemokine receptors would indicate recent stimulation by chemoatracting cytokines. Furthermore, with the aim of ex vivo mimic the microenvironment found in the proliferative centers, we co-cultured primary CLL cells from 27 patients with the BM-derived stromal cell line UE6E7T-2, 1μg/mL soluble CD40L and 1.5μg/mL CpG ODN, and analyzed the effects in terms of proliferation, modulation of surface molecules and development of chemoresistance. Of note, already 24 hours after co-culture, CLL cells increased their size, resembling stimulated B cells, and tended to be located in sparse clusters that included dividing cells, as observed after immunocytochemistry studies. In this setting, proliferative responses assessed by Ki-67 expression analyzed by FC were already observed after 24 hours (mean % Ki-67 positive cells: 1.48±0.28 in co-culture vs. 0.56±0.11 in suspension, P<0.01) and further increased after 48 hours (mean % Ki-67 positive cells: 2.89±0.51 in co-culture vs. 0.25±0.10 in suspension, P<0.01) and 72 hours (mean % Ki-67 positive cells: 7.68±2.11 in co-culture vs. 0.81±0.24 in suspension, P<0.001). Next, we analyzed the expression levels of ZAP-70, CD38 and the chemokine receptors CXCR4, CXCR5 and CCR7. Interestingly, the modulation of the expression of these molecules in co-cultured CLL cells correlated with the previously mentioned differential expression observed between the Ki-67 positive vs. negative compartments, indicating that the ex vivo co-culture highly resembles the conditions found in proliferative niches (Figure 1). Finally, in order to assess the role of microenvironment in chemoresistance, CLL cells were cultured in suspension and in co-culture for 48 hours and subsequently treated with increasing doses of fludarabine for 24 hours. Interestingly, the co-culture of CLL cells inhibited at such extend the capacity of fludarabine to induce apoptosis that it was not possible to calculate its lethal dose 50, whereas lethal dose 50 for fludarabine in CLL cells in suspension was 416μM (95%CI 125.5-1379).In conclusion, these results suggest that culturing CLL cells in the previously described conditions promotes proliferation and the acquisition of a phenotype which bears many similarities to the one found in actively proliferating CLL cells circulating in PB. This study provides a model for a co-culture system which might serve as a basis for the development and testing of new drugs that target the proliferative and drug resistant CLL cell compartment. [Display omitted] Disclosures:No relevant conflicts of interest to declare.