Peripheral Blood Chronic Lymphocytic Leukemia Research Articles

EMT-like Activation in CLL Provides Novel Therapeutic Target

Introduction: There is growing interest in the role of Epithelial to Mesenchymal Transition (EMT) regulators in immune cell development, but the functional role of EMT in B-cell biology and transformation remains obscured. The outcomes of the "classical" EMT activation of epithelial cells are known; committed migratory cells do not divide and become resistant to apoptotic stimuli. Importantly, EMT supports migratory cells' "stem cell-like" properties, allowing differentiation into more than one cell type and/or terminal differentiation and senescence. Chronic lymphocytic leukaemia (CLL) cells engage with antigen and microenvironment stimuli in tissue sites that lead to proliferation, followed by their egress from the tissue sites into the peripheral blood. This lifecycle implies functional changes altering CLL cell movement. Here we show EMT-like activation in CLL that correlates with disease progression and transformation. We identified a targetable candidate and show that it can be inhibited by small molecule ligands. Results: Our analysis of available expression datasets showed the expression of various EMT factors in healthy B cells. The single-cell analysis of lymph nodes confirms strong EMT-like activation in healthy B-cells (GSE159929), confirming the idea that genes involved in classical EMT would be expressed differently depending on the stage of B-cell differentiation. The following GENEVESTIGATOR analysis of B cell malignancies highlighted high EMT-like activation in CLL cases. However, differential expression (DE) analysis of peripheral blood (PB) samples from 44 CLL patients and 24 healthy B-cell donors identified six EMT factors aberrantly expressed in CLL cells compared to healthy B cells indicating significant changes in CLL EMT-like activation. At the transcription level, ZEB1 expression was higher and ZEB2 expression lower in PB CLL cells. The main stimulatory events in CLL cells appear restricted to affected tissue sites, primarily in lymph nodes (LN) where CLL cells migrate to reactivate and proliferate. Further, EMT factor protein expression was confirmed in CLL LN tissue microarrays. Strikingly, transformation into a more aggressive form (Richter syndrome) strongly correlates with an increase in ZEB2 but not ZEB1 protein expression. Interestingly, analysis of the published transcriptome data set of CLL cells co-cultured with nurse-like cells shows an increase in ZEB2 and a decrease in ZEB1 expression after 14 days (GSE13811). An apparent reliance of the EMT-like activation in CLL on the environmental stimuli has been demonstrated in CLL cells stimulated with BCR activation (anti-IgM) or mitogen (PMA), implying protection from cell death and activation of migration. Additionally, we observed higher S100A4 expression in CLL cells compared to mature B-cells from healthy individuals. Previously it has been shown that this calcium-dependent protein can control cell movement by direct interaction with non-muscle myosin and via regulation of stress fibre formation, thus facilitating cellular protrusions and increasing cell migration and invasion. Our analysis has shown the S100A4 positive correlation with CLL markers of poor prognosis, Zap70 and CD38. The S100A4 knockdown with siRNA dramatically impeded CLL migration in trans-well assay (Figure 1). Biochemical inhibitor screening for disruption of S100A4 interaction with myosin II identified several promising leads active in the low micromolar range in both biochemical and cell assays. Conclusions: Our findings improve the understanding of CLL pathobiology. Normally, healthy naïve B cells give rise to multiple B-cell subtypes with specific functions and locations. EMT-like activation likely contributes to B-cell development at different stages of differentiation and B-cell malignant transformation. Our investigation shows that the EMT-like activation is dysregulated in CLL cells compared to healthy B cells. The EMT expression signature changes upon B-cell receptor stimulation. These results suggest that targeting some of the aberrantly expressed EMT components such as S100A4 in CLL can be exploited, prolonging remission by, for example, impeding CLL returning to the tissue sites after an egress due to the treatment with Ibrutinib depriving these cells of vital microenvironmental support. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Enhanced Costimulatory Signaling Improves CAR T-cell Effector Responses in CLL.

CLL cells insufficiently activate CAR T cells, driven by low levels of costimulatory molecules on the tumor. LN-derived CLL cells are more costimulatory and mediate enhanced CAR T-cell killing. This costimulatory phenotype can be modeled via CD40 L activation, and the activated tumor promotes stronger CAR T-cell responses.

Nurselike cells sequester B cells in disorganized lymph nodes in chronic lymphocytic leukemia via alternative production of CCL21

AbstractTumor microenvironment exerts a critical role in sustaining homing, retention, and survival of chronic lymphocytic leukemia (CLL) cells in secondary lymphoid organs. Such conditions foster immune surveillance escape and resistance to therapies. The physiological microenvironment is rendered tumor permissive by an interplay of chemokines, chemokine receptors, and adhesion molecules as well as by direct interactions between malignant lymphocytes and stromal cells, T cells, and specialized macrophages referred to as nurselike cells (NLCs). To characterize this complex interplay, we investigated the altered architecture on CLL lymph nodes biopsies and observed a dramatic loss of tissue subcompartments and stromal cell networks as compared with nonmalignant lymph nodes. A supplemental high density of CD68+ cells expressing the homeostatic chemokine CCL21 was randomly distributed. Using an imaging flow cytometry approach, CCL21 mRNA and the corresponding protein were observed in single CD68+ NLCs differentiated in vitro from CLL peripheral blood mononuclear cells. The chemokine was sequestered at the NLC membrane, helping capture of CCR7-high-expressing CLL B cells. Inhibiting the CCL21/CCR7 interaction by blocking antibodies or using therapeutic ibrutinib altered the adhesion of leukemic cells. Our results indicate NLCs as providers of an alternative source of CCL21, taking over the physiological task of follicular reticular cells, whose network is deeply altered in CLL lymph nodes. By retaining malignant B cells, CCL21 provides a protective environment for their niching and survival, thus allowing tumor evasion and resistance to treatment. These findings argue for a specific targeting or reeducation of NLCs as a new immunotherapy strategy for this disease.

