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Chronic Lymphocytic Leukemia (CLL) has a complex pattern of driver mutations and much of its clinical diversity remains unexplained. We devised a method for simultaneous subgroup discovery across multiple data types and applied it to genomic, transcriptomic, DNA methylation and ex-vivo drug response data from 217 Chronic Lymphocytic Leukemia (CLL) cases. We uncovered a biological axis of heterogeneity strongly associated with clinical behavior and orthogonal to the known biomarkers. We validated its presence and clinical relevance in four independent cohorts (n=547 patients). We find that this axis captures the proliferative drive (PD) of CLL cells, as it associates with lymphocyte doubling rate, global hypomethylation, accumulation of driver aberrations and response to pro-proliferative stimuli. CLL-PD was linked to the activation of mTOR-MYC-oxidative phosphorylation (OXPHOS) through transcriptomic, proteomic and single cell resolution analysis. CLL-PD is a key determinant of disease outcome in CLL. Our multi-table integration approach may be applicable to other tumors whose inter-individual differences are currently unexplained.

The expression of ZAP-70 in a subset of chronic lymphocytic leukemia (CLL) patients strongly correlates with a more aggressive clinical course, although the exact underlying mechanisms remain elusive. The ability of ZAP-70 to enhance B-cell receptor (BCR) signaling, independently of its kinase function, is considered to contribute. We used RNA-sequencing and proteomic analyses of primary cells differing only in their expression of ZAP-70 to further define how ZAP-70 increases the aggressiveness of CLL. We identified that ZAP-70 is directly required for cell survival in the absence of an overt BCR signal, which can compensate for ZAP-70 deficiency as an antiapoptotic signal. In addition, the expression of ZAP-70 regulates the transcription of factors regulating the recruitment and activation of T cells, such as CCL3, CCL4, and IL4I1. Quantitative mass spectrometry of double–cross-linked ZAP-70 complexes further demonstrated constitutive and direct protein-protein interactions between ZAP-70 and BCR-signaling components. Unexpectedly, ZAP-70 also binds to ribosomal proteins, which is not dependent on, but is further increased by, BCR stimulation. Importantly, decreased expression of ZAP-70 significantly reduced MYC expression and global protein synthesis, providing evidence that ZAP-70 contributes to translational dysregulation in CLL. In conclusion, ZAP-70 constitutively promotes cell survival, microenvironment interactions, and protein synthesis in CLL cells, likely to improve cellular fitness and to further drive disease progression.

Acalabrutinib is a highly selective, potent, next-generation, covalent Bruton tyrosine kinase inhibitor with minimal off-target activity. Matching-adjusted indirect comparisons (MAICs) were performed to estimate the safety and efficacy of acalabrutinib compared to other targeted therapies for treatment-naïve patients with chronic lymphocytic leukemia (CLL). Individual patient data for acalabrutinib (ELEVATE-TN trial) were matched to aggregate baseline characteristics for comparators. After matching, acalabrutinib (with or without obinutuzumab) showed improved safety outcomes, except for increased risk of neutropenia (

Chronic lymphocytic leukemia (CLL) is an indolent B cell malignancy invariably infiltrating the bone marrow. Although treatment options for patients with advanced disease have significantly improved in the past years, the disease remains incurable and after emergence of therapy resistant disease patients succumb to infections due to secondary bone marrow failure. The underlying mechanisms impairing normal hematopoiesis in patients with CLL are poorly defined., We would like to express our deepest gratitude to patients who donated blood for this research. Samples were obtained with assistance from the Cambridge Blood and Stem Cell Biobank, funded by the Cambridge Cancer Centre and Cambridge Stem Cell Institute. This work was funded by the Cancer Research UK (CRUK; C49940/A17480). I.R. is a senior CRUK fellow. E.L. is supported by a Sir Henry Dale fellowship from Wellcome/Royal Society (107630/Z/15/Z). Research in E.L.’s laboratory is supported by Wellcome, BBSRC, EHA and Royal Society. Research in I.R. and E.L. laboratories is supported by core support grants by Wellcome and MRC to the Wellcome-MRC Cambridge Stem Cell Institute.

