Resistance to ABT-199 induced by microenvironmental signals in chronic lymphocytic leukemia can be counteracted by CD20 antibodies or kinase inhibitorsA major clinical problem in chronic lymphocytic leukemia (CLL) is development of chemoresistance, which can be caused by genetic lesions but is also strongly influenced by the leukemic microenvironment. Two compartments can be distinguished in CLL; the blood, where quiescent CLL cells accumulate, and the lymphoid microenvironment within the lymph nodes (LN), spleen and bone marrow, where surrounding cells provide external signals that drive CLL proliferation and survival.1 CLL survival is correlated with NF-kB-mediated upregulation of various protective Bcl-2 family members, notably Bcl-XL, Bfl-1 and Mcl-1 in LN samples compared to peripheral blood (PB). 2,3Recently, novel therapeutics that target microenvironmental signals or Bcl-2 family members have entered clinical trials and practice.1,4 One prominent strategy for CLL and other cancers is to target the apoptosis machinery directly by so-called BH3-mimetics. The Bcl-2-specific compound ABT-199 or Venetoclax is highly cytotoxic for CLL cells and shows improved clinical efficacy and induces no thrombocytopenia as opposed to its predecessor Navitoclax.4 Peripheral blood lymphocyte counts, lymph node size and bone marrow involvement all diminished early after treatment.5 A second strategy employs kinase inhibitors that target critical signal transduction pathways controling cell growth, adhesion and survival. Inhibitors of haematologica 2015; 100:e302 LETTERS TO THE EDITOR Figure 1. Bcl-XL is expressed in CLL cells in LN tissue. One representative CLL LN sample out of 4 stained is shown and stained for (A) Isotype control staining and nuclear staining with DAPI in blue, scale-bar represent 10 mm. (B) CD20 in green and Bcl-XL in red and nuclear staining with DAPI in blue (C and D). CLL cells (Online Supplementary Table S1; patients #23-25) were co-cultured with NIH3T3 fibroblasts transfected with empty vector (3T3) (C) or co-cultured with NIH3T3 fibroblasts transfected with hCD40L (3T40L) (D) for three days. After detachment, cytospins were made and stained for CD20, Bcl-XL and DAPI. Imaging was performed using a Leica TCS SP8-X confocal microscope. One CLL sample is shown of a total of three analyzed, scale-bar represent 10 mm. (A and B) Paraffin-embedded LN samples from CLL patients were incubated with primary antibody anti-CD20 (eBioscience, San Diego, CA, USA) and anti-Bcl-XL (Cell Signaling, Boston, MA, USA) and subsequently incubated with Alexa Fluor 488 labeled goat anti-mouse and Alexa Fluor 594 labeled goat anti-rabbit antibodies (Invitrogen, Camarillo, CA, USA) and counterstained with DAPI. Immunofluorescent imaging (40x) was performed using a Leica DMRA fluorescence microscope. A C D B© F e r r a t a S t o r t i F o u n d a t i o n the B-cell receptor (BCR)-associated kinases Bruton's tyrosine kinase (Btk) and phosphatidylinositol-3-kinase (PI3K) show strong clinical activity and were recently approved f...
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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