Identification of a Novel Proliferating Cell Fraction in Chronic Lymphocytic Leukaemia with High Expression of IgM and Chemokine Receptors

Chronic lymphocytic leukaemia (CLL) is characterised by the accumulation of malignant CD5+ B cells in the peripheral blood (PB), secondary lymphoid tissues and bone marrow. Currently considered an incurable disease, B cell receptor (BCR) signalling plays a key role in the disease aetiology as evidenced by the therapeutic success of BCR signalling inhibitors such as ibrutinib. Previous studies using incorporation of 2H-labelling of DNA in vivo demonstrated sub-clonal heterogeneity in PB CLL cell fractions sorted based on reciprocal densities of chemokine C-X-C motif receptor 4 (CXCR4) and CD5. The CXCR4 loCD5 hi fraction was shown to be enriched in recently born proliferating cells while the CXCR4 hiCD5 lo fraction consists of resting, quiescent cells thought to reflect their migratory and BCR signalling histories in tissue. Whilst these proliferating/resting fractions have since been more closely examined, the remaining bulk PB CLL population has been left relatively unexplored leaving other therapeutically relevant cell fractions undetected.Here, we have comprehensively analysed the phenotype of subpopulations of PB cells from 11 CLL patients using flow cytometry to identify activated and proliferating cell fractions. CD19 +CD5 +cells were divided into 9 fractions based on CXCR4/CD5 densities and to permit comparisons between fractions, each cell fraction was defined as containing 1-2% of the total clonal CD19 +CD5 + population. Surprisingly, we detected enrichment for Ki67+ proliferating cells and high expression of AID in the cell fraction with highest expression levels of both CXCR4 and CD5 (CXCR4 hiCD5 hi), demonstrating that CXCR4 loCD5 hi cells are not the only proliferating fraction in the blood. Moreover, we could detect mitotic cells in the CXCR4 hiCD5 hi fraction using imaging flow cytometry of a nuclear stain. This CXCR4 hiCD5 hi fraction showed the highest surface expression levels of IgM, CD86, CCR7, CXCR3 and CXCR5 of all the fractions assessed (p<0.05), indicating they are highly activated and primed for migration to lymph nodes (LNs) for further activation and proliferation.Proliferation of CLL cells is highest in secondary lymphoid tissues, however the phenotype of proliferating cells in tissue is unknown. To examine the phenotype of proliferating CLL cells in LNs, we analysed a fine-needle aspirate obtained from an enlarged cervical node using flow cytometry and compared this to a matched PB sample. Flow cytometric gates set on the PB sample were used to define and quantify LN cell fractions. Expression levels of both Ki67 and surface IgM were highest in the CXCR4 hiCD5 hi fraction which was expanded to 20% of the CD19 +CD5 + population in the LN whilst CXCR4 loCD5 hi cells (accounting for 2% of the bulk LN population) expressed very low surface IgM and Ki67 levels, suggesting CXCR4 hiCD5 hi cells may be the most proliferative cells in CLL.The CXCR4 loCD5 hi cell fraction has been shown to be a key target of ibrutinib, however the impact of ibrutinib on the CXCR4 hiCD5 hi fraction is unknown. Administration of ibrutinib to PB CLL cells for 48hr in vitro resulted in selective targeted depletion of the CXCR4 loCD5 hi fraction, as evidenced by induction of apoptotic markers in this compartment; conversely, persistent cells after 48hr ibrutinib administration in vitro were exclusively of the CXCR4 hi phenotype.In conclusion, we have identified a potentially dangerous fraction of proliferating cells in the PB of CLL patients with high expression of CXCR4, CD5, IgM, CCR7, CXCR3 and CXCR5 open for both migration to tissue and reception of BCR signals. Furthermore, CXCR4 hiCD5 hi cells in the periphery may closely mirror tissue-resident activated cell phenotypes and may represent critical targets for therapeutic intervention, particularly in high-risk CLL patients refractory to BCR inhibitor therapies. DisclosuresPatten: ROCHE: Research Funding; GILEAD SCIENCES: Honoraria, Research Funding; NOVARTIS: Honoraria; JANSSEN: Honoraria; ASTRA ZENECA: Honoraria; ABBVIE: Honoraria.

Deep Profiling of Chronic Lymphocytic Leukaemia (CLL) and Healthy Immune Cells By Mass Cytometry Resolves Impacts of Venetoclax Pressure

Deep Profiling of Chronic Lymphocytic Leukaemia (CLL) and Healthy Immune Cells By Mass Cytometry Resolves Impacts of Venetoclax Pressure

Targeting Metabolic Alterations in CLL Microenvironment; Inhibition of Glutamine Import Attenuates Venetoclax Resistance

Introduction: For chronic lymphocytic leukemia (CLL), especially in the lymph node (LN) setting where cells receive proliferative and pro-survival signals, in-depth studies of altered metabolism and its relationship with therapeutic responses are still lacking. Venetoclax, a BCL-2 inhibitor currently in wide clinical use for CLL, has shown high efficiency yet emerging resistance is a growing clinical problem. In cell line models, induced resistance to Venetoclax was accompanied by profound metabolic changes 1. This is in accordance with our earlier findings on metabolic and apoptotic changes that CLL cells undergo within the LN environment 2. In the current study, we performed RNA sequencing and applied fluxomics with 13C 6-glucose and 13C 5-glutamine to investigate in detail the metabolic routes in LN CLL. This led to studies to manipulate glutamine metabolism in a venetoclax resistance model.Methods: Peripheral blood (PB) samples from CLL patients were in vitro stimulated for 24 hrs by CD40 or B cell receptor (BCR), which are two potential key signals in LN. RNAseq analysis was compared with microarray data of paired PB/LN patient samples 3. For fluxomics, CLL cells were cultured for 2 hrs in medium containing either 5 mM 13C 6-glucose or 1 mM 13C 5-glutamine. Incorporation of 13C in metabolic intermediates was analyzed by LC-MS. For glutamine blockade, CLL cells were stimulated in presence of specific inhibitors of glutamine/glutamate metabolism or amino acid transporters. Cells were then treated with venetoclax, and viability was measured.Results: Gene expression profiles demonstrated that CLL cells obtained from LN tissue as well as after in vitro CD40 or BCR stimulation showed increased expression of gene sets involved in glycolysis, oxidative phosphorylation / citric acid cycle (OXPHOS/TCA) and amino acid metabolism as well as Myc activation. This confirmed that in vitro stimulation can be used to model the CLL LN setting. For unstimulated PB CLL cells, fluxomics data demonstrated low uptake of either glucose or glutamine, with 13C labelling close to zero for most metabolites. In contrast, both CD40 or BCR stimulation increased the uptake and utilization of glucose and glutamine. 13C labelling from glucose was detected in all glycolytic intermediates analyzed in both CD40- and BCR-stimulated CLL cells. Glucose was catalyzed to lactate and also partly converted to acetyl-CoA, which entered the TCA cycle. Additionally, labelling from glucose was also increased in several metabolites of the pentose phosphate pathway (PPP) suggesting it entered nucleotide synthetic routes. Compared to glucose, the contribution of glutamine was much higher in the TCA cycle in both BCR and CD40-stimulated cells. All intermediates of the TCA cycle were highly enriched with 13C from glutamine (Figure 1A). Combined, these data revealed that glutamine is the key metabolite to fuel the TCA cycle in LN CLL cells, and prompted us to study effects of glutamine blockade in conditions of Venetoclax resistance. It was found that venetoclax resistance induced by CD40 or BCR stimulation was clearly attenuated by glutamine uptake inhibition. CLL cells became re-sensitized to Venetoclax in both CD40- or BCR-stimulated samples, with an approximate 100-fold shift in IC50 (Figure 1B).Conclusions: Our study highlights the role of glutamine, in addition to glucose, in the metabolic reprogramming that CLL cells undergo in the LN (Figure 1C). These processes show potential for therapeutic targeting. Inhibition of glutamine import could contribute to dampen tumor microenvironment-induced Venetoclax resistance.

