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. 2021 Aug 1;106(8):2251-2256.
doi: 10.3324/haematol.2020.271304.

Rationale for the combination of venetoclax and ibrutinib in T-prolymphocytic leukemia

Christoph Kornauth  1 , Charles Herbaux  2 , Bernd Boidol  3 , Chantal Guillemette  4 , Patrick Caron  4 , Marius E Mayerhöfer  5 , Stéphanie Poulain  6 , Olivier Tournilhac  7 , Tea Pemovska  1 , Stephen J F Chong  2 , Emiel Van der Kouwe  1 , Lukas Kazianka  1 , Georg Hopfinger  8 , Daniel Heintel  9 , Roland Jäger  10 , Markus Raderer  11 , Ulrich Jäger  1 , Ingrid Simonitsch-Klupp  12 , Wolfgang R Sperr  1 , Stefan Kubicek  3 , Matthew S Davids  2 , Philipp B Staber  13
Affiliations

Rationale for the combination of venetoclax and ibrutinib in T-prolymphocytic leukemia

Christoph Kornauth et al. Haematologica. .
No abstract available

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Figures

Figure 1.
Figure 1.
Ibrutinib synergizes with venetoclax in T-prolymphocytic leukemia via inhibition of ITK and enhances Bcl2-dependent apoptotic priming. (A) Single cell suspensions of blood or bone marrow samples were subjected to combinatorial drug screens with venetoclax and 24 potential combination partners. Deviation from the Bliss Independence score was evaluated for each combination. For BH3-profiling single cells were stained for cytochrome and analyzed by flow cytometry. (B) Heatmap demonstrating deviation from the Bliss independence score for each combination and individual patients (n=7). Synergy is denoted in red while antagonism is shown in blue. Clear boxes represent missing data for Patient 5 whose material was only screened for a subset of drugs due to sample availability. The bar plot in the lower half shows the synergy score as a mean over all patients. Drug synergy was calculated using the Synergy finder R package by integrating data of three independent runs (Online Supplementary Figure S1B). (C) A representative three-dimensional drug synergy plot (data from Patent 7) for ibrutinib and cisplatin with venetoclax. (D) Annexin V assay showing viability compared to that with a DMSO control for ibrutinib (n=12 different T-PLL patient samples), ITK-inhibitor BMS-509744 (n=6), and BTK-inhibitor acalabrutinib (n=12, Wilcoxon-Mann-Whitney test, ***P<0.001, *P<0.05, all compounds tested at 10 μM with drug exposure for 24 h under NKtert co-culture). (E) Annexin V assay demonstrating viability of T-PLL samples under NKtert co-culture treated with venetoclax alone (n=10), or in combination with ibrutinib (n=10), BMS-509744 (n=6), and acalabrutinb (n=10) compared to DMSO-Ctrl. (t-test,*P≤0.05, ***P<0.001, ibrutinib, acalabrutinib and BMS-509744 were used at a dose of 10 mM with drug exposure for 24 h, venetoclax was used at a dose of 100 nM with drug exposure for 4 h both as a single agent and in combination. After annexin V/Hoechst staining, cells were fixed with paraformaldehyde and analyzed using a BD Fortessa with a 96-well HTS plate-reader. NKTert cells were excluded using forward and side scatter parameters. Primary antibodies were directed against cytochrome c (Alexa Fluor 488-labeled, 6H2.B4/612308, Biolegend), CD19 (PE/Cy7-labeled, H13B19/302216, BioLegend), anti–CD5 (PE-labeled, UCHT2/300608, Biolegend). The analysis was performed on the CD5+CD19– cell fraction. (F) BH3-profiling in primary T-PLL samples treated with ibrutinib: cytochrome C release compared to control for overall mitochondrial priming and specific BCL2-dependence is shown for samples treated with either DMSO or ibrutinib 10 mM for 24 h (t-test,**P≤0.01). T-PLL: T-prolymphocytic leukemia; DMSO: dimethylsulfoxide; Ctrl. Control.
Figure 2.
Figure 2.
The combination of ibrutinib and venetoclax is clinically active in T-prolymphocytic leukemia. (A, B) Clinical follow-up of two patients treated with the combination of ibrutinib and venetoclax, patient A (A) and patient B (B). The WBC count and LDH concentration are plotted as blue and red lines, respectively. The lower part of each panel represents drug serum levels as black dots and drugs given as gray rectangles. Drug levels were determined by mass spectrometry. The red arrow denotes the time point at which the serum ibrutinib concentration dropped below the level of detection with a concomitant rise of WBC. (C, D) In vivo BH3 profiling of primary patients’ samples during co-treatment: patient A (C) and patient B (D). (E, F) Western blot analysis of primary cells showing changes in protein levels of ITK and phospho-ITK during co-treatment of patient A (E) and patient B (F). Primary antibodies were directed against phospho-ITK (Tyr512, Life Technologies, #PA564523), ITK (Cell Signaling Technology, #2380S) and b-actin (Santa Cruz Biotechnology, #SC-47778) (G) Proposed mechanism. Monotherapy with venetoclax may lead to drug resistance via upregulation and activation of ITK and reduced apoptotic priming. ITK inhibition might increase Bcl-2-dependent apoptotic priming and restore the activity of venetoclax. WBC: white blood cell; LDH: lactate dehydrogenase.
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References

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