Blood Of Chronic Lymphocytic Leukemia Patients Research Articles

Relevance of fatty acid metabolism in proliferating CLL cells

Chronic lymphocytic leukemia (CLL) cells undergo, during their life, iterative cycles of re-activation and subsequent clonal expansion. We previously demonstrated that the antidiabetic drug metformin, known to also inhibit oxidative phosphorylation (OXPHOS), inhibits cell cycle entry of leukemic cells derived ex-vivo from the peripheral blood of CLL patients and stimulated in vitro by cell culture systems that recreate a microenvironment to drive their proliferation (Bruno et al, Oncotarget, 2015). However, overtly proliferating CLL cells were resistant to the cytostatic effects of metformin. Since metformin switched the energetic metabolism of activated, not yet proliferating, CLL cells from OXPHOS to accelerated glycolysis, in the present study we asked whether combining metformin with glycolysis impairment could inhibit also proliferating CLL cells. Still, CLL cells recovered from a transitory block and rescued in vitro proliferation. What kind of energetic reprogramming was involved in the resistance of proliferating CLL cells to glucose utilization? Recent studies highlight on the role of fatty acid utilization of CLL cells. We asked 1) whether inhibitors of lipid metabolism could impair proliferation of in vitro stimulated CLL cells; 2) whether impairing glucose energetic pathways could act synergistically with beta oxidation inhibitors.We found that inhibitors of critical steps of fatty acid metabolisms, such as carnitine-palmitoyl transferase 1A (CPT1A) -rate-limiting enzyme for fatty acid import into mitochondria- or Peroxisome Proliferator-Activated Receptor (PPAR)-alpha -regulator of beta-oxidation- administered at clinically achievable doses, were ineffective on quiescent CLL cells and on CLL cells stimulated by the microenvironment during the first stages of activation. Conversely, remarkable susceptibility to undergo apoptosis was observed at later stages of cell activation and during overt proliferation. Synergism with impairment of other energetic pathways occurred depending on the stage of activation of the in vitro stimulated CLL cells.The results suggest that energetic metabolic pathways could be relevant targets for CLL treatment, provided that the complex metabolic reprogramming network during the transition of leukemic cells from quiescence to proliferation, and back, are clearly elucidated. This work was supported by grants from AIRC IG15426.

The Retinoid Derivative Drug, Acitretin, Modulates CD38 Expression on MEC-1 Cells and Primary Chronic Lymphocytic Leukemia (CLL) Cells and Reduces Migration in Response to the Chemokine CXCL12

Background: CD38 is a cell surface receptor and ectoenzyme expressed in a variety of haematological malignancies. In CLL, CD38 expression is a marker of poor prognostic disease and is now attracting growing interest as a target molecule for anti CLL therapy.Retinoids modulate CD38 expression due to the presence of the Retinoic Acid Response Element (RARE) DNA sequence in the promotor region of the CD38 gene. In this study we demonstrate that the retinoic acid derivative, acitretin, upregulates the expression of CD38 on the MEC-1 cell line and on primary CLL cells from CD38 positive patients. We propose that, by doing so, Acitretin may render these cells more susceptible to the actions of anti CD38 monoclonal antibody drugs and thus have a potential role as an adjunct to such therapies.Methods: Primary CLL cells were freshly isolated from whole blood of CLL patients using the FICOLL density centrifugation technique. The CLL like cell line MEC-1 was obtained from DSMZ, Germany.MEC-1 cells and cells from three CD38 negative and five CD38 positive patients were treated with 10micM acitretin for 24, 48 and 72hrs. CD38 expression was measured by FACS analysis at baseline and at the different time points. Results are expressed as fold increase in Mean Fluorescence Intensity (MFI).Migration assays were performed using 2x10^6 cells per well incubated with 10microM acitretin overnight in 1%FBS. Cells were subsequently transferred to chamber inserts overlying 10%FBS media containing 200ng/ml CXCL12 in all wells, except negative controls. Cells were then allowed to migrate for 4hrs. Migration was calculated as percentage of cells in lower chamber to upper chamber.Results: Acitretin increased the expression of CD38 on MEC-1 cells by a mean of 7.4 fold and on cells from CD38 positive patients by a mean of 3.4 folds (n=5). Acitretin, however, did not modulate CD38 expression levels on primary CLL cells from CD38 negative patients (n=3).Migration of primary CLL cells from 11/14 patients toward the CXCL12 chemokine was reduced in response to Acitretin treatment as compared to untreated controls. Mean migration of treated cells was 12.3% versus 27.3% mean migration in untreated controls.Conclusion: Our findings re-affirm the potent effect of retinoid drugs as upregulators of CD38 expression and suggest that their use in CLL therapeutics in conjunction with anti CD38 monoclonal antibodies should be explored. We also propose that acitretin appears to exert a favourable blocking effect on CLL cell migration towards micro-environmental chemokines such as CXCL12, warranting further evaluation. DisclosuresMurphy:Celgene: Honoraria.

Chronic Lymphocytic Leukemia Patients and Eµ-TCL1 Mice Share a Phenotype of Functional Granulocyte-like and Dysfunctional Monocyte-like Myeloid Derived Suppressor Cells

