Chronic Lymphoid Leukemia Cells Research Articles

ZAP-70 augments tonic B-cell receptor and CCR7 signaling in IGHV–unmutated chronic lymphocytic leukemia

AbstractExpression of ZAP-70 in a subset of patients with chronic lymphocytic leukemia (CLL) positively correlates with the absence of immunoglobulin heavy-chain gene (IGHV) mutations and is indicative of a more active disease and shorter treatment-free survival. We recently demonstrated that ZAP-70 regulates the constitutive expression of CCL3 and CCL4, activation of AKT, and expression of MYC in the absence of an overt B-cell receptor (BCR) signal, bona fide functions of BCR activation. We, here, provide evidence that these features relate to the presence of a constitutive tonic BCR signal, exclusively found in IGHV-unmutated CLL and dependent on the ZAP-70–mediated activation of AKT and its downstream target GSK-3β. These findings are associated with increased steady-state activation of CD19 and SRC. Notably this tonic BCR signal is not present in IGHV-mutated CLL cells, discordantly expressing ZAP-70. Results of quantitative mass spectrometry and phosphoprotein analyses indicate that this ZAP-70–dependent, tonic BCR signal regulates CLL cell migration through phosphorylation of LCP1 on serine-5. Indeed, we show that CCL19- and CCL21-induced chemotaxis is regulated by and dependent on the expression of ZAP-70 through its function to enhance CCR7 signaling to LCP1. Thus, our data demonstrate that ZAP-70 converges a tonic BCR signal, exclusively present in IGHV-unmutated CLL and CCR7-mediated chemotaxis.

The Marine Product Elatol Is a Novel Inhibitor of Mitochondrial Translation Triggering the Integrated Stress Response and Apoptosis in Leukemia and Lymphoma Cells

Background: Leukemia stem cells (LSCs) show unique, targetable dependence on activity of mitochondrial ribosomes (mitoribosomes). In addition, aggressive B-cell lymphomas may have gene expression signatures of increased dependence on oxidative phosphorylation (OXPHOS) and show sensitivity to mitoribosome inhibitors such as the FDA approved antibiotic tigecycline. Discovery and optimization of new mitoribosome inhibitors, however, is relatively unexplored in drug development for hematologic malignancies despite demonstrated therapeutic potential. Here, we assess the marine natural product elatol as a novel mitochondrial translation inhibitor. Methods and Results: We have previously reported that elatol, the major compound from the red alga Laurencia microcladia, is effective against several non-Hodgkin lymphomas and primary chronic lymphoid leukemia (CLL) cells. In vitro studies showed elatol inhibits eIF4A1 helicase activity, suppressing cytoplasmic cap-dependent translation initiation. Further assessments using 35S-methionine incorporation in newly synthesized polypeptides in HEK293T cells with or without single-digit micromolar concentrations of elatol for short time periods revealed strong attenuation of mitochondrial protein synthesis with no effect on mitochondrial transcription. Based on previously published work highlighting the effectiveness of tigecycline in leukemia and our data supporting elatol cytotoxicity in hematologic malignancies, we assessed elatol cytotoxicity and mitochondrial translation inhibition in several leukemia and lymphoma cell lines in comparison to tigecycline. All but one line decreased viability by 50% or more (compared to vehicle control) after elatol incubation with EC50 values near 1 μM. This potency was 10-40x higher than for tigecycline in side-by-side comparisons across several lines when compared at 72h. The compound cooperated to activate a heightened integrated stress response (ISR) through DELE1 cleavage by the mitochondrial stress-activated protease OMA1. This in turn suppressed glycolytic capacity, resulting in adenosine triphosphate (ATP) depletion and subsequent cell death. We found elatol does not affect integrity of small and large mitoribosome subunits through sedimentation property analysis using sucrose gradients. Although the specific target on the mitochondrial translation apparatus remains elusive, we found that its mechanism of action differs from that of chloramphenicol, which inhibits translation elongation. Conclusions: We have performed proof-of-concept studies using HEK293T and HeLa cell lines, isolated mitochondria from HEK293T, and leukemia and lymphoma cell lines to reveal that elatol is a potent inhibitor of mitochondrial protein synthesis at concentrations that do not affect cytoplasmic protein synthesis and that this mechanism differs from chloramphenicol. Elatol triggers the OMA1-DELE1-ATF4 mitochondrial stress pathway and apoptosis of leukemia and lymphoma cells. Tigecycline's compelling preclinical data in combination with kinase inhibitors informed design of a pending clinical trial (NCT02883036). Elatol's greatly improved potency provides a potential starting point for further optimization of this paradigm.

MiRNA-16-1 Suppresses Mcl-1 and Bcl-2 and Sensitizes Chronic Lymphocytic Leukemia Cells to BH3 Mimetic ABT-199.

ObjectiveChronic lymphoid leukemia (CLL) is the most common type of leukemia among adults. Increased levels of Mcl-1 and Bcl-xL is linked to resistance to Bcl-2 inhibitors including ABT-199. In this study, we investigated the effect of miRNA-16-1 on apoptosis and sensitivity of the CLL cells to ABT-199. Materials and Methods In this experimental study, the Mcl-1 and Bcl-2 expression were measured using qualitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting. The effect of treatments on cell survival and growth were explored with MTT assay and Trypan blue assay, respectively. The drug interaction was evaluated using combination index analysis. Apoptosis was assessed by ELISA cell death and caspase-3 activity assays. Results MiRNA-16-1 markedly inhibited the expression of Mcl-1 and Bcl-2 in a time dependent manner (P<0.05, relative to blank control). Pretreatment with miRNA-16-1 synergistically suppressed the cell growth and survival and reduced the half-maximal inhibitory concentration (IC50) value of ABT-199. Moreover, miRNA-16-1 markedly augmented the apoptotic effect of ABT-199 in CLL cells (P<0.05). ConclusionOur findings propose that miRNA-16-1 act in concert with ABT-199 to exert synergistic anticancer efficacy against CLL, which is attributed to the inhibition of Bcl-2 and Mcl-1. This may propose a promising strategy for CLL resistant patients.