Pathogenesis of chronic lymphocytic leukaemia (CLL) is contingent upon antigen receptor (BCR) expressed by malignant cells of this disease. Studies on somatic hypermutation of the antigen binding region, receptor expression levels and signal capacity have all linked BCR on CLL cells to disease prognosis. Our previous work showed that the src-family kinase Lck is a targetable mediator of BCR signalling in CLL cells, and that variance in Lck expression associated with ability of BCR to induce signal upon engagement. This latter finding makes Lck similar to ZAP70, another T-cell kinase whose aberrant expression in CLL cells also associates with BCR signalling capacity, but also different because ZAP70 is not easily pharmacologically targetable. Here we describe a robust method of measuring Lck expression in CLL cells using flow cytometry. However, unlike ZAP70 whose expression in CLL cells predicts prognosis, we find Lck expression and disease outcome in CLL are unrelated despite observations that its inhibition produces effects that biologically resemble the egress phenotype taken on by CLL cells treated with idelalisib. Taken together, our findings provide insight into the pathobiology of CLL to suggest a more complex relationship between expression of molecules within the BCR signalling pathway and disease outcome.

The crucial dependence of chronic lymphocytic leukemia (CLL) cells on signals derived from the B cell receptor (BCR) has encouraged the development of new inhibitors, which interfere with BCR signaling and demonstrate clinical benefits in nearly all patients. In addition, signaling through Toll-like receptor (TLR) 9 of the innate immune system has been shown to further contribute to the activation of CLL cells. However, responses to TLR9 engagement are not uniform, but diametrically opposed with cell death in some patients and cell proliferation in others. We now provide evidence that heterogeneous responses to TLR agonists are related to differences in the ability of CLL cells to activate the BCR-associated kinase Syk. Notably, expression of ZAP-70 appears to be of crucial importance for TLR9-mediated activation of Syk. We show that the activation of Syk provides an antiapoptotic signal, which is independent of Mcl-1, Bcl-2, and Bcl-XL, but related to the degradation of the proapoptotic Bim. Mechanistically, TLR9-mediated antiapoptotic signals in ZAP-70-positive CLL trigger secretion of immunoglobulin M, which then serves as (auto-) antigen for a prosurvival BCR signal. Thus, our data show that single activation of the innate immune receptor TLR9 is sufficient to fully engage BCR signaling in ZAP-70-positive CLL, protecting malignant cells from apoptosis. We conclude that the integration of TLR signaling into an adaptive immune response can further promote survival of CLL cells and may contribute to the unfavorable prognosis of ZAP-70-positive CLL.

As new generations of targeted therapies emerge and tumor genome sequencing discovers increasingly comprehensive mutation repertoires, the functional relationships of mutations to tumor phenotypes remain largely unknown. Here, we measured ex vivo sensitivity of 246 blood cancers to 63 drugs alongside genome, transcriptome, and DNA methylome analysis to understand determinants of drug response. We assembled a primary blood cancer cell encyclopedia data set that revealed disease-specific sensitivities for each cancer. Within chronic lymphocytic leukemia (CLL), responses to 62% of drugs were associated with 2 or more mutations, and linked the B cell receptor (BCR) pathway to trisomy 12, an important driver of CLL. Based on drug responses, the disease could be organized into phenotypic subgroups characterized by exploitable dependencies on BCR, mTOR, or MEK signaling and associated with mutations, gene expression, and DNA methylation. Fourteen percent of CLLs were driven by mTOR signaling in a non-BCR-dependent manner. Multivariate modeling revealed immunoglobulin heavy chain variable gene (IGHV) mutation status and trisomy 12 as the most important modulators of response to kinase inhibitors in CLL. Ex vivo drug responses were associated with outcome. This study overcomes the perception that most mutations do not influence drug response of cancer, and points to an updated approach to understanding tumor biology, with implications for biomarker discovery and cancer care.

