Circulating Chronic Lymphocytic Leukemia Cells Research Articles

Measurement of Trogocytosis: Quantitative Analyses Validated with Rigorous Controls.

Trogocytosis is a process in which receptors on acceptor cells remove and internalize cognate ligands from donor cells. Trogocytosis has a profound and negative impact on mAb-based cancer immunotherapy, as seen in the treatment of chronic lymphocytic leukemia (CLL) with CD20 mAbs, such as rituximab (RTX) and ofatumumab (OFA). Our clinical observations of RTX/OFA-mediated loss of the CD20 target from circulating CLL cells have been replicated in our in vitro studies. Here we describe flow cytometry and fluorescence microscopy experiments, which demonstrate that acceptor cells, such as monocytes/macrophages that express FcγR, remove and internalize both antigen and donor cell-bound cognate IgG mAbs for several different mAb-donor cell pairs. Fluorescent mAbs and portions of the plasma cell membrane are transferred from donor cells to acceptor cells, which include the THP-1 monocytic cell line as well as freshly isolated monocytes. We describe rigorous controls to validate the reactions and eliminate dissociation or internalization as alternative mechanisms. Trogocytosis is likely to contribute to neutropenia, thrombocytopenia, and liver damage associated with use of antibody-drug conjugates. The methods we have described should allow for examination of strategies focused on blocking trogocytosis and its adverse effects. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Trogocytosis of mAb-opsonized donor cells mediated by adherent THP-1 cells Alternate Protocol: Application of fluorescence microscopy to examine THP-1 cell-mediated trogocytosis Support Protocol 1: Alexa labeling of mAbs and determination of F/P ratios Support Protocol 2: Standard washing procedure Support Protocol 3: Labeling and opsonization of cells Basic Protocol 2: Trogocytosis mediated by human monocytes as acceptor cells Support Protocol 4: Isolation of human monocytes Basic Protocol 3: Trogocytosis mediated by THP-1 cells in solution Support Protocol 5: Retinoic acid treatment of THP-1 cells Support Protocol 6: Culturing of SCC-25, BT-474, MOLT-4 and THP-1 cell lines.

The CXCR4dim/CD5bright Proliferative Fraction Reappears in Relapsed CLL Under Ibrutinib, Enriched in BTK Mutations

BTK inhibitors deeply affect the proliferative capacity, homing and migrations of chronic lymphocytic leukemia (CLL) cells, and cause their redistribution from nodal compartments to the blood stream. Reciprocal expression of CXCR4 and CD5 on circulating CLL cells is an established marker discriminating the proliferative fraction (CD5high/CXCR4dim, PF), recently egressed from the lymph node, from the resting fraction (CXCR4high/CD5dim, RF) of older, quiescent cells navigating the blood stream. We monitored the dynamics of the PF under the course of ibrutinib therapy in 31 CLL cases recruited in the IOSI-EMA-001 study (NCT02827617) with sequential blood sampling (156 samples, median 6 samples/case, range 3-7). PF was monitored through fixed gates, designed on the pre-treatment sample around the population visually detaching from the main cluster (panel A). Longitudinal analysis showed a depletion of the PF over time, from a median of 17.7% at pre-treatment to 4.75, 0.4, 1.5, 0.6, 1.5% at subsequent timepoints (0.5, 6, 12, 18, 24 months; range 0-50; p < 0.001, U test). Loss of proliferative potential was confirmed by concurrent loss of Ki67 expression. We retrospectively analyzed the PF from a second cohort of 99 ibrutinib-treated CLL cases from the real world, referred for routine immunophenotyping (278 samples, median 3 samples/case, range 2-7). Median PF of pre-ibrutinib samples was 12.0% (range 0.7-50%), with a significant drop to 2.7% after 1 year of treatment and to 1.0% after 2 years (p<0.001, U test). Out of 99 cases, 55 discontinued ibrutinib within 5 years (16 toxicity, 38 progression/relapse, 1 death; median time of treatment 29.7 months, range 1-58.6 months). Median PF in samples collected after ibrutinib discontinuation was 1.15% after 2 years of treatment, and increased to 13.3, 7.10, 6.30, 12.0% after 3, 4, 5 and >5 years. Conversely, samples collected under ibrutinib treatment presented a more stable trend of median PF (3.0 and 3.5% after 3 and 4 years respectively). Interestingly, of 27 patients with blood samples collected during the third year of therapy, 11 failed ibrutinib due to relapse/progression within the following two years: these cases presented a higher median PF (10.0%) than cases who stayed on ibrutinib (median PF 1.8%, p=0.045). Mutations of BTK/PLCG2 were detected in 28/99 cases (28.2%); among these, 18 (64.2%) discontinued ibrutinib due to progression/relapse (17/18) or toxicity (1/18), whereas 10 cases, although mutated, remained on treatment. Median time of appearance of BTK/PLCG2 mutations was 48 months (range 16-70). Median VAF was 22.8 (range 1.2-99.0) and 3.5 (range 1.7-17.6) for BTK and PLCG2, respectively. We isolated the PF and RF fractions from 10 cases developing BTK/PLCG2 mutations, who showed reappearance of the PF after prolonged ibrutinib treatment (median 50.7 months). Sequencing revealed 1.7 mean BTK mutations per sample (range 1-4), with a median VAF higher in the PF (23.0%, range 0.3-92.3) than in the RF (5.15%, range 0.1-84.8; p=0.0027, paired rank test); overall, the PF VAF was almost 3 times larger than the RF (average 2.82, range 0.01-38). Accompanying PLCG2 mutations were present in 6 cases, generally more frequent (average 3.7 mutations per case, range 1-8) but with lower burden (median VAF 1.7%, range 0.1-4.3) than BTK mutations; again, average the PF fraction had higher VAF than the RF (average PF: 2.0, range 0.1-6.2; average RF: 1.35, range 0.4-2.8; p=0.0142) (panel B). Finally, we performed mRNA-seq on 6 cases relapsed after ibrutinib, isolating the RF and the newly reconstituted PF, the latter enriched in BTK mutations, with matching fractions isolated from pre-ibrutinib samples. Differential expression signature of PF vs. RF at pre-treatment was concordant with published gene sets (e.g. Calissano et al. Mol Med 2011); application of this signature to post-ibrutinib PF/RF fractions drove co-clustering of 12 out of 13 fractions with their respective pre-treatment counterparts, suggesting that the reconstituted PF fraction at relapse is functionally similar to the original pre-treatment PF. Overall, our data support the hypothesis that, in patients undergoing ibrutinib treatment, BTK/PLCG2 mutations arise within the lymph nodes, the proliferative compartment of CLL; clinically, longitudinal monitoring of the CXCR4/CD5 fractions by flow cytometry may provide a simple tool helping to intercept CLL progression under ibrutinib therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

