Regulation of Bcl-XL by non-canonical NF-κB in the context of CD40-induced drug resistance in CLL

AbstractIn chronic lymphocytic leukemia (CLL), the lymph node (LN) microenvironment delivers critical survival signals by inducing the expression of anti-apoptotic Bcl-2 members Bcl-XL, Bfl-1, and Mcl-1, resulting in apoptosis blockade. We determined previously that resistance against various drugs, among which is the clinically applied BH3 mimetic venetoclax, is dominated by upregulation of the anti-apoptotic regulator Bcl-XL. Direct clinical targeting of Bcl-XL by, e.g., Navitoclax is however not desirable due to induction of thrombocytopenia. Since the actual regulation of Bcl-XL in CLL in the context of the LN microenvironment is not well elucidated, we investigated various candidate LN signals to drive Bcl-XL expression. We found a dominance for NF-κB signaling upon CD40 stimulation, which results in activation of both the canonical and non-canonical NF-κB signaling pathways. We demonstrate that expression of Bcl-XL is first induced by the canonical NF-κB pathway, and subsequently boosted and continued via non-canonical NF-κB signaling through stabilization of NIK. NF-κB subunits p65 and p52 can both bind to the Bcl-XL promoter and activate transcription upon CD40 stimulation. Moreover, canonical NF-κB signaling was correlated with Bfl-1 expression, whereas Mcl-1 in contrast, was not transcriptionally regulated by NF-κB. Finally, we applied a novel compound targeting NIK to selectively inhibit the non-canonical NF-κB pathway and showed that venetoclax-resistant CLL cells were sensitized to venetoclax. In conclusion, protective signals from the CLL microenvironment can be tipped towards apoptosis sensitivity by interfering with non-canonical NF-κB signaling.

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Possible Mechanisms Of Resistance To The Novel BH3-Mimetic ABT-199 In In Vitro Lymph Node Models Of CLL – The Role Of Abl and Btk

Blood ◽

10.1182/blood.v122.21.4188.4188 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4188-4188 ◽

Cited By ~ 3

Author(s):

Rachel Thijssen ◽

Christian R Geest ◽

Martin FM de Rooij ◽

Nora Liu ◽

Bogdan I Florea ◽

...

Keyword(s):