Chronic lymphocytic leukemia (CLL) has a high prevalence in western countries and remains incurable to date. Here, we provide evidence that the multikinase inhibitor sorafenib induces apoptosis in primary CLL cells. This strong pro-apoptotic effect is not restricted to any subgroup of patients, based on Binet stage and the expression of ZAP70 or CD38. Mechanistically, sorafenib-induced cell death is preceded by a rapid downregulation of Mcl-1 through the inhibition of protein translation. Subsequently, the cell intrinsic apoptotic pathway is activated, indicated by destabilization of the mitochondrial membrane potential and activation of caspase-3 and -9. In contrast to sorafenib, the monoclonal vascular epidermal growth factor (VEGF)-antibody bevacizumab failed to induce apoptosis in CLL cells, suggesting that sorafenib induces cell death irrespectively of VEGF signalling. Notably, although sorafenib inhibits phosphorylation of the Scr-kinase Lck, knock-down of Lck did not induce apoptosis in CLL cells. Of note, the pro-apoptotic effect of sorafenib is not restricted to cell-cycle arrested cells, but is also maintained in proliferating CLL cells. In addition, we provide evidence that sorafenib can overcome drug resistance in CLL cells protected by microenvironmental signals from stromal cells. Conclusively, sorafenib is highly active in CLL and may compose a new therapeutic option for patients who relapse after immunochemotherapy.

International audience; Although B-cell chronic lymphocytic leukemia (CLL) is treatable, it remains an incurable disease and most patients inevitably suffer relapse. Many therapeutic options exist for those requiring therapy, including monoclonal antibodies and stem cell transplantation, but remissions tend to last shorter in the course of the disease. Targeting the cell cycle has recently been realized to be an attractive therapeutic approach in solid and hematological malignancies, and the proliferative nature of B-CLL is increasingly accepted. Here, we report data on a phase II pilot trial with the oral mammalian target of rapamycin (mTOR) inhibitor RAD001 5 mg/daily in patients with advanced B-CLL who had progressive disease after at least two lines of treatment. After treatment of seven patients, this trial was stopped because of toxicity concerns, although some degree of activity was observed (one partial remission, three patients with stable disease). Interestingly, cyclin E expression decreased in responding patients. Further strategies of mTOR inhibition by RAD001 in B-CLL should focus on different treatment schedules, adequate anti-infectious prophylaxis, or combinations with cytotoxic drugs.

In the present work we investigated the role and biological significance of mitogen activated protein kinases (MAPK) in B-cell chronic lymphocytic leukaemia (B-CLL). The MAPK p38 was constitutively activated in B-CLL, but not in normal peripheral B cells. In addition, we demonstrated that the upstream kinases of p38, MKK3/6 were also constitutively activated in B-CLL cells. Furthermore, we determined by EMSA that the p38 MAP kinase pathway was not linked to the constitutive high expression of NF-kappaB, a critical survival factor of B-CLL cells. Recently, it has been shown that serum levels of angiogenic factors like VEGF, bFGF and MMP-9 are elevated in the serum of CLL patients and correlate with an unfavorable prognosis. We showed that the constitutive expression of MMP-9 was dependent on p38-activity and inhibition of p38 strongly downregulated MMP-9 expression. Coculture of B-CLL cells and stromal cells can protect spontaneous apoptosis of leukemic B cells. To determine the role of permanently activated p38 and MMP-9 expression, we cocultured B-CLL cells with bone marrow stromal cells. Survival of B-CLL cells on stroma was severely impaired when p38 was inhibited. Furthermore, blockade of MMP-9 activity also antagonized the antiapoptotic effect of stromal cells.