B-cell Receptor Signaling Induced Metabolic Alterations in Chronic Lymphocytic Leukemia Can Be Partially Bypassed by TP53 Abnormalities.

It has been unclear what role metabolism is playing in the pathophysiology of chronic lymphocytic leukemia (CLL). One reason is that the study of CLL metabolism is challenging due to the resting nature of circulating CLL cells. Also, it is not clear if any of the genomic aberrations observed in this disease have any impact on metabolism. Here, we demonstrate that CLL cells in proliferation centers exhibit upregulation of several molecules involved in glycolysis and mitochondrial metabolism. Comparison of CXCR4/CD5 intraclonal cell subpopulations showed that these changes are paralleled by increases in the metabolic activity of the CXCR4lowCD5high fraction that have recently egressed from the lymph nodes. Notably, anti-IgM stimulation of CLL cells recapitulates many of these metabolic alterations, including increased glucose uptake, increased lactate production, induction of glycolytic enzymes, and increased respiratory reserve. Treatment of CLL cells with inhibitors of B-cell receptor (BCR) signaling blocked these anti-IgM-induced changes in vitro, which was mirrored by decreases in hexokinase 2 expression in CLL cells from ibrutinib-treated patients in vivo. Interestingly, several samples from patients with 17p-deletion manifested increased spontaneous aerobic glycolysis in the unstimulated state suggestive of a BCR-independent metabolic phenotype. We conclude that the proliferative fraction of CLL cells found in lymphoid tissues or the peripheral blood of CLL patients exhibit increased metabolic activity when compared with the bulk CLL-cell population. Although this is due to microenvironmental stimulatory signals such as BCR-engagement in most cases, increases in resting metabolic activity can be observed in cases with 17p-deletion.

NOTCH1 Mutation in CLL Enhances Cell Cycle G1/S Transition through Specific Cyclin Overexpression: Preclinical Ground for CDK4/6 Targeted Inhibition

Introduction: The partially understood biological consequences of the NOTCH1 acquired lesion, seems to be distinctive enough among chronic lymphocytic leukemia (CLL) patients, as clinical studies have repeatedly found specific features: intermediate prognosis, anti-CD20 poorer responses, and a higher frequency of trisomy 12 and Richter transformation. Though located in a different domain, the activating nature of NOTCH1 mutation in T lymphoblastic leukemia relies on cell cycle regulators. In fact, pivotal studies, from the pre-next generation sequencing era, showed dysregulation of cyclins-gene expression, as driver of the unique CLL features. Thus, our goal was to revisit the cell cycle in CLL, but focusing now in the NOTCH1 mutated subset (NOTCH1MUT), hypothesizing that biological differences versus wild type cases (NOTCH1WT) would explain the clinical ones, and exploiting potential differences with targeted molecules in vitro.Methods: From 2010 to 2019, presentation bone marrow aspirates or blood samples DNA was collected during the diagnostic workout from 378 CLL patients, all of them annotated by next generation sequencing. G 0/early-G 1 effectors gene expression was measured by RT-qPCR in negatively immunoselected circulating CLL cells. A siRNA approach was selected for silencing by electroporation 7 NOTCH1WT and 2 NOTCH1MUT cases. Cell cycle and apoptosis flow cytometry assays were performed on cultured fresh primary cells from n? NOTCH1MUT and 4 NOTCH1WT cases, before and after exposure to different concentrations of palbociclib, a CDK4/6 inhibitor.Results: We found that 37/378 (9.8%) of patients harbored a NOTCH1 mutation. NOTCH1MUTcases presented with higher lymphocyte counts [NOTCH1MUT 17.2 x10 9/L vs. NOTCH1WT 9.7 x10 9/L; p=0.042], trisomy 12 (35.1% vs. 11.4%; p<0.001) and a higher frequency of an unmutated IGHV status (70% vs. 21%; p<0.001). Of note, NOTCH1MUT patients had poorer responses to anti-CD20 based schemes than NOTCH1WT patients (35.7 vs. 69.8% complete response; p). We found that NOTCH1MUT cases showed a relevant increase of 38-fold change (FC) for CCND3, 27-FC for CDK4 and CCND2, 11-FC for CCND1 and 9-FC for CDK6 gene expression in negatively immunoselected circulating CLL cells at diagnosis. In addition, NOTCH1MUT cases displayed a statistically significant higher percentage of cells in the S phase than the wild type cases (21% vs. 1%, p=0.004). Though significance was not met, NOTCH1MUT cases showed a higher percentage of events within G 2-M (28% vs. 26 %). Next, we incorporated the flow cytometry assay to in vitro palbociclib treated CLL cells from 3 NOTCH1MUT and 4 NOTCH1WT cases. Five days after culture stimulation, cells were exposed to 38 and 76 μM (dose range for reaching maximum CLL cells sensitivity plateau) of the drug for 48 hours. As stated above, NOTCH1MUT cases were characterized by a much higher proportion of cells in S phase at baseline (21%), which was reduced in a dose dependent manner to an 8% and a 6% after exposure to palbociclib, respectively. The standard 48-72 hours drug assay may not be the most suitable for slow growth tumors as CLL and, in particular, for testing cell cycle inhibitors. Thus, we designed an assay for two cell cycles based on the average population doubling time of the primary cell culture experiments (0.6 in 72 hours), and using the mean steady state plasma concentration of palbociclib achieved clinically: 1 μM. After 120 hours, the baseline 15% of cells in S phase was reduced to an 0.64% after exposure to palbociclib 1 μM in NOTCH1MUT cases and a 1.8x-increase in the percentage dead cells was noted, compared with NOTCH1WT cases.Conclusions: Compared with NOTCH1WT CLL cases, we describe an overexpression of effectors of early phase in NOTCH1MUT. This profile made NOTCH1MU cells more suited to enter and traverse through the cell cycle and could explain, in part, the proliferative clinical-biological features of this subset of patients and opening a window for exploiting therapeutically these differences. Ours experiments in vitro with palbociclib sets the ground for the clinical research. [Display omitted] DisclosuresJerez: BMS: Consultancy; Novartis: Consultancy; GILEAD: Research Funding.