Immune imbalance is a common characteristic of patients with chronic lymphocytic leukemia (CLL). This feature is shared with Eμ-TCL1 transgenic mice that, like CLL patients, exhibit an expansion of CD5+ B cells with associated non-B-cell defects. In patients and in mice, T-cell responses are often ineffective. This alteration is generally considered due to the direct effects of the leukemic cells. The expansion of myeloid derived suppressor cells (MDSCs), which play a major role in helping tumor cells escape immune surveillance by inhibiting T-cell responses, is promoted by many cancers. MDSCs are a heterogeneous population of cells that are subdivided into monocyte-like (m-MDSC) and granulocyte-like (g-MDSC) subsets, both in humans and mice. There we have investigated the extent that patients with CLL have expansions of MDSCs, what are their types and functions, and how these correlate with the Eμ-TCL1 mice model.Using flow cytometry on cryopreserved PBMCs, we found that the absolute numbers of MDSCs (HLA-DRlo/CD11b+/CD33+) in 49 untreated CLL patients were significantly higher than 15 healthy controls (HCs) (966 446 vs. 163 578 cells/ml, P<0.001). Moreover, we observed that the absolute numbers of MDSCs significantly correlated with CLL B-cell counts in the blood (P =0.005, Spearman r=0.423). Of note, the distribution between m-MDSCs (CD14+) and g-MDSCs (CD15+) was dramatically different, with CLL patients exhibiting significantly higher numbers and percentages of g-MDSCs than HCs (702 296 vs. 26 818 cells/ml, P<0.001; 50.89 vs. 16.98%, P<0.001).In line with these results, when we explored the MDSC populations (CD11b+/GR1+) in Eμ-TCL1 mice of 5-16 months of age with leukemia cell blood counts ranging from 0.1 to 100 x 106 cell/ml. This analysis indicated a positive correlation between MDSCs and leukemic CD19+ CD5+ cells (P =0.003; Spearman r=0.328). Furthermore, the dot-plot analysis of GR1 and CD11b showed three well defined cell populations: one monocytic (Ly6-C+) and two granulocytic (Ly6-G+ CD11blo and Ly6-G+ CD11bhi). As in patients, the g-MDSC population was larger than the m-MDSC population (884 100 vs. 454 700,P =0.016). However in this case, the m-MDSCs correlated with the numbers of circulating leukemic cells (P<0.001; Spearman r=0.463) and the g-MDSCs did not. The latter was the case even when they were subdivided into both CD11blo and CD11bhi subgroups. A similar pattern was observed when analyzing single cell suspensions from murine spleens.When we evaluated the ability of MDSCs to inhibit autologous T-cell proliferation in CLL patients (n=7), we observed a consistent reduction of proliferation only when co-culturing with g-MDSCs(P =0.034). In contrast, the effects of m-MDSCs on T-cell expansion were varied and insignificant statistically. In 5 CLL samples, we induced m-MDSCs (im-MDSCs) from purified CD33+ cells in vitro with GM-CSF, IL10, and IL6; the im-MDSCs effectively suppressed T-cell proliferation in 4 of 5 cases at an average inhibition of 33% (range: 10-79%). Thus, dysfunctional m-MDSC suppression was not inherent and functional suppression could be achieved by stimulation of CLL precursor cells. Similarly in 3 independent experiments performed with MDSCs from Eμ-TCL1 mice (12-14 months of age), we observed a reduction of in vitro proliferation with g-MDSCs (P =0.049) and not with m-MDSCs. In addition, for those Eμ-TCL1 animals for which sufficient sample was available, we subdivided the g-MDSC population into the two subpopulations based on CD11b density; the CD11blo subset present less nuclear segmentation and higher suppressive activity.In summary, absolute numbers of MDSCs in the blood of CLL patients and Eμ-TCL1 mice are elevated and correlate with the levels of expansion of the leukemia. The major subtype in both situations was g-MDSCs.These g-MDSCs were functionally competent suppressors, whereas m-MDSCs were impaired in this function. In CLL patients, this m-MDSC suppressor defect could be corrected by in vitro stimulation with growth factors that support monocyte differentiation. The high similarity between CLL patients and Eμ-TCL1 mice in relation to MDSC number and function suggest that an imbalance in g-MDC vs. m-MDSC function may affect CLL development and expansion, altering interactions with members of the microenvironment such as T cells. DisclosuresNo relevant conflicts of interest to declare.

Single Cell Analysis of IG Genes in CLL: Cases with Multiple IGH Rearrangements Are Constituted of Several Independent Clones Even When Indistinguishable By Flow Cytometry

Abstract Current evidence suggests that in 2-5% of chronic lymphocytic leukemia (CLL) cases, multiple B cell clones - each expressing unique productive immunoglobulin gene rearrangements (P-IGH)- can be detected. However, only in 35% of them, coexistence of at least two leukemic clones was revealed by flow-cytometry (Plevova et al, Haematologica 2014). In the remaining 65% of cases, presence of multiple clones was presumed based on the number of immunoglobulin gene rearrangements detected, but underlying biological cause, also possibly including lack of allelic exclusion, was not satisfactorily proven at the level of single cells. Therefore, we developed a technique for simultaneous analysis of IGH, IGK and IGL rearrangements at the single cell level and performed further investigation of cases with multiple P-IGH. In years 2006-2015, patients with multiple clonal P-IGHs were searched among 1670 CLL patients examined for IGHV mutation status. Identified cases were submitted to flow-cytometric measurement of surface markers CD5, CD19, CD20, CD23, CD43, CD45, sIgK, sIgL and FMC7 on sorted cells where possible. Based on this examination, patients with clonal P-IGHs exceeding the number of CLL populations distinguished by flow-cytometry were tested using single cell analysis. Patients with corresponding number of P-IGHs and CLL populations (i.e. confirmed as biclonal CLL according to the flow-cytometry and PCR-based IGH detection) served as a control of the technique accuracy. Single cell analysis technique was developed at our department to detect transcribed IGH, IGK and IGL simultaneously in individual cells. B cells from peripheral blood of CLL patients were separated by gradient centrifugation with depletion of non-B cells. Single CD19+ cells were then sorted using FACS Aria III into 96-well plates containing lysis buffer, followed by 2 rounds of multiplex nested RT-PCR, capillary electrophoresis and Sanger sequencing. For each patient, 1-3 plates were analyzed. To consider an IG rearrangement clonal, it had to be detected at least in 3 wells. We detected multiple clonal P-IGHs in 76/1670 (4,6%) CLL patients analyzed. Expression of surface markers was assessed by flow cytometry in 37/76 patients: In 24/37(65%), the number of P-IGHs exceeded number of distinguished populations. Single cell analysis was performed in 16/24 cases, 15/16 patients displayed only one homogeneous CLL population by flow cytometry and 1/16 patient displayed two distinguished populations but three P-IGHs. Two patients with corresponding number of P-IGHs and CLL populations were used as a control. The median of wells tested per patient was 92 and P-IGH detection efficacy 83%. In 12/16 tested patients, as well as in two controls, no cell with more than one P-IGH was detected, confirming the expansion of multiple B-cell clones in all of them. Also, based on the structure of detected clonal IGH rearrangements in each case, a possibility of VH replacement was excluded. In 2 cases, we observed intraclonal diversification within one of the present clones (expressing either IGHV1-69, or IGHV3-72), a rare phenomenon described in CLL. In the remaining 4/16 cases we failed to detect one of the expected P-IGHs likely due to its underrepresentation in a sample, which is supported by results obtained from quantitative PCR with allele-specific primers in a bulk sample. Light chains were successfully detected in 10/12 analyzed cases and two controls; in 4 cases, single cell analysis revealed transcribed clonal IG rearrangements previously undetected in bulk samples. Importantly, each identified clonal IGK/IGL was repeatedly detected exclusively with only one distinct clonal P-IGH, thus constituting independent B cell receptors in independent leukemic clones. We confirm and substantially extend the notion that oligoclonality is the major cause of multiple P-IGH detection in CLL. Obtained information on P-IGH and P-IGK/L pairing will help in further investigation of IG receptors in oligoclonal CLL, as the biological background of oligoclonality in CLL still remains to be elucidated. Supported by grants IGA NT13493-4/2012, MUNI/A/1180/2014 and AZV 15-30015A. Disclosures No relevant conflicts of interest to declare.

