B-cell Chronic Lymphocytic Leukemia Cells Research Articles

Diverse B-cell tumors associated with t(14;19)(q32;q13)/IGH::BCL3 identified by G-banding and fluorescence in situ hybridization.

We characterized 5 B-cell tumors carrying t(14;19)(q32;q13) that creates the IGH::BCL3 fusion gene. The patients' ages ranged between 55 and 88 years. Two patients presented with progression or recurrence of B-cell chronic lymphocytic leukemia (B-CLL)/small lymphocytic lymphoma (SLL), two with diffuse large B-cell lymphoma (DLBCL) of non-germinal center B-like phenotype, and the remaining one with composite angioimmunoblastic T-cell lymphoma and Epstein-Barr virus-positive DLBCL. The presence of t(14;19)(q32;q13) was confirmed by fluorescence in situ hybridization (FISH), showing colocalization of 3' IGH and 3' BCL3 probes on der(14)t(14;19) and 5' BCL3 and 5' IGH probes on der(19)t(14;19). One B-CLL case had t(2;14)(p13;q32)/IGH::BCL11A, and 2 DLBCL cases had t(8;14)(q24;q32) or t(8;11;14)(q24;q11;q32), both of which generated IGH::MYC by FISH, and showed nuclear expression of MYC and BCL3 by immunohistochemistry. The IGH::BCL3 fusion gene was amplified by long-distance polymerase chain reaction in 2 B-CLL/SLL cases and the breakpoints occurred immediately 5' of BCL3 exon 1 and within the switch region associated with IGHA1. The 5 cases shared IGHV preferentially used in B-CLL cells, but the genes were unmutated in 2 B-CLL/SLL cases and significantly mutated in the remaining 3. B-cell tumors with t(14;19)(q32;q13) can be divided into B-CLL/SLL and DLBCL groups, and the anatomy of IGH::BCL3 in the latter may be different from that of the former.

Studying the DNA damage response pathway in hematopoietic canine cancer cell lines, a necessary step for finding targets to generate new therapies to treat cancer in dogs.

Dogs present a significant opportunity for studies in comparative oncology. However, the study of cancer biology phenomena in canine cells is currently limited by restricted availability of validated antibody reagents and techniques. Here, we provide an initial characterization of the expression and activity of key components of the DNA Damage Response (DDR) in a panel of hematopoietic canine cancer cell lines, with the use of commercially available antibody reagents. The techniques used for this validation analysis were western blot, qPCR, and DNA combing assay. Substantial variations in both the basal expression (ATR, Claspin, Chk1, and Rad51) and agonist-induced activation (p-Chk1) of DDR components were observed in canine cancer cell lines. The expression was stronger in the CLBL-1 (B-cell lymphoma) and CLB70 (B-cell chronic lymphocytic leukemia) cell lines than in the GL-1 (B-cell leukemia) cell line, but the biological significance of these differences requires further investigation. We also validated methodologies for quantifying DNA replication dynamics in hematopoietic canine cancer cell lines, and found that the GL-1 cell line presented a higher replication fork speed than the CLBL-1 cell line, but that both showed a tendency to replication fork asymmetry. These findings will inform future studies on cancer biology, which will facilitate progress in developing novel anticancer therapies for canine patients. They can also provide new knowledge in human oncology.

Selection of a Nuclease-Resistant RNA Aptamer Targeting CD19.

Simple SummaryHaematological malignancies show a constantly growing incidence, accounting for 6.5% of new cancer cases worldwide. Among them, B-cell neoplasms often show resistance to conventional chemotherapy that is also associated with numerous adverse effects. Therefore, in order for the treatment outcome to be improved, the development of new safe and effective targeted therapeutic approaches represents a main challenge. In this regard, nucleic acid aptamers are very attractive molecules. Indeed, they show high affinity and specificity for their target, increased tumour penetration, and low toxicity. Recently, CD19 has emerged as a key surface marker of malignant B cells, suitable for the development of new compounds for malignant B-cell targeting. Here, we isolated an RNA aptamer targeting the human CD19 antigen on malignant B cells that was able to rapidly internalise into target cells. Therefore, it represents a useful carrier for secondary reagents and a promising tool for the development of new safe and effective targeted therapies for B-cell malignancy treatment.The transmembrane glycoprotein cluster of differentiation 19 (CD19) is a B cell–specific surface marker, expressed on the majority of neoplastic B cells, and has recently emerged as a very attractive biomarker and therapeutic target for B-cell malignancies. The development of safe and effective ligands for CD19 has become an important need for the development of targeted conventional and immunotherapies. In this regard, aptamers represent a very interesting class of molecules. Additionally referred to as ‘chemical antibodies’, they show many advantages as therapeutics, including low toxicity and immunogenicity. Here, we isolated a nuclease-resistant RNA aptamer binding to the human CD19 glycoprotein. In order to develop an aptamer also useful as a carrier for secondary reagents, we adopted a cell-based SELEX (Systematic Evolution of Ligands by EXponential Enrichment) protocol adapted to isolate aptamers able to internalise upon binding to their cell surface target. We describe a 2′-fluoro pyrimidine modified aptamer, named B85.T2, which specifically binds to CD19 and shows an exquisite stability in human serum. The aptamer showed an estimated dissociation constant (KD) of 49.9 ± 13 nM on purified human recombinant CD19 (rhCD19) glycoprotein, a good binding activity on human B-cell chronic lymphocytic leukaemia cells expressing CD19, and also an effective and rapid cell internalisation, thus representing a promising molecule for CD19 targeting, as well as for the development of new B-cell malignancy-targeted therapies.