Drug Resistance ◽

Lymph Node ◽

Chronic Lymphocytic Leukemia ◽

Clinical Application ◽

Kinase Inhibitors ◽

Conflicts Of Interest ◽

Solid Phase ◽

Lymphocytic Leukemia ◽

Bh3 Mimetic

Abstract The new BH3-mimetic ABT-199 antagonizes Bcl-2 and avoids the thrombocytopenia associated with clinical application of its predecessor ABT-263 (navitoclax). Chronic lymphocytic leukemia (CLL) cells are highly sensitive to ABT-199 and the first clinical results show clear reductions in peripheral and bone marrow CLL cells and in lymph node size. In the lymph node, CLL cells receive pro-survival signals that upregulate Bcl-XL, Mcl-1 and Bfl-11. These Bcl-2 family members are not targeted by ABT-199, which poses the potential risk of remaining clones with residual viability. Here, we aimed to define the signals that determine sensitivity for ABT-199 and ABT-737 in an in vitro lymph node model of CLL. We applied CD40 and cytokine stimulation in combination with kinase inhibitors that are known to change microenviroenmental signals and increase drug resistance in CLL. Stimulation via CD40 plus IL-4 or IL-21 differentially affected the expression of Mcl-1, Bcl-XL, Bfl-1 and Noxa and this correlated with strong alterations in sensitivity to ABT-737 and ABT-199 (see table 1 for LC 50 values). As reported before2, in vitro CD40 stimulation reduced sensitivity to ABT-737 by 100-fold, and this was further decreased by IL-4. Strikingly, CD40+IL-4 stimulation in primary CLL cells resulted in full resistance to 10 μM ABT-199, probably due to very high levels of Bcl-XL.Table 1The LC50 of ABT-737 or ABT-199 for CLL cells stimulated with CD40L and IL-21 or IL-4 (averaged values n=8)StimulationLC50 (μM)ABT-737ABT-1993T3 (control)0.0050.0013T40L0.781> 103T40L + IL-210.1950.210 3T40L + IL-46.772> 103T40L + IL-21 + IL-40.4269.121 We next sought ways to circumvent resistance against ABT-199 induced in our in vitro model. We showed previously that the broad spectrum kinase inhibitor dasatinib prevented CD40-mediated resistance to various drugs, including ABT-7373. We therefore first characterized the targets of dasatinib in primary CLL by solid-phase pull-down, mass-spectrometry and competition binding. Abl and Btk were identified as dominant and specific interactors of dasatinib. Importantly, resistance for BH3-mimetics could be overcome by dasatinib (see table 2) and the Abl inhibitor imatinib, but not by the more selective Btk inhibitor ibrutinib. Conversely, BCR- and chemokine-controlled adhesion could be abolished by dasatinib and ibrutinib, but not by imatinib. Thus, Abl and Btk function in two key pro-survival arms; chemoresistance and localization in the protective environment.Table 2The LC50 of ABT-737 or ABT-199 for CLL cells stimulated with CD40L in combination with Dasatinib (averaged values n=4)StimulationLC50 (μM)ABT-737ABT-1993T30.0050.0013T40L0.781> 103T40L + 100 nM Dasatinib0.0810.066 3T40L + 1000 nM Dasatinib0.0370.020 The observed resistance to ABT-199 induced in our in vitro a co-culture system designed to simulate the CLL microenvironment does not reflect the observations from clinical trials in patients. Nevertheless, long-term clinical application of ABT-199 in CLL might select for resistant clones at protective niches. Our data suggest that this may be overcome by combination treatment with kinase inhibitors that either directly abrogate anti-apoptotic signals or cause egress from lymph node sites and prevent the resistance mechanism from coming into play. 1. Smit LA, Hallaert DY, Spijker R et al. Differential Noxa/Mcl-1 balance in peripheral versus lymph node chronic lymphocitic leukemia cells correlates with survival capacity. Blood 2007;109:1660-1668. 2. Vogler M, Butterworth M, Majid A et al. Concurrent up-regulation of BCL-XL and BCL2A1 induces approximately 1000-fold resistance to ABT-737 in chronic lymphocytic leukemia. Blood 2009;113:4403-4413. 3. Hallaert DY, Jaspers A, van Noesel CJ et al. c-Abl kinase inhibitors overcome CD40-mediated drug resistance in CLL; Implications for therapeutic targeting of chemoresistant niches. Blood 2008;112:5141-5149. Disclosures: No relevant conflicts of interest to declare.

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Mapping the Targets of Dasatinib in Chronic Lymphocytic Leukemia Reveals Distinct Roles for Abl and Btk in Drug Resistance and Adhesion, and Explains Clinical Effects On Lymph Node Reduction

Blood ◽

10.1182/blood.v120.21.3900.3900 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3900-3900

Author(s):

Eric Eldering ◽

Christian R Geest ◽

Martin FM de Rooij ◽

Nora Liu ◽

Bogdan I Florea ◽

...

Keyword(s):

Drug Resistance ◽

Lymph Node ◽

Chronic Lymphocytic Leukemia ◽

Broad Spectrum ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Lymphocytic Leukemia ◽

Clinical Effects ◽

Open Label

Abstract Abstract 3900 In the lymph node (LN) microenvironment, chronic lymphocytic leukemia (CLL) cells are protected from apoptosis by upregulation of anti-apoptotic proteins. In vitro, this can be mimicked via CD40-stimulation of CLL cells, which also provides resistance to various chemotherapeutics. Novel drugs that target kinases involved in B cell signalling, including the broad spectrum kinase inhibitor dasatinib, are currently in clinical development for CLL. We have shown previously that dasatinib prevents CD40-mediated anti-apoptotic changes in CLL (Hallaert et Blood 2008). However, the kinase(s) involved remain unidentified. Here, we coupled dasatinib to an affinity matrix and pulled down its targets from CD40-stimulated CLL cells. By mass-spectrometry and Western blotting, Abl and Btk were identified as dominant targets of dasatinib. Functional analysis revealed that CD40-mediated anti-apoptotic signals and drug-resistance could be overcome both by dasatinib and the Abl inhibitor imatinib, but not by the novel Btk inhibitor PCI-32765 (ibrutinib), whereas BCR- and chemokine-controlled adhesion could be abolished by dasatinib and ibrutinib, but not by imatinib. Thus, dasatinib combines two key aspects that are clinically relevant: inhibition of Abl overrides chemoprotective survival signals, whereas inhibition of Btk impairs integrin-mediated adhesion of CLL cells in the microenvironmental niche. This combined inhibition of Abl and Btk was put to an initial test in an open-label phase 2 trial of dasatinib combined with fludarabine in twenty refractory CLL patients. As might be expected based on the in vitro data, reductions in lymph node size were observed in most patients. A LN reduction of ≥20% provided a significant improved PFS (256 days) and OS (510 days) as compared to non-responders (80 days and 158 days respectively). Details of the clinical study will be presented separately. In conclusion, in agreement with in vitro molecular studies, dasatinib seems to have clinical efficacy in heavily pretreated refractory CLL patients. Combined, these data encourage further studies on a broad-spectrum kinase inhibitor like dasatinib in combination with other classes of drugs in relapsed and refractory CLL. Disclosures: No relevant conflicts of interest to declare.