Richter transformation of chronic lymphocytic leukaemia: a British Society for Haematology Good Practice Paper.

Richter transformation of chronic lymphocytic leukaemia: a British Society for Haematology Good Practice Paper.

Rituximab induces rapid blood repopulation by CLL cells mediated through their release from immune niches and complement exhaustion

The in vivo rituximab effects in B cell malignancies are only partially understood. Here we analyzed in a large chronic lymphocytic leukemia (CLL) cohort (n = 80) the inter-patient variability in CLL cell count reduction within the first 24 h of rituximab administration in vivo, and a phenomenon of blood repopulation by malignant cells after anti-CD20 antibody therapy. Larger CLL cell elimination after rituximab infusion was associated with lower pre-therapy CLL cell counts, higher CD20 levels, and the non-exhausted capacity of complement-dependent cytotoxicity (CDC). The absolute amount of cell-surface CD20 molecules (CD20 density x CLL lymphocytosis) was a predictor for complement exhaustion during therapy. We also describe that a highly variable decrease in CLL cell counts at 5 h (88 %–2%) following rituximab infusion is accompanied in most patients by peripheral blood repopulation with CLL cells at 24 h, and in ∼20 % of patients, this resulted in CLL counts higher than before therapy. We provide evidence that CLL cells recrudescence is linked with i) CDC exhaustion, which leads to the formation of an insufficient amount of membrane attack complexes, likely resulting in temporary retention of surviving rituximab-opsonized cells by the mononuclear-phagocyte system (followed by their release back to blood), and ii) CLL cells regression from immune niches (CXCR4dimCD5bright intraclonal subpopulation). Patients with major peripheral blood CLL cell repopulation exhibited a longer time-to-progression after chemoimmunotherapy compared to patients with lower or no repopulation, suggesting chemotherapy vulnerability of CLL cells that repopulate the blood.

Abstract 2368: miR-29-TRAF4 axis is a novel regulator of CD40 signaling in malignant B cells

Abstract The synchronous activation of BCR and CD40 signaling via B-T cell interactions is required for proliferation of normal (Luo et al, 2018) and some malignant B cells, especially in chronic lymphocytic leukemia (CLL) and follicular lymphoma (FL). In CLL/FL cells, proliferation occurs mainly in lymph nodes, but not in bone marrow or peripheral blood which lack access to proper B-T cell interactions. Here we have analyzed the mRNA and miRNAs profile in the proliferative intraclonal CLL cell subpopulation that has recently exited lymph node niches (CXCR4dimCD5bright cells; Calissano et al,2011; Pavlasova et al,2016) to reveal molecules potentially participating in synchronous regulation of CD40 and BCR pathway. This has identified 36 miRNAs and 1370 mRNAs differentially expressed in CLL cells exiting lymph nodes as compared to resting non-proliferative CLL cells (CXCR4brightCD5dim cells). Next, we overlapped the 36 miRNAs with their predicted target mRNAs with putative function in CD40/BCR (TargetScan, KEGG) which were anti-correlated to at least one miRNA (miRNAs typically decrease mRNA stability). This revealed among others anti-correlation of lower miR-29 with higher TRAF4 levels in immune niches, which was validated in paired lymph node biopsies vs peripheral blood CLL cells (P&amp;lt;0.01). The negative regulation of TRAF4 by miR-29 was confirmed by transfection of primary CLL and MEC1 cells with synthetic miR-29, miR-29 inhibitor and by a luciferase assay with binding site from TRAF4 3'UTR. Several other miR-29 targets involved in other pathways have been identified as additional targets. TRAF4 is an understudied member of a protein family regulating receptor-induced immune cell activation, however, its role in CD40 pathway or its interactome is unknown. Co-immunoprecipitation and mass spectrometry in B cells pre- and post- stimulation by CD40 ligand revealed TRAF4 interaction with members of the CD40 pathway and a novel function in positive regulation of CD40-induced NFκB activity. In B cells, TRAF4 silencing or its over-expression affected IKKα/β phosphorylation following CD40 ligation or co-culture with activated T cells (all P&amp;lt;0.01). Notably, BCR inhibition lead in vivo to miR-29 upregulation by interfering with its negative regulator MYC induced by BCR. This leads to TRAF4 repression and drastically impairs CD40 signaling (P&amp;lt;0.001), which at least partially explains the observed anti-proliferative effects of BCR inhibitors. Altogether, we have described a novel MYC-miR-29-TRAF4 axis that regulates CD40 signaling in B cells, and acts to synchronize BCR activation with CD40 pathway. Supported by: The ERC under the European Union's Horizon 2020 research and innovation program (grant agreement No 802644), MH CZ, grant nr. NV18-03-00054 and NU20-03-00292. All rights reserved. Czech Science Foundation (20-02566S), MEYS CZ under the project CEITEC 2020 (LQ1601), MH CZ-DRO (FNBr,65269705) and MUNI/A/1595/2020. Citation Format: Sonali Sharma, Vaclav Seda, Eva Vojackova, Gabriela Mladonicka Pavlasova, Daniel Filip, Laura Ondrisova, Veronika Sandova, Lenka Kostalova, Pedro F. Zeni, Marek Borsky, Sarka Pospisilova, Medhat Shehata, Laura Z. Rassenti, Ulrich Jaeger, Michael Doubek, Matthew S. Davids, Jennifer R. Brown, Jiri Mayer, Thomas J. Kipps, Marek Mraz. miR-29-TRAF4 axis is a novel regulator of CD40 signaling in malignant B cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2368.

