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Letters to the Editor
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 treat- ed 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.
venetoclax, whereas cisplatin appeared the most antago- nistic (Figure 1B and C, Online Supplementary Figure S1B). The activity of ibrutinib as a single agent was very mod- est (Online Supplementary Figure S1A and B), consistent with previous reports.6 In addition, independent annexin V/Hoechst assays of primary T-PLL cells on NK-Tert stro- mal support demonstrated modest single-agent activity of ibrutinib (Figure 1D, Online Supplementary Figure S2A). Moreover, selective inhibition of Bruton tyrosine kinase (BTK) by acalabrutinib had no effect on viability (Figure 1D). In contrast, ibrutinib has substantial inhibitory activ- ity on the intracellular mediator of T-cell receptor signal- ing IL-2-inducible T-cell kinase (ITK; half maximal inhibitory concentration [IC50]: 2.2 nM), which has been reported to play a functional role in T-PLL.5,7 In line with previous observations, single-agent treatment with BMS- 509744, a specific ITK inhibitor, had no effect on viability in primary T-PLL samples (Figure 1D).8
To elucidate the mechanism of the combinatorial effect, we treated primary T-PLL samples with venetoclax alone and in combination with ibrutinib, BMS509744 or acalabrutinib. In contrast to BTK-specific inhibition, only drugs that inhibit ITK (ibrutinib, and BMS509744) enhanced Bcl2-inhibitor-induced cell death (Figure 1E). We performed dynamic BH3-profiling of primary T-PLL samples treated ex vivo with ibrutinib to elucidate changes in apoptotic priming. The assay measured cytochrome C release upon stimulation with BH3- mimetics as a readout for a cellular tendency towards apoptosis.9 The data demonstrated enhanced overall mitochondrial priming for apoptosis and a shift to increased functional dependence on Bcl-2 for survival upon ibrutinib treatment (Figure 1F, Online Supplementary Figure S2B).
Based on our in vitro drug synergy findings, we initia- ted combined treatment with venetoclax and ibrutinib in two r/r-T-PLL patients after alemtuzumab-based therapy. Both patients presented with active disease after multiple treatment lines with no further standard treatment options available. We employed tandem mass spectrometry coupled to liquid chromatography (LC-MS/MS) to measure venetoclax and ibrutinib serum levels and in vivo BH3 profiling to evaluate overall and Bcl2-dependent apoptotic priming during treatment. The expression of phospho-ITK, ITK and BH3 family mem- bers was evaluated by immunoblotting of patients’ cells obtained during treatment (Figure 2, Online Supplementary Figure S3).
Patient A (male, aged 78 years) was admitted with r/r- T-PLL after three previous treatment lines (alemtuzumab monotherapy, alemtuzumab + FCM [fluradabine,
cyclophosphamide and mitoxantrone], rechallenged with alemtuzumab monotherapy). He presented with dysp- nea, a white blood cell (WBC) count of 519x109/L, elevat- ed lactate dehydrogenase (LDH; 5,230 U/L), fever, absolute lymphocytosis (472x109/L) and neutrophilia (42x109/L) (Figure 2A, Online Supplementary Figure S3A), and splenomegaly (diameter: 26 cm). Fluorescence in situ hybridization analyses of interphase nuclei revealed mul- tiple cytogenetic aberrations (TCL1 and TCRA/D translo- cations, heterozygous deletions of several 6q and 13q loci, trisomies 8 and 12, duplication of the MLL locus as well as MYC amplifications), suggesting the presence of a complex aberrant karyotype. Venetoclax treatment was commenced with a daily ramp-up from 20 mg to 800 mg, which was well-tolerated. However, the clinical response was limited with the WBC count still above 300x109/L and LDH above 3,000 U/L after 2 weeks. When co-treat- ment with ibrutinib at a dose of 420 mg was initiated on day 16, both the WBC count and LDH decreased steadily (Figure 2A). The patient’s overall clinical condition improved, and the spleen size decreased to 22 cm after 20 days of co-treatment. Serum levels of venetoclax and ibrutinib were continuously monitored. Interruption of ibrutinib on day 24 was associated with a rise of WBC count that declined after ibrutinib was reintroduced. The course of patient A was complicated by influenza A infection and subsequent bacterial pneumonia requiring multiple admissions to hospital, and mechanical respira- tory support resulting in discontinuation of anti-T-PLL therapy. After resolution of the pneumonia, T-PLL thera- py was reinitiated, however treatment adherence dropped, when the patient returned to his local treatment team who eventually switched to best supportive care.
Patient B (female, aged 75 years) had r/r-T-PLL that relapsed after initial therapy with CHOP (cyclophos- phamide, doxorubicin, vincristine and prednisone) and was refractory to alemtuzumab. She presented with dys- pnea, pleural effusion, a WBC count of 300x109/L, elevat- ed LDH (581 U/L), and absolute neutrophilia (27x109/L) during alemtuzumab treatment (Figure 2B, Online Supplementary Figure S3B). The cytogenetic report demonstrated a complex karyotype including inv(14) and isochromosome 8q. Ibrutinib was started at a dose of 420 mg once daily before venetoclax daily at a dose increased from 20 mg to 400 mg. The combination of ibrutinib and venetoclax led to a rapid reduction of WBC count and LDH as well as an improvement of clinical status (Figure 2B). On day 26, the patient experienced a minimal sub- arachnoidal hemorrhage (grade 2). Ibrutinib was with- held but venetoclax continued. As serum levels of ibruti- nib dropped to undetectable levels, a concomitant
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