Identification of novel, clonally stable, somatic mutations targeting transcription factors PAX5 and NKX2-3, the epigenetic regulator LRIF1, and BRAF in a case of atypical B-cell chronic lymphocytic leukemia harboring a t(14;18)(q32;q21).

Diagnosis of B-cell chronic lymphocytic leukemia (B-CLL) is usually straightforward, involving clinical, immunophenotypic (Matutes score), and (immuno)genetic analyses (to refine patient prognosis for treatment). CLL cases with atypical presentation (e.g., Matutes ≤ 3) are also encountered, and for these diseases, biology and prognostic impact are less clear. Here we report the genomic characterization of a case of atypical B-CLL in a 70-yr-old male patient; B-CLL cells showed a Matutes score of 3, chromosomal translocation t(14;18)(q32;q21) (BCL2/IGH), mutated IGHV, deletion 17p, and mutations in BCL2, NOTCH1 (subclonal), and TP53 (subclonal). Quite strikingly, a novel PAX5 mutation that was predicted to be loss of function was also seen. Exome sequencing identified, in addition, a potentially actionable BRAF mutation, together with novel somatic mutations affecting the homeobox transcription factor NKX2-3, known to control B-lymphocyte development and homing, and the epigenetic regulator LRIF1, which is implicated in chromatin compaction and gene silencing. Neither NKX2-3 nor LRIF1 mutations, predicted to be loss of function, have previously been reported in B-CLL. Sequencing confirmed the presence of these mutations together with BCL2, NOTCH1, and BRAF mutations, with the t(14;18)(q32;q21) translocation, in the initial diagnostic sample obtained 12 yr prior. This is suggestive of a role for these novel mutations in B-CLL initiation and stable clonal evolution, including upon treatment withdrawal. This case extends the spectrum of atypical B-CLL with t(14;18)(q32;q21) and highlights the value of more global precision genomics for patient follow-up and treatment in these patients.

BIRD-2, a BH4-domain-targeting peptide of Bcl-2, provokes Bax/Bak-independent cell death in B-cell cancers through mitochondrial Ca2+-dependent mPTP opening

Anti-apoptotic Bcl-2 critically controls cell death by neutralizing pro-apoptotic Bcl-2-family members at the mitochondria. Bcl-2 proteins also act at the endoplasmic reticulum, the main intracellular Ca2+-storage organelle, where they inhibit IP3 receptors (IP3R) and prevent pro-apoptotic Ca2+-signaling events. IP3R channels are targeted by the BH4 domain of Bcl-2. Some cancer types rely on the IP3R-Bcl-2 interaction for survival. We previously developed a cell-permeable, BH4-domain-targeting peptide that can abrogate Bcl-2′s inhibitory action on IP3Rs, named Bcl-2 IP3 receptor disrupter-2 (BIRD-2). This peptide kills several Bcl-2-dependent cancer cell types, including diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukaemia (CLL) cells, by eliciting intracellular Ca2+ signalling. However, the exact mechanisms by which these excessive Ca2+ signals triggered by BIRD-2 provoke cancer cell death remain elusive. Here, we demonstrate in DLBCL that although BIRD-2 activates caspase 3/7 and provokes cell death in a caspase-dependent manner, the cell death is independent of pro-apoptotic Bcl-2-family members, Bim, Bax and Bak. Instead, BIRD-2 provokes mitochondrial Ca2+ overload that is rapidly followed by opening of the mitochondrial permeability transition pore (mPTP). Inhibiting mitochondrial Ca2+ overload using Ru265, an inhibitor of the mitochondrial Ca2+ uniporter complex counteracts BIRD-2-induced cancer cell death. Finally, we validated our findings in primary CLL patient samples where BIRD-2 provoked mitochondrial Ca2+ overload and Ru265 counteracted BIRD-2-induced cell death. Overall, this work reveals the mechanisms by which BIRD-2 provokes cell death, which occurs via mitochondrial Ca2+ overload but acts independently of pro-apoptotic Bcl-2-family members.