A Pilot Study of Lower Doses of Ibrutinib in Patients (pts) with Chronic Lymphocytic Leukemia (CLL)

A Pilot Study of Lower Doses of Ibrutinib in Patients (pts) with Chronic Lymphocytic Leukemia (CLL)

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

INTRODUCTION. The Bcl-2 inhibitor Venetoclax provides profound reductions in circulating chronic lymphocytic leukemia (CLL) cells in the majority of patients. The lymph node (LN) responses are less complete, which may be linked with pro-survival signals in the LN. Bcl-XL expression is prominently increased in LN compared to blood. Since Bcl-XL is a prime determinant of resistance to Venetoclax, its regulation is of clinical significance. In the present study, we investigated how candidate LN signals from T helper cytokines IL-21 or IL-4 affect CD40-signalling, specifically in relation to sensitivity/resistance to Venetoclax.RESULTS In an expression screen of changes in apoptotic regulators following CD40 stimulation, IL-4 and IL-21 had opposing effects specifically on Bcl-XL expression. This was associated with CD40-induced resistance to Venetoclax which was augmented by IL-4 and reversed by IL-21. We investigated the signalling pathway(s) involved and found that upon IL-4 or IL-21 stimulation, STAT3 and -6 were differentially phosphorylated, and pharmacological inhibition of upstream JAK kinases restored Bcl-XL expression. Molecularly, IL-21 or IL-4 signals did not significantly affect (non-)canonical NF-κB activation measured by DNA-binding of p65 or p52. 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 synergy (STAT6) or competition (STAT3) with NF-κB. We next applied novel IKKα and NIK inhibitors to inhibit NF-kB signalling during CD40 stimulation. Especially IKKα inhibition specifically blocked Bcl-XL expression, without affecting overall viability of the CLL cells or normal T cells.Importantly, both types of NF-κB inhibitors significantly reversed CD40-induced resistance for Venetoclax. Averaged LD50 values for Venetoclax increased approximately 2500-fold from 1.8 nM to 4.6 μM after 24 hrs CD40 stimulation, but in the presence of IKKα inhibitor only to 7.4 nM (N=8). This value remains well below the average plasma Venetoclax level in CLL patients undergoing treatment.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 signals, providing novel therapeutic clues in case of emerging resistance to targeted drugs such as Venetoclax. DisclosuresKater:Celgene: Consultancy, Research Funding; Johnson & Johnson: Research Funding; Abbvie: Research Funding. Eldering:Roche: Research Funding; Gilead: Research Funding; Celgene: Research Funding.

αvβ3 Integrin Expression and Mitogenic Effects by Thyroid Hormones in Chronic Lymphocytic Leukemia.

Background: Chronic lymphocytic leukemia (CLL) is the most common adult leukemia. The thyroid hormones, T3 and T4, bind the αvβ3 integrin and activate phosphorylates ERK (pERK). These tumor-promoting actions were reported in a number of malignancies, but not in CLL. Methods: Primary cells from 22 CLL patients were verified for disease markers (CD5/CD19/CD23) and analyzed for αvβ3 by flow cytometry (FC), ImageStream, Western blots (WB), and immunohistochemistry (IHC) in archival bone marrow (BM, n = 6) and lymph node (LN, n = 5) tissues. Selected samples (n = 8) were incubated with T3 (1–100 nM) or T4 (0.1–10 µM) for 30 min, and the expression levels of αvβ3, pERK and PCNA (cell proliferation marker) were determined (WB). Results: αvβ3 was detected on the membrane of circulating CLL cells and in the BM but not in the LN. T3 and T4 enhanced αvβ3 protein levels in primary CLL cells. Similarly, pERK and PCNA were rapidly induced in response to T3 and T4 exposure. Conclusions: αvβ3 integrin is expressed on primary CLL cells and is induced by thyroid hormones. We further suggest that the hormones are mitogenic in these cells, presumably via αvβ3-mediated signaling.

In vivo, ex vivo and in vitro dasatinib activity in chronic lymphocytic leukemia.

