Ibrutinib Sensitizes AML Cells to ROS Inducers Via a BTK-Independent Mechanism

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2226-2226 ◽  
Author(s):  
Lianne E Rotin ◽  
Marcela Gronda ◽  
Neil Maclean ◽  
Feng-Hsu Lin ◽  
Jeff Wrana ◽  
...  
Keyword(s):  
Cell Viability ◽  
B Cell ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Synergistic Effects ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Fold Increase ◽  
Synergistic Cytotoxicity ◽  
Btk Inhibitor

Abstract Ibrutinib is a small-molecule Bruton’s tyrosine kinase (BTK) inhibitor yielding impressive clinical responses in B-cell malignancies, where BTK contributes to disease development and progression. We noted that BTK is expressed and constitutively active in acute myeloid leukemia (AML) cell lines and in a subset of AML patients. We therefore sought to determine BTK’s potential role in AML. Using BTK-pY223 expression on immunoblot as a marker of BTK activity, we demonstrated that ibrutinib doses as low as 1μM were sufficient to inhibit BTK activity in the AML cells TEX and OCI-AML2. Yet, these cell lines were insensitive to ibrutinib compared to the B-cell lymphoma Daudi cell line (IC50 10.4μM and 23.7μM versus 1.1μM, respectively). Although inactive as a single agent, we sought to identify drug combinations that sensitized AML cells to pharmacologically relevant concentrations of ibrutinib by conducting a combination chemical screen with our in-house known drug library in TEX and OCI-AML2 cells. According to excess-over-Bliss additivism criteria, we determined that in both cell lines, the poly(ADP-ribose) glycohydrolase inhibitor ethacridine lactate was the most synergistically cytotoxic, producing an 8-fold reduction in the IC50 of ibrutinib. Synergistic cytotoxicity was also observed in a subset of primary AML cells, but not normal hematopoietic cells. Mechanistically, the combination’s synergistic cytotoxicity resulted from excessive ROS production (>10-fold increase, versus a <2-fold increase with either drug alone in TEX and OCI-AML2), since α-tocopherol addition to combination-treated cells rescued cell viability from 24% to 85% and from 4% to 84% in TEX and OCI-AML2, respectively. Combining ibrutinib with other ROS-inducing agents such as parthenolide and the first-line AML therapy daunorubicin produced similar synergistic effects in AML cells,with α-tocopherol also rescuing cell viability. However, the synergistic effects were likely not related to inhibition of BTK, as knockdown of BTK with shRNA did not sensitize TEX cells to ethacridine or daunorubicin and thus did not recapitulate the effects of ibrutinib. We therefore conclude that the BTK inhibitor ibrutinib lacks single agent anti-AML activity, but synergizes with ROS-inducing agents including daunorubicin by inhibiting targets beyond BTK. Thus, clinical trials of ibrutinib in combination with standard chemotherapy could be warranted in AML. Disclosures No relevant conflicts of interest to declare.

Differential Role of the B-Cell Receptor Pathway in Diffuse Large Cell B Cell Lymphoma: Temsirolimus Has Additive Effects in Combination with the BTK Inhibitor PCI-32765 and PI3K Inhibitor Cal101 but Antagonizes Bortezomib in GCB Subtype

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1664-1664 ◽  
Author(s):  
Anna-Katharina Zoellner ◽  
Stephan Bayerl ◽  
Marc Weinkauf ◽  
Georg Hess ◽  
Wolfgang Hiddemann ◽  
...  
Keyword(s):  
Cell Growth ◽  
B Cell ◽  
Cell Lines ◽  
Synergistic Effects ◽  
B Cell Lymphoma ◽  
Large Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Additive Effects ◽  
Btk Inhibitor