BDNF belongs to the nurse-like cell secretome and supports survival of B chronic lymphocytic leukemia cells

Evading apoptosis and sustained survival signaling pathways are two central hallmarks of B-cell chronic lymphocytic leukemia (B-CLL) cells. In this regard, nurse-like cells (NLC), the monocyte-derived type 2 macrophages, deliver stimulatory signals via B-cell activating factor (BAFF), a proliferation-inducing ligand (APRIL), and the C-X-C Motif Chemokine Ligand 12 (CXCL12). Previously, we demonstrated that brain-derived neurotrophic factor (BDNF) protects B-CLL cells from spontaneous apoptosis by activating the oncogenic complex NTSR2-TrkB (neurotensin receptor 2-tropomyosin-related kinase receptor B), only overexpressed in B-CLL cells, inducing anti-apoptotic protein Bcl-2 (B-cell lymphoma 2) expression and Src kinase survival signaling pathways. Herein, we demonstrate that BDNF belongs to the NLC secretome and promotes B-CLL survival. This was demonstrated in primary B-CLL co-cultured with their autologous NLC, compared to B-CLL cells cultured alone. Inhibition of BDNF in co-cultures, enhances B-CLL apoptosis, whereas its exogenous recombinant activates pro-survival pathways in B-CLL cultured alone (i.e. Src activation and Bcl-2 expression), at a higher level than those obtained by the exogenous recombinant cytokines BAFF, APRIL and CXCL12, the known pro-survival cytokines secreted by NLC. Together, these results showed that BDNF release from NLC trigger B-CLL survival. Blocking BDNF would support research strategies against pro-survival cytokines to limit sustained B-CLL cell survival.

Excessive amount and activity of μ-calpain affects apoptotic machinery in chronic B-cell leukemia cells and influences the course of the disease.

B-cell Chronic Lymphocytic Leukemia (B-CLL) is the most common hematological disorder among middle-aged/elderly people in the Western countries. We have shown earlier that B-CLL cells exhibit elevated total amount and available activity of µ-calpain, belonging to a family of ubiquitous, strongly Ca-dependent proteases, involved in the control of proliferation and apoptosis. In this study we attempted to estimate a potential clinical value of μ-calpain in relation to B-CLL clinical staging in patients with extremely high lymphocytosis and studied the molecular mechanisms associating calpain activity with clinical progress of the disease. We observed significant correlations between the amounts of intracellular μ-calpain and clinical staging of the disease, with RAI stage 1 corresponding to the highest calpain amounts in the leukemic cells. There was also a positive, statistically significant correlation between the amount of μ-calpain and phosphorylated (p)ZAP-70 in B-CLL lymphocytes. Calpain activity in the B-CLL cells is associated with decreased activities of pro-apoptotic caspases -3 and -9, and reciprocally with an increased amount of anti-apoptotic Bcl-2. Together, all of these findings make calpain activity in B-CLL cells a promising target modifying the properties of these cells and facilitating therapy. Finally, the proportion of CD19+ B cells with elevated μ-calpain and pZap-70 was markedly reduced in patients after successful therapy.

Cytokine Release Ensuing Interaction Between Human Peripheral Blood Mononuclears and Epstein-Barr Virus Transformed B-CLL Cell Line.

B-cell chronic lymphocytic leukemia (B-CLL) is a common form of leukemia affecting mostly elderly individuals. The course of the disease is usually unremarkable, but because it may proceed with impaired immune defense, B-CLL might be complicated with infections and even death. The leukemic microenvironment containing a number of immune cells, mainly lymphocytes and macrophages capable to produce various molecules including inflammatory cytokines, plays an important role in the development and outcome of the disease. We studied the capacity of Epstein-Barr virus (EBV)-transformed B-cell chronic lymphocytic leukemia (B-CLL) cell line (EHEB) cells, an EBV-transformed line established from a B-CLL patient, to affect the production of inflammatory cytokines by human peripheral blood mononuclear cells (PBMC). PBMC isolated from peripheral blood of healthy donors were incubated either with EHEB cells or with their supernatants and the production of the following cytokines: tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, interferon (IFN)-γ, IL-1ra, and IL-10 were detected using the enzyme-linked immunosorbent assay method. Direct contact of PBMC incubated with EHEB cells induced a marked increase of TNFα, IL-1β, IL-6, IFNγ, and IL-10 release by the immune cells. Yet, incubation of PBMC with EHEB cells' supernatant resulted in a mild production of the same cytokines. The noticeable increased production of inflammatory cytokines by PBMC following direct contact with EHEB cells and to a lesser degree with their supernatants implies the existence of an immune dialogue between these two types of cells. The results support the concept that not only leukemic cells, but also peripheral blood mononuclears could serve as a therapeutic target for B-CLL.