Dasatinib inhibits the breakpoint cluster region-Abelson murine leukemia 1 (BCR-ABL1) gene along with other kinases known to be overexpressed and abnormally active in patients with chronic lymphocytic leukemia (CLL). The current study used primary leukemic cells obtained from 53 patients with CLL that were treated with dasatinib. A 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay and Annexin V staining was performed to assess the cytotoxic effects of dasatinib treatment. The XTT assay revealed that the median cytotoxicity of dasatinib was 8.30% (range, 0.00–77.89%). Due to high dispersion of dasatinib activity, patients were divided into sensitive (n=27; 50.94%; median cytotoxicity, 22.81%) and resistant groups (n=26; 49.06%; median cytotoxicity, 0.00%). A median cytotoxicity of 8.30% was selected as a cut off value. Using Annexin V staining and flow cytometry on exemplary sensitive and resistant CLL samples, it was revealed that 17.71 and 1.84% of cells were apoptotic, respectively. The current study presented a case of a patient with concomitant occurrence of CLL and chronic myeloid leukemia (CML) with a major molecular response after dasatinib treatment. A simultaneous reduction of circulating CLL cells indicated in vivo anti-CLL activity induced by dasatinib. After an in vitro culture of the patient's mononuclear cells with subsequent dasatinib treatment, a higher percentage of CLL cells undergoing apoptosis was obsevered when compared with untreated samples (38.19 vs. 21.99%, respectively). Similarly, the percentage of CLL apoptotic cells (ΔΨmlow) measured by chloromethyl-X-rosamine was higher after incubation with dasatinib (7.28%) than in the negative control (2.86%). In conclusion, dasatinib induced antileukemic effects against CML and CLL cells. The results of the current study indicated that dasatinib may induce apoptosis ex vivo, in vitro and in vivo in CLL.

VLS-101, a ROR1-Targeting Antibody-Drug Conjugate, Demonstrates a Predictable Safety Profile and Clinical Efficacy in Patients with Heavily Pretreated Mantle Cell Lymphoma and Diffuse Large B-Cell Lymphoma

VLS-101, a ROR1-Targeting Antibody-Drug Conjugate, Demonstrates a Predictable Safety Profile and Clinical Efficacy in Patients with Heavily Pretreated Mantle Cell Lymphoma and Diffuse Large B-Cell Lymphoma

Modulation of the Tumor Suppressor Protein PP2A Using a Small Molecule Agonist Overcomes Multi-Drug Resistance through Mitochondrial Permeability Transition Pore (MPTP) Dependent Induction of Apoptosis in Chronic Lymphocytic Leukemia

The advent of molecularly targeted therapies has revolutionized the treatment of Non-Hodgkin's Lymphoma (NHL), including Chronic Lymphocytic Leukemia (CLL). Venetoclax (VEN, an inhibitor of Bcl-2) and ibrutinib (IBR, an inhibitor of BTK) generated excellent clinical responses in CLL patients singly and even more effectively in combination (Portell et al. Blood, 2019; Tam et al.NEJM, 2018). Despite its efficacy, the majority of VEN or VEN+IBR responses are partial and resistance usually develops. We previously reported that treatment with microenvironmental agonists ex vivo can generate anti-apoptotic resistance to the VEN+IBR combination in CLL cells via NF-kB-dependent upregulation of multiple anti-apoptotic proteins (Mcl-1 and Bcl-xL) (Jayappa et al. Blood Adv, 2017). Here, we report that circulating CLL cells of lymph node origin (CD69positive) exhibit resistance to several BH-domain antagonists (inhibitors of Bcl-2, Mcl-1, and Bcl-xL) when used as single agents. Apoptosis resistance was shown to occur at a pre-mitochondrial level by insufficient activation of Bax/Bak proteins (Fig. 1A-B). Supportive of these findings, ex vivo treatment of primary CLL cells with agonists (sCD40L, TLR9 agonist CpG-ODN, and IL10) known to be expressed in the cancer microenvironment in vivoalso resulted in apoptosis restriction due to defective Bax/Bak activation. Our molecular studies suggest that this resistance is driven by the upregulation of anti-apoptotic proteins, which generates an intracellular environment in which single-drug treatments allow pro-apoptotic proteins (e.g. Bim) to swap between upregulated anti-apoptotic proteins (Mcl-1/Bcl-xL/Bcl-2), leading to defective Bax/Bak activation. Hence, therapies aimed at depleting this reservoir must block multiple anti-apoptotic proteins simultaneously or bypass Bax/Bak-dependent apoptosis. Several cancers, including NHL, have evolved mechanisms to suppress the activity of Protein Phosphatase 2A (PP2A), a serine/threonine phosphatase known to regulate cell survival/proliferation. In our current study involving various cancer cell lines (~250), a small molecule agonist of PP2A (SMAP, TRC-382) showed broad activity across blood cancer cell lines. A further pharmacologically optimized SMAP compound (DT061), also known to be safe in animal models (Tohmé et al. JCI Insight, 2019), was effective even in blood cancer cell lines and agonist treated CLL samples resistant to several BH-domain antagonists (inhibitors on Mcl-1, Bcl-xL, and Bcl-2), suggesting that PP2A activation could overcome pre-mitochondrial apoptosis restriction. DT061 was able to overcome drug resistance in patient-derived CD69positive CLL cells through the induction of Bax/Bak-independent apoptosis (Fig. 1C). Supportive of this finding, DT061 was also able to induce apoptosis in Bax/Bak double knockout CLL cell line (MEC1) (Fig. 1D). To determine the mechanisms underlying SMAP-induced apoptosis, we examined additional pathways capable of triggering apoptosis such as mitochondrial permeability transition pore (MPTP), calcium channels, and VDAC1 using selected small molecule inhibitors. Only inhibitors blocking MPTP (NIM811 or cyclopsorin-A/CspA) significantly impaired the DT061- but not VEN-induced apoptosis in primary CLL cells (Fig. 2A-B). Additionally, our analysis using the CalceinAM/CoCl2 method revealed that DT061 was able to induce MPTP opening in primary CLL cells, which was inhibited in the presence of NIM811 or CspA, suggesting that DT061 induces apoptosis through MPTP activation (Fig. 2C). In summary, we identify, in the circulation of treatment-naïve and treated CLL patients, microenvironmentally-activated leukemic B cells that exhibit apoptosis resistance at the level of Bax/Bak activation. Therapies aimed at depleting this reservoir of drug-resistant leukemic B cells must trigger a process of Bax/Bak independent apoptosis. We demonstrate that PP2A activation using the SMAP (DT061) induces apoptosis in drug resistant CLL cells bypassing the Bax/Bak pathway. The apoptosis induction was dependent on MPTP activation. Collectively, this work highlights the existence of an anti-apoptotic multi-drug resistant pool of CLL cells in patients, and validates a novel pharmaceutically tractable pathway to deplete this reservoir. Disclosures Williams: Janssen: Research Funding; Pharmacyclics: Research Funding; TG Therapuetics: Research Funding; Celgene: Honoraria; Gilead: Honoraria; TG Therapeutics: Honoraria; Abbvie: Honoraria; Kite: Honoraria; Xian Janssen: Honoraria. Portell:Xencor: Research Funding; Roche/Genentech: Consultancy, Research Funding; Infinity: Research Funding; TG Therapeutics: Research Funding; AbbVie: Research Funding; Pharmacyclics: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Bayer: Consultancy; BeiGene: Consultancy, Research Funding; Kite: Consultancy, Research Funding; Acerta/AstraZeneca: Research Funding. Narla:The Icahn School of Medicine at Mount Sinai: Patents &amp; Royalties: nternational Application Numbers: PCT/US15/19770, PCT/US15/19764; and US Patent: US 9,540,358 B2; Mount Sinai: Other: Mount Sinai is actively seeking commercial partners for the further development of the technology. G.N. has a financial interest in the commercialization of the technology.; RAPPTA Therapeutics: Consultancy, Current equity holder in private company.