Synthesis, in vitro and structural aspects of cap substituted Suberoylanilide hydroxamic acid analogs as potential inducers of apoptosis in Glioblastoma cancer cells via HDAC /microRNA regulation

Glioblastoma multiforme (GBM) is a heterogeneous, aggressive brain cancer characterized by chemo-resistance and cancer stemness. Histone deacetylases (HDACs) are a group of enzymes that regulate chromatin epigenetics which were in turn found to be controlled by microRNAs (miRs). The drug employed in chemotherapy for the treatment of GBM is Temozolomide (TMZ). Unfortunately, many GBM patients exhibit chemo-resistance to this drug. Here we have synthesized various Suberoyl anilide hydroxamic acid (SAHA) analogs with many substitutions at the cap site majority of which not yet studied. These SAHA analogs have exhibited profound cytotoxicity at 2 μM, and 4 μM concentrations in GBM cancer cell line U87MG, and 1 μM, and 2 μM concentrations in breast cancer cell line MCF-7. Surprisingly, these analogs have exhibited cytotoxic effects in chronic lymphoid leukemia cells (Raji) at 64 μM, and 128 μM concentrations due to mutated p53. Among all the synthesized analogs 3-Chloro-SAHA, 3-Chloro-4-fluoro SAHA have exhibited effective cytotoxicity in all cancer cells. These potent analogs inhibited HDAC-8 enzyme activity by 2-folds in U87MG, and MCF-7 cell lines and 7-folds decrease in HDAC-8 activity was observed in Raji cell line. These analogs decreased the expression of HDAC-2, HDAC-3 genes and enhanced the expression of p53 tumor suppressor. Interestingly, these compounds decreased the expression of Rictor, the main component of the mTORC2 complex involved cancer cell metabolism. Furthermore, these molecules have decreased oncogenic microRNA expression such as miR-21 and enhanced the expression of tumor suppressor microRNAs such as miR-143. The HDAC binding ability of these molecules was highly significant and have exhibited the ability to cross blood-brain barrier (BBB), and followed the Lipinski rule of five. Thus, these molecules need to be taken up further to clinics for better therapy against GBM either singly or combination therapy.

CD19 CAR T cells for adults with relapsed or refractory acute lymphoblastic leukaemia

CD19-directed chimeric antigen receptor (CAR) T cells first showed potency in adults with chronic lymphocytic leukaemia 1 Porter DL Levine BL Kalos M Bagg A June CH Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med. 2011; 365: 725-733 Crossref PubMed Scopus (2417) Google Scholar and with acute lymphoblastic leukaemia 2 Brentjens RJ Davila ML Riviere I et al. CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci Transl Med. 2013; 5177ra38 Crossref PubMed Scopus (1390) Google Scholar a decade ago, but clinical development of CAR T cells in these two disease populations stalled. Chronic lymphocytic leukaemia did not respond to CAR T cells as frequently as hoped, in part because of challenges with making the product from patients whose T cells were not as fit, either due to the underlying disease or exposure to years of chemotherapy. 3 Porter DL Hwang WT Frey NV et al. Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia. Sci Transl Med. 2015; 7303ra139 Crossref PubMed Scopus (1024) Google Scholar , 4 Frey NV Gill S Hexner EO et al. Long-term outcomes from a randomized dose optimization study of chimeric antigen receptor modified T cells in relapsed chronic lymphocytic leukemia. J Clin Oncol. 2020; 38: 2862-2871 Crossref PubMed Scopus (36) Google Scholar In adults with acute lymphoblastic leukaemia, severe toxic effects led to delays in order to test modified dosing strategies, 5 Frey NV Shaw PA Hexner EO et al. Optimizing chimeric antigen receptor T-cell therapy for adults with acute lymphoblastic leukemia. J Clin Oncol. 2020; 38: 415-422 Crossref PubMed Scopus (65) Google Scholar or even complete halting of trials. 6 Cancer DiscoveryJCAR015 in ALL: a root-cause investigation. Cancer Discov. 2018; 8: 4-5 Google Scholar The toxic effects were a result of the high potency of CAR T cells, which led to remarkable cytokine elevations and permeability of the blood–brain barrier that resulted in cerebral oedema in a series of high-profile cases. 6 Cancer DiscoveryJCAR015 in ALL: a root-cause investigation. Cancer Discov. 2018; 8: 4-5 Google Scholar Toxicity seemed to be particularly lethal with more chemotherapy lymphodepletion, with CAR T cells bearing the CD28 costimulatory signalling domain (so-called sprinter CAR T cells) or in the setting of a coincident infection, which is a fairly frequent occurrence in this patient population. Children and young adults with relapsed or refractory acute lymphoblastic leukaemia seemed to tolerate these cytokine elevations much better than adults, 7 Grupp SA Kalos M Barrett D et al. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med. 2013; 368: 1509-1518 Crossref PubMed Scopus (2268) Google Scholar and the first pivotal study and approval from the US Food and Drug Administration (FDA) were secured in this population with a CAR T-cell product (tisagenlecleucel) bearing the 4-1BB costimulatory domain (so-called marathoner CAR T cells). 8 Maude SL Laetsch TW Buechner J et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N Engl J Med. 2018; 378: 439-448 Crossref PubMed Scopus (1950) Google Scholar The FDA has since approved both CD28-bearing and 4-1BB-bearing CAR T cells targeting the CD19 antigen for adults with large-cell lymphoma (CD28-bearing: axicabtagene cilolecuel; 4-1BB-bearing: tisagenlecleucel and lisocabtagene maraleucel), mantle cell lymphoma (CD28-bearing: brexucabtagene autoleucel), and follicular lymphoma (CD28-bearing: axicabtagene cilolecuel). KTE-X19 for relapsed or refractory adult B-cell acute lymphoblastic leukaemia: phase 2 results of the single-arm, open-label, multicentre ZUMA-3 studyKTE-X19 showed a high rate of complete remission or complete remission with incomplete haematological recovery in adult patients with relapsed or refractory B-precursor acute lymphoblastic leukaemia, with the median overall survival not reached in responding patients, and a manageable safety profile. These findings indicate that KTE-X19 has the potential to confer long-term clinical benefit to these patients. Full-Text PDF