The Transcription Factor IRF4 Is Crucial for CLL Progression and Regulates Survival and Proliferation in a Microenvironment Related Manner

Introduction: Chronic Lymphocytic Leukemia (CLL) is characterized by an accumulation of CD19+ CD5+ CLL cells in the peripheral blood and the lymphoid compartments. The disease is still incurable despite recent advances in the development of novel therapy concepts (e.g. inhibitors of BCR signaling). Although it is not entirely clear how CLL develops, proliferation centers located in the lymph node and the bone marrow are thought to be the crucial niche that provides the surrounding in which CLL cells originate and re-establish the clone after therapy, leading to relapse in almost 100% of CLL patients. In analogous normal lymphoproliferative environments the transcription factor IRF4 plays an important role in controlling B cell maturation, differentiation, proliferation and survival. A SNP in the human IRF4 3’UTR was linked to CLL susceptibility (DiBernardo Nat.Gen. 2008) and in the mouse model germline deletion of IRF4 in all lineages contributed to CLL development in the New Zealand Black and in the VH11 mouse model (Shukla Blood 2013, Ma J.Biol.Chem. 2013).Methods: CLL samples were collected from the peripheral blood of CLL patients according to the Declaration of Helsinki and with written informed consent. IRF4 mRNA expression analyses were performed in purified CD19+ CLL cells using TaqMan Real Time PCR. In vitro studies were performed in soloculture without stimulation or in coculture systems mimicking the lymph node microenvironment, as previously described (Asslaber Br.J.Haematol. 2013). IRF4 knockdown was performed using lipofection and IRF4 specific siRNAs or non-targeting control siRNAs. IRF4 protein expression was measured by intracellular flow cytometry. Proliferation was detected by Ki-67 intracellular staining. All intracellular stainings were performed after gating on viable CD19+ CD5+ CLL cells. Cell death was assessed by Annexin V / 7AAD staining or using a fixable viability dye.Results: Overall, IRF4 mRNA expression was decreased in CLL patients (N=101) when compared to healthy donors (N=8, P=0.04). In the CLL cohort low IRF4 mRNA expression (cut-off determined by ROC analysis and Youden Index calculation) was significantly correlated with earlier time to treatment (median treatment-free survival: 40 month in the IRF4LOW and 69 month in the IRF4HIGH CLL patient group (P<0.01)). This was proved to be independent of mutation state and other CLL risk factors in multivariate analysis. Using in vitro knockdown of IRF4 in solo- and coculture models we found that lowered expression of IRF4 was associated with increased CLL cell survival and abrogated spontaneous apoptosis (N=11, P<0.01). Moreover, we detected a failure of CLL cells to respond to CD40L signals derived from the microenvironment after IRF4 knockdown, which was associated with abrogated proliferation (N=9, P<0.01). Indeed microenvironmental stimuli (e.g. CD40L ligation or addition of activated T cells) were required to maintain IRF4 expression in vitro (N=17, P < 0.001) which also corresponds to our observation that IRF4 positive CLL cells were highly overrepresented in the CXCR4dim CD5brightCLL population (N = 55, P < 0.0001) which is considered to be the proliferative CLL cell fraction emigrated from the lymph node (Calissano Mol. Med. 2011).Conclusion: We demonstrate that low IRF4 expression is an independent negative prognostic marker for CLL progression. In the microenvironmental context IRF4 plays a dual role, with high expression limiting CLL survival on the one hand but being a crucial mediator of CD40L mediated proliferation on the other hand. While our data suggest that IRF4 is upregulated in response to microenvironmental stimuli the association of low IRF4 levels with bad prognosis suggests that IRF4 levels may serve to limit CLL growth by enhancing cell death responses. The exact mechanisms by which IRF4 impacts CLL pathophysiology is currently being investigated in vivo using B-cell specific IRF4-deficient CLL mouse models. DisclosuresNo relevant conflicts of interest to declare.

Natural Killer Cell Dysfunction in Chronic Lymphocytic Leukemia Is Associated with Loss of the Mature KIR3DL1+ Subset

Background: B-cell chronic lymphocytic leukemia (CLL) is a common blood cancer characterized by high prevalence of malignant B cells in peripheral blood. Small lymphocytic lymphoma (SLL) is considered to be a different presentation of the same disease, with the malignant B cells primarily localized in lymph nodes. Natural killer (NK) cells are innate immune effectors that can spontaneously identify and kill malignant cells, especially hematopoietic cancers. In peripheral blood of CLL patients, NK cells are chronically exposed to significant tumor burden, which is predicted to influence their phenotype and function. Effective NK cell function may be particularly beneficial in CLL patients, since commonly-used monoclonal antibody therapies (e.g. rituximab, alemtuzumab) rely at least partially on ADCC-mediated by NK cells.Methods: We performed a prospective analysis of biomarkers on fresh peripheral blood lymphocytes from 25 untreated CLL patients, 10 untreated SLL and 17 age-matched healthy controls by 10-color flow cytometry. All subjects signed IRB approved informed consent forms. Our study analyzed 180 distinct biomarker parameters, with a particular focus on NK and T cells. Differences in biomarker expression between patients with SLL, CLL, and healthy controls were compared by Wilcoxon rank-sum test.Results: Absolute numbers of NK and T cells per µl of blood were significantly higher in CLL patients, and this correlated with increased B cell numbers. As indicators of immune suppression, the frequency of regulatory T cells was significantly increased in CLL samples, as were levels of PD-1 expression on T cells and CD56dim NK cells. NK cells in CLL expressed higher levels of CD27, which is characteristic of a less mature phenotype, and CD56dim cells expressed lower levels of NKG2D. Compared to healthy controls, CLL samples displayed a marked reduction in degranulation by CD56dim NK cells in response to transformed 721.221 B cells, either with or without rituximab. CD56dim NK cells from CLL patients were also less viable under resting conditions or when challenged with target cells, especially in ADCC responses. We further observed a striking reduction in the frequency and viability of KIR3DL1+ NK cells, which progressed over time in most CLL patients. Surprisingly, CLL patients with the highest levels of PD-1 expression on NK cells possessed genes for both KIR3DL1 and its ligand, HLA-Bw4. Our findings were also clearly evident in a CLL patient compared to her healthy monozygotic twin, thereby providing compelling support for the results in the full patient cohort. The altered expression levels of nearly all of the NK cell biomarkers and degranulation were less pronounced in blood samples from SLL patients, presumably due to low tumor burden in peripheral blood.Conclusions: CLL patients have increased numbers of NK cells in peripheral blood, but these NK cells are less mature, are significantly depleted of the KIR3DL1+ subset, and have deficits in degranulation response, reduced expression of NKG2D activating receptor, increased expression of inhibitory PD-1, and enhanced susceptibility to activation-induced death when challenged with tumor targets and rituxumab. Our findings support the hypothesis that immune dysfunction in CLL may be due in part to a selective loss of mature KIR3DL1+ NK cells, possibly upon encountering overwhelming tumor burden in peripheral blood, and CLL patients may benefit from therapeutic strategies that augment NK cell function. DisclosuresNo relevant conflicts of interest to declare.