CLL intraclonal fractions exhibit established and recently acquired patterns of DNA methylation

AbstractIntraclonal subpopulations of circulating chronic lymphocytic leukemia (CLL) cells with different proliferative histories and reciprocal surface expression of CXCR4 and CD5 have been observed in the peripheral blood of CLL patients and named proliferative (PF), intermediate (IF), and resting (RF) cellular fractions. Here, we found that these intraclonal circulating fractions share persistent DNA methylation signatures largely associated with the mutation status of the immunoglobulin heavy chain locus (IGHV) and their origins from distinct stages of differentiation of antigen-experienced B cells. Increased leukemic birth rate, however, showed a very limited impact on DNA methylation of circulating CLL fractions independent of IGHV mutation status. Additionally, DNA methylation heterogeneity increased as leukemic cells advanced from PF to RF in the peripheral blood. This frequently co-occurred with heterochromatin hypomethylation and hypermethylation of Polycomb-repressed regions in the PF, suggesting accumulation of longevity-associated epigenetic features in recently born cells. On the other hand, transcriptional differences between paired intraclonal fractions confirmed their proliferative experience and further supported a linear advancement from PF to RF in the peripheral blood. Several of these differentially expressed genes showed unique associations with clinical outcome not evident in the bulk clone, supporting the pathological and therapeutic relevance of studying intraclonal CLL fractions. We conclude that independent methylation and transcriptional landscapes reflect both preexisting cell-of-origin fingerprints and more recently acquired hallmarks associated with the life cycle of circulating CLL cells.

STAT3-Induced Wnt5a Provides Chronic Lymphocytic Leukemia Cells with Survival Advantage.

The wingless and integration site growth factor-5a (Wnt5a) is a ligand of the receptor tyrosine kinase-like orphan receptor-1 (ROR1). Because both Wnt5a and ROR1 are expressed in circulating chronic lymphocytic leukemia (CLL) cells, and because in other cell types, STAT3, which is constitutively activated in CLL, induces Wnt5a signaling, we wondered whether STAT3 induces the expression of Wnt5a in CLL cells. Sequence analysis detected four putative STAT3 binding sites in close proximity to the Wnt5a gene promoter's start codon. Chromatin immunoprecipitation and EMSA revealed that STAT3 binds to the Wnt5a gene promoter, and a luciferase assay showed that STAT3 activates the Wnt5a gene. Additionally, transfection of peripheral blood CLL cells with STAT3 short hairpin RNA downregulated Wnt5a mRNA and protein levels, suggesting that STAT3 binds to the Wnt5a gene promoter and induces the expression of Wnt5a in CLL cells. Flow cytometry and confocal microscopy determined that both Wnt5a and its receptor ROR1 are coexpressed on the surface of CLL cells, and Western immunoblotting showed an inverse correlation between Wnt5a and ROR1 protein levels, implying that, regardless of CLL cells' ROR1 levels, blocking the interaction between Wnt5a and ROR1 might be beneficial to patients with CLL. Indeed, transfection of CLL cells with Wnt5a small interfering RNA reduced Wnt5a mRNA and protein levels and significantly increased the spontaneous apoptotic rate of CLL cells. Taken together, our data unravel an autonomous STAT3-driven prosurvival circuit that provides circulating CLL cells with a microenvironment-independent survival advantage.