Continuous in vivo Metabolism by NMR.

Dense time-series metabolomics data are essential for unraveling the underlying dynamic properties of metabolism. Here we extend high-resolution-magic angle spinning (HR-MAS) to enable continuous in vivo monitoring of metabolism by NMR (CIVM-NMR) and provide analysis tools for these data. First, we reproduced a result in human chronic lymphoid leukemia cells by using isotope-edited CIVM-NMR to rapidly and unambiguously demonstrate unidirectional flux in branched-chain amino acid metabolism. We then collected untargeted CIVM-NMR datasets for Neurospora crassa, a classic multicellular model organism, and uncovered dynamics between central carbon metabolism, amino acid metabolism, energy storage molecules, and lipid and cell wall precursors. Virtually no sample preparation was required to yield a dynamic metabolic fingerprint over hours to days at ~4-min temporal resolution with little noise. CIVM-NMR is simple and readily adapted to different types of cells and microorganisms, offering an experimental complement to kinetic models of metabolism for diverse biological systems.

Therapeutic CD94/NKG2A blockade improves natural killer cell dysfunction in chronic lymphocytic leukemia

ABSTRACTNatural killer (NK)-cell count is predictive of chronic lymphoid leukemia (CLL) disease progression and their dysfunction is well documented, but the etiology of this is currently lacking. CLL cells have been shown to over-express HLA-E, the natural ligand for NKG2A expressed on NK-cells that generates a distinct negative signal relative to direct NK-cell cytotoxicity in other disease models. Utilizing a novel anti-NKG2A monoclonal blocking antibody (mAb), monalizumab, we explored the in vitro preclinical activity of targeting the NKG2A receptor, and the NKG2A/HLA-E interaction as a mechanism of tumor evasion in CLL patients. Our work confirmed overexpression of HLA-E on CLL B-cells and demonstrated NKG2A expression on CD56+/16+ NK-cells from CLL patients. We also demonstrate that blocking NKG2A on CLL NK-cells was sufficient to restore direct cytotoxicity ability of NK-cells against HLA-E-expressing targets without impacting NK-cell mediated antibody-dependent cellular cytotoxicity. Additionally, we proved the specificity of monalizumab in blocking NKG2A through Fc-blocking mechanisms. This paper provides justification for the potential clinical utility of monalizumab in the treatment of patients with CLL.

Metabolic plasticity in CLL: adaptation to the hypoxic niche.

Metabolic transformation in cancer is increasingly well understood. However, little is known about the metabolic responses of cancer cells that permit their survival in different microenvironments. We have used a nuclear magnetic resonance based approach to monitor metabolism in living primary chronic lymphoid leukemia (CLL) cells and to interrogate their real-time metabolic responses to hypoxia. Our studies demonstrate considerable metabolic plasticity in CLL cells. Despite being in oxygenated blood, circulating CLL cells are primed for hypoxia as measured by constitutively low level hypoxia-inducible factor (HIF-1α) activity and modest lactate production from glycolysis. Upon entry to hypoxia we observed rapid upregulation of metabolic rates. CLL cells that had adapted to hypoxia returned to the ‘primed' state when re-oxygenated and again showed the same adaptive response upon secondary exposure to hypoxia. We also observed HIF-1α independent differential utilization of pyruvate in oxygenated and hypoxic conditions. When oxygenated, CLL cells released pyruvate, but in hypoxia imported pyruvate to protect against hypoxia-associated oxidative stress. Finally, we identified a marked association of slower resting glucose and glutamine consumption, and lower alanine and lactate production with Binet A0 stage samples indicating that CLL may be divided into tumors with higher and lower metabolic states that reflect disease stage.

Neutralization of (NK-cell-derived) B-cell activating factor by Belimumab restores sensitivity of chronic lymphoid leukemia cells to direct and Rituximab-induced NK lysis.

Natural killer (NK) cells are cytotoxic lymphocytes that substantially contribute to the therapeutic benefit of antitumor antibodies like Rituximab, a crucial component in the treatment of B-cell malignancies. In chronic lymphocytic leukemia (CLL), the ability of NK cells to lyse the malignant cells and to mediate antibody-dependent cellular cytotoxicity upon Fc receptor stimulation is compromised, but the underlying mechanisms are largely unclear. We report here that NK-cells activation-dependently produce the tumor necrosis factor family member 'B-cell activating factor' (BAFF) in soluble form with no detectable surface expression, also in response to Fc receptor triggering by therapeutic CD20-antibodies. BAFF in turn enhanced the metabolic activity of primary CLL cells and impaired direct and Rituximab-induced lysis of CLL cells without affecting NK reactivity per se. The neutralizing BAFF antibody Belimumab, which is approved for treatment of systemic lupus erythematosus, prevented the effects of BAFF on the metabolism of CLL cells and restored their susceptibility to direct and Rituximab-induced NK-cell killing in allogeneic and autologous experimental systems. Our findings unravel the involvement of BAFF in the resistance of CLL cells to NK-cell antitumor immunity and Rituximab treatment and point to a benefit of combinatory approaches employing BAFF-neutralizing drugs in B-cell malignancies.