A new class of cell cycle inhibitors is currently entering clinical trials. These drugs exert their activity by inhibition of cyclin-dependent kinases (cdk) and induce cell cycle arrest and apoptosis in cancer cells. Roscovitine, a cdk2-inhibitor that is in preclinical evaluation, induced apoptosis in B-CLL cells at doses that were not cytotoxic for normal human B cells. At 20 microM, Roscovitine induced apoptosis in 21 of 28 B-CLL samples and was equally effective in zap-70-positive or -negative samples. Caspase-3 was cleaved in B-CLL cells exposed to Roscovitine and the pancaspase inhibitor z.VAD.fmk-blocked Roscovitine-induced apoptosis. Expression of the proapoptotic protein Bak was increased and Bax cleavage and conformational change was observed in Roscovitine-treated B-CLL cells. Antiapoptotic proteins Mcl-1 and XIAP were downregulated, but the expression of Bcl-2 remained unchanged. In contrast to previous reports in cancer cell lines, Roscovitine treatment was not accompanied by nuclear accumulation of p53. Cyc202 (R-Roscovitine) is in early clinical trials in cancer patients. Given its powerful effects on zap-70-positive and -negative B-CLL cells, but not on normal lymphocytes, Roscovitine might be an attractive drug to be tested in this incurable disease.

International audience; Although B chronic lymphocytic leukemia (B-CLL) cells express the alpha chain of the interleukin-2 (IL-2) receptor CD25, little is known about the effect of IL-2 on apoptosis in B-CLL cells. We have shown previously that stimulation of B-CLL cells with a CpG-oligonucleotide induces IL-2 high affinity receptors. In our current work, we analyzed the effect of IL-2 on apoptosis in resting B-CLL cells and in our model of activated B-CLL cells (CD25 high cells). IL-2 had modest antiapoptotic activity in resting B-CLL cells. In contrast, IL-2 was much more potent to prevent apoptosis in activated cells. Prevention of cell death was also associated with the maintenance of the mitochondrial membrane potential. While only limited regulation of apoptosis controlling proteins was observed in resting B-CLL cells, IL-2 had strong effects on MCL-1, Bcl-xl, and survivin expression and inhibited Bax cleavage in CD25 high cells. Interestingly, expression of Bcl-2 was reduced. Addition of IL-2 to activated B-CLL cells caused rapid phosphorylation of Akt, while IL-2 failed to significantly phosphorylate Akt in resting B-CLL cells. Pharmacological inhibition of Akt by LY294002 restored sensitivity of activated B-CLL cells to fludarabine. IL-2 might be an important survival factor in activated B-CLL cells and might contribute to disease progression by upregulation of several critical antiapoptotic proteins.

ZAP-70 is a key signaling molecule in T cells. It couples the antigen-activated T-cell receptor to downstream signaling pathways. Its expression in leukemic B-cells derived from a subgroup of patients with chronic lymphocytic leukemia (CLL) is associated with an aggressive course of the disease. However, its implication for the pathogenesis of aggressive CLL is still unclear. In this study, we show that the expression of ZAP-70 enhances the signals associated with the B-cell receptor, recruiting protein kinase C-betaII (PKC-betaII) into lipid raft domains. Subsequently, PKC-betaII is activated and shuttles from the plasma membrane to the mitochondria. We unravel that the antiapoptotic protein Bcl-2 and its antagonistic BH3-protein Bim(EL) are putative substrates for PKC-betaII. PKC-betaII-mediated phosphorylation of Bcl-2 augments its antiapoptotic function by increasing its ability to sequester more pro-apoptotic Bim(EL.) In addition, the phosphorylation of Bim(EL) by PKC-betaII leads to its proteasomal degradation. These changes confer leukemic cells to a more antiapoptotic state with aggressiveness of the disease. Most importantly, these molecular changes can be therapeutically targeted with the small molecule inhibitor Enzastaurin. We provide evidence that this compound is highly active in leukemic cells and augments the cytotoxic effects of standard chemotherapeutic drugs.