Ibrutinib and Venetoclax Target Distinct Subpopulation of CLL Cells: Rationale for Drug Combination and Implication of Minimal Residual Disease Eradication

Ibrutinib (Ibr) is a specific inhibitor of Bruton tyrosine kinase, a component of the B-cell receptor (BCR) signaling. Venetoclax (Veneto) inhibits the anti-apoptotic protein BCL2. Both drugs are highly effective as monotherapy against chronic lymphocytic leukemia (CLL) (Byrd NEJM, Roberts, NEJM, 2015 and Roberts, Blood, 2019) and clinical trials using the combination therapy are ongoing. An interesting clinical observation is that the tumor cell sensitivity to these two drugs differ between different anatomic compartments. While lymph node-resident CLL cells are more sensitive to ibr, circulating CLL cells in the peripheral blood are more sensitive to the action of veneto. When these two drugs are used in combination to treat relapsed/refractory (Hillman ASH 2018 and Kater EHA 2019) or previously untreated CLL patients (Jain NEJM 2019), rate of complete response significantly increases compared to single drug alone. Moreover, negativity of bone marrow minimal residual disease continues to improve over time. However, the reason behind these observed compartmental responses is largely unknown, and we investigated the differential response using an ex vivo model that promotes CLL proliferation (CLL proliferation model). A better understanding of how these two drugs synergize would eventually help develop other rational combination strategies. We established the ex vivo model using fibroblasts derived from the bone marrow of a CLL patient. In this model, CLL cells closely interact with the bone marrow fibroblasts (BMF) (Fig.1) and divide and proliferate for several generations, as measured by CFSE labeling. The culture, in some cases, may continue for 10 weeks before cell death ensues. With this ability to promote long-term cell proliferation, the model recapitulates one of the most salient features observed histologically in the "proliferation centers" of the CLL lymph nodes. With the proliferation model, we first tested the effects of ibrutinib on cell division/proliferation as well as cell viability in samples taken from 30 CLL patients consisting of 22 ibr-naïve (thus sensitive) and 8 ibr-relapsed (thus resistant) patients. We demonstrate that there was lack of a significant inhibition of ibr on cell viability. In comparison, Ibr markedly inhibited cell proliferation in cells from ibr-naïve/sensitive patients (p&amp;lt;0.0001), while such inhibition was much less prominent in cells from ibr-relapsed/resistant patients (p=0.053). Overall, our data demonstrate that only the dividing subpopulation of CLL responds to ibr, and this response is highly correlated with patients' clinical response to the drug while cell viability responses are not. With the CLL proliferation model, we also evaluated how veneto works. Veneto, as expected, markedly decreased cell viability at clinically achievable concentrations. CFSE profiles of the remaining live cells, however, revealed an interesting pattern of distribution, showing that veneto induces cell death but it preferentially kills the resting CLL population (Fig. 2). In other words, the resting subpopulation of CLL, instead of the dividing one, preferentially responds to veneto as compared to ibr. Combination of the two drugs, as expected, worked most effectively, significantly reducing the total number of live cells, both resting and dividing subpopulations, in all cases. Analyses of CFSE profiles became less meaningful in many of these cases due to the small number of live cells remaining after the combination treatment. In conclusion, our study, comparing the actions of ibr and veneto in a clinically relevant CLL proliferation model, demonstrates for the first time that the drugs target distinct subpopulations of CLL cells with different proliferative capacity. The data also suggest that single drug therapy may leave a subpopulation behind that would potentially rise to cause a future relapse. Our study provides a strong laboratory rationale explaining why combining these two drugs has the potential to eradicate CLL disease. The findings are also in line with the clinical observations regarding the compartmental response; ibrutinib works primarily on the lymph nodes where CLL cells divide in the "proliferation centers" and veneto acts preferentially on the circulating CLL cells that are mostly non-dividing and resting. Disclosures Wang: Incyte Reaserch Institute: Employment. Ma:Abbvie: Research Funding; Xeme: Research Funding; Novartis: Research Funding; Bioverativ: Consultancy; Janssen: Consultancy, Speakers Bureau; Beigene: Research Funding; Kite: Consultancy; Gilead: Research Funding; Pharmacyclics: Consultancy, Research Funding, Speakers Bureau; Acerta: Research Funding; Astra Zeneca: Consultancy, Research Funding, Speakers Bureau; Genentech: Consultancy; Incyte: Research Funding; Juno: Research Funding. Larson:Celgene: Consultancy; Agios: Consultancy; Novartis: Honoraria, Other: Contracts for clinical trials.

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

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.

Wnt5a Induces ROR1-Dependent Upregulation of MMP9 and Enhanced Invasiveness in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) cells recirculate between blood and lymphoid tissue compartments, where they receive growth/survival signals from accessory cells within the lymphoid-tissue microenvironment. Such trafficking requires leukemia cells to pass through endothelial barriers and to transverse the extracellular matrix (EM), a process that is facilitated by matrix metallopeptidases (MMP). We co-cultured CLL cells with marrow mesenchymal stromal cells (MSCs) at a physiologic oxygen tension (e.g., 5% O2 in N2). Under such conditions we found that MSCs could enhance leukemia-cell expression of MMP9, a 92 kDa type IV collagenase and key MMP involved in EM degradation. Unexpectedly, however, we found circulating CLL cells with high-level expression of the onco-embryonic protein, ROR1 (designated ROR1Pos), also had high-level expression of MMP9 despite not having immediate contact with such accessory cells. Moreover, we found that circulating ROR1Pos CLL (N = 12) had significantly greater levels of MMP9 than blood CLL cells with low-to-negligible levels of ROR1 (ROR1Neg CLL, N = 10) (P &lt; 0.001). Short-term culture of ROR1Pos CLL in serum-free media significantly reduced their expressed levels of MMP9, unless we added exogenous Wnt5a, a non-canonical Wnt factor and ligand for ROR1 that we found expressed at significantly higher levels in the plasma of patients with CLL than in age-matched adults (Yu et al., J Clin Invest, 2015). Treatment of ROR1Pos CLL cells with Wnt5a enhanced their expression and release of MMP9 and their capacity to invade Matrigel in a Boyden-Chamber Assay; such effects could not be inhibited by inhibitors of B-cell receptor/chemokine signaling (e.g. Ibrutinib), but could be blocked by cirmtuzumab, a humanized mAb specific for ROR1 that can inhibit leukemia-cell ROR1-signaling in patients with CLL (Choi et al., Cell Stem Cell, 2018). Silencing expression of MMP9 with siRNA or treatment with a MMP9-specific inhibitor (CAS 1177749-58-4) could inhibit the capacity of Wnt5a to enhance the invasive capability of CLL cells, indicating that MMP9 plays a major role in facilitating CLL-cell invasiveness. Targeted siRNA-mediated silencing of cytoskeletal proteins HS1/cortactin, which complex with ROR1 in response to Wnt5a (Hasan et al., Leukemia, 2018; Hasan et al., Leukemia, 2017), also impaired the capacity of Wnt5a to enhance expression and release of MMP9 in ROR1Pos CLL. Collectively, these studies indicate that Wnt5a can induce ROR1-signaling leading to enhanced expression of MMP9, which can facilitate invasion of the EM. Moreover, inhibitors of ROR1 signaling, such as cirmtuzumab, can repress Wnt5a-induced CLL-cell expression of MMP9, potentially impairing the homing of ROR1Pos CLL to their protective niches within the lymphoid microenvironment. Disclosures Kipps: AstraZeneca, Inc.: Membership on an entity's Board of Directors or advisory committees; Verastem: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Velos-Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Jannsen Pharmaceutical Companies of Johnson &amp; Johnson: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Ibrutinib Therapy Releases Leukemic Surface IgM from Antigen Drive in Chronic Lymphocytic Leukemia Patients.