CCR2 Expression Signature Can Classify and Predict Outcome in a Subpopulation of Chronic Lymphocytic Leukemia (CLL) Patients

Despite the recent advances in chronic lymphocytic leukemia (CLL) treatment with the development of novel targeted agents such as Bruton's tyrosine kinase (BTK) inhibitor ibrutinib and the BCL2 antagonist Venetoclax, the disease remains incurable for most patients, and resistance mechanisms to these novel agents have already been identified. Therefore, identifying new therapeutic vulnerabilities to treat and prevent resistant tumors is of great importance. Stromal cells provide CLL cells a protective niche which increases resistance to drug-induced apoptosis. Therefore, a current challenge is to develop new strategies by targeting not only CLL cells, but also the microenvironment. We found that soluble factors can recapitulate much of the protective function of stromal cells. To understand the role of microenvironmental factors on CLL cells, we took advantage of the conditioned media (CM) collected from the stromal cell line NK-tert, which protects CLL cells from spontaneous cell death. First, we identified three major cytokines (IL6, IL8, CCL2) from the conditioned media using Proteome Profiler Human XL Cytokine Array. Next, we evaluated the effect of each cytokine on peripheral blood (PB) CLL survival (n=5, treatment naïve, unselected regarding genetic background). CLL cells were cultured ex-vivo in CM and treated with neutralizing antibody against either Il6, IL8 or CCL2 for 48hrs and found a significant decrease in cell viability in presence of anti-CCL2 antibody (median % decrease in cell viability over isotype control is 50%, p&amp;lt;0.0001). Similarly, we also found an increase in cell viability when CLL cells were cultured in RPMI in presence of recombinant-CCL2 protein than those are cultured in RPMI only, suggesting that CCL2 has an important role in CLL survival. BH3 profiling quantifies apoptotic signaling based on cytochrome c loss as a measure of mitochondrial outer membrane permeabilization (MOMP) by flow cytometry in response to BH3-only synthetic peptides that mimic pro-apoptotic BCL-2 family proteins, applied to digitonin permeabilized cells. We performed BH3 profile on PB derived -CLL samples (n=5, treatment naive) cultured in CM and exposed to either isotype control or anti-CCL2 antibody. Anti-CCL2 treated CLL samples showed an increase in MOMP in response to BIM, PUMA, MS1 and HRK peptides compared to CLL samples exposed to isotype control. This suggests that CCL2 suppresses apoptotic signaling in CLL cells. Furthermore, Dynamic BH3 profiling (DBP), a measure of drug-induced apoptotic signaling as previously described (Montero et al., Cell, 2015) showed that a combination of anti-CCL2 and venetoclax increased dependence on BCL-2 (BAD peptide)MCL-1(MS1 peptide) as well as BCL-xL (HRK peptide) in CLL cells. To our surprise, we also found a subgroup of treatment naive PB-CLL samples (CM independent) that undergo minimal spontaneous cell death in the absence of stroma support. In addition to this, IL-6, IL8, CCL2 neutralizing antibodies showed no effect on their survival. These CM independent CLL samples(n=5) are less sensitive to venetoclax treatment compared to the CLL samples that need stroma support (CM dependent). Moreover, baseline BH3 profile showed that CM independent CLL cells are less primed for apoptosis compared to CM dependent CLL cells, and exposure to venetoclax and anti-CCL2 antibody did not increase any drug-mediated apoptotic priming. Next, we checked mRNA expression level of the CCL2 receptor, CCR2 from CM dependent and CM independent CLL samples and found that CM dependent CLL cells have higher expression of CCR2, suggesting a distinct signature discriminating CM dependent and independent patients. In summary, our study shows that CCL2 may serve as a novel biomarker to target in CLL, especially in relapsed/refractory patients. Disclosures Davids: Verastem: Consultancy, Research Funding; TG Therapeutics: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Surface Oncology: Research Funding; MEI Pharma: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Gilead Sciences: Consultancy; Merck: Consultancy; Bristol Myers Squibb: Research Funding; Janssen: Consultancy; Genentech: Consultancy, Research Funding; Eli Lilly: Consultancy; Celgene: Consultancy; AstraZeneca: Consultancy, Research Funding; BeiGene: Consultancy; Ascentage Pharma: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy; AbbVie: Consultancy; Zentalis: Consultancy; Sunesis: Consultancy; Syros Pharmaceuticals: Consultancy; Research to Practice: Honoraria. Letai:Chugai: Other: Lecture Fees; Novartis: Research Funding; AbbVie: Consultancy; AstraZeneca: Consultancy; Zentalis: Membership on an entity's Board of Directors or advisory committees; Flash Therapeutics: Membership on an entity's Board of Directors or advisory committees; Dialectic: Membership on an entity's Board of Directors or advisory committees.

Abnormal Expression of BTLA and CTLA-4 Immune Checkpoint Molecules in Chronic Lymphocytic Leukemia Patients.

Chronic lymphocytic leukemia (CLL) is characterized by the peripheral accumulation of neoplastic B cells and is frequently complicated by the systemic immunosuppression associated with an impairment in B and T lymphocyte activation. We hypothesized that the expression of immune checkpoint suppressors B and T lymphocyte attenuator (BTLA) and cytotoxic T lymphocyte antigen (CTLA-4) is disturbed in both lymphocyte subpopulations in CLL. The expression of CTLA-4 and BTLA mRNA was determined by real-time PCR, while CTLA-4 protein expression (surface or intracellular) was estimated in BTLA+ lymphocytes by flow cytometry. In CLL patients, we observed a higher gene transcript level of BTLA and CTLA-4 than in healthy individuals in both freshly isolated and PMA stimulated B and T cells. Remarkably, lower amounts of both inhibitory proteins were found in peripheral blood (PB) CLL B cells, whereas normal BTLA and elevated CTLA-4 were found in T cells. Consistently, there was a prevalence of CTLA-4+ cells within circulating BTLA+ T cells cells of patients confronting PB healthy cells. After in vitro stimulation, the only change found in CLL patients was a decrease in BTLA expression in B and T lymphocytes. In contrast, healthy lymphocytes responded more vigorously as regards the BTLA and CTLA expression with substantially higher frequency of CD69+ cells under the stimulating condition compared to corresponding cells from the CLL group. Our results indicate that CLL development is associated with the affected expression of BTLA and CTLA-4 checkpoint receptors in PB and its impaired expression might be associated with lowering of the threshold for B cell activation and proliferation, while upregulated CTLA-4 expression in CLL peripheral BTLA+ T cells may contribute to suppressed T cell effector functions. This hypothesis needs to be validated in future studies, which would allow us to explain how the increased or decreased expression of these molecules affects the cell function.