The differences in thermal profiles between normal and leukemic cells exposed to anticancer drug evaluated by differential scanning calorimetry

Chronic lymphocytic leukemia (CLL) is a heterogenous disease with an imbalance between apoptosis and cell proliferation. Therefore, the main goal in CLL therapy is to induce apoptosis and effectively support this process in transformed B lymphocytes. In the current study, we have compared differential scanning calorimetry (DSC) profiles of nuclei isolated from CLL cells and normal mononuclear cells exposed to cladribine or fludarabine combined with mafosfamide (CM; FM), and additionally to CM combined with monoclonal antibody—rituximab (RCM) for 48 h, as well as in culture medium only (controls). Under current study, the mononuclear cells from peripheral blood (PBMCs) of healthy individuals have been included. The obtained results have shown the presence of thermal transition at 95 ± 5 °C in most of nuclear preparations (92.2 %) isolated from blood of CLL patients. This thermal characteristic parameter was changed after drug exposure, however, to a different extent. These thermal changes were accompanied by the decrease of cell viability, an elevation of apoptosis rate and the changes in expression/proteolysis of poly(ADP-ribose)polymerase-1—main marker of apoptosis. Importantly, in DSC profiles of nuclear preparations of PBMCs from blood of healthy donors exposed to investigated drug combinations and control CLL cells, the lack of such changes was observed. Our results confirmed that DSC technique complemented with other biological approaches could be helpful in tailoring therapy for CLL patients.

Matrix metalloproteinase-9 is involved in chronic lymphocytic leukemia cell response to fludarabine and arsenic trioxide.

BackgroundMatrix metalloproteinase-9 (MMP-9) contributes to chronic lymphocytic leukemia (CLL) pathology by regulating cell migration and preventing spontaneous apoptosis. It is not known if MMP-9 is involved in CLL cell response to chemotherapy and we address this in the present study, using arsenic trioxide (ATO) and fludarabine as examples of cytotoxic drugs.MethodsWe used primary cells from the peripheral blood of CLL patients and MEC-1 cells stably transfected with an empty vector or a vector containing MMP-9. The effect of ATO and fludarabine was determined by flow cytometry and by the MTT assay. Expression of mRNA was measured by RT-PCR and qPCR. Secreted and cell-bound MMP-9 was analyzed by gelatin zymography and flow cytometry, respectively. Protein expression was analyzed by Western blotting and immunoprecipitation. Statistical analyses were performed using the two-tailed Student's t-test.ResultsIn response to ATO or fludarabine, CLL cells transcriptionally upregulated MMP-9, preceding the onset of apoptosis. Upregulated MMP-9 primarily localized to the membrane of early apoptotic cells and blocking apoptosis with Z-VAD prevented MMP-9 upregulation, thus linking MMP-9 to the apoptotic process. Culturing CLL cells on MMP-9 or stromal cells induced drug resistance, which was overcome by anti-MMP-9 antibodies. Accordingly, MMP-9-MEC-1 transfectants showed higher viability upon drug treatment than Mock-MEC-1 cells, and this effect was blocked by silencing MMP-9 with specific siRNAs. Following drug exposure, expression of anti-apoptotic proteins (Mcl-1, Bcl-xL, Bcl-2) and the Mcl-1/Bim, Mcl-1/Noxa, Bcl-2/Bax ratios were higher in MMP-9-cells than in Mock-cells. Similar results were obtained upon culturing primary CLL cells on MMP-9.ConclusionsOur study describes for the first time that MMP-9 induces drug resistance by modulating proteins of the Bcl-2 family and upregulating the corresponding anti-apoptotic/pro-apoptotic ratios. This is a novel role for MMP-9 contributing to CLL progression. Targeting MMP-9 in combined therapies may thus improve CLL response to treatment.

Quantitative and Qualitative Analysis Of Regulatory T Cells (Tregs) Derived From The Peripheral Blood Of Chronic Lymphocytic Leukemia Patients

IntroductionT regulatory cells are immunosuppressive cells, which are considered to play an important role in the regulation of immune response to cancer, by restraining autoreactive lymphocytes. Several studies, mostly in solid tumors, revealed that the number of Treg cells increases as the disease progresses and that Treg cells act by suppressing anti-tumor immune response, through the targeting of other immune cells, such as T cells, B cells and dendritic cells. Chronic lymphocytic leukemia (CLL) is a lymphoid malignancy, characterized by both, immunodeficiency and autoimmune disorders. Accumulated data indicate the role of T cells in the pathogenesis and development of CLL and reveal an increased number of Treg cells in CLL patients. The scope of this study is the analysis of the functional role of Tregs derived from the peripheral blood of CLL patients, mainly on B-CLL cells, and its correlation with well known prognostic factors. MethodsTreg cells derived from mononuclear cells of 28 untreated B-cell CLL patients with a median age 62 (44-88) and 17 healthy donors were analyzed through Flow cytometry. Patients were classified according to Rai classification as Rai I:19, Rai II:4, Rai III:5 and according to Binet as Binet A: 24, Binet B:3 and Binet C:1. The following antibodies were used for the fluorescence-activated cell sorter (FACS) analysis:1. CD45Ro-FITC/CD45RA-PE/CD4-ECD/CD25-PC5/CD127-PC72. CD1a-FITC/CD137-PE/CD4-ECD/CD25-PC5/CD127-PC73. CD95-FITC/cyCD152-PE/CD4-ECD/CD25-PC5/CD127-PC74. beads/FoxP3-PE/CD4-ECD/CD25-PC5/CD127-PC75. Annexin V-FITC/CD4-ECD/CD25-PC5/CD127-PC7Moreover, peripheral blood was obtained from 15 patients with B-cell CLL. Mononuclear cells were isolated using Ficoll-Paque gradient centrifugation. CD4+CD25+(Treg cells), CD4+CD25- (T effectοr cells, Teff), CD5+CD19+ (B-CLL) and CD5+CD19-(Normal B, NB) cells were separated using magnetic antibody cell sorting. To test the functionality of the assayed Tregs, the isolated cell populations were cultured in a 96-well plate (Tregs, Teff, B-CLL, NB cells, Tregs:Teff in a 4:1 ratio, B-cll:Tregs in 1:20 ratio, B-cll:Teff in 1:20 ratio, NB cells:Tregs in 1:20 ratio, NB cells:Teff in 1:20 ratio) and their proliferative capacity was measured using the BrdU assay. ResultsFACS analysis of the Treg cells resulted at the following observations: (1) The co-expression of the CD45RA-CD45RO markers was significantly higher in patients' samples than in controls (p=0.047). (2) No significant differences were observed between patients and controls, regarding the expression of the CD1α marker, as well as the expression of CD95 and CD152 markers. (3) The Treg absolute cell number (cells/μL), estimated either as the number of CD4+CD25+ CD127- cells or as the number of CD4+ CD25+FoxP3+cells, was statistically significantly higher in patients' samples than in controls (CD127- p=0.047, FoxP3+ p= 0.036). (4) Annexin V expression in Treg cells from B- CLL patients was significantly lower compared to controls (p=0.027). Following the purification and culturing of T and B cells from B-cell CLL patients' samples, functional analysis of the different cell populations was performed using the BrdU proliferation assay. We observed that Tregs were able to significantly suppress the proliferation of the Teff cells (p=0.002). After the co-culturing of NB cells (CD5+CD19-)and Tregs (CD4+CD25+) we found that NB cells seemed to significantly increase the proliferation of Treg cells, compared to the proliferation capacity of the Tregs when cultured alone (p=0.047). Moreover, we observed that Teff (CD4+CD25-) were able to significantly suppress the proliferation of B-CLL cells (CD5+CD19+), when co-cultured (B-CLL: Teff, 1:20 ratio) (p=0.05). ConclusionsIn B-cell CLL patients, Treg cells are significantly higher and present with lower apoptotic levels compared to healthy donors' samples. The functional analysis of Treg cells indicates that they can effectively suppress the proliferation of T effector cells. Moreover, T effector cells seem to suppress the proliferation of B-CLL cells, while NB cells increase the proliferation of Treg cells. These observations could probably indicate that at the early stages of the disease, where NB cells are more aberrant, Treg cells' activity is induced, leading to Teff cells' suppression and therefore, to an indirect induction of B-CLL cells' proliferation. Disclosures:No relevant conflicts of interest to declare.