Abstract Abstract 1664 Background: Diffuse large cell B cell lymphoma (DLBCL) is the most common malignant lymphoma, with the molecular subtypes activated b cell-like (ABC) and germinal center b cell-like (GCB) lymphoma showing distinct biological and clinical characteristics. The B-cell receptor signal pathway including the kinases BTK and PI3K, both upstream of mTOR, play an important role in the pathogenesis of DLBCL. In addition, our previous analyses showed that the proteasome inhibitor Bortezomib also interacts with this pathway. Methods: Established GCB (DB, WILL-2, SU-DHL-4, SU-DHL-5, HT, ULA) and ABC (U2932, HBL-1) cell lines were cultivated under standard cell culture conditions. In all cell lines dose finding experiments with various concentrations of temsirolimus, BTK inhibitor PCI-32765, PI3K delta inhibitor Cal101 and bortezomib were performed in triplicates. Viable cell lines were counted with an automated cell viability analyzer using trypan blue exclusion test after 0, 24, 48 and 72 hours. Results: Interestingly, all cell lines showed significantly reduced cell growth (in comparison to untreated cell lines) after treatment with temsirolimus (10nM) 72 hours after treatment [53,1%, range 44,4%-69.4%]. In contrast, PCI-32765 (2,5nM) significantly reduced cell growth only in the ABC cell lines HBL-1 [50,2% ± 6,6%] and U2932 [69,7% ± 7,3%] but none of the GCB cell lines. Finally, Cal101 5μM reduced cell growth in both ABC cell lines [U2932 63,1% ± 9,6%; HBL-1 63,2% ± 6,6%] whereas heterogeneous growth reduction was detected in GCB cell lines [ULA 45,6% ± 3,9%; HT 51,9% ± 6,1%; SU-DHL-5 35, 6% ± 3,0%; Will-2 76,5% ±9,3%; DB 74,8% ± 8,7%]. Nonetheless, combination of temsirolimus with PCI-32765 showed synergistic effects in the GCB cell lines DHL-5 [27,2% ± 3,4%] and Will-2[53,4% ± 2,5%] and additive effects in ABC cell lines [HBL-1 28,8% ± 2,4%; U2932 24,9% ± 1,6%]. Combination of temsirolimus with Cal101 also showed additional effects in both, ABC and GCB cell lines. Furthermore the combination of PCI-32765 with Cal101 showed synergistic effects in the GCB cell lines SUDHL-5 [14,0% ± 7,1% and Will-2 [57,8% ± 3,0%] and additive effects in ABC cell lines [HBL-1 28,9% ± 4,7%; U2932 36,6% ± 11,2%]. Cell cycle analysis detected a BTK and PI3K induced G1 phase arrest. In contrast, temsirolimus [T] combined with bortezomib [B] induced antagonistic effects in the GCB cell lines ULA [TB 62,6% ± 0,7%, T 58,5% ±,9%, B 53,9% ± 1,1%] and SU-DHL-5 [TB 51,9% ± 2,67%, T 53,4% ± 1,7%, B 57,1% ± 0,5%]. Interestingly, in the ABC cell lines HBL-1 [TB 37,4% ± 2,8%,T 52,3% ± 2,8%, B 62,7% ± 4,0%] and U2932 [TB 29,3% ± 3,6%,T 61,8% ± 1,8%, B 43,3% ± 3,7%] additive effects were seen. Furthermore, combination of bortezomib with Cal101 [C] showed antagonistic effects in U2932 [BC 95,8% ± 4,1%], ULA [BC 42,8% ± 7,7%] and SU-DHL5 [BC 47,9% ± 4,0%]. In contrast, combination of bortezomib with PCI-32765 additively impaired cell growth in HBL-1, ULA, SU-DHL5 and DB cell lines. Conclusions: ABC-DLBCL and GCB-DLBCL show significantly reduced cell growth after treatment with the mTOR inhibitor temsirolimus. In consistency with reported data (Davis 2010) GCB cell lines were not sensitive towards PCI-32765. However, combinations with temsirolimus showed additional or synergistic effects also in GCB cell lines. In contrast, combination of bortezomib with temsirolimus or CAL101 was antagonistic in some GCB cell lines. Different genetic mutations involving CD79b and regulators of NF-kB which have been identified in the majority of ABC and some GCB cell lines (HBL-1, U2923 and SUDHL-5) may be responsible for the heterogeneous response towards inhibitors of the B-cell receptor pathway. Disclosures: Hess: Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Dreyling:Pfizer: Research Funding, Speakers Bureau, scientific advisory.

scholarly journals GSK-3β inhibitor, 9-ING-41, reduces cell viability and halts proliferation of B-cell lymphoma cell lines as a single agent and in combination with novel agents

Oncotarget ◽  
2017 ◽  
Vol 8 (70) ◽  
pp. 114924-114934 ◽  
Author(s):  
Reem Karmali ◽  
Vineela Chukkapalli ◽  
Leo I. Gordon ◽  
Jeffrey A. Borgia ◽  
Andrey Ugolkov ◽  
...  
Keyword(s):  
Cell Viability ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Lymphoma Cell ◽  
Novel Agents ◽  
Gsk 3Β

scholarly journals Evaluation of Idelalisib with B-Cell Receptor or Orthogonal Pathway Inhibitors in Diffuse Large B-Cell Lymphoma Cell Lines in Vitro and In Vivo

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1845-1845 ◽  
Author(s):  
Sarah Meadows ◽  
Anella Yahiaoui ◽  
Rick Sorensen ◽  
Zhi-Hua Cui ◽  
Robert Brockett ◽  
...  
Keyword(s):  
Cell Viability ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Tumor Regression ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
Bet Inhibitor ◽  
Large B Cell Lymphoma