In chronic lymphocytic leukemia (CLL), disease progression associates with surface IgM (sIgM) levels and signaling capacity. These are variably downmodulated in vivo and recover in vitro, suggesting a reversible influence of tissue-located antigen. Therapeutic targeting of sIgM function via ibrutinib, an inhibitor of Bruton tyrosine kinase (BTK), causes inhibition and tumor cell redistribution into the blood, with significant clinical benefit. Circulating CLL cells persist in an inhibited state, offering a tool to investigate the effects of drug on BTK-inhibited sIgM. We investigated the consequences of ibrutinib therapy on levels and function of sIgM in circulating leukemic cells of patients with CLL. At week 1, there was a significant increase of sIgM expression (64% increase from pretherapy) on CLL cells either recently released from tissue or persisting in blood. In contrast, surface IgD (sIgD) and a range of other receptors did not change. SIgM levels remained higher than pretherapy in the following 3 months despite gradual cell size reduction and ongoing autophagy and apoptotic activity. Conversely, IgD and other receptors did not increase and gradually declined. Recovered sIgM was fully N-glycosylated, another feature of escape from antigen, and expression did not increase further during culture in vitro. The sIgM was fully capable of mediating phosphorylation of SYK, which lies upstream of BTK in the B-cell receptor pathway. This specific IgM increase in patients underpins the key role of tissue-based engagement with antigen in CLL, confirms the inhibitory action of ibrutinib, and reveals dynamic adaptability of CLL cells to precision monotherapy.See related commentary by Burger, p. 2372.

Ibrutinib reprograms the glucocorticoid receptor in chronic lymphocytic leukemia cells.

Glucocorticoid (GC) receptor (GR) phosphorylation and signature genes were studied in chronic lymphocytic leukemia (CLL) cells to help place GCs within modern treatment algorithms. In contrast to normal B and T cells, transcription of GC-regulated genes was not rhythmic and the synthetic GC dexamethasone (DEX) could not inhibit toll-like receptor (TLR)-responses in CLL cells. This intrinsic GC-resistance was associated with aberrant GR-phosphorylation on activating Ser211 and inhibitory Ser226 sites. Ibrutinib increased transcription of the GR-signature gene GILZ in circulating CLL cells along with GR(pS211)/GR(pS226) ratios and lytic sensitivity to DEX that were not reversed by the competitive antagonist mifepristone in vitro. However, ibrutinib could not improve GR-responses in circulating CLL cells activated with IL2 and TLR7/8 agonists to mimic conditions in pseudofollicle microenvironments. Addition of the janus kinase inhibitor ruxolitinib to block ibrutinib-insensitive signals increased GILZ transcription in pseudofollicle conditions in vitro and in a clinical trial (NCT02912754), and also increased GR(S211)/GR(S226) ratios and DEX-mediated killing in patient samples in vitro. These observations suggest that intrinsic resistance to endogenous GCs is characteristic of CLL cells and ibrutinib may help increase the therapeutic activity of GCs by non-canonical activation of GR.

CLL Cells with 13q- Are Characterized By High BCL2 Levels and CLL Cells with Trisomy-12 By Increased Activation of STAT3 and NF-κB

CLL Cells with 13q- Are Characterized By High BCL2 Levels and CLL Cells with Trisomy-12 By Increased Activation of STAT3 and NF-κB

Reduction of Tumor Burden Rather Than Off-Target Effects Drives Changes in T-Cell Number and Profile during Prolonged Ibrutinib Treatment in Relapsed or Refractory Chronic Lymphocytic Leukemia Patients