Abstract 1033: Distinct response of chronic lymphocytic leukemia cells to PI-3Kδ inhibitor GS-1101 is related to MAPK signaling and likely associated with IGHV mutation.

Abstract Background: PI3Kδ is expressed and regulates survival and proliferation of malignant B-cells. The PI-3Kδ inhibitor, GS-1101, has promising therapeutic efficacy in chronic lymphocytic leukemia (CLL) through inhibiting B-cell receptor signaling, PI3K signaling and chemokine networks. It is currently in clinical trials either as monotherapy or in combination with others therapies. Here we report the exploration of the signaling molecules which are related to distinct response of CLL cells to GS-1101. Methods: Cells isolated from peripheral blood of CLL patients were treated with GS-1101 and analyzed by flow cytometry based apoptotic assay. Human Phospho-Kinase Array (R&amp;D Systems) was performed with CLL cells which had distinct response to GS-1101. RNA or genomic DNA was isolated and analyzed for IGHV somatic hypermutation (InVivoScribe Technologies). Sequences containing more than 2% deviation from the germline sequence were considered as somatically hypermutated. Results: To date, 20 CLL cases have been tested for their in vitro response to GS-1101. In comparison to the control group, GS1101-exposed CLL cells displayed a wide range of apoptotic cells (3 to 59%). 3 cases with high levels apoptosis in response to GS-1101 were subjected to Human Phospho-Kinase Array screening. These showed down regulation of p-ERK1/2(T202/Y204), p-P38α(T180/Y182), MEK1/2 (S218/S222), among tested 46 kinases’ phosphorylation sites, upon GS-1101 exposure. Two of the three cases also showed down-regulation of p-STAT5(Y699). In contrast, no alteration or minor up-regulation of the above 4 targets was observed in a GS-1101 non-responsive case. Currently we are performing immunoblotting assay to evaluate/confirm the alteration of above targets in multiple CLL cases and preliminary p-P38 expression data is consistent with that of array assay. 15 of the tested 20 CLL samples had high quality of IGHV sequence data from both directions which cover the entire variable region of IGH (FR1, CDR1, FR2, CDR2 and FR3). Nine cases were IGHV unmutated with a mean of GS1101 caused apoptotic induction of 25.3% (range 5-59%); while the mean of apoptotic induction of the 6 cases with somatic hypermutation was 12.5% (range 3 - 32%). The p value of the unmutated verse mutated group was 0.046 (T-test). Conclusion: Our preliminary studies suggest that GS-1101-induced apoptosis is associated with alterations in the MAPK signaling pathway. Combination of GS-1101 with MAPK inhibitor may be rational for further synergy study. GS-1101 may have differential activity against IGHV unmutated CLL cases compared to IGHV mutated ones. Ongoing studies with additional cases will further evaluate this observation. Profiling CLL cells for IGHV mutation status might be a strategy to predict CLL patients’ response to GS-1101 in clinical trials. Citation Format: Xiaoxian Zhao, Juraj Bodo, Eric D. Hsi. Distinct response of chronic lymphocytic leukemia cells to PI-3Kδ inhibitor GS-1101 is related to MAPK signaling and likely associated with IGHV mutation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1033. doi:10.1158/1538-7445.AM2013-1033

In vivo and ex vivo responses of CLL cells to purine analogs combined with alkylating agent

BackgroundThe heterogeneity of chronic lymphocytic leukemia (CLL) is thought to be due to differences in the expression of factors that regulate apoptosis and cell cycle, giving rise to diverse apoptotic disturbances and tumor properties. Therefore, the primary goal in CLL treatment is to overcome resistance to apoptosis and efficiently trigger this process in leukemic cells. MethodsMononuclear cells were obtained from the blood of CLL patients by Histopaque-1077 sedimentation. CLL cell samples from the blood of drug treated patients, (cladribine or fludarabine with cyclophosphamide; CC or FC), as well as the cell samples of untreated patients exposed to the used drug combinations (CM, FM) or mafosfamide alone for 48h were fractionated into nuclear and cytoplasmic fractions or were lysed. DNA fragmentation was evaluated by agarose electrophoresis and also cytometrically as sub-G1 population. The expression of apoptosis related proteins and H1.2 histone translocation were evaluated in lysates and nuclear and cytoplasmic fractions, respectively with appropriate antibodies. ResultsCladribine (C) and fludarabine (F) combined with cyclophosphamide/mafosfamide in vivo, as well as ex vivo trigger apoptosis in CLL cells. These drug combinations (CC; FC/CM; FM) induce leukemic cell apoptosis confirmed by DNA fragmentation, sub-G1 cell number, down-regulation of anti-apoptotic proteins (Mcl-1, Bcl-2), and H1.2 histone translocation in comparison with appropriate control cells, however, to a different degree. ConclusionsThe kinetics and rate of drug-induced apoptosis in leukemic cells under ex vivo experiments differ between patients, mirroring the differences noticed during in vivo treatment. Individual model cell samples indicate comparable susceptibility to the used drug combinations under in vivo and ex vivo conditions.