Erratum: Viability and stress protection of chronic lymphoid leukemia cells involves overactivation of mitochondrial phosphoSTAT3Ser727

Erratum: Viability and stress protection of chronic lymphoid leukemia cells involves overactivation of mitochondrial phosphoSTAT3Ser727

Activation-Induced Release of the TNF-Family Member BAFF By NK Cells Facilitates Resistance of Chronic Lymphoid Leukemia Cells to Direct and Rituximab-Induced NK Lysis

NK cells are cytotoxic lymphocytes that play an important role in the immunosurveillance of leukemia and, due to their ability to mediate antibody-dependent cellular cytotoxicity (ADCC), substantially contribute to the therapeutic benefit of antitumor antibodies like Rituximab. Available data indicate that the ability of NK cells to mediate ADCC is compromised in Chronic Lymphoid Leukemia (CLL), but the underlying mechanisms are still unclear. The TNF family member B cell activating factor (BAFF) was described to be aberrantly produced in mature B cell malignancies and contributes to disease pathophysiology e.g. by acting as a growth and survival signal for CLL cells. Here we report that NK cells express and release BAFF, and NK cell activation, notably including FcγRIIIa stimulation by Rituximab, results in increased secretion of BAFF (but not its close relative APRIL). Expression on the cell surface was neither detectable in resting nor in activated state. NK cell-derived BAFF enhanced the metabolic activity of primary CLL cells and protected CLL cells from chemotherapy-induced cell death. Moreover, exposure to BAFF profoundly diminished direct and Rituximab-induced lysis of primary CLL cells by allogeneic and autologous NK cells, while NK activation and degranulation per se remained unaffected. Notably, sensitivity of CLL cells to both chemotherapeutic treatment as well as direct lysis and Rituximab-induced ADCC of NK cells could be restored by the anti-BAFF antibody Belimumab (Benlysta®), which is approved for the treatment of systemic lupus erythematosus. Thus, our data provide evidence for the involvement of BAFF in the resistance of CLL cells to chemotherapy. Moreover, our results offer a functional explanation for the reportedly compromised ability of NK cells to combat lymphoid as compared to myeloid leukemias as well as their impaired ability to mediate ADCC upon Rituximab treatment in CLL patients. Our findings point to a possible benefit of combinatory approaches employing Rituximab and Belimumab for chemo-immunochemotherapy of B cell malignancies. DisclosuresNo relevant conflicts of interest to declare.

Development of a High-Throughput Screening Assay with Nurse-like Cell-Based Microenviroment in Chronic Lymphoid Leukemia Cells.

Background and Objectives: B-cell chronic lymphoid leukemia (CLL) is a lymphoproliferative disorder where specific microenvironment between B-cells and nurse-like Cells (NLC) seem to be involved in disease progression providing cell survival, proliferation and drug resistance. Consequently, functional screening platforms that can assess drug candidates within this microenvironment are needed. Our aim is to show the ability of the Exvitech® automated flow cytometry platform to screen agents that interfere with the microenvironmentxs protective scenario such as ibrutinib or idelalisib or standard CLL drugs such as fludarabine or prednisolone. This approach will allow us to select candidates in an in vitro assay and could personalize the treatment according the response to the drugs.Patients and methods: We have adapted Jan Burger’s published assay1. Peripheral blood mononuclear cells (PBMCs) from not previously treated CLL patients were isolated by density gradient centrifugation over Ficoll-Pacque and were used fresh (N=14) or cryopreserved (N=6). B-cells were assessed to be >90% viable by flow cytometry. NLC co-cultures were stablished by suspending PBMCs from CLL patients in complete RPMI medium with 10% FBS to a concentration of 2x107/ml. Cells were incubated for 14 days in 96-well plates and presence of NLC was confirmed by microscopy. After that, viability was investigated in B-cells treated with 8 concentrations of ibrutinib, idelalisib, fludarabine and prednisolone after 72h of incubation with annexin-V and the appropriate CLL flow cytometry markers. Drug response was evaluated as a depletion survival index of the B-cell population relative to the average of the control wells with NLC but without drug.Results: As expected, depletion of B-cells cultured without NLCs were significant greater than with NLC after the 72h incubation supporting the assay where NLCs protect B-CLL cells from in vitro spontaneous apoptosis. In a similar way, viability of fresh samples with NLC was higher than the corresponding frozen samples (84% vs 25%), though both could be used. Our results show a lower pharmacological median potency, measured as a higher EC50, when we work with NLC versus without NLC for ibrutinib (10µM vs 4µM), idelalisib (17µM vs 0.4µM) and prednisolone (3.5µM vs 1.5µM). However, the effect of fludarabine seems to be independent of the presence of the NLC in the cell culture (7µM vs 6µM). This is consistent with the protective role of microenvironment; more pronounced for ibrutinib and idelalisib. Interestingly with NLC, for each drug there is a significant interpatient variability (Figure 1); each line correspond to a different patient, reflecting the possibility that patients might be more sensitive or resistant to a certain drug in this particular scenario. There is a higher degree of patient sample stratification for idelalisib, fludarabine or prednisolone, where there are still an important % of B-cells alive for some patients after drug exposure at high concentration, supporting the notion of drug resistance. Synergism between some of these drugs was evaluated in 3 samples, with some samples being more synergistic than other, requiring a larger number of samples.Conclusions: Cellular and molecular interactions between B-cells and the microenvironment represented here with the NLC, have become an attractive target for CLL therapy. Because novel drugs such as ibrutinib or idelalisib are transforming CLL therapy targeting the microenvironment, novel technologies that could predict its effect are necessary. Here we have adapted a Nurse-Like Cell assay mimicking the microenvironment published by Burger1 to our ExviTech platform. The automated platform enables scaling of the data points acquired with this assay supporting characterization of drug activity by pharmacological dose response curves, as well as exploring synergistic interactions. As showed in the results and illustrated in Figure 1, there is a interpatient variability of the pharmacological profile for the studied drugs, if clinically validated, could help guiding a personalized treatment selection; measuring the drug activity inside this particular microenvironment responsible of drug resistance.1.- Burger JA et al. Blood. 2000 Oct 15;96(8):2655-63. [Display omitted] DisclosuresPrimo:Vivia Biotech: Employment. Martinez:Vivia Biotech: Membership on an entity’s Board of Directors or advisory committees. Gorrochategui:Vivia Biotech: Employment. Espinosa:Vivia Biotech: Employment. Arroyo:Vivia Biotech: Employment. Ballesteros:Vivia Biotech: Employment. Hernandez:Vivia Biotech: Employment.