Antiapoptotic Proteins mcl-1 and bcl-2 as well as Growth Factors FGF and VEGF Influence Survival of Peripheral Blood and Bone Marrow Chronic Lymphocytic Leukemia Cells

Abstract Apoptosis inhibition in chronic lymphocytic leukemia (CLL) is one of the most important mechanism in the disease onset, progression and therapy response and is dependent of interaction with different microenvironments. Aim of our paper is to determine expression of antiapoptoic proteins mcl-1 and bcl-2 in CLL cells isolated from two different compartments (peripheral blood and bone marrow) and its relation to percent of apoptotic cells and concentration of growth factors (FGF and VEGF). Our results showed that peripheral blood CLL lymphocytes have lower apoptotic rate then those isolated from bone marrow, though bone marrow CLL lymphocytes express higher levels of antipoptotic proteins bcl-2 and mcl-1. In bone marrow FGF concentration is 10-fold higher then in patients plasma but has an limited impact on mcl-1 expression. In contrary, VEGF concentration is higher in peripheral blood and corelate with percent of apoptotic cells and mcl-1 expression in this compartment. CLL cells derived from two different microenvironmets acts differently when tested for apoptosis „ex vivo“. In peripheral blood apoptosis is strongly connected with expression of antiapoptoic proteins (mcl-1 and bcl-2) and growth factors, but not in bone marrow.

CD49d promotes disease progression in chronic lymphocytic leukemia: new insights from CD49d bimodal expression

AbstractCD49d is a remarkable prognostic biomarker of chronic lymphocytic leukemia (CLL). The cutoff value for the extensively validated 30% of positive CLL cells is able to separate CLL patients into 2 subgroups with different prognoses, but it does not consider the pattern of CD49d expression. In the present study, we analyzed a cohort of 1630 CLL samples and identified the presence of ∼20% of CLL cases (n = 313) characterized by a bimodal expression of CD49d, that is, concomitant presence of a CD49d+ subpopulation and a CD49d− subpopulation. At variance with the highly stable CD49d expression observed in CLL patients with a homogeneous pattern of CD49d expression, CD49d bimodal CLL showed a higher level of variability in sequential samples, and an increase in the CD49d+ subpopulation over time after therapy. The CD49d+ subpopulation from CD49d bimodal CLL displayed higher levels of proliferation compared with the CD49d− cells; and was more highly represented in the bone marrow compared with peripheral blood (PB), and in PB CLL subsets expressing the CXCR4dim/CD5bright phenotype, known to be enriched in proliferative cells. From a clinical standpoint, CLL patients with CD49d bimodal expression, regardless of whether the CD49d+ subpopulation exceeded the 30% cutoff or not, experienced clinical behavior similar to CD49d+ CLL, both in chemoimmunotherapy (n = 1522) and in ibrutinib (n = 158) settings. Altogether, these results suggest that CD49d can drive disease progression in CLL, and that the pattern of CD49d expression should also be considered to improve the prognostic impact of this biomarker in CLL.

Linking Microenvironmental Signals to Metabolic Switches and Drug Responses in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) has become a paradigm for a cancer that depends on signals from the microenvironment. In lymph nodes (LN), CLL cells receive from surrounding cells proliferative and pro-survival signals, which can protect against chemotherapy and also the Bcl-2 inhibitor venetoclax (VEN). To avoid drug resistance, combination of VEN with the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib (IBR) is currently explored. IBR has the potency to drive CLL cells out of the LN to eventually die by VEN-induced apoptosis. The activation status in the LN likely affects tumor metabolism, though it is yet unclear how, and whether such putative metabolic changes can be linked to VEN resistance. In this study,we aimed to investigate the altered metabolism of CLL cells in the tumor microenvironment (TME), what signals determine these changes, and how to counteract VEN resistance at the metabolic level. We compared the metabolic status of CLL cells in LN biopsy material and paired peripheral blood (PB) CLL cells. Both higher mitochondrial mass and glucose uptake (flow cytometry) were found in CLL cells derived from LN as compared to PB. To determine which TME signals affect metabolism, we mimicked 3 LN signals: CD40, B cell receptor (BCR) and toll-like receptor (TLR) signaling.Invitrostimulation of PB CLL was followed by mitochondrial mass and glucose uptake (flow cytometry), oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measured on Seahorse XF Analyzer. The datademonstrated that CD40 but surprisingly not BCR or TLR signaling recapitulated the metabolic changes observed in LN cells. Next, two sets of metabolomics were performed for samples both in vitro and in vivo. Firstly, PB samples of 10 patients were stimulated with/without CD40L for 48 hours and analyzed by mass-spec for metabolic intermediates. In parallel, metabolomics was performed on PB samples from 13 patients sampled before and after 3 months of ibrutinib treatment. The data show various metabolic pathways are activated in the TME, particularly citric acid (TCA) cycle, pyruvate metabolism, glycolysis, and fatty acid metabolism. Apart from these general changes, the highest-ranking shifts in metabolites point to involvement of amino acids to fuel the TCA cycle, which in turn drives oxidative phosphorylation (OXPHOS). Overall, CD40 signaling results in increased glycolysis, but more dominantly, increased OXPHOS, while IBR in fact has opposing effects (Figure 1), indicating that TME-driven metabolic alterations are mitigated by IBR treatment. In order to link the altered metabolic state to VEN resistance, PB-derived cells were treated with OXPHOS inhibitors during CD40 stimulation. Remarkably, OXPHOS inhibition by oligomycin (ATP synthase inhibitor) did not affect CLL activation, yet counteracted strongly for VEN resistance. Of several BCL-2 family members induced by CD40 ligation, both anti-apoptotic MCL-1 and BCL-XL were downregulated by oligomycin. These data suggest that OXPHOS inhibition affects CD40 signals to counteract VEN resistance. In conclusion, metabolic changes of CLL in LN are recapitulated by CD40 signal, while IBR treatment shows opposite effects, together providing indirect insight into the LN metabolism. In LN, most of the key metabolic pathways are enhanced, particularly OXPHOS. Finally, we found OXPHOS inhibition reverses CLL VEN resistance. Our findings link CLL metabolism in LN microenvironment to VEN resistance, and may provide novel clues for therapeutic targeting in the treatment of VEN resistance patients. Disclosures Forconi: Roche: Honoraria; Gilead Sciences: Research Funding; Abbvie: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Menarini: Consultancy; Novartis: Honoraria; Janssen-Cilag: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau. van der Windt:Genmab: Employment. Eldering:Celgene: Research Funding; Janssen Pharmaceutical Companies: Research Funding; Roche: Research Funding.