In this study, we analyzed the changes occurring in the peripheral blood mononuclear cells (PBMC) during long-term treatment with the Bruton's tyrosine kinase inhibitor ibrutinib in relapsed or refractory (R/R) chronic lymphocytic leukemia (CLL) patients (pts). PBMC populations were assessed by flow-cytometry including CLL cells, Natural Killer (NK) cells, T-cell memory subsets, helper subpopulations (Ths) and regulatory T cell (Tregs) subsets. Peripheral blood samples were collected before start of treatment and at week (wk) 4, 10, 16, 22 and at 8, 12, 24 and 36 months (mo) of treatment.Eleven pts were included in the study, 8 males and 3 females, with median age 73 years. Nine age- and sex-matched healthy individuals were included as controls. Comparisons between pts and controls were done with the non-parametric Mann-Whitney U test; between follow-up time points and baseline in pts by Wilcoxon rank test for paired comparisons. Simple regression analyses and the Pearson´s rank test were used to estimate the relationship between different cell populations.Eight pts had high-risk and 3 intermediate-risk disease according to the modified Rai staging system; 8/11 had 17p deletion or TP53 mutation. Three pts had mutated IgHV genes, 3 unmutated, 2 had borderline mutational status (97-98% identity), and 3 were not assessed. Median number of previous treatments was 1 (range 1-4) and median lymphocyte count 45 x 109/L (range 3.1-109.5). At 12 mo, overall response rate was 100%. All pts achieved partial response, with 63% fulfilling all complete response criteria (Hallek M et al, Blood 2018), but for minimal residual disease negativity. Eight pts were still on treatment at 24 mo, 7 pts at 36 mo. Treatment discontinuation was due to: death (n=1), allogeneic transplantation (n=1), disease progression (n=1) and toxicity (n=1). In 3 pts, ibrutinib dose was reduced due to toxicity; the remaining pts stayed on full-dose (420 mg/day p.o.) during the whole treatment period. At study entry, eight pts had hypogammaglobulinemia but none had ongoing IgG replacement therapy. Of these, 5 started IgG replacement therapy during treatment. All 11 patients experienced G2 infections.After an initial increase in circulating CLL cells due to redistribution lymphocytosis, a gradual reduction occurred during treatment becoming significant at wk 16 (p<0.005) (Fig. 1A). As we previously reported (Palma M et al, ASH abs 4322, 2017), at study entry CD8+cells were significantly higher compared to controls (p=0.0005). CD8+cells started to decrease at wk 10 (p=0.02) and normalized (i.e. reached values not significantly different from controls) by 12 mo (Fig. 1B). On the other hand, CD4+T cells, at baseline not significantly different from controls, decreased also gradually from wk 16, reaching by 24 mo levels below normal (p=0.02) (Fig.1D).We show here that the reduction of CD8+ cells correlated with that of CLL cells (r=0.45, p< 0.0001) (Fig. 1C) and that the correlation between the number of CD4+ cells and CLL cells was even more significant (r=0.66, p< 0.0001) (Fig.1E). The expression of the immune checkpoints PD-1 and CTLA-4, at baseline aberrantly high on both CD4+ and CD8+ cells and mainly in the antigen-experienced subsets, decreased gradually during treatment.Among the helper subsets, Th1 (CCR6-/ CXCR3+) cells were at baseline higher compared to controls (p=0.0003), while Th2 (CCR6-/ CXCR3-) did not significantly differ (Fig.2A-B). As expected, this resulted in a Th1/Th2 ratio significantly higher than in controls (median 2.6 vs 0.26, p<0.0001) (Fig.2C). During treatment, both Th1 and Th2 cells decreased. Th1 normalized by 12 mo, while Th2 cells decreased below normal levels at 12 mo (p=0.006). However, Th1/Th2 ratio never normalized during 36 mo of treatment. The strongest direct correlation was between the decrease of Th1 cells and CLL cells (r=0.68, p< 0.0001). During treatment, NK cells decreased and also Tregs (CD4+/CD25+/ CCR4+/CD127low), in particular the Ag-experienced non-activated subset (CD45RO+HLA-DR-).In conclusion, the reduction of tumor burden directly correlated with the reduction of all T-cell subsets, but most significantly with those subsets involved in the antitumor immune response, i.e. CD8+cells and, among the CD4+subset, Th1 cells. Whether these changes also compromised the anti-infection immunity is difficult to assess, due to the fact that most of the pts had also underlying hypogammaglobinemia. [Display omitted] DisclosuresPalma:Takeda Pharma AB: Research Funding. Österborg:Gilead: Consultancy, Research Funding; Beigene: Research Funding; Pharmacyclics: Research Funding; Janssen: Research Funding; Abbvie: Research Funding. Mellstedt:Kancera AB: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sandoz: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria; Astra-Zeneca: Equity Ownership; Novo Nordisk: Equity Ownership; Global Health Sciences: Speakers Bureau.

Potent anti-leukemic activity of a specific cyclin-dependent kinase 9 inhibitor in mouse models of chronic lymphocytic leukemia.

Onset of progression even during therapy with novel drugs remains an issue in chronic lymphocytic leukemia (CLL). Thus, there is ongoing demand for novel agents. Approaches targeting cyclin-dependent kinases (CDK) have reached the clinical trial stage. CDK9 mediating RNA transcriptional elongation is the evolving pivotal CLL CDK inhibitor target. However, more CDK9 selective compounds are desirable. Here, we describe the CDK9 inhibitor LDC526 displaying a low nanomolar biochemical activity against CDK9 and an at least 50-fold selectivity against other CDKs. After demonstrating in vitro MEC-1 cell line and primary human CLL cell cytotoxicity we evaluated the LDC526 in vivo effect on human CLL cells transplanted into NOD/scid/γcnull (NSG) mice. LDC526 administration (75 mg/kg) for 5 days resulted in a 77% reduction of human CLL cells in NSG spleens compared to carrier control treatment. Next, we longitudinally studied the LDC526 impact on circulating CLL cells in the TCL1 transgenic mouse model. LDC526 (50 mg/kg) administration for two days led to a 16-fold reduction of blood CLL cell numbers. Remarkably, residual CLL cells exhibited significantly increased intracellular BCL-2 levels. However, the LDC526 cytotoxic effect was not restricted to CLL cells as also declining numbers of normal B and T lymphocytes were observed in LDC526 treated TCL1 mice. Taken together, our in vivo data provide a strong rational for continued LDC526 development in CLL therapy and argue for the combination with BCL-2 inhibitors.