Clinico-Biological Implications of Modified Levels of Cytokines in Chronic Lymphocytic Leukemia: A Possible Therapeutic Role.

B-cell chronic lymphocytic leukemia (B-CLL) is the main cause of mortality among hematologic diseases in Western nations. B-CLL is correlated with an intense alteration of the immune system. The altered functions of innate immune elements and adaptive immune factors are interconnected in B-CLL and are decisive for its onset, evolution, and therapeutic response. Modifications in the cytokine balance could support the growth of the leukemic clone via a modulation of cellular proliferation and apoptosis, as some cytokines have been reported to be able to affect the life of B-CLL cells in vivo. In this review, we will examine the role played by cytokines in the cellular dynamics of B-CLL patients, interpret the contradictions sometimes present in the literature regarding their action, and evaluate the possibility of manipulating their production in order to intervene in the natural history of the disease.

Features of immunophenotypic finding B-cell lymphoproliferative diseases by flow cytometry

Aim. To assess the information content of conventional and additional immunophenotypic markers (CD200, CD305) in the differential diagnosis B-cell lymphoproliferative diseases by flow cytometry.
 Methods. An immunophenotypic study using 4-color flow cytometry was performed in 204 patients with different variants of B-cell non-Hodgkin's lymphomas. The study material included peripheral blood and bone marrow. The expression of CD45, CD19, CD20, CD22, CD79b, CD79a, CD5, CD10, CD23, FMC7, CD43, CD38, CD11c, CD103, CD25, CD 200, CD 305, light chains of immunoglobulins (kappa/lambda) using monoclonal antibodies (Becton Dickinson, USA) was evaluated. The intensity of antigen expression was assessed using mean fluorescence intensity (y. e.).
 Results. Conventional FMC7-positive expression revealed only half patients with different variants of leukemization of non-Hodgkin's lymphomas, whereas atypical positive expression of CD23 was observed in patients with marginal spleen lymphoma and follicular lymphoma in 27.3 and 28.6% of cases, respectively. In mantle cell lymphoma, expression of CD200 in B-cell was detected in a significantly smaller number of observations, accompanied by a significant decrease in the average intensity of CD200 fluorescence compared to B-cell chronic lymphocytic leukemia (B-CLL) cells. The mean fluorescence intensity (MFI) of CD305 in hairy cell leukemia is significantly higher than in splenic marginal zone lymphoma (SMZL) with villous lymphocytes.
 Conclusion. Different levels of the information content of some conventional markers were revealed in differential immunophenotypic diagnosis of B-cell lymphoproliferative diseases by flow cytometry; the use of additional markers CD200 and CD305 was highly informative in differential diagnostics between different variants of B-cell lymphoproliferative diseases with similar immunophenotypic and morphological characteristics of lymphoid elements.

Cladribine Induces ATF4 Mediated Apoptosis and Synergizes with SAHA in Diffuse Large B-Cell Lymphoma Cells.

Cladribine is a purine nucleoside analog used to treat B-cell chronic lymphocytic leukemia and hairy cell leukemia, also functions as an inhibitor of DNA synthesis to block the repair of the damaged DNA. The therapeutic role of cladribine against diffuse large B-cell lymphoma cells (DLBCL) is still undefined. In the present study, we demonstrated that cladribine inhibited cell proliferation and induced G1 phase arrest in human DLBCL cells. Furthermore, we showed that cladribine induced apoptosis by decreasing the expression of c-FLIPL and increasing the expression of DR4 and the cleaved form of caspase8. Cladribine also upregulated the expression of Bax, and downregulated the expression of Mcl-1 and Bcl-2 in a dose-dependent manner. It also activated endoplasmic reticulum (ER) stress, and ATF4 expression was required for cladribine induced apoptosis. Also, we showed that suberoylanilide hydroxamic acid (SAHA) enhanced the pro-apoptotic role of cladribine. Collectively, cladribine activated extrinsic and intrinsic apoptotic signaling pathways via stimulating ER stress signaling pathway and eliciting synergistic effect with SAHA in DLBCL cells.