Viability and stress protection of chronic lymphoid leukemia cells involves overactivation of mitochondrial phosphoSTAT3Ser727.

Chronic lymphoid leukemia (CLL) is characterized by the accumulation of functionally defective CD5-positive B lymphocytes. The clinical course of CLL is highly variable, ranging from a long-lasting indolent disease to an unpredictable and rapidly progressing leukemia requiring treatment. It is thus important to identify novel factors that reflect disease progression or contribute to its assessment. Here, we report on a novel STAT3-mediated pathway that characterizes CLL B cells-extended viability and oxidative stress control. We observed that leukemic but not normal B cells from CLL patients exhibit constitutive activation of an atypical form of the STAT3 signaling factor, phosphorylated on serine 727 (Ser727) in the absence of detectable canonical tyrosine 705 (Tyr705)-dependent activation in vivo. The Ser727-phosphorylated STAT3 molecule (pSTAT3Ser727) is localized to the mitochondria and associates with complex I of the respiratory chain. This pSer727 modification is further controlled by glutathione-dependent antioxidant pathway(s) that mediate stromal protection of the leukemic B cells and regulate their viability. Importantly, pSTAT3Ser727, but neither Tyr705-phosphorylated STAT3 nor total STAT3, levels correlate with prolonged in vivo CLL B cells survival. Furthermore, STAT3 activity contributes to the resistance to apoptosis of CLL, but not normal B cells, in vitro. These data reveal that mitochondrial (Mt) pSTAT3Ser727 overactivity is part of the antioxidant defense pathway of CLL B cells that regulates their viability. Mt pSTAT3Ser727 appears to be a newly identified cell-protective signal involved in CLL cells survival. Targeting pSTAT3Ser727 could be a promising new therapeutic approach.

Abstract 148: Release of the TNF-family member BAFF by NK cells contributes to the resistance of chronic lymphoid leukemia cells to direct and Rituximab-induced NK reactivity

Abstract NK cells are cytotoxic lymphocytes that are particularly important for the immunosurveillance of leukemia. Due to their ability to mediate antibody-dependent cellular cytotoxicity (ADCC), NK cells substantially contribute to the therapeutic benefit of antitumor antibodies like Rituximab, which is a crucial component in the treatment of B cell malignancies. Available data indicate that the ability of NK cells to target malignant cells and to mediate ADCC is compromised in Chronic Lymphoid Leukemia (CLL), but the underlying mechanisms are still unclear (Jaglowski et al., Blood 2010). With the TNF family member B cell activating factor (BAFF) that regulates, among others, the proliferation and survival of B cells, we believe to have identified a factor which contributes to these clinical observations. We report that NK cells express BAFF on the mRNA level and release the protein in a soluble form with the levels depending on activation state. Notably, relevant BAFF surface expression was neither detected on resting nor activated NK cells. Using a tetrazolium salt based colorimetric assay we found that NK cell-derived BAFF enhances the metabolic activity of primary CLL cells. In addition, BAFF protects CLL cells from chemotherapy-induced cell death e.g. upon treatment with Fludarabine and Cyclophosphamide. Notably, the BAFF-specific antibody Belimumab, which is approved for the treatment of systemic lupus erythematosus, was found to block the leukemia cell-protective effects of BAFF and to restore the sensitivity of the leukemia cells to chemotherapy. Furthermore, BAFF was found to substantially reduce direct NK cell lysis and also Rituximab-mediated ADCC in cultures with primary CLL cells as measured by Europium cytotoxicity assays. Our data on the capacity of BAFF to enhance the metabolic activity of B-CLL cells and their resistance to NK cell cytotoxicity suggest its involvement in the compromised ability of NK cells to combat lymphoid as compared to myeloid leukemias (Pende et al., Blood 2005) and the reportedly impaired ability of NK cells to mediate ADCC upon Rituximab treatment in CLL. Furthermore, our findings point to a possible benefit of combinatory approaches employing Rituximab and Belimumab for immunotherapy of B cell malignancies. Citation Format: Julia Wild, Benjamin J. Schmiedel, Andreas Maurer, Stefanie Raab, Pascal Schneider, Helmut R. Salih. Release of the TNF-family member BAFF by NK cells contributes to the resistance of chronic lymphoid leukemia cells to direct and Rituximab-induced NK reactivity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 148. doi:10.1158/1538-7445.AM2014-148

Mantle-Cell Lymphoma (MCL) Cells Are Highly Sensitive To ABT-199 But Their Sensitivity May Be Altered By The Microenvironment Via The Up-Regulation Of Bcl-Xl and Bcl2A1