Receptor Tyrosine Kinase-Like Orphan Receptor 1 (ROR1) - a Putative Diagnostic Marker for Chronic Lymphocytic Leukemia (CLL).

Abstract 1587Poster Board I-613In chronic lymphocytic leukemia (CLL) WNT signaling is constitutively active and several members of this signaling pathway are uniformely upregulated in these cells. Apart from classical WNT receptors like FZD and LRP6, receptor tyrosine kinase-like orphan receptor 1 (ROR1) has been shown to function as a receptor for WNT proteins, too. Furthermore, it could recently be demonstrated that ROR1 is frequently expressed on the surface of CLL cells and might therefore serve as a therapeutic target in this disease. However, so far only little is known about the expression status of this protein in different patients. Moreover, a diagnostic antibody for flow cytometric investigations is lacking. Thus, the aim of our study was to i) establish a directly labelled anti-ROR1 antibody for flow cytometry, ii) to confirm previous results on ROR1 expression in CLL, iii) to investigate ROR1 expression in different cell compartments and iv) correlate our findings to known markers of risk and disease progression. Peripheral blood of CLL patients as well as healthy volunteers was subjected to flow cytometric analysis. Besides standard determination of leukocyte subpopulations ZAP70 and CD38 status was assessed according to current diagnostic recommendations. In addition, ROR1 surface expression was first detected by flow cytometry using a specific primary antibody directed against ROR1 and a fluorescent labelled secondary antibody. Using this experimental setting we found that ROR1 is expressed on 63.4% of all neoplastic CLL cells and also on 30.5% of T cells in the peripheral CLL blood. In contrast, no ROR1 expression could be detected on NK cells, B cells, CD8+- or CD4+-T cells of healthy individuals. To improve the analytical technique the ROR1 antibody was directly conjugated with Phycoerythrin (PE) and the experiments were repeated. With the conjugated antibody we detected ROR1 expression on 97.1% of neoplastic CLL cells and virtually on no T lymphocytes. ROR1 expression levels correlated neither with the expression of ZAP70 nor with CD38. Again, we could not detect ROR1 expression on peripheral blood cells of our healthy volunteers. Taken together, ROR1 expression appears to be highly restricted to CLL cells. If in addition to CD5 and CD19 ROR1 detection is included into diagnostic flow cytometric panels the specificity and sensitivity of immunophenotypic CLL diagnostics may be greatly enhanced. DisclosuresHallek:Roche: Consultancy, Honoraria, Research Funding.

Evaluation of circulating endothelial cells as noninvasive marker of angiogenesis in patients with chronic lymphocytic leukemia

An increased angiogenesis has been documented in bone marrow and lymph nodes of patients with chronic lymphocytic leukemia (CLL). There is accumulating evidence that circulating endothelial cells (CECs) play an important role in angiogenesis. The aim of our study was to compare the number of CECs in peripheral blood of CLL patients and healthy donors and to correlate these numbers with bone marrow microvessel density (MVD) and known prognostic factors in CLL. Proportions of resting CECs (rCECs), activated CECs (aCECs), apoptotic CECs (apoCECs) and circulating precursor endothelial cells (CPECs) were estimated by flow cytometry in 104 untreated CLL patients and 29 healthy blood donors. The MVD was analysed in the bone marrow biopsy in 21 CLL patients and 11 controls using the ‘hot spot analysis’ of vessels' density (×200 HPS). We found significantly higher numbers of CPECs, rCECs, aCECs and apoCECs in CLL patients than in healthy controls (p < 0.001 for all comparisons). Furthermore, the rCECs number was higher in advanced versus low clinical stage CLL (median 17.5 cells/µL vs. 13.5 cells/µL, p = 0.05). The MVD was significantly higher in the bone marrow of CLL patients when compared with controls (p = 0.016). However, we did not find any correlation between MVD and different CECs populations. In conclusion, the number of CECs is increased in CLL and correlates with stage of the disease, but does not seem to directly reflect the intensity of neovascularisaton in the bone marrow.

Characterization of Microvesicles in B-Cell Chronic Lymphocytic Leukemia (CLL): A Potential Mediator in CLL B Cell Disease Progression?.

While cellular communication and interactive signaling typically relies on multiple mechanisms including secretion of various growth factors, cytokines, bioactive lipids, and adhesion molecules, we have recently focused on cell-to-cell communication that involves circular membrane vesicles designated as microvesicles (MVs). These are normal constituents of blood plasma released by leukocytes, endothelium, platelets and erythrocytes and are generated by cell activation and growth. In normal serum, the order of MV abundance is platelet-derived (80%), endothelial (10%) and leukocyte-derived (10%) but MV can also be released by malignant cells. The size varies from 0.1–1.0 μm and, MVs express antigens characteristic of the cell of origin, carry other membrane and cytoplasmic constituents and exhibit negatively charged phospholipids (phosphatidylserine) detectable by Annexin V binding. To begin to characterize MV and their potential for communication in CLL, we isolated and characterized MVs from the plasma of chronic lymphocytic leukemia B-cell (CLL B) patients (Rai 0-IV). After isolation from platelet-free plasma, MVs were characterized by flow cytometry for forward and side scatter using 1μm fluorescent beads and stained with Annexin V. Our results suggest that MVs are heterogeneous in size with the majority of MVs within 1μm. Transmission electron microscopy confirmed the size heterogeneity of MVs and exhibited the morphology of the membrane-bound vesicles after phosphotungstic acid staining onto parlodium-coated 300 mesh copper grids. Flow cytometric analysis further demonstrated the presence of CD61 (platelet/megakaryocyte marker), CD19, CD5, CD52, and CD20 on the surface of MV, albeit in differential amounts depending on the stage of the disease and the total leukocyte count. For example, in Rai stage IV (n= 5), we found lesser numbers of MV carrying platelet-derived marker CD61 and more CD19 (41–79%), and CD5 compared to normal MV; 1–5% MV from healthy controls expressed CD19 while Rai stage 4 patients MV expressed CD19 from 41–79%. These results suggest that a significant number of MVs are generated from the leukemic B-lymphocytes in more advanced Rai stage. However, we also found that patients with a higher blood lymphocyte count but lower Rai-stage (Rai I/II) contain moderate levels of CD19-bearing MVs (13–43%). Importantly, presence of MVs carrying CD52/CD20 in the blood of CLL patients may undermine the therapeutic effectiveness of alemtuzumab and rituximab. To this end, we observed that MVs could be incorporated into the CLL B cells as evident from the expression of the platelet antigen CD61 on CLL B cell surfaces after co-culture of CD61-bearing MVs with CLL B cells. Global proteomic analysis of MVs isolated from an advanced stage (Rai IV) CLL B patient detected the presence of about 700 proteins that normally reside in the nucleus, cytoplasm or membrane. In summary, we have isolated and characterized plasma MV from CLL B patients and found them to be more likely generated from CLL B cells in relation to disease stage or total lymphocyte count. These MVs contain a multitude of proteins and are able to integrate into bystander CLL B cells. We also found that these MVs carry a large set of proteins, which could potentially not only modulate cell-cell communication but also interrupt monoclonal antibody directed therapy. Further studies are underway to dissect their roles in CLL B malignancy.