Treatment with Acalibrutinib, Ibrutinib and CD19 CAR T Cells Restore the Number of Granulocytic Myeloid Derived Supressor Cells in CLL-Bearing Mice

The complex crosstalk between malignant chronic lymphocytic leukemia (CLL) cells and the tumor microenvironment (TME) is not fully understood. CLL is associated with an inflammatory TME and T cells exhibit exhaustion and multiple functional defects, fully recapitulated in Eµ-TCL1 (TCL1) mice and induced in healthy mice by adoptive transfer (AT) of murine CLL cells, making it an ideal model to test novel immunotherapies for this disease. Myeloid-derived suppressor cells (MDSCs), a non-leukemic cell type within the TME, are immature myeloid cells with the ability to suppress T cell function and promote Treg expansion. In humans, CLL cells can induce conversion of monocytes to MDSCs provoking their accumulation in peripheral blood (PB). MDSCs include two major subsets granulocytic (Gr) and monocytic (M)-MDSC. In mice, Gr-MDSCs are defined as CD11b+Ly6G+Ly6Clo and M-MDSC as CD11b+Ly6G-Ly6Chi. Both murine and human MDSCs express BTK. We observed that in CLL-bearing mice, MDSCs cells are lost in PB as disease progresses. Treatment with both BTK inhibitors (BTKi), ibrutinib (Ibr) and acalabrutinib (Acala), result in shift of T cell function from Th2 towards Th1 polarity and increase MDSC populations in vivo. We aimed to determine whether combination treatment with BTKi and chimeric antigen receptor (CAR) T cells renders recovery of the MDSC population in CLL-bearing mice. To address this question we designed a two-part experiment, aiming to mimic the clinically relevant scenario of pre-treatment of CLL with BTKi to improve CAR T cell function. Part 1 of our experiment consisted of 4 groups (n=12) of 2.5 month old C57/Bl6 mice. Three groups had AT with 30x106 TCL1 splenocytes. A fourth group of WT mice remained CLL-free as a positive control and donors for WT T cells. When PB CLL load reached &gt;10% (day 14) animals were randomized to either Ibr or Acala at 0.15 mg/l in 2% HPBC or no treatment for 21 days. All animals from part 1 were culled at day 35 post-AT and splenic cells were isolated, analyzed and used to manufacture CAR T cells. WT, CLL, Ibr and Acala treated T cells were activated and transduced with a CD19-CD28 CAR to treat mice in part 2. Here, 50 WT mice were given AT with 20x106 TCL1 splenocytes for CLL engraftment. All mice were injected with lymphodepleting cyclophosphamide (100mg/kg IP) one day prior to IV CAR injection. At day 21 post-AT, mice were treated with WT CAR, CLL CAR, IbrCAR, AcalaCAR or untransduced T cells. MDSC sub-populations were monitored weekly in PB and SP were analysed by flow cytometry. As malignant CD19+CD5+ cells expands in PB, the overall myeloid (CD19-CD11b+) cell population was not affected, but MDSCs significantly decreased (p&lt;0.0001). Treatment with Acala, but not Ibr restores total MDSCs. However, MDSC impairment occurs in the Gr- but not M- MDSC population and both Acala and Ibr restores this population (Figure 1a). When we examined the spleen, treatment with both Ibr (p&lt;0.001) and Acala (p&lt;0.001) reduced CD5+CD19+ cells, whereas neither BTKi affected the overall myeloid (CD19-CD11b+) cell population. Gr-MDSCs were restored by both treatments whilst M-MDSCs were only restored after Ibr treatment (p&lt;0.001 in each case). In part 2 of this experiment we observed that treatment with all CAR-T cell groups provokes the clearance of all CD19+CD5+ cells. The overall CD19-CD11b+ population stays the same across all mice groups 35 days after treatment in PB with any group of CAR and untransduced T cells. Overall MDSC population is maintained following all CAR T cells compared to CLL-bearing mice (p&lt;0.0001) and it is the Gr- but not the M- MDSC population which is recovered in PB (Figure 1b). These parts of the experiments can of course be influenced by treatment with cyclophosphamide. We conclude that novel therapies for CLL treatment have an effect not only in CLL cells but also in non-malignant cell components of the TME. In this animal model of CLL, the rapid expansion of CLL cells in PB and secondary lymphoid organs provokes loss of MDSC, particularly the Gr-MDSC subpopulation is affected. Treatment with BTKi and CAR T cells provokes clearance of CLL cells in PB and spleen allowing MDSC recovery; suggesting this may be BTK and ITK independent. We continue to explore secondary lymphoid organs to further characterize the shift of the CLL microenvironment from an immunosuppressive to an immune effective one and its impact on immune function in this model. Disclosures Sanderson: Kite/Gilead: Honoraria. Gribben:Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Acerta/Astra Zeneca: Consultancy, Honoraria, Research Funding.