Despite improvement in the treatment of Mantle-cell lymphoma (MCL), relapse invariably occurs and innovative strategies are needed. Bcl-2 inhibitors such as ABT-737 and ABT-263 (navitoclax), which target both Bcl-2 and Bcl-xL, demonstrated antitumor activity in B-cell malignancies. However, the clinical development of navitoclax is limited by a deep thrombocytopenia, which is induced by inhibition of Bcl-xL in platelets. To overcome this toxicity, ABT-199, the first-in-class orally bioavailable Bcl-2-selective BH3 mimetic, has been developed and showed promising antitumor activity in B-cell lymphoma while sparing platelets. In the present study, the apoptotic efficiency of ABT-199 in comparison with that of ABT-737 was evaluated in seven MCL cell lines. We found two MCL cell lines sensitive to ABT-199 (LD50 of 100 and 200 nM), one intermediate (LD50 of 1000nM) and 4 resistant (LD50 from 5000 to 10000 nM). Surprisingly, LD50 values of the 2 sensitive cell lines (MINO, GRANTA-519) were slightly higher for ABT-199 than for ABT-737. We further demonstrated that the Bcl-2/Mcl-1 ratio determined by RT-PCR is a predictive biomarker for ABT-199 sensitivity. To further determine the role of Mcl-1 in ABT-199 resistance, Mcl-1 siRNA were transfected in Z-138 and JEKO-1 cells. Mcl-1 silencing sensitized these 2 cell lines to low dose ABT-199 confirming the importance of Mcl-1 in ABT-199 resistance as previously shown for ABT-737. Moreover, in Z-138 cells, which highly express Bcl-xL, we showed that Bcl-xL silencing sensitized them to ABT-199. These results show that in addition to Mcl-1, Bcl-xL might also confer resistance to ABT-199-induced apoptosis in MCL. This could explain the slight difference of sensitivity of MCL cells between ABT-199 and ABT-737. In contrast to MCL cell lines, we found so far that ABT-199 efficiency killed all tested circulating primary cells from MCL patients (n=7) with LD50 values inferior to 10 nM. Because MCL cells reside mainly in lymph nodes, we wondered whether mimicking the microenvironment could impact the sensitivity of MCL cells to BH3 mimetics like it was previously demonstrated for chronic lymphoid leukemia cells. Thus, the ABT-199 sensitive MINO and GRANTA-519 cells were cultured on CD40L-expressing fibroblasts L in order to mimic the lymph node microenvironment. Both cell lines and primary cells became resistant to ABT-199 within 24h. Investigation of the underlying mechanism revealed a strong up-regulation of both Bcl-xL and Bcl2A1 protein expression. By contrast, culture of MCL cells with parental CD40L- fibroblasts or in conditioned medium from CD40L+ L fibroblasts culture failed to induce ABT-199 resistance. These results highlight the implication of the CD40L pathway in ABT-199 resistance through the up-regulation of Bcl-xL and Bcl2A1 in MCL. In conclusion, while circulating primary MCL cells are highly sensitive to ABT-199, it would be important to address the impact of microenvironment on long-term survival of MCL cells within lymph nodes under ABT-199 treatment. Disclosures:No relevant conflicts of interest to declare.

Mechanistic Insights into the Antileukemic Activity of Hyperforin

Hyperforin is a prenylated phloroglucinol present in the medicinal plant St John's wort (Hypericum perforatum). The compound has many biological properties, including antidepressant, anti-inflammatory, antibacterial and antitumor activities. This review focuses on the in vitro antileukemic effects of purified hyperforin and related mechanisms in chronic lymphoid leukemia (CLL) and acute myeloid leukemia (AML) - conditions that are known for their resistance to chemotherapy. Hyperforin induces apoptosis in both CLL and AML cells. In AML cell lines and primary AML cells, hyperforin directly inhibits the kinase activity of the serine/threonine protein kinase B/AKT1, leading to activation of the pro-apoptotic Bcl-2 family protein Bad through its non-phosphorylation by AKT1. In primary CLL cells, hyperforin acts by stimulating the expression of the pro-apoptotic Bcl-2 family member Noxa (possibly through the inhibition of proteasome activity). Other hyperforin targets include matrix metalloproteinase-2 in AML cells and vascular endothelial growth factor and matrix metalloproteinase-9 in CLL cells - two mediators of cell migration and angiogenesis. In summary, hyperforin targets molecules involved in signaling pathways that control leukemic cell proliferation, survival, apoptosis, migration and angiogenesis. Hyperforin also downregulates the expression of P-glycoprotein, a protein that is involved in the resistance of leukemia cells to chemotherapeutic agents. Lastly, native hyperforin and its stable derivatives show interesting in vivo properties in animal models. In view of their low toxicity, hyperforin and its derivatives are promising antileukemic agents and deserve further investigation in vivo.

Bimodal Induction of NK Cell Reactivity Against Acute Myeloid (AML) and Chronic Lymphoid Leukemia (CLL) by Fc-Engineered GITR-Fc Fusion Proteins.