Circulating Endothelial Cells as Noninvasive Marker of Angiogenesis in Patients with Chronic Lymphocytic Leukemia.

There is accumulating evidence that circulating endothelial cells (CECs) play an important role in angiogenesis. An increased neovascularisation has been documented in bone marrow and lymph nodes of patients with chronic lymphocytic leukemia (CLL). The aim of our study was to compare the number of CECs in peripheral blood of CLL patients and healthy donors and to correlate these numbers with bone marrow microvessel density (MVD). In addition we checked if the number of CECs correlates with the response to treatment. We analyzed the total (CECs), activated endothelial cells (aCECs), apoptotic endothelial cells (apoCECs) and endothelial precursors (CPECs) count by the four colour flow cytometry in the peripheral blood of 80 untreated CLL patients and 29 healthy donors. We also evaluated the MVD in the bone marrow biopsy in 21 CLL patients and 11 controls, using the hot spot analysis of vessels density (x200 HPS). In 34 patients treated with purine analogs (Cladribine or Fludarabine) as first line treatment, we analyzed the influence of the initial number of CECs on treatment results. We found significantly higher numbers of CECs, aCECs, apoCECs and CPECs in CLL patients than in healthy controls (p=0.0001, p=0.001, p=0.0001 and p=0.001 respectively). The total number of CECs was significantly higher in the group of patients with advanced disease (Rai stage 3–4) as compared to patients in Rai stage 0–2 (p=0.02). A correlation was found between CECs and apoCECs number and the β2-microglobulin level (r=0.4, p=0.01 and r=0.3, p=0.01 respectively). The MVD was significantly higher in the bone marrow of CLL patients (median 60, range 27–147) as compared to controls (median 20, range 10–60) (p=0.01). In addition, the number of aCECs correlated with the MVD in CLL patients (r= 0.5, p= 0.02). Moreover, the patients with CECs numbers above median had significantly lower probability to achieve response to treatment with purine analogs (p=0.048). In conclusion, the number of CECs is increased in CLL patients and correlates with vessel density and tumor stage. These findings indicate that analysis of CECs may be a suitable, noninvasive marker of angiogenesis in CLL patients. Evaluation of CECs could be useful to identify CLL patients with low probability of response to purine analogs that may benefit from alternative therapy including anti-angiogenic agents.

Generation of alpha-Type-1 Polarized Dendritic Cells as a Potent Immunogen in Patients with Chronic Lymphocytic Leukemia.

We tested the feasibility of generating alpha-type-1 polarized DC (αDC1) from the blood of chronic lymphocytic leukemia (CLL) patients and the in vitro effectiveness of autologous tumor-loaded αDC1s in inducing CTLs against CLL. CD14+ cells were isolated from the peripheral blood of CLL patients and were cultured in rhu GM-CSF and IL-4. On day 6, DC maturation was induced using either a standard cytokine cocktail (in IL-1β, TNFα, IL-6, and PGE2) or αDC1-polarizing cocktail (in IL-1β, TNFα, IFNα, IFNγ, and poly-I:C). In either case, the maturing DCs were loaded with gamma-irradiated autologous CLL cells. αDC1s from CLL patients expressed substantially higher levels of several costimulatory molecules (CD83, CD86, CD80, CD11c and CD40) than standard DCs (sDCs) while CCR7 showed lower expression. Gamma-irradiated CLL cells were more efficienly taken up by DCs compared to UVC-irradiated CLL cells, but the ability of tumor antigen uptake was similar between αDC1 and sDCs. The capacity of IL-12p70 secretion in DCs (baseline and after stimulation with CD40L-transfected J558 cells) was significantly elevated in αDC1s (10–60 times) compared to sDCs. αDC1s also induced significantly higher numbers of functional CD8+ T cells against CLL cells, as determined by IFN-γ ELISPOT, using anti-MHC class I blocking mAb (W6/32) to verify the specificity of CTL responses. Our data demonstrate that αDC1s are a potent alternative to standard DCs as inducers of T cells for adoptive immunotherapy in patients with CLL and a clinical trial will be initiated.

CD74 Is a Novel Survival Receptor in Chronic Lymphocytic Leukemia.

Scientific Background: Previous studies have shown that chronic lymphocytic leukemia (CLL) lymphocytes express relatively large amounts of CD74 mRNA compared to normal B cells. We have recently demonstrated in a murine model that CD74 stimulation with anti-CD74 antibody leads to an induction of a signaling cascade resulting in NF-κ B activation, entry of the stimulated cells into the S phase, elevation of DNA synthesis, cell division, and augmented expression of BCL-XL. These findings therefore demonstrated that surface CD74 functions as a survival receptor. In the current study we aimed to determine whether activation of cell surface CD74 in B-CLL cells leads to induction of a signaling cascade resulting in cell survival.Meterials and methods: B cells were purified from the peripheral blood of CLL patients of different stages. CD74 stimulation was achieved using anti-CD74 or MIF (a natural occurring ligand of CD74). IL-8 expression and function was determined by RT-PCR, western blot, ELISA and Annexin V staining.Results: In all CLL patients there was a significantly increased expression of cell surface CD74. Activation of cell surface CD74 initiates a signaling cascade that results in secretion of Interleukin 8 (see Fig1), which in turn regulates cell survival(see Fig 1,2).Conclussion: Our data show that over-expression of CD74 in CLL is an important survival mechanism, operational from the very early stages of the disease, and inherent in all further stages. This survival mechanism thus appears to be an early and significant event in the pathogenesis of the disease. Our findings open prospects for novel therapeutic strategies aimed at this survival pathway.Legends: [Display omitted] [Display omitted]

Induction of Chronic Lymphocytic Leukemia (CLL) Apoptosis by Nitric Oxide Synthase (NOS) Inhibitors: Drug Efficacy Correlates with Lipid Solubility and NOS1 Dissociation Constant.