A multicenter phase 1 study of plerixafor and rituximab in patients with chronic lymphocytic leukemia

CXCR4 directs chronic lymphocytic leukemia (CLL) trafficking within protective tissue niches, and targeting CXCR4 with plerixafor may enhance drug sensitivity. We performed a phase 1 dose escalation study of plerixafor (NCT00694590) with rituximab in 24 patients with relapsed/refractory CLL. Patients received rituximab 375 mg/m2 on days 1, 3, and 5, followed by bi-weekly rituximab plus dose-escalated plerixafor for 4 weeks. The maximum tolerated dose of plerixafor was 320 µg/kg. The most common toxicities were fatigue (13 patients, 57%), nausea (11, 48%), chills (10, 43%), and diarrhea and dyspnea (seven, 30% each). No patients developed symptomatic hyperleukocytosis or tumor lysis syndrome. A median 3.3-fold increase (range 1.2–12.4) in peripheral blood CLL was seen following the first dose of plerixafor, confirming CLL cell mobilization. The overall response rate was 38% and correlated with higher doses of plerixafor. Plerixafor is well-tolerated in patients with CLL; further tumor sensitization studies with CXCR4 antagonists are warranted.

PS1125 LINKING MICROENVIRONMENTAL SIGNALS TO METABOLIC SWITCHES AND IBRUTINIB RESPONSE IN CHRONIC LYMPHOCYTIC LEUKEMIA

Background:Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous B cell malignancy, with accumulation of clonal CD5+ B cells in blood, lymph nodes (LN) and bone marrow. CLL has become a paradigm for a cancer that depends on signals from the microenvironment. This is evidenced by the clinical success of the Btk inhibitor ibrutinib, which drives CLL cells out of the LN to eventually die by neglect. In peripheral blood, CLL cells are quiescent, non‐cycling, with low metabolic activity. In contrast, in LN CLL cells receive various signals from surrounding cells, crucial for proliferation, and protection from chemotherapeutic drugs. These acquired advantages are possibly linked with altered metabolism, but it is unclear if and how CLL cells change their metabolism in the LN environment.Aims:We aimed to investigate the altered metabolism of CLL cells in the tumor microenvironment, and what signals determine these changes.Methods:Glucose uptake, glycolysis, mitochondrial mass, oxidative phosphorylation and lipid uptake were measured by flow cytometer or Seahorse XF96 analyzer, and 90 metabolites were analyzed by LC mass‐spectrometry.Results:We first established that the metabolic activity profile of circulating recent LN emigrants (CXCR4high/CD5dim PB lymphocytes) resembled that of actual LN residents (obtained by a core biopsy), which showed both higher mitochondrial mass and glucose uptake than paired peripheral blood CLL cells. In vitro stimulation of isolated blood‐derived CLL samples demonstrated that CD40 but not BCR signaling recapitulate these metabolic changes. Next, 10 patient samples were stimulated with/without CD40L for 48 hours and analyzed by mass‐spec for metabolic intermediates. To draw a further parallel with the situation in LN, metabolomics was performed on 13 patients sampled before and after 3 months of ibrutinib treatment. Combined, the data shows that various metabolic pathways are activated in the tumor microenvironment, particularly TCA cycle, pyruvate metabolism, glycolysis, and fatty acid metabolism. Apart from these overall changes, the highest ranking shifts in metabolites point to involvement of amino acids to fuel the TCA cycle. Moreover, aberrant patterns in metabolic changes after ibrutinib were noted in some patients, possibly linked with clinical response.Summary/Conclusion:CD40 signaling leads to increased oxidative phosphorylation and glycolysis in the CLL microenvironment. Opposite changes were observed in ibrutinib treated samples, together providing indirect insight into the metabolism in the CLL LN.

PF358 FUNCTIONAL HIERARCHY AMONG PRO-SURVIVAL BCL-2 MEMBERS IN DETERMINING RESISTANCE TO VENETOCLAX IN CLL

Background: The Bcl-2 inhibitor Venetoclax rapidly induces apoptosis in chronic lymphocytic leukemia (CLL) cells in peripheral blood (PB) whereas lymph node (LN) responses are less complete. This may be linked to pro-survival signals in the LN, where micro-environmental signals strongly induce Bcl-XL, Mcl-1 and Bfl-1 which are not targeted by Venetoclax. In some patients resistance occurs over time which sometimes can be explained by acquired mutations, but in most cases the mechanism(s) remain unknown. Aims: We hypothesize that changes in expression or shifts in functional interactions among Bcl-2 family members are key to clinical response and refractory disease. Methods: Intracellular FACS staining for Bcl-2, Mcl-1 and Bcl-XL in circulating recent LN emigrants (CD19+/CXCR4high/CD5dim) versus returning (CD19+/CXCR4llow/CD5high) lymphocytes in patient samples treated with Venetoclax. To investigate the functional impact of expression of Bcl-2 family members, profiling with additional BH3 mimetics against Bcl-XL (A1331852) and Mcl-1 (S63845) was applied in a CLL LN model. Primary CLL cells were co-cultured with fibroblasts expressing CD40L in order to mimic T cell help in the LN. Results: Under Venetoclax treatment, levels of Bcl-2 itself and other family members may increase in some patients, early in clinical response. During short-term in vitro incubation with Venetoclax or other BH3 mimetics, we established that Mcl-1 and Bfl-1 can increase but not Bcl-2 or Bcl-XL. The effect on Mcl-1 and Bfl-1 can be explained by a stabilization of these normally short-lived proteins. In vitro CD40 activation renders CLL cells fully resistant to single Venetoclax treatment. Using this model, we find that dual or triple BH3 mimetic combinations targeting Bcl-2, Bcl-XL and Mcl-1 restored killing. Specifically, whereas the IC50 of dual Bcl-2+Mcl-1 targeting is >1uM, combined inhibition of Bcl-2+Bcl-XL has an IC50 around 0.3uM. Bcl-2 and Bcl-XL thus seem to be the most dominant players. However, even when Bcl-2, Bcl-XL and Mcl-1 were inhibited using triple BH3 mimetic treatment, Venetoclax resistance was not fully reversed to unstimulated control (IC50 1 versus 10 nM), highlighting a potential role for Bfl-1. To further dissect the functional roles, Bim co-immunoprecipitation was performed. In PB CLL cells, Bim only binds to Bcl-2 and upon Venetoclax treatment it is released and targets Bax/Bak to kills cells. After CD40 stimulation, Bim shifts predominantly to Bcl-XL and less to Mcl-1. Only when applying triple BH3 mimetic combinations targeting Bcl-2, Bcl-XL and Mcl-1, Bim starts to bind Bfl-1. These observations suggest a hierarchical model in which Bcl-2 is most important and where upon Venetoclax treatment, Bcl-XL and Mcl-1 come into play. When all of these are targeted by triple BH3 mimetics, Bfl-1 is engaged to intercept apoptosis. Summary/Conclusion: We applied BH3 mimetics in primary CLL and showed that combined targeting of Bcl-2 members can abrogate resistance to Venetoclax. Moreover, we dissected the roles of the different Bcl-2 family members and constructed a hierarchical model of Bcl-2 family members, providing new insights into Venetoclax resistance which may have clinical utility.