Abstract Background: Idelalisib, a selective oral inhibitor of PI3Kd, is approved for the treatment of chronic lymphocytic leukemia (CLL) in combination with rituximab and as monotherapy for patients with follicular lymphoma who have received at least 2 prior therapies. Despite remarkable clinical efficacy, complete responses are rare, highlighting the need to identify more effective therapies, including combinations of novel agents. GS-4059 (ONO-4059) is an investigational next generation Bruton's tyrosine kinase (BTK) inhibitor with improved selectivity compared with ibrutinib. We report here the results of the combination of a PI3Kd inhibitor and GS-4059 in a diffuse large B-cell lymphoma (DLBCL) xenograft model, demonstrating supportive data for our ongoing combination trial in B-cell malignancies (NCT02457598). Additionally, we investigated preclinical orthogonal combination approaches for DLBCL. Methods: Growth inhibition was assessed using CellTiter-Glo Assay after 96 h incubation with idelalisib and GS-4059. CB17-SCID mice were irradiated, implanted subcutaneously with TMD8, and treated BID PO with the PI3Kd inhibitor GS-649443, GS-4059, or coformulated combination when tumors reached 200 mm3. Lysates from tumors or cell cultures were analyzed by Simple Western (Protein Simple). Synergy for antiproliferative effects was assessed using Chalice software (Horizon Discovery, Inc., Lehar et al., Nature Biotech, 2009). Results: Idelalisib and GS-4059 potently inhibited the ABC subtype DLBCL cell line TMD8, which is a B-cell receptor (BCR)-dependent line that exhibits chronic activated B-cell signaling due to mutations in CD79A/CD79B and MYD88 (Kim Y. et al., Hum Pathol, 2014). When a clinically relevant single concentration of idelalisib or GS-4059 was added in combination to a dose responsive effect of the other, a shift in EC50 on cell viability was seen. GS-4059 (50 nM) shifted the EC50 of idelalisib from 141 nM to 5 nM, a 28-fold shift. Idelalisib (1 µM) shifted the EC50 of GS-4059 from 27 nM to 2 nM, a 14-fold shift. Evaluation of downstream signaling pathways implicated in malignant B-cell survival and proliferation showed enhanced inhibition of pAkt S437, pBTK Y223, pErk1/2 T202/Y204, and MYC with a combination of idelalisib and GS-4059, more than either single agent alone. When TMD8 xenografts were treated with a PI3Kd tool compound, GS-649443, GS-4059 or a combination of the 2 inhibitors, a statistically significant decrease in tumor volume was seen as well as tumor regression, when compared with single agent effects (Figure 1A). Evaluation of TMD8 tumor lysates showed strong suppression of pAkt S437, pBTK Y223, pS6RP S235/236, and MYC in tumors treated with both GS-649443 and GS-4059 (Figure 1B). pS6RP S235/236 and MYC, in formalin-fixed paraffin-embedded (FFPE) TMD8 tumors, were profoundly inhibited in tumors treated with combination therapy compared to the monotherapies (Figure 1C). Since the combination of a PI3Kd inhibitor and GS-4059 led to TMD8 tumor regression, an effect correlated to strong down-modulation of MYC, the combination of idelalisib with a bromodomain and extra-terminal (BET) family inhibitor was explored as a potential new orthogonal combination approach for DLBCL. A panel of DLBCL cell lines was evaluated for inhibition of cell viability by idelalisib in combination with GS-5829, a BET inhibitor currently being evaluated in a phase 1 clinical trial. At clinically relevant concentrations, the combination of idelalisib and GS-5829 showed synergistic effects on cell viability in 2 of 6 ABC subtype, 4 of 5 GCB subtype, and 2 of 2 double-hit DLBCL cell. As compared with combination with other agents that inhibit the BCR pathway (GS-4059) or the Bcl-2 pathway (ABT-199), the broadest activity across cell lines was seen with the combination of idelalisib and GS-5829. Conclusion: Idelalisib and GS-4059 demonstrated synergistic inhibition of the TMD8 xenograft with concomitant inhibition of MYC. Screening of other targeted agent combinations in a panel of DLBCL lines revealed broad preclinical activity for the BET inhibitor GS-5829 in combination with idelalisib. This represents a potential orthogonal approach for a new therapeutic strategy for the treatment of B-cell malignancies. Figure 1A Figure 1A. Figure 1B Figure 1B. Figure 1C Figure 1C. Disclosures Meadows: Gilead Sciences: Employment. Yahiaoui:Gilead Sciences: Employment. Sorensen:Gilead Sciences: Employment. Cui:Gilead Sciences: Employment. Brockett:Gilead Sciences: Employment. Keegan:Gilead Sciences: Employment. Tannheimer:Gilead Sciences: Employment.