The clinical course of chronic lymphocytic leukemia is variable. While some patients have indolent disease, others require aggressive treatment within a short time after diagnosis. Differences in the expression of proteins regulating cell cycle and apoptosis may be responsible for the heterogeneous course of the disease. Recently, protein ZAP 70 [zeta-chain (T-cell receptor) associated protein kinase 70 kDa] has been found to be differentially expressed within two biologic subgroups, characterized by the presence or absence of somatic mutations in specific immunoglobulin heavy-chain variable region genes. In the present work, we analyzed highly purified B-CLL cells from 60 patients for ZAP 70 expression and the expression of cyclin E, bcl-2, bax, and mcl-1 as well as the ratios of bcl-2/bax and mcl-1/bax. The results indicate that cyclin E is expressed significantly higher in ZAP 70-positive as in ZAP 70-negative samples. We did not observe significant differences within the expression of Bcl-2 family member proteins. We conclude that higher cyclin E expression in samples of ZAP 70-positive patients may reflect a larger proliferating compartment in vivo compared to ZAP 70-negative patients and that cyclin E may add prognostic information in this context for patients with B-CLL.

Chronic lymphocytic B-cell leukemia (B-CLL) is an incurable disease characterized by the accumulation of monoclonal mature B cells, although disease progression relies upon cycling B-CLL cells in proliferation centers in central lymph organs. Rapamycin and its analogs are immunosuppressant drugs that exert their activity by specific inhibition of the mammalian target of rapamycin (mTOR). mTOR inhibition induces cell cycle arrest not only in normal lymphocytes but also in malignant cells. Therefore, rapamycins have recently entered the field of cancer treatment. In the present review we discuss how progression through the cell cycle is regulated in B-CLL cells and how rapamycin and its analogs can be used as target therapies against proliferating B-CLL cells. We also focus on additional effects of rapamycin, such as targeting the interaction between malignant B cells and the microenvironment.

Although peripheral blood B-CLL cells are arrested in G0 phase of the cell cycle, a proliferating pool of cells in proliferation centers might be involved in disease progression. We have previously described an in vitro model of this proliferating pool of cells using B-CLL cells stimulated with immunostimulatory oligonucleotides (CpG-ODN) and interleukin-2. Lactacystin is a specific inhibitor of the proteasome and is a potent apoptosis inductor in resting peripheral B-CLL cells. In the present study, we investigated the effect of proteasome inhibition in proliferating B-CLL cells.The effect of proteasome inhibition was analyzed using thymidine incorporation, annexin V assays, and TUNEL staining. Immunoblots were performed to evaluate expression of proteins involved in cell cycle and apoptosis regulation.Lactacystin blocked cell cycle progression in activated B-CLL cells and inhibited degradation of p27. Upregulation of cyclin D2 and D3 in activated B-CLL cells was inhibited while the expression of cdk2, cdk4, and cyclin E remained unchanged. Activated B-CLL cells were more susceptible to apoptosis induction as compared to resting B-CLL cells. Apoptosis induction was accompanied by cleavage of Bax, procaspase 8, procaspase 9, and procaspase 3. However, a broad-spectrum caspase inhibitor (z-VAD.fmk) only partially inhibited cell death although DNA degradation was completely inhibited.Proteasome inhibition is highly effective in proliferating B-CLL cells and induces apoptosis using a caspase-dependent and -independent pathway.