PIM kinase inhibitor, AZD1208, inhibits protein translation and induces autophagy in primary chronic lymphocytic leukemia cells.

The PIM1, PIM2, and PIM3 serine/threonine kinases play a role in the proliferation and survival of cancer cells. Mice lacking these three kinases were viable. Further, in human hematological malignancies, these proteins are overexpressed making them suitable targets. Several small molecule inhibitors against this enzyme were synthesized and tested. AZD1208, an orally available small-molecule drug, inhibits all three PIM kinases at a low nanomolar range. AZD1208 has been tested in clinical trials for patients with solid tumors and hematological malignancies, especially acute myelogenous leukemia. The present study evaluated the efficacy and biological actions of AZD1208 in chronic lymphocytic leukemia (CLL) cells. CLL cells had higher levels of PIM2 protein and mRNAs than did normal lymphocytes from healthy donors. Treatment of CLL lymphocytes with AZD1208 resulted in modest cell death, whereas practically no cytotoxicity was observed in healthy lymphocytes. To determine the mechanism by which AZD1208 inhibits PIM kinase function, we evaluated PIM kinase pathway and downstream substrates. Because peripheral blood CLL cells are replicationally quiescent, we analyzed substrates involved in apoptosis, transcription, and translation but not cell cycle targets. AZD1208 inhibited protein translation by decreasing phosphorylation levels of 4E-binding protein 1 (4E-BP1). AZD1208 induced autophagy in replicationally-quiescent CLL cells, which is consistent with protein translation inhibition. These data suggest that AZD1208 may elicit cytotoxicity in CLL cells through inhibiting translation and autophagy induction.

SHIP1 Inhibition As Novel Therapeutic Approach in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is one of the most common B cell malignancies in the Western world. Malignant B cells are blocked from differentiating into immunoglobulin producing-plasma cells and clonally accumulate in the spleen, lymph nodes, bone marrow and peripheral blood. CLL is characterized by immunosuppression throughout all disease stages, which is mediated by increased numbers of myeloid-derived suppressor cells (MDSCs), regulatory T cells (Jitschin and Braun et al., Blood 2014) and direct inhibitory effects of the malignant CLL cells on T cells (Christopoulos etal., Blood 2011).Over the past decade, significant improvement in understanding the pathogenesis of CLL has highlighted the importance of active B cell receptor (BCR) signaling. This has revealed promising targeted treatment options, including the small molecule inhibitors targeting the phosphatidylinositol-3-kinase (PI3K) signaling pathway. Idelalisib and Duvelisib are under clinical investigation for CLL treatment, however, treatment-related toxicities are limiting their application and none of these approaches are curative, highlighting the importance of functional anti-tumor immune responses in CLL for prolonged treatment efficacy.Here, we are testing a novel approach that aims to selectively target CLL B cells and simultaneously restore an appropriate immune cell function. The phosphatase SH2-domain-containing inositol 5ʹ-phosphatase 1 (SHIP1) serves as negative feedback molecule and downregulates PI3K signaling in B cells by dephosphorylating the 5'phosphate of Phosphatidylinositol (3,4,5)-trisphosphate. We hypothesize that CLL cells rely on such negative regulators for optimal survival and can only tolerate a maximum signaling level. We are therefore testing whether SHIP1 inhibition induces hypersignaling and thereby CLL cell death. Furthermore, we are investigating whether SHIP1 inhibition simultanously stimulates immune responses, as it has been shown to induce expansion of murine hematopoietic and mesenchymal stem cell compartments (Brooks et al., Stem cells 2014). 3α-Aminocholestane (3AC) is a small molecule inhibitor of SHIP1 and can be used for pharmacological inhibition.First, we investigated the expression and phosphorylation levels of SHIP1 in CLL. We found SHIP1 to be expressed at various levels in CLL peripheral blood and strongly phosphorylated compared to age-matched healthy donors. Besides, SHIP1 transcription is upregulated in lymph nodes as compared to peripheral blood, which is in line with the assumption of increased BCR signaling in secondary lymphoid organs. We then set out to investigate the consequences of SHIP1 phosphatase inhibition. Similarly, to recent findings in acute lymphoblastic leukemia (Chen et al., Nature 2015), pharmacological inhibition of SHIP1 lead to rapid cell death of CLL cells. We further investigated the mode of cell death and observed specific features of apoptosis, namely caspase 3/7 activation and phosphatidylserine exposure on the outer cell membrane. This has been tested on primary CLL patient samples and T cell leukemia/lymphoma 1 (TCL1)-driven murine CLL cells and was not observed or significantly less pronounced in other lymphoma cell lines or healthy primary B cells. To confirm the specificity of the observed effects, we genetically activated AKT with a GFP reporter in the TCL1-driven mouse model in vivo and in vitro. By tracking GFP-expressing CLL cells, we observed an initial expansion followed by rapid cell death in vitro. When we induced AKT activation in vivo, GFP+ CLL cells were not detectable in the peripheral blood, total CLL count declined upon induction and we found decreased tumor burden in the secondary lymphoid organs, particularly in the lymph nodes. In addition to the direct effects on CLL cells, we sought to investigate the impact of SHIP1 inhibition on other immune cell functions. We observed that SHIP1 inhibition lowers the activity threshold of T cells: When we stimulated a reporter cell line with suboptimal doses of anti-CD3, 3AC treatment significantly enhanced the response rate.Therefore, we propose SHIP1 as a novel interesting target in CLL. In contrast to kinase inhibition and downregulation of the BCR signaling strength, phosphatase inhibition and BCR signaling overaction provides an attractive new treatment strategy for elimination of malignant CLL cells and stimulation of immune responses. DisclosuresNo relevant conflicts of interest to declare.