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Dual TORK/DNA-PK inhibition blocks critical signaling pathways in chronic lymphocytic leukemia

Blood ◽

10.1182/blood-2016-02-700328 ◽

2016 ◽

Vol 128 (4) ◽

pp. 574-583 ◽

Cited By ~ 40

Author(s):

Rachel Thijssen ◽

Johanna ter Burg ◽

Brett Garrick ◽

Gregor G. W. van Bochove ◽

Jennifer R. Brown ◽

...

Keyword(s):

Drug Resistance ◽

Chronic Lymphocytic Leukemia ◽

Signaling Pathways ◽

Lymphocytic Leukemia ◽

Clinical Effects ◽

Key Points

Key Points TORK/DNA-PK inhibition induces cytotoxicity and blocks signaling pathways important for CLL survival, proliferation, and drug resistance. Preliminary clinical effects of TORK/DNA-PK inhibition show 7 of 8 CLL patients with decreased lymphadenopathy.

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Differential Noxa/Mcl-1 balance in peripheral versus lymph node chronic lymphocytic leukemia cells correlates with survival capacity

Blood ◽

10.1182/blood-2006-05-021683 ◽

2006 ◽

Vol 109 (4) ◽

pp. 1660-1668 ◽

Cited By ~ 106

Author(s):

Laura A. Smit ◽

Delfine Y.H. Hallaert ◽

René Spijker ◽

Bart de Goeij ◽

Annelieke Jaspers ◽

...

Keyword(s):

Lymph Node ◽

Chronic Lymphocytic Leukemia ◽

Lymph Nodes ◽

Peripheral Blood ◽

Proteasome Inhibition ◽

Lymphocytic Leukemia ◽

Protein Levels ◽

Cd40 Stimulation ◽

Apoptosis Gene

Abstract The gradual accumulation of chronic lymphocytic leukemia (B-CLL) cells is presumed to derive from proliferation centers in lymph nodes and bone marrow. To what extent these cells possess the purported antiapoptotic phenotype of peripheral B-CLL cells is unknown. Recently, we have described that, in B-CLL samples from peripheral blood, aberrant apoptosis gene expression was not limited to protective changes but also included increased levels of proapoptotic BH3-only member Noxa. Here, we compare apoptosis gene profiles from peripheral blood B-CLL (n = 15) with lymph node B-CLL (> 90% CD5+/CD19+/CD23+ lymphocytes with Ki67+ centers; n = 9). Apart from expected differences in Survivin and Bcl-xL, a prominent distinction with peripheral B-CLL cells was the decreased averaged level of Noxa in lymph nodes. Mcl-1 protein expression showed a reverse trend. Noxa expression could be reduced also in vitro by CD40 stimulation of peripheral blood B-CLL. Direct manipulation of Noxa protein levels was achieved by proteasome inhibition in B-CLL and via RNAi in model cell lines. In each instance, cell viability was directly linked with Noxa levels. These data indicate that suppression of Noxa in the lymph node environment contributes to the persistence of B-CLL at these sites and suggest that therapeutic targeting of Noxa might be beneficial.