Birinapant Enhances Bendamustine-Induced Apoptosis In Activated B Cell-Diffuse Large Cell Lymphoma Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5150-5150
Author(s):  
Indira D Joshi ◽  
Mitchell R Smith
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Single Agent ◽  
Annexin V ◽  
B Cell Lymphoma ◽  
Lymphoma Cell ◽  
Low Level ◽  
Induced Apoptosis

Abstract Birinapant (TL32711), a Smac mimetic in clinical testing, potently targets Inhibitor of Apoptosis Proteins (IAPs, including cIAPs and XIAP) to unblock intrinsic and extrinsic pathways, enabling caspase-dependent apoptosis via multiple signals. Birinapant also inactivates canonical NF-kB signaling through cIAPs. We investigated the pro-apoptotic effects of birinapant, alone and in combination with bendamustine (BDM), an active lymphoma therapeutic agent, in a panel of B cell lymphoma cell lines representing germinal center/follicular (GC) vs. activated B cell (ABC) subtypes. We hypothesized that the efficacy of this potential combination therapeutic strategy might differ between GC and ABC lymphoma types, as ABC are reported to be NF-kB-dependent. We used the following EBV negative cell lines: WSU-FSCCL t(14:18)+ follicular lymphoma (FL), FC-TxFL2 t(14:18)+ transformed FL, and SU-DHL4 GC-type diffuse large B cell lymphoma (DLBCL) as examples of GC origin lymphomas. U2932 and TMD8 cell lines represent ABC-type DLBCL.  Apoptosis was determined by annexin V staining and confirmed by caspase-3 activation, each assessed by flow cytometric methods following 48 h incubation. Birinapant had little effect (<5% annexin V+ cells) as a single agent on any of these B cell lymphoma cell lines at ≤ 100 nM, though a low level of apoptosis (7-12% annexin V+ cells) was detectable at 10-20 µM in GC types. Addition of birinapant 30-60 minutes prior to BDM did not further enhance the already high level (>50% annexin V+) of apoptosis induced by 10 uM BDM in WSU-FSCCL and FC-TxFL2,  and only slightly enhanced the low level of BDM-induced apoptosis in the GC DLBCL cell line DHL-4 (to 10-15%). In the ABC DLBCL cell lines, however, whereas 10uM BDM induced <5% annexin V+ cells for U2932 and 10-15% for TMD8, addition  of 100 nM birinapant 30-60 minutes prior to 10 uM BDM induced 35-40% annexin V+ cells in each of these ABC-DLBCL cell lines. This enhancement was schedule-dependent, not observed when birinapant was added after BDM. Thus, the cell lines representing FL and transformed FL are sensitive to BDM at clinically-achievable concentrations, without further enhancement by birinapant. The 3 DLBCL lines were relatively insensitive to BDM compared with FL cells, but BDM-induced apoptosis was markedly enhanced when birinapant was added before (but not after) BDM in the 2 ABC type DLBCL lines. Further explorations into the mechanism of birinapant sensitization of ABC-DLBCL to BDM, issues of dose and schedule, and role of NF-kB-dependency are ongoing. These data suggest that therapeutic trials of BDM plus birinapant would be of interest in ABC type DLBCL. Disclosures: No relevant conflicts of interest to declare.

Lumiliximab (anti-CD23 antibody) mediates apoptosis and antitumor activity in chronic lymphocytic leukemia (CLL) cells and CD23+ lymphoma cell lines

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 3039-3039 ◽  
Author(s):  
N. Pathan ◽  
J. Byrd ◽  
K. Hariharan ◽  
P. Chu ◽  
A. Molina
Keyword(s):  
Antitumor Activity ◽  
B Cell ◽  
Cell Lines ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
In Vivo Evaluation ◽  
Synergistic Cytotoxicity ◽  
Human Lymphoma