In B-cell chronic lymphocytic leukemia (B-CLL), malignant cells seem to be arrested in the G0/early G1phase of the cell cycle, and defective apoptosis might be involved in disease progression. However, increasing evidence exists that B-CLL is more than a disease consisting of slowly accumulating resting B cells: a proliferating pool of cells has been described in lymph nodes and bone marrow and might feed the accumulating pool in the blood. Rapamycin has been reported to inhibit cell cycle progression in a variety of cell types, including human B cells, and has shown activity against a broad range of human tumor cell lines. Therefore, we investigated the ability of rapamycin to block cell cycle progression in proliferating B-CLL cells. We have recently demonstrated that stimulation with CpG-oligonucleotides and interleukin-2 provides a valuable model for studying cell cycle regulation in malignant B cells. In our present study, we demonstrated that rapamycin induced cell cycle arrest in proliferating B-CLL cells and inhibited phosphorylation of p70s6 kinase (p70s6k). In contrast to previous reports on nonmalignant B cells, the expression of the cell cycle inhibitor p27 was not changed in rapamycin-treated leukemic cells. Treatment with rapamycin prevented retinoblastoma protein (RB) phosphorylation in B-CLL cells without affecting the expression of cyclin D2, but cyclin D3 was no longer detectable in rapamycin-treated B-CLL cells. In addition, rapamycin treatment inhibited cyclin-dependent kinase 2 activity by preventing up-regulation of cyclin E and cyclin A. Interestingly, survivin, which is expressed in the proliferation centers of B-CLL patients in vivo, is not up-regulated in rapamycin-treated cells. Therefore, rapamycin interferes with the expression of many critical molecules for cell cycle regulation in cycling B-CLL cells. We conclude from our study that rapamycin might be an attractive substance for therapy for B-CLL patients by inducing a G1 arrest in proliferating tumor cells.

In the present study we analyzed the role of phophatidylinositol-3 kinase (PI-3K) in B chronic lymphocytic leukemia (B-CLL) cells. PI-3K is activated by many stimuli and is linked to several different signaling pathways. We demonstrated that inhibition of PI-3K by a specific inhibitor, LY294002, induced apoptosis in B-CLL cells in vitro. This effect was specific for the inhibition of PI-3K because inhibition of other signaling pathways such as extracellular signaling-regulated kinase (ERK), p38, or p70S6 kinase did not affect spontaneous apoptosis. Furthermore, PI-3K was constitutively activated in freshly isolated B-CLL cells. Corresponding to enhanced apoptosis, LY294002 down-regulated expression of the antiapoptotic proteins X-linked inhibitor of apoptosis protein (XIAP) and Mcl-1. Next, we investigated which factors downstream of PI-3K were activated in B-CLL cells. We demonstrated that protein kinase B/Akt is expressed in all tested CLL samples but no activation of Akt was detected. In contrast, we observed a constitutive activation of protein kinase Cdelta (PKCdelta) in freshly isolated B-CLL cells. PKCdelta is linked to PI-3K and is phosphorylated at Thr505 in response to PI-3K activation. We further demonstrated that tyrosine phosphorylation and activity of PKCdelta were dependent on PI-3K activity in B-CLL cells. Inhibition of PKCdelta by the specific inhibitor Rottlerin strikingly enhanced apoptosis. In contrast, peripheral blood B cells of healthy donors were resistant to inhibition of PI-3K or PKCdelta. We conclude that activated PI-3K might be important in the pathogenesis of B-CLL, and survival signals might be mediated via PKCdelta. Therefore, inhibition of PI-3K or PKCdelta may be an innovative approach to treat B-CLL.

SummaryTumor cell survival critically depends on heterotypic communication with benign cells in the microenvironment. Here, we describe a survival signaling pathway activated in stromal cells by contact to B cells from patients with chronic lymphocytic leukemia (CLL). The expression of protein kinase C (PKC)-βII and the subsequent activation of NF-κB in bone marrow stromal cells are prerequisites to support the survival of malignant B cells. PKC-β knockout mice are insusceptible to CLL transplantations, underscoring the in vivo significance of the PKC-βII-NF-κB signaling pathway in the tumor microenvironment. Upregulated stromal PKC-βII in biopsies from patients with CLL, acute lymphoblastic leukemia, and mantle cell lymphoma suggests that this pathway may commonly be activated in a variety of hematological malignancies.