AbstractAbstract 2143NK cells play an important role in anti-tumor immunity and largely contribute to the efficacy of therapeutic strategies like allogenic stem cell transplantation in AML and application of Rituximab that induces antibody-dependent cellular cytotoxicity (ADCC) in CLL. Recently, we demonstrated that the TNF family member GITR ligand (GITRL) is expressed on leukemia cells in a high proportion of AML and CLL patients and impairs direct and Rituximab-induced reactivity of NK cells which constitutively express its counterpart GITR (e.g., Buechele et al., Leukemia 2012). Here we developed a strategy to reinforce NK anti-leukemia reactivity by combining disruption of NK-inhibitory GITR-GITRL interaction with induction of ADCC against the GITRL-expressing leukemia cells using GITR-Ig fusion proteins with modified Fc moieties. Fc parts were engineered by amino acid exchange as previously described (Lazar et al., PNAS 2006; Armour et al., Eur. J. Immunol. 1999). Compared to wild type GITR-Ig (GITR-Fc-WT), our mutants (S239D/I332E and E233P/L234V/L235A/deltaG236/A327G/A330S) displayed highly enhanced (GITR-Fc-ADCC) and abrogated (GITR-Fc-KO) affinity to the Fc(gamma)RIIIa receptor (CD16) expressed on NK cells, respectively. In functional analyses of NK cells and primary leukemia cells, GITR-Fc-KO, which does not induce ADCC, already increased NK reactivity due to disruption of GITR-GITRL interaction. Treatment with GITR-Fc-WT further enhanced NK reactivity due to modest induction of ADCC, while GITR-Fc-ADCC induced highly increased NK-mediated target cell lysis, degranulation and cytokine production in a target-antigen dependent manner. With CLL cells, combined treatment with GITR-Fc-ADCC fusion protein and Rituximab caused additive effects, resulting in significantly enhanced NK cell ADCC. Notably, the effects of our fusion proteins were observed both in an allogenic setting and when employing NK cells of patients with autologous leukemia cells as targets. Our results demonstrate that Fc-engineered GITR-Fc-ADCC fusion protein may combine both neutralization of the NK-inhibitoryeffects of GITR-GITRL interaction and targeting GITRL-expressing malignant cells for NK anti-tumor reactivity and thus constitute an attractive immunotherapeutic means for the treatment of AML and CLL. Disclosures:No relevant conflicts of interest to declare.

Mechanistic Insights into the Antileukemic Activity of Hyperforin

Hyperforin is a prenylated phloroglucinol present in the medicinal plant St John's wort (Hypericum perforatum). The compound has many biological properties, including antidepressant, anti-inflammatory, antibacterial and antitumor activities. This review focuses on the in vitro antileukemic effects of purified hyperforin and related mechanisms in chronic lymphoid leukemia (CLL) and acute myeloid leukemia (AML) - conditions that are known for their resistance to chemotherapy. Hyperforin induces apoptosis in both CLL and AML cells. In AML cell lines and primary AML cells, hyperforin directly inhibits the kinase activity of the serine/threonine protein kinase B/AKT1, leading to activation of the pro-apoptotic Bcl-2 family protein Bad through its non-phosphorylation by AKT1. In primary CLL cells, hyperforin acts by stimulating the expression of the pro-apoptotic Bcl-2 family member Noxa (possibly through the inhibition of proteasome activity). Other hyperforin targets include matrix metalloproteinase-2 in AML cells and vascular endothelial growth factor and matrix metalloproteinase-9 in CLL cells - two mediators of cell migration and angiogenesis. In summary, hyperforin targets molecules involved in signaling pathways that control leukemic cell proliferation, survival, apoptosis, migration and angiogenesis. Hyperforin also downregulates the expression of P-glycoprotein, a protein that is involved in the resistance of leukemia cells to chemotherapeutic agents. Lastly, native hyperforin and its stable derivatives show interesting in vivo properties in animal models. In view of their low toxicity, hyperforin and its derivatives are promising antileukemic agents and deserve further investigation in vivo.

Mechanistic Insights into the Antileukemic Activity of Hyperforin

Hyperforin is a prenylated phloroglucinol present in the medicinal plant St John's wort (Hypericum perforatum). The compound has many biological properties, including antidepressant, anti-inflammatory, antibacterial and antitumor activities. This review focuses on the in vitro antileukemic effects of purified hyperforin and related mechanisms in chronic lymphoid leukemia (CLL) and acute myeloid leukemia (AML) – conditions that are known for their resistance to chemotherapy. Hyperforin induces apoptosis in both CLL and AML cells. In AML cell lines and primary AML cells, hyperforin directly inhibits the kinase activity of the serine/threonine protein kinase B/AKT1, leading to activation of the pro-apoptotic Bcl-2 family protein Bad through its non-phosphorylation by AKT1. In primary CLL cells, hyperforin acts by stimulating the expression of the pro-apoptotic Bcl-2 family member Noxa (possibly through the inhibition of proteasome activity). Other hyperforin targets include matrix metalloproteinase-2 in AML cells and vascular endothelial growth factor and matrix metalloproteinase-9 in CLL cells – two mediators of cell migration and angiogenesis. In summary, hyperforin targets molecules involved in signaling pathways that control leukemic cell proliferation, survival, apoptosis, migration and angiogenesis. Hyperforin also downregulates the expression of P-glycoprotein, a protein that is involved in the resistance of leukemia cells to chemotherapeutic agents. Lastly, native hyperforin and its stable derivatives show interesting in vivo properties in animal models. In view of their low toxicity, hyperforin and its derivatives are promising antileukemic agents and deserve further investigation in vivo. Keywords: Apoptosis, drug resistance, hyperforin, leukemia, proliferation, signaling