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Cross-Talk between Cytokine and NF-ĸb Signaling in the CLL Microenvironment Can Affect Sensitivity for Venetoclax

Blood ◽

10.1182/blood-2019-126788 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5449-5449

Author(s):

Marco Haselager ◽

Rachel Thijssen ◽

Arnon P. Kater ◽

Eric Eldering

Keyword(s):

Lymph Node ◽

Chronic Lymphocytic Leukemia ◽

Cross Talk ◽

Binding Sites ◽

Research Funding ◽

Time Course ◽

Lymphocytic Leukemia ◽

Cytokine Signaling ◽

T Helper ◽

Cd40 Stimulation

INTRODUCTION. The Bcl-2 inhibitor Venetoclax provides profound reductions in circulating chronic lymphocytic leukemia (CLL) cells in the majority of patients. However, lymph node (LN) responses are less robust, which may be linked to an acquired resistance imposed by pro-survival signals. Prime among these is CD40 stimulation leading to activation of NF-kB, and induction of Bcl-XL expression1. Bcl-XL is a prime determinant of resistance to Venetoclax2 and regulatory mechanisms of its expression are of clinical significance. Cytokines IL-21 and IL-4 are secreted by T helper cells and abundant in the CLL lymph node microenvironment. Importantly, IL-21 and IL-4 play an important role in CLL survival and proliferation3. In the present study, we investigated how signals from T helper cytokines IL-21 or IL-4 affect Bcl-XL expression as a model for the CLL LN microenvironment, specifically in relation to Venetoclax resistance. RESULTS. Following CD40 stimulation, IL-21 and IL-4 show opposing effects on Bcl-XL expression. Correspondingly, this was associated with CD40-induced resistance to Venetoclax which was augmented by IL-4 and reversed by IL-21. We subsequently investigated the rewiring between CD40 activation, differential cytokine signaling and Bcl-XL expression. IL-21 or IL-4 stimulation correspond with differential STAT3 or -6 phosphorylation and STAT3 and -6 have predicted binding sites near the known p65 and p52 binding sites in the Bcl-XL promoter region. Using reporter assays with Bcl-XL promotor constructs we demonstrate competition (through IL-21-induced STAT3) or synergy (through IL-4 induced-STAT6) with CD40-mediated activation of the NF-kB pathway. By applying in situ proximity ligation (isPLA) in primary CLL cells, we showed direct interaction of both (non-)canonical p65 and p52 with STAT3 and STAT6. Moreover, time-course analyses indicated that STAT3 drives NF-kB out of the nucleus, whereas STAT6 keeps NF-kB inside the nucleus, and this distinction controls Bcl-XL expression. These observations suggest that cross-talk between JAK/STAT signaling and NF-kB signaling happens by direct binding to the Bcl-XL promoter and by limiting NF-kB availability at the Bcl-XL promoter. CONCLUSIONS. These data show that protective signals from the CLL microenvironment can be tipped towards apoptosis sensitivity by interfering with JAK/STAT and NF-kB signaling, providing novel therapeutic clues in case of emerging resistance to targeted drugs such as Venetoclax. 1 J. Tromp, S. Tonino, J. Elias, A. Jaspers, D. Luijks, A. Kater, R. van Lier, M. van Oers, E. Eldering. Dichotomy in NF-kB signaling and chemoresistance in IGHV mutated versus unmutated CLL cells upon CD40/TLR9 triggering. Oncogene 2010. 2 R. Thijssen, E. Slinger, K. Weller, C. Geest, T. Beaumont, M. van Oers, A. Kater, E. Eldering. Resistance to ABT-199 induced by microenvironmental signals in chronic lymphocytic leukemia can be counteracted by CD20 antibodies or kinase inhibitors. Haematologica 2015. 3 C. Schleiss, W. Ilias, O. Tahar, Y. Güler, L. Miguet, C. Mayeur-Rousse, L. Mauvieux, L. Fornecker, E. Toussaint, R. Herbrecht, F. Bertrand. M. Maumy-Bertrand, T. Martin, S. Fournel, P. Georgel, S. Bahram, L. Vallat. BCR-associated factors driving chronic lymphocytic leukemia cells in proliferation ex vivo. Scientific Reports 2019. Disclosures Eldering: Celgene: Research Funding; Roche: Research Funding; Janssen Pharmaceutical Companies: Research Funding.

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