3039 Background: Given the success of treating CLL with antibody therapies, interest in those directed at alternative B-cell antigens remains high. Lumiliximab is a chimeric macaque and human anti-CD23 monoclonal antibody whose antigen is expressed on almost all CLL cells. Methods: We examined lumiliximab’s ability to mediate direct apoptosis, antibody-dependent cellular cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC) against primary CLL cells and CD23+ B-cell lines. Apoptosis was measured with a flow-cytometry based assay for active caspase-3. ADCC was determined by 51Cr-release assay. CDC assays were performed in the presence of 30% autologous plasma from patients and quantified by propidium iodide staining. Western blotting analysis was used to monitor protein expression before and after treatment with lumiliximab. The CD23+ human lymphoma SKW6.4 cell line was used for in vivo evaluation of lumiliximab in a disseminated human lymphoma model. Results: Lumiliximab mediates apoptosis, ADCC, and CDC in CD23+ B-cell lines. However, in primary CLL cells, the primary mechanism of cell killing appears to be mediated via apoptosis. Apoptosis induced by lumiliximab occurs mainly through the intrinsic pathway used by other CLL therapies. Lumiliximab decreased expression of Bcl-2 and XIAP and inhibited Akt activation in CLL cells. Lumiliximab when combined in vitro or in vivo with rituximab or fludarabine effectively mediates synergistic cytotoxicity against primary CLL cells and CD23+ B-cell lines. Significant antitumor activity was also observed with lumiliximab vs a control antibody in a SCID mouse model of human B-cell lymphoma (P <.01). More importantly, lumiliximab + rituximab or lumiliximab + fludarabine results in prolonged survival vs lumiliximab, rituximab, or fludarabine single-agent treatment. Conclusions: These results indicate that lumiliximab induces apoptosis by activating caspases and downregulating antiapoptotic proteins, and suggest that in combination with rituximab or chemotherapy, lumiliximab synergistically enhances antitumor activity in CLL or other B-cell malignancies in which this antigen is overexpressed. No significant financial relationships to disclose.

NIK Is Involved In the Activation of the Classical and Alternative NF-κB Pathways In Diffuse Large B Cell Lymphoma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3099-3099
Author(s):  
Lina Odqvist ◽  
Margarita Sánchez-Beato ◽  
Santiago Montes-Moreno ◽  
Ken H Young ◽  
Francesco Acquadro ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Alternative Pathway ◽  
B Cell Lymphoma ◽  
Classical Pathway ◽  
Strong Positive Correlation ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Abstract 3099 Deregulated NF-κB activity plays a role in the lymphoma pathogenesis, and has been proposed to constitute a cardinal feature of some subtypes of diffuse large B cell lymphoma (DLBCL). The NF-κB-Inducing Kinase (NIK) is essential for the activation of the alternative NF-κB pathway by inducing the phosphorylation of the NF-κB member p100, which leads to its processing to p52 and its subsequent nuclear translocation. A role for NIK in the classical NF-κB pathway as well has been shown, suggesting NIK as an attractive therapeutic target in lymphomas. Here, we study the frequency and extent of alternative and classical NF-κB activation in diffuse large B cell lymphoma, and the implication of NIK in both pathways. The activation of the classical and alternative NF-κB pathways was present in 28 and 34% of DLBCL cases respectively, as assessed by nuclear expression of p50 (classical pathway) and p52 (alternative pathway) by immunohistochemistry in a series of 301 samples. Activation of both NF-κB pathways was observed in germinal centre B-cell like (GC) and activated B-cell like (ABC) subtypes, with a slight predominance, although not significant, in ABC subtype. In contrast, the levels of p52 and p50 were significantly higher in ABC-DLBCL cell lines than those of GC subtype. The activation of both pathways was mostly overlapped and there was a strong positive correlation between nuclear p52 and p50 (p<0.001). Eighteen % of the cases expressed both p50 and p52 while only 8 and 16% expressed exclusively p50 or p52, respectively. Activation of the alternative NF-κB pathway was strongly associated with Epstein-Barr virus (EBV), since 93% of EBV+ cases expressed nuclear p52 (p<0.001). In our study, no TRAF3 deletions were detected in a panel of 25 DLBCL samples, although absence of TRAF3 was observed in one DLBCL cell line. Since NIK acts as a bottleneck in the activation of the alternative pathway but has also been described to play a role in the classical pathway, we wanted to analyze the effect of the knockdown of NIK on both pathways. Using small interference RNA in two lymphoma cell lines, we observed that the silencing of NIK had an effect on both pathways, decreasing the processing of p100 as well as p105. Taken together, our results show that the activation of NF-κB distinguishes a subset of DLBCL cases, comprising both ABC and GC subtypes, suggest a frequent overlap between the classical and alternative NF-κB pathway in DLBCL, and identify a possible role for NIK in the activation of both pathways. Disclosures: No relevant conflicts of interest to declare.