Cancer Battlefield: Six Characters in Search of an Author

ImmunotherapyVol. 4, No. 8 EditorialCancer battlefield: six characters in search of an authorLeslie Huye & Barbara SavoldoLeslie HuyeCenter for Cell & Gene Therapy, Departments of Pediatrics, Baylor College of Medicine, The Methodist Hospital & Texas Children’s Hospital, Houston, TX 77030, USASearch for more papers by this author & Barbara SavoldoCenter for Cell & Gene Therapy, Departments of Pediatrics, Baylor College of Medicine, The Methodist Hospital & Texas Children’s Hospital, Houston, TX 77030, USASearch for more papers by this authorPublished Online:4 Sep 2012https://doi.org/10.2217/imt.12.77AboutSectionsView ArticleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit View articleKeywords: chimeric T cell receptorimmune evasionimmunotherapytumor microenvironmentReferences1 Dudley M, Wunderlich J, Yang JC et al. Adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma. J. Clin. Oncol.23(10),2346–2357 (2005).Crossref, Medline, CAS, Google Scholar2 Dudley ME, Yang JC, Sherry R et al. Adoptive cell therapy for patients with metastatic melanoma: evaluation of intensive myeloablative chemoradiation preparative regimens. J. Clin. Oncol.26(32),5233–5239 (2008).Crossref, Medline, CAS, Google Scholar3 Quintarelli C, Dotti G, Hasan ST et al. High-avidity cytotoxic T lymphocytes specific for a new PRAME-derived peptide can target leukemic and leukemic-precursor cells. Blood117(12),3353–3362 (2011).Crossref, Medline, CAS, Google Scholar4 Bonini C, Brenner MK, Heslop HE, Morgan RA. Genetic modification of T cells. Biol. Blood Marrow Transplant.17(Suppl. 1),S15–S20 (2011).Crossref, Medline, CAS, Google Scholar5 Kohn DB, Dotti G, Brentjens R et al. CARS on track in the clinic. Mol. Ther.19(3),432–438 (2011).Crossref, Medline, CAS, Google Scholar6 Topfer K, Kempe S, Muller N et al. Tumor evasion from T cell surveillance. J. Biomed. Biotechnol.2011,918471 (2011).Crossref, Medline, Google Scholar7 Marshall NA, Christie LE, Munro LR et al. Immunosuppressive regulatory T cells are abundant in the reactive lymphocytes of Hodgkin lymphoma. Blood103(5),1755–1762 (2004).Crossref, Medline, CAS, Google Scholar8 Solinas G, Germano G, Mantovani A, Allavena P. Tumor-associated macrophages (TAM) as major players of the cancer-related inflammation. J. Leukoc. Biol.86(5),1065–1073 (2009).Crossref, Medline, CAS, Google Scholar9 Gabrilovich DI, Nagaraj S. Myeloid-derived suppressor cells as regulators of the immune system. Nat. Rev. Immunol.9(3),162–174 (2009).Crossref, Medline, CAS, Google Scholar10 Barnas JL, Simpson-Abelson MR, Yokota SJ, Kelleher RJ, Bankert RB. T cells and stromal fibroblasts in human tumor microenvironments represent potential therapeutic targets. Cancer Microenviron.3(1),29–47 (2010).Crossref, Medline, CAS, Google Scholar11 Motz GT, Coukos G. The parallel lives of angiogenesis and immunosuppression: cancer and other tales. Nat. Rev. Immunol.11(10),702–711 (2011).Crossref, Medline, CAS, Google Scholar12 Porter DL, Levine BL, Kalos M, Bagg A, June CH. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N. Engl. J. Med.365(8),725–733 (2011).Crossref, Medline, CAS, Google Scholar13 Muranski P, Boni A, Wrzesinski C et al. Increased intensity lymphodepletion and adoptive immunotherapy – how far can we go? Nat. Clin. Pract. Oncol.3(12),668–681 (2006).Crossref, Medline, CAS, Google Scholar14 Chinnasamy D, Yu Z, Kerkar SP et al. Local delivery of interleukin-12 using T cells targeting VEGF receptor-2 eradicates multiple vascularized tumors in mice. Clin. Cancer Res.18(6),1672–1683 (2012).Crossref, Medline, CAS, Google Scholar15 Pegram HJ, Lee JC, Hayman EG et al. Tumor-targeted T cells modified to secrete IL-12 eradicate systemic tumors without need for prior conditioning. Blood119(18),4133–4141 (2012).Crossref, Medline, CAS, Google Scholar16 Chmielewski M, Kopecky C, Hombach AA, Abken H. IL-12 release by engineered T cells expressing chimeric antigen receptors can effectively Muster an antigen-independent macrophage response on tumor cells that have shut down tumor antigen expression. Cancer Res.71(17),5697–5706 (2011).Crossref, Medline, CAS, Google Scholar17 Bollard CM, Rossig C, Calonge MJ et al. Adapting a transforming growth factor β-related tumor protection strategy to enhance antitumor immunity. Blood99(9),3179–3187 (2002).Crossref, Medline, CAS, Google Scholar18 Vanneman M, Dranoff G. Combining immunotherapy and targeted therapies in cancer treatment. Nat. Rev. Cancer12(4),237–251 (2012).Crossref, Medline, CAS, Google Scholar19 De Angelis B, Dotti G, Quintarelli C et al. Generation of Epstein–Barr-virus-specific cytotoxic T lymphocytes resistant to the immunosuppressive drug tacrolimus (FK506). Blood114(23),4784–4791 (2009).Crossref, Medline, CAS, Google Scholar20 Huye LE, Nakazawa Y, Patel MP et al. Combining mTor inhibitors with rapamycin-resistant T cells: a two-pronged approach to tumor elimination. Mol. Ther.19(12),2239–2248 (2011).Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByStrategies to Enhance Migration and Persistence of Chimeric Antigen Receptor (CAR)-T Cells into Tumors31 August 2016 Vol. 4, No. 8 Follow us on social media for the latest updates Metrics Downloaded 150 times History Published online 4 September 2012 Published in print August 2012 Information© Future Medicine LtdKeywordschimeric T cell receptorimmune evasionimmunotherapytumor microenvironmentFinancial & competing interests disclosureThis work was supported by the William Lawrence and Blanche Hughes Foundation, by NIH-NCI Lymphoma SPORE P50 CA126752, NIH R01CA131027 and by a Leukemia and Lymphoma Society Translational Research grant. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.PDF download