The viability of CLL cells may be dependent on the autocrine production of nitric oxide because nitric oxide synthase (NOS) inhibitors induce CLL cell apoptosis and CLL cells express inducible NOS (NOS2). Our previous study indicated that the non-specific NOS inhibitor NMMA induced CLL cell apoptosis but only at high concentrations (> 1 mM) (Levesque et al., Leukemia 17:442, 2003). Therefore, we performed the current study to identify NOS inhibitors that induce CLL cell apoptosis at lower concentrations and to understand factors that promote NOS inhibitor-induced CLL cell toxicity. We isolated and enriched CLL cells from the blood of CLL patients and cultured the CLL cells in media containing various concentrations of 21 different NOS inhibitors. We determined CLL cell viability following culture with each NOS inhibitor. We found that NOS inhibitors with specificity for neuronal NOS (NOS1) induced CLL cell death at concentrations lower than non-specific NOS inhibitors and lower than inducible NOS (NOS2) specific inhibitors. There was a weak correlation (r2 = 0.29, p = 0.1608) of the NOS1 (but not NOS2) half-maximal inhibitory concentration (IC50) of each NOS inhibitor for purified recombinant NOS and its ability to induce CLL cell death. We confirmed the specificity of the NOS inhibitors by inhibition of purified recombinant NOS1 and NOS2 enzyme activity, and we confirmed that NOS1 specific inhibitors induced CLL cell death by apoptosis. Because there was only a weak correlation of the NOS1 IC50 with NOS inhibitor induced CLL cell death, we considered whether other factors such as the Kd and hydrophobicity of each compound correlated with CLL cell death. We found that there was a direct correlation between the NOS1 (but not NOS2) dissociation constant (Kd) of NOS inhibitors and CLL cell death (r2 = 0.77, p = 0.0041) and a direct correlation of the partitioning coefficient (a measure of hydrophobicity) of each NOS inhibitor and its ability to induce CLL cell death (r2 = 0.68, p < 0.0001). Therefore, NOS inhibitors that bound tightly to NOS1 and were hydrophobic induced CLL cell death at lower concentrations. There was variable expression of CLL cell NOS1 mRNA (6 of 28 samples positive) and we were unable to demonstrate CLL cell expression of NOS1 protein by immunoblotting. This suggests that if NOS1 is present in CLL cells, it exists at very low levels. Taken together, we believe that low level NO production promotes CLL cell viability and that inhibition of CLL NOS induces CLL cell apoptosis. Importantly, our studies provide direction for the rational design and selection of NOS inhibitors that may be useful as CLL therapeutics.

Jasmonates–a new family of anti-cancer agents

Since salicylate, a plant stress hormone, suppresses the growth of various types of cancer cells, it was deemed of interest to investigate whether the jasmonate family of plant stress hormones is endowed with anti-cancer activities. Cell lines representing a wide spectrum of malignancies, including prostate, breast and lung, exhibit sensitivity to the cytotoxic effects of methyl jasmonate (MJ). Jasmonates induced death in leukemic cells isolated from the blood of chronic lymphocytic leukemia (CLL) patients and increased significantly the survival of lymphoma-bearing mice. Among the naturally occurring jasmonates, MJ is the most active, while the synthetic methyl-4,5-didehydrojasmonate, was approximately 29-fold more active than MJ. The cytotoxic activity of MJ is independent of transcription and translation. Studies have suggested several mechanisms of action. It appears that while prolonged exposures to relatively low concentrations of jasmonates induce growth arrest and re-differentiation in myeloid leukemia cells, higher concentrations of MJ induce direct perturbation of cancer cell mitochondria, leading to the release of cytochrome c and eventual cell death. A most important characteristic of jasmonates is their ability to selectively kill cancer cells while sparing normal cells. Even within a mixed population of normal and leukemic cells derived from the blood of CLL patients, MJ killed preferentially the leukemic cells. In conclusion, jasmonates present a unique class of anti-cancer compounds which deserves continued research at the basic and pharmaceutical levels in order to yield novel chemotherapeutic agents against a range of neoplastic diseases.

Motexafin Gadolinium, a Redox Mediator, Induces Apoptosis in Chronic Lymphocytic Leukemia B Cells and Is Additive or Synergistic with Fludarabine.

Motexafin gadolinium (MGd, Xcytrin®) is a tumor-selective redox mediator that oxidizes intracellular reducing metabolites and protein thiols, thereby generating reactive oxygen species (ROS) through a process known as redox cycling. B-cell chronic lymphocytic leukemia (CLL) has elevated levels of the antioxidant ascorbate, and is known to be sensitive to redox stress. Since MGd can generate ROS through oxidation of ascorbate, we reasoned that MGd would be cytotoxic to CLL cells. Mononuclear cells were isolated from the blood of CLL patients (n=15) characterized by Rai Stage, CD38, FISH and immunoglobulin heavy chain variable (Ig VH) mutational status. CLL cells were incubated in vitro over 2–5 days with MGd at concentrations ranging from 0–50 uM. Cytotoxicity was assessed by flow cytometry using annexin V binding/propidium iodide uptake. Accumulation of MGd in CLL cells was demonstrated by flow cytometry using drug fluorescence (>650 nm). MGd-induced cytotoxicity was found to be dose-dependent over a range of 2.5 to 50 uM in cells from 11 of 15 CLL patients. CLL cells from 11/15 patients treated with 50 uM MGd and 4/15 patients treated with 10 uM MGd had cytotoxicity levels at least 2 fold above baseline. MGd uptake was similar in sensitive and resistant CLL cells, implying that mechanisms other than uptake are responsible for the differential responses. CLL cells that were sensitive to MGd treatment showed an increase in caspase-3 activity and cleavage of the caspase substrate PARP was found by immunoblot suggesting that MGd induced apoptosis. The induction of apoptosis was found to be exclusive of Rai stage, CLL FISH defects, CD38 expression or Ig VH mutational status. Cells from 5 CLL patients were studied for drug synergy using seven concentrations of MGd (7.5 – 50 uM) and the active moiety F-ara-A of fludarabine (0.25 –2.0 uM). The cells were treated with each drug individually or both drugs for 48 hours prior to analysis for cytotoxicity. MGd was found to exhibit additive activity (N=4) or synergistic (N=1) effects on CLL B cytotoxicity when combined with fludarabine. Thus, our pre-clinical results reveal that MGd is cytotoxic to CLL cells in vitro through induction of apoptosis. Furthermore, MGd shows additive or synergistic activity when combined with fludarabine. These results support the evaluation of MGd in ongoing CLL clinical trials utilizing this agent alone or in combination with fludarabine.