Constitutively Active STAT6 Represses BCL6 in Primary Mediastinal B Cell Lymphoma.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2417-2417
Author(s):  
Olga Ritz ◽  
Jochen K Lennerz ◽  
Karolin Rommel ◽  
Karola Dorsch ◽  
Elena Kelsch ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Ectopic Expression ◽  
B Cell Lymphoma ◽  
Proximal Promoter ◽  
Constitutively Active

Abstract Abstract 2417 Primary mediastinal B-cell lymphoma (PMBL) is a subtype of diffuse large B-cell lymphoma (DLBCL) that affects predominantly young women (Swerdlow et al. 2008). Despite improvements due to addition of rituximab, which has become state of the art treatment, 20% of PMBL patients succumb to disease progression or relapse. Notably, here are currently no registered trials that are actively recruiting PMBL-patients and a better understanding of the underlying pathobiology may identify novel therapeutic targets and provide an alternative to dose escalation (Steidl and Gascoyne 2011). BCL6 is a key germinal center B-cell transcription factor that suppresses genes involved in lymphocyte activation, differentiation, cell cycle arrest and DNA damage response gene. BCL6 is aberrantly expressed in certain DLBCL subgroups and BCL6 overexpression is sufficient for lymphomagenesis in mice (Cattoretti et al. 2005). In cellular- and murine DLBCL models, targeting of BCL6 via retroinverted BCL6 peptid inhibitor (RI-BPI) appears effective (Polo et al. 2004; Cerchietti et al. 2010). In conjunction with the relatively restricted expression pattern of BCL6, these data collectively suggest BCL6 as a candidate for targeted therapy in BCL6-positive lymphomas. Despite substantial work on BCL6 in lymphomas, the function of BCL6 in PMBL is unknown. To address the BCL6 function in PMBL, we performed BCL6 depletion by siRNA in all three available PMBL cell lines: K1106, U-2940 and MedB-1. We found that BCL6 acts pro-proliferative and anti-apoptotic; however, PMBL models were only partially dependent on and not addicted to BCL6. Given that BCL6 expression in all PMBL cell lines is variable with a notable fraction of BCL6-negative cells, we argued that increasing the fraction of BCL6-positive cells might increase the level of BCL6-dependence. Since IL-4/STAT6 signaling upregulates BCL6 in mouse lymphocytes (Schroder et al. 2002), we treated PMBL cell lines with IL-4 (or IL-13) and, as expected, observed increased phosphorylated (p)STAT6 levels. Surprisingly, the pSTAT6 increase was not associated with higher – but with drastically lower BCL6 protein levels. Moreover, in untreated cells, co-localization studies for pSTAT6- and BCL6 demonstrated staining in mutually exclusive subsets of cells (Figure 1A), suggesting negative interaction between BCL6 and pSTAT6. Other STAT family members were already shown to participate in the transcriptional regulation of BCL6. Thus, we examined binding of STAT6 to the proximal promoter of BCL6 in all PMBL cell lines using shift assay and chromatin immunoprecipitation. We found that STAT6 can bind all five GAS binding sites within the BCL6 promoter in vitro and in all PMBL cell lines STAT6 was bound to proximal BCL6 promoter in vivo. Furthermore, transient STAT6 depletion by siRNA and/or ectopic expression of constitutively active STAT6 confirms that pSTAT6 is sufficient for transcriptional repression of BCL6. Co-localization studies in primary patient samples demonstrated mutually exclusive BCL6/pSTAT6 distribution as a visual hallmark of the repression mechanism (Figure 1B, C). Thus, our data demonstrate for the first time that constitutively active STAT6 transcriptionally represses BCL6 in PMBL. In conjunction with functional data, the delineated repression mechanism may prevent addiction to one single oncogenic pathway (i.e. BCL6) in PMBL. Figure 1. Mutually exclusive distribution of BCL6 and pSTAT6 in PMBL Figure 1. Mutually exclusive distribution of BCL6 and pSTAT6 in PMBL Disclosures: No relevant conflicts of interest to declare.

5-Aza-2-Deoxycytidine Regulates Wnt Beta-Catenin Pathway in B Cell Lymphoma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4810-4810
Author(s):  
Xinyu Li ◽  
Lingyu Geng ◽  
Xiangxiang Zhou ◽  
Kang Lu ◽  
Peipei Li ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cell ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Beta Catenin ◽  
Cell Functions ◽  
Protein Expressions

Abstract Introduction: The Wnt/beta-catenin pathway is aberrantly activated in B cell lymphomas, unphosphorylated beta-catenin accumulates and translocates into the nucleus, regulates the expression of c-myc, cyclinD1 and many other target genes which govern fundamental cell functions, such as proliferation, cell cycle regulation and apoptosis. Methylation is a highlight of epigenetic regulation research which also occurred in lymphoma, but the concrete mechanism of how the demethylation drug 5-aza-2-deoxycytidine affect Wnt/beta-catenin pathway is still unknown. This study was designed to illuminate the implications on Wnt/beta-catenin pathway via demethylation 5-aza in B cell lymphoma. Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from samples of 30 primary CLL patients. The PBMCs contained more than 90% of CD19+ B lymphocytes, which were detected by flow cytometry and were referred to as primary CLL cells. The activation of Wnt/beta-catenin pathway and DNMT-1 of B cell lymphoma cells lines (MEC-1, LY8, Jeko-1, Grant519, mino and sp53) and the 30 patients were detected by qPCR and western blot. The expressions of beta-catenin in 20 cases of B cell lymphoma tissues were measured by IHC. The B cell lines and PBMCs from 10 primary CLL patients were given 5-aza-2-deoxycytidine in different concentrations, the effects in the pathway and apoptosis were observed by WB and flow cytometry. Results: The expressions of beta-catenin, c-myc, cyclinD1 and DNMT-1 were aberrantly higher in all cell lines we used ( MEC-1,LY8, Jeko-1, Grant519, mino and sp53 Fig.1-A,B), most primary CLL patients (Fig.1-C), and B cell lymphoma tissues (Fig.1-D). The protein expressions of beta-catenin in MEC-1 were higer than primary CLL patients. 0, 0.5, 1.0, 2.0¦ÌM 5-aza-2-deoxycytidine were given to the B cells lines and PBMCs from primary CLL patients for 48h, beta-catenin were found accumulated, but c-myc and cyclinD1 in the downstream were reduced (Fig.2-A,B,C,D). For further understanding of aberrant accumulation ofbeta-catenin, we extracted the nuclear protein of MEC-1, nuclear beta-catenin protein expressions were found decreased and cytoplasmic were increased (Fig.2-E). After 5-aza treatment, the apoptosis rate increased and caspase pathway were activated (Fig.2-A,F). Conclusions: The enhanced expressions of beta-catenin, c-myc, cyclinD1 in the B cell lines and the B cell lymphoma samples indicated the Wnt/beta-catenin was aberrantly activated. After 5-aza treatment with the cell lines (MEC-1, Jeko-1, LY8) and primary CLL cells, the abnormal accumulation of beta-catenin protein was observed which was discrepancy with previous reports, but the decrease of c-myc and cyclinD1 suggested the pathway was inhibited, apoptosis also occurred. The increase of totalbeta-catenin protein was supposed to be an stress reaction of the 5-aza treatment, however, the redundant beta-catenin protein in B cell lymphoma was speculated to be combined with demethylated genes and resulted in dormancy of this pathway. Our results indicated that 5-aza played a demethylation role through Wnt/beta-catenin pathway in B cell lymphoma. The data are of interest in the context of epigenetic-based therapy in B cell lymphoma. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

Synergism Between PDE4 and PI3Kδ Inhibitors in DLBCL: Improved Clinical Activity with the Potential for Lower Toxicity

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2938-2938 ◽  
Author(s):  
Jeffrey Cooney ◽  
Long Wang ◽  
An-Ping Lin ◽  
Daifeng Jiang ◽  
Avvaru Suhasini ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Ectopic Expression ◽  
Xenograft Model ◽  
B Cell Lymphoma ◽  
Regulatory Subunit ◽  
Pde4 Inhibitor ◽  
Structural Constraints ◽  
Pi3k Activity

Abstract Aberrant activation of the B cell receptor (BCR) is a hallmark of mature B-cell tumors. A better understanding of this process will spearhead effective clinical translation. The initiation and amplification of BCR signaling are well-defined events, and the successful deployment of BTK and PI3Kδ inhibitors in the clinic capitalizes on this knowledge. Conversely, the intricacies of the termination of BCR signals are less well-understood, and to date no rational therapeutic approach has been developed that exploit this aspect of the oncogenic BCR. Cyclic-AMP (cAMP) is a second messenger with marked growth suppression properties towards immune cells, including neoplastic mature B lymphocytes. In earlier work, we showed that inhibition of phosphodiesterase 4 (PDE4), the enzyme that hydrolyzes cAMP, downmodulates the activity of classical effectors of BCR signals, including SYK and PI3K. Herein, we attempted to gain further mechanistic understanding on how cAMP suppresses the proximal BCR activity, and built on this information to pre-clinically test therapeutic strategies that simultaneously attack the BCR at its amplification and termination points. Using diffuse large B cell lymphoma (DLBCL) as a model, we focused our attention on the interplay between cAMP and LCK, as we unexpectedly found that this cAMP-regulated canonical T-cell kinase is also widely expressed in DLBCL. Working with LCK-positive PDE4-low/null DLBCL cell lines, we found a marked increase in the phosphorylation of the inhibitory Y505 of LCK following elevation of intra-cellular cAMP. Next, we showed that ectopic expression of wild-type (WT) PDE4B, but not of a phosphodiesterase-inactive (PI) mutant, abrogated the cAMP-mediated, CSK-dependent, phosphorylation of LCK. Active LCK can phosphorylate PI3K's p85 regulatory subunit, thus freeing the catalytic domain from its structural constraints to promote lipid kinase activity. Thus, we tested whether the cAMP-mediated inhibition of LCK, by suppressing p85 phosphorylation, down-modulated PI3K activity. In LCK-positive PDE4B-null DLBCL, we showed that cAMP readily decreased the phosphorylation of p85 that followed BCR engagement; using the WT and PI PDE4B genetic models, we demonstrated that PDE4B expression abrogated cAMP effects and led to sustained PI3K activity following BCR engagement. These data suggested that inhibition of PDE4, by unleashing the negative effects of cAMP on LCK/p85, could accelerate the termination of PI3K activation that follows BCR engagement. If this hypothesis was correct, then the combination of PI3K and PDE4 inhibitors by attacking the BCR at its amplification and termination points, respectively, may synergistically suppress the growth of DLBCL. In in vitro studies with multiple DLBCL cell lines (WSU-NHL, OCI-Ly7, OCI-Ly18, OCI-Ly3, HBL-1, and OCI-Ly10) we showed that the combination of the FDA-approved PDE4 inhibitor roflumilast with idelalisib synergistically suppresses DLBCL growth (combination index < 1). This synergism was associated with a significant suppression of PI3K and AKT activities (p<0.05, cells treated with the drug combination vs. single agents). We expanded this observation to an in vivo xenograft model of human DLBCL, and showed that mice treated with roflumilast and idelalisib had a significantly smaller tumor burden than those receiving single agents (p< 0.01, two cohorts, n=47 mice). We also found a greater suppression of PI3K activity in the xenografts from mice treated with the combination of PDE4 and PI3Kδ inhibitors (p< 0.0001), as well as increased apoptosis. Together, these data further delineated how cAMP suppresses the BCR and showed that the rational combination PDE4 and PI3Kδ inhibitors synergistically suppresses DLBCL growth. These results are particularly important given the recent evidence of inflammatory/immune toxicity associated with the use of idelalisib, which we propose could be countered by the well-established anti-inflammatory properties of PDE4 inhibitors. Thus, we hypothesize that combining PDE4 and PI3Kδ inhibitors will enhance anti-lymphoma activity while decreasing clinical toxicity. This concept is ripe for clinical testing as we have recently completed a phase Ib trial showing that roflumilast is safe and active in patients with advanced B cell malignancies. Disclosures No relevant conflicts of interest to declare.

scholarly journals SLAMF7 in Primary Effusion Lymphoma, Target for Individualized Therapy?

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5300-5300
Author(s):  
Hilmar Quentmeier ◽  
Claudia Pommerenke ◽  
Wilhelm G Dirks ◽  
Vivien Hauer ◽  
Max Koeppel ◽  
...  
Keyword(s):  
B Cell ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Primary Effusion Lymphoma ◽  
B Cell Lymphoma ◽  
B Cell Differentiation ◽  
Expression Array ◽  
Non Hodgkin Lymphoma ◽  
Novel Therapeutic Strategies

Abstract Primary effusion lymphoma (PEL) is a rare, aggressive form of B-cell lymphoma. With a median survival time of around six months the prognosis for PEL patients is poor. Therefore, there is a medical need for novel therapeutic strategies. We performed expression array analysis to find potential targets for antibody-based therapy. Unsupervised clustering analysis revealed that PEL cell lines grouped separate from cell lines derived from other B-non Hodgkin lymphoma (B-NHL) entities. Notably, PEL and Hodgkin Lymphoma (HL) cell lines clustered on one arm, separate from all cell lines representing less-differentiated B-NHL variants. PEL and HL cell lines were characterized by a set of common up- and downregulated genes. Typical for PEL and HL was the expression of CCND2 and the absence of Brutons tyrosine kinase and of B-cell markers including CD19, CD20, CD79A and CD79B. Highly expressed in PEL - but not in HL - were CD138, IL-10, SLAMF7 and PRDM1. PRDM1/BLIMP1 is a master regulator of terminal B-cell differentiation. Originally described as repressor, BLIMP1 can also enhance transcription of SLAMF7 in multiple myeloma (MM) and of IL-10 in type 1 regulatory T-cells. Thus, coexpression of the three genes suggests a causal relationship between transcriptionally active PRDM1/BLIMP1 and its targets SLAMF7 and IL-10 also in PEL. Expression of SLAMF7 in PEL is especially noteworthy because a monoclonal antibody targeting SLAMF7 (elotuzumab) has been approved for treatment of patients with MM. We observed that SLAMF7 is comparably expressed in PEL and in MM cell lines. If the results on cell lines can be translated to primary PEL, i.e. if PEL tumor cells express SLAMF7, the patients might benefit from an antibody-based targeted therapy against this antigen. Disclosures No relevant conflicts of interest to declare.

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