Mevalonate pathway inhibitors in the treatment of B-cell chronic lymphocytic leukemia.

6561 Background: B-cell chronic lymphocytic leukemia (CLL) cells are arrested in G0/G1 phase of the cell cycle and are resistant to programmed cell death, hypothesized to contribute to the resistance of CLL cells to standard chemotherapy with curative intent. Methods: Mec-2 cells and Wac-3 cells are CLL cells that have been shown to be resistant to fludarabine and rituximab. We tested a novel enzyme inhibitor’s ability to render CLL cells sensitive to fludarabine and rituximab. Results: BIBB515, a lanosterol synthase inhibitor, at a concentration of 10μM, was able to reduce the cell viability from 82% in controls to 65% after 72 hours. Fludarabine 10μM alone did not reduce the cell viability, 82 % in controls compared to 80%. BIBB515+ fludarabine treatment for 72 hours, at the dose of 10μM each decreased the cell viability to 37%. Cell proliferation by MTT assay was 0.66±0.010 in control compared to 0.37±0.01 in BIBB515+fludarbine and 0.21±0.01 in BIBB515+ fludarabine+ rituximab. There is a 68% down