Abstract 2318: Combinatorial IGK-CD19 CAR primarily targets IgK+ malignant B-cells and is less prone to serum IgG inhibition

Abstract The first Chimeric antigen receptor (CAR) T-cell therapies have been approved for treatment of B-cell malignancies. This is mainly due to the success of CAR T cells targeting B-lymphocyte antigen CD19, which has led to astonishing results in clinical trials. Since CD19 is a general B-cell antigen, CAR-T cells eliminate all B-lineage cells, including non-malignant B cells. Therefore, the patients suffer from the impaired humoral immune response, increasing susceptibility to severe infections.Since B-cell lymphoma and chronic lymphocytic leukemia cells have a clonally restricted expression of Immunoglobulin (Ig) light chains, either Ig-kappa or Ig-lambda, Ig-kappa positive tumor cells can be targeted while sparing normal Ig-lambda positive B-cells. In this respect, we isolated the sequence encoding the antigen-binding parts of an anti-Ig kappa antibody and designed a second-generation CAR construct (IGK CAR). Expression of IGK CAR in expanded peripheral blood T cells and subsequent testing of the CAR T cells in various in vitro assay with target cells, demonstrated cytokine production and potent killing of Ig-kappa expressing B-cell lines such as BL-41, whereas no response was observed against Ig lambda positive B-cell lines such as Granta-519. We compared IGK CAR with a clinical CD19 CAR (fmc63) and observed similar potency in target cell killing. Previous reports have shown that the presence of free immunoglobulins present in human serum could inhibit IGK CAR T cells, and our tests confirmed this. To improve IGK CAR T cells in the presence of IgGs while maintaining the specificity, we utilized a combinatorial CAR system, where the signaling domains were split. Our design demonstrated efficient killing of Ig-kappa positive cells and was less sensitive to free IgG as compared to IGK CAR T cells. Additionally, we observed a trade-off between specificity and cytotoxic potential. Increasing one individual component of the combinatorial system made the cells less prone to serum IgG inhibition but demonstrated somewhat higher cytotoxic activity against Ig-kappa negative targets. Our fully adjustable design, therefore, brings another perspective to the field by regulating the individual expression levels according to the treatment needs. Hence, enabling T cells to be either more aggressive or specific depending on the treatment efficiency and on the on-target toxicity in patients. Taken together, our in vitro data demonstrate that IGK-CD19 CAR combination is as potent as IGK or CD19 CAR T cells, and provides an alternative by combining their benefits into one design and thus reduces on-target toxicity. Citation Format: Hakan Köksal, Pierre Dillard, Sólrún M. Maggadóttir, Gunnar Kvalheim, Erlend B. Smeland, June H. Myklebust, Else Marit Inderberg, Sébastien Walchli. Combinatorial IGK-CD19 CAR primarily targets IgK+ malignant B-cells and is less prone to serum IgG inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2318.

Mechanism for IL-15-Driven B Cell Chronic Lymphocytic Leukemia Cycling: Roles for AKT and STAT5 in Modulating Cyclin D2 and DNA Damage Response Proteins.

Clonal expansion of B cell chronic lymphocytic leukemia (B-CLL) occurs within lymphoid tissue pseudofollicles. IL-15, a stromal cell-associated cytokine found within spleens and lymph nodes of B-CLL patients, significantly boosts in vitro cycling of blood-derived B-CLL cells following CpG DNA priming. Both IL-15 and CpG DNA are elevated in microbe-draining lymphatic tissues, and unraveling the basis for IL-15-driven B-CLL growth could illuminate new therapeutic targets. Using CpG DNA-primed human B-CLL clones and approaches involving both immunofluorescent staining and pharmacologic inhibitors, we show that both PI3K/AKT and JAK/STAT5 pathways are activated and functionally important for IL-15→CD122/ɣc signaling in ODN-primed cells expressing activated pSTAT3. Furthermore, STAT5 activity must be sustained for continued cycling of CFSE-labeled B-CLL cells. Quantitative RT-PCR experiments with inhibitors of PI3K and STAT5 show that both contribute to IL-15-driven upregulation of mRNA for cyclin D2 and suppression of mRNA for DNA damage response mediators ATM, 53BP1, and MDC1. Furthermore, protein levels of these DNA damage response molecules are reduced by IL-15, as indicated by Western blotting and immunofluorescent staining. Bioinformatics analysis of ENCODE chromatin immunoprecipitation sequencing data from cell lines provides insight into possible mechanisms for STAT5-mediated repression. Finally, pharmacologic inhibitors of JAKs and STAT5 significantly curtailed B-CLL cycling when added either early or late in a growth response. We discuss how the IL-15-induced changes in gene expression lead to rapid cycling and possibly enhanced mutagenesis. STAT5 inhibitors might be an effective modality for blocking B-CLL growth in patients.

IL-15-driven clonal expansion of TLR9-primed human B-CLL is characterized by activation of AKT and STAT5, downstream elevation of cyclin D2 and repression of DNA damage response mediators, ATM and TP53BP

Abstract Clonal expansion of B cell chronic lymphocytic leukemia (B-CLL) is linked to mutagenesis and occurs within lymphoid tissues. CpG DNA (ODN-2006) and IL-15, a cytokine found within stromal cells of B-CLL-infiltrated tissues, manifest striking synergy in promoting in vitro growth of B-CLL cells (J Immunol 195:901–923, 2015). Synergy is in part explained by an ODN-induced, NF-kB (p65)-dependent elevation of IL-15Rα and IL-2/15Rβ (CD122) mRNA and protein (J Immunol 201:1570–1585, 2018). Most recently, we examined the downstream consequences of IL-15 signaling through experiments involving intracellular immunofluorescence staining, pharmacologic inhibitors, RT-PCR, and western blotting. Levels of activated pAKTSer473 and pSTAT5Tyr694 were significantly elevated soon after IL-15 exposure to ODN-primed B-CLL cells; furthermore, STAT5 remained activated throughout cycling. Pharmacologic inhibitors of PI-3K or STAT5 blocked IL-15-driven growth, indicating the functional relevance of these pathways. RT-PCR experiments +/− these inhibitors show that IL-15 signaling in B-CLL is characterized by PI-3K & STAT5-dependent rise in cyclin-D2 and repression of two important DNA damage response (DDR) mediators, ATM and TP53BP1, known to dampen growth and facilitate DNA repair. Repression of the latter two proteins was confirmed by western blotting and immunofluorescence studies. We conclude that IL-15 signaling in TLR9-primed B-CLL can provoke a rapid burst in growth via mechanisms resembling those recently described in CD8 T cells. Nonetheless, because dividing B-CLL cells additionally upregulate the mutagenic enzyme, AICDA (Blood 120: 4802–4811, 2012), these simultaneous events may augment B-CLL mutagenesis.

Protoporphyrin IX is a dual inhibitor of p53/MDM2 and p53/MDM4 interactions and induces apoptosis in B-cell chronic lymphocytic leukemia cells

p53 is a tumor suppressor, which belongs to the p53 family of proteins. The family consists of p53, p63 and p73 proteins, which share similar structure and function. Activation of wild-type p53 or TAp73 in tumors leads to tumor regression, and small molecules restoring the p53 pathway are in clinical development. Protoporphyrin IX (PpIX), a metabolite of aminolevulinic acid, is a clinically approved drug applied in photodynamic diagnosis and therapy. PpIX induces p53-dependent and TAp73-dependent apoptosis and inhibits TAp73/MDM2 and TAp73/MDM4 interactions. Here we demonstrate that PpIX is a dual inhibitor of p53/MDM2 and p53/MDM4 interactions and activates apoptosis in B-cell chronic lymphocytic leukemia cells without illumination and without affecting normal cells. PpIX stabilizes p53 and TAp73 proteins, induces p53-downstream apoptotic targets and provokes cancer cell death at doses non-toxic to normal cells. Our findings open up new opportunities for repurposing PpIX for treating lymphoblastic leukemia with wild-type TP53.

Role of Calcium Signaling in GA101-Induced Cell Death in Malignant Human B Cells.

GA101/obinutuzumab is a novel type II anti-CD20 monoclonal antibody (mAb), which is more effective than rituximab (RTX) in preclinical and clinical studies when used in combination with chemotherapy. Ca2+ signaling was shown to play a role in RTX-induced cell death. This report concerns the effect of GA101 on Ca2+ signaling and its involvement in the direct cell death induced by GA101. We reveal that GA101 triggered an intracellular Ca2+ increase by mobilizing intracellular Ca2+ stores and activating Orai1-dependent Ca2+ influx in non-Hodgkin lymphoma cell lines and primary B-Cell Chronic Lymphocytic Leukemia (B-CLL) cells. According to the cell type, Ca2+ was mobilized from two distinct intracellular compartments. In Raji, BL2, and B-CLL cells, GA101 induced a Ca2+ release from lysosomes, leading to the subsequent lysosomal membrane permeabilization and cell death. Inhibition of this calcium signaling reduced GA101-induced cell death in these cells. In SU-DHL-4 cells, GA101 mobilized Ca2+ from the endoplasmic reticulum (ER). Inhibition of ER replenishment, by blocking Orai1-dependent Ca2+ influx, led to an ER stress and unfolded protein response (UPR) which sensitized these cells to GA101-induced cell death. These results revealed the central role of Ca2+ signaling in GA101’s action mechanism, which may contribute to designing new rational drug combinations improving its clinical efficacy.

Abstract A035: Combinatorial IGK-CD19 CAR primarily targets IgK+ malignant B-cells and is less prone to serum IgG inhibition

Abstract The first chimeric antigen receptor (CAR) T-cell therapies have been approved for treatment of B-cell malignancies. This is mainly due to the success of CAR T-cells targeting B-lymphocyte antigen CD19, which has led to astonishing results in clinical trials. Since CD19 is a general B-cell antigen, CAR-T-cells eliminate all B-lineage cells, including nonmalignant B cells. Therefore, the patients suffer from the impaired humoral immune response, increasing susceptibility to severe infections. Since B-cell lymphoma and chronic lymphocytic leukemia cells have a clonally restricted expression of Immunoglobulin (Ig) light chains, either Ig-kappa or Ig-lambda, Ig-kappa positive tumor cells can be targeted while sparing normal Ig-lambda positive B-cells. In this respect, we isolated the sequence encoding the antigen-binding parts of an anti-Ig kappa antibody and designed a second-generation CAR construct (IGK CAR). Expression of IGK CAR in expanded peripheral blood T-cells and subsequent testing of the CAR T-cells in various in vitro assay with targeT-cells demonstrated cytokine production and potent killing of Ig-kappa expressing B-cell lines such as BL-41, whereas no response was observed against Ig lambda positive B-cell lines such as Granta-519. We compared IGK CAR with a clinical CD19 CAR (fmc63) and observed similar potency in targeT-cell killing. Previous reports have shown that the presence of free immunoglobulins present in human serum could inhibit IGK CAR T-cells, and our tests confirmed this. To improve IGK CAR T-cells in the presence of IgGs while maintaining the specificity, we utilized a combinatorial CAR system, where the signaling domains were split. Our design demonstrated efficient killing of Ig-kappa positive cells and was less sensitive to free IgG as compared to IGK CAR T-cells. Additionally, we observed a trade-off between specificity and cytotoxic potential. Increasing one individual component of the combinatorial system made the cells less prone to serum IgG inhibition but demonstrated somewhat higher cytotoxic activity against Ig-kappa negative targets. Our fully adjustable design, therefore, brings another perspective to the field by regulating the individual expression levels according to the treatment needs, hence enabling T-cells to be either more aggressive or specific depending on the treatment efficiency and on the on-target toxicity in patients. Taken together, our in vitro data demonstrate that IGK-CD19 CAR combination is as potent as IGK or CD19 CAR T-cells, and provides an alternative by combining their benefits into one design and thus reduces on-target toxicity. Citation Format: Hakan Köksal, Pierre Dillard, Sólrún Melkorka Maggadóttir, Gunnar Kvalheim, Erlend Bremertun Smeland, June Helen Myklebust, Else Marit Inderberg, Sébastien Wälchli. Combinatorial IGK-CD19 CAR primarily targets IgK+ malignant B-cells and is less prone to serum IgG inhibition [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A035.

CXCR4- and BCR-triggered integrin activation in B-cell chronic lymphocytic leukemia cells depends on JAK2-activated Bruton's tyrosine kinase.

Bruton's tyrosine kinase (BTK) regulates the B-cell receptor (BCR) signaling pathway, which, in turn, plays a critical role in B-cell chronic lymphocytic leukemia (B-CLL) pathogenesis. The BTK-specific inhibitor Ibrutinib blocks BCR signaling and is now approved as effective B-CLL therapy. Chemokines, such as the homeostatic chemokine CXCL12, play a central role in B-CLL pathogenesis and progression, by regulating CLL cell interaction with the stromal microenvironment, leading to cells survival and proliferation. In this study, we investigated, in normal versus CLL B-lymphocytes, the role of BTK in signal transduction activated by the CXCL12-CXCR4 signaling axis and its involvement in rapid integrin activation. We show that BTK is rapidly activated by CXCL12 in healthy as well as CLL B-lymphocytes, with a kinetic of tyr-phosphorylation coherent with rapid adhesion triggering. BTK inhibition prevents CXCL12-induced triggering of lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) integrins. Furthermore, BTK inhibition blocks the activation of the small GTP-binding protein RhoA, controlling integrin affinity. Very importantly, we show that BTK tyr-phosphorylation and activation by CXCL12 depends on upstream activation of JAK2 tyrosine kinase. A comparative analysis of 36 B-CLL patients demonstrates that JAK2-dependent BTK regulatory role on integrin activation by CXCL12 is fully conserved in CLL cells. Finally, we show that the JAK2-BTK axis also regulates signaling to integrin activation by BCR. Thus, BTK and JAK protein tyrosine kinases (PTKs) manifest a hierarchical activity both in chemokine- as well as BCR-mediated integrin activation and dependent adhesion, potentially suggesting the possibility of combined therapeutic approaches to B-CLL treatment.

Mechanistic Insights into CpG DNA and IL-15 Synergy in Promoting B Cell Chronic Lymphocytic Leukemia Clonal Expansion.

Malignant cell growth within patients with B cell chronic lymphocytic leukemia (B-CLL) is largely restricted to lymphoid tissues, particularly lymph nodes. The recent in vitro finding that TLR-9 ligand (oligodeoxynucleotide [ODN]) and IL-15 exhibit strong synergy in promoting B-CLL growth may be particularly relevant to growth in these sites. This study shows IL-15-producing cells are prevalent within B-CLL-infiltrated lymph nodes and, using purified B-CLL cells from blood, investigates the mechanism for ODN and IL-15 synergy in driving B-CLL growth. ODN boosts baseline levels of phospho-RelA(S529) in B-CLL and promotes NF-κB-driven increases in IL15RA and IL2RB mRNA, followed by elevated IL-15Rα and IL-2/IL-15Rβ (CD122) protein. IL-15→CD122 signaling during a critical interval, 20 to 36-48 h following initial ODN exposure, is required for optimal induction of the cycling process. Furthermore, experiments with neutralizing anti-IL-15 and anti-CD122 mAbs indicate that clonal expansion requires continued IL-15/CD122 signaling during cycling. The latter is consistent with evidence of heightened IL2RB mRNA in the fraction of recently proliferated B-CLL cells within patient peripheral blood. Compromised ODN+IL-15 growth with limited cell density is consistent with a role for upregulated IL-15Rα in facilitating homotypic trans IL-15 signaling, although there may be other explanations. Together, the findings show that ODN and IL-15 elicit temporally distinct signals that function in a coordinated manner to drive B-CLL clonal expansion.

PO-032 Displacement of hexokinase 2 from mitochondria induces mitochondrial Ca2 +overload and caspase-independent cell death in cancer cells

IntroductionHexokinase 2 (HK2) phosphorylates glucose for starting its utilisation in glycolysis and pentose phosphate pathway. In many cancer cell types these processes are enhanced and HK2 expression is strongly induced and mainly localised to the outer mitochondrial membrane, where it also exerts an anti-apoptotic activity. Genetic ablation in mouse highlights HK2 importance in tumour formation. Therefore, HK2 is a good target for antineoplastic strategies, but HK2 inhibitors can have important side effects as they affect glucose metabolism. Here we have developed an antineoplastic strategy based on HK2 detachment from mitochondria in order to induce tumour cell death without inhibiting hexokinase enzymatic activity.Material and methodsPeptide design and synthesis; hexokinase enzymatic activity assays. Measurements of mitochondrial membrane potential, intracellular Ca2+ levels, cell death and in vitro and in vivo tumorigenic assays on human and mouse cancer cell models (CT26 colon cancer cells, 4 T1 breast cancer cells, HeLa cervix carcinoma cells and primary human B-CLL cells).Results and discussionsWe have observed that in cancer cells HK2 locates at contact sites between mitochondria and endoplasmic reticulum called MAMs (mitochondria-associated membranes). We could selectively detach HK2 from MAMs by using a peptide that does not perturb hexokinase enzymatic activity. This treatment rapidly induces opening of the Inositol-3-Phospate-Receptor and the ensuing Ca2+ transfer from endoplasmic reticulum to mitochondria. As a consequence, a Ca2+ overload occurs in mitochondria, leading to permeability transition pore opening, mitochondrial membrane depolarization and apoptosis in a caspase-independent way. Peptide administration reduces allografic growth of breast and colon cancer cells without any noxious effect on healthy tissues, and elicits death of B-cell chronic lymphocytic leukaemia (B-CLL) cells freshly obtained by patients and in vivo.ConclusionWe have reported that HK2 locates in MAMs of cancer cells, where it acts as an important player in the control of their survival. Targeting HK2 with a peptide-based strategy constitutes a novel and promising anti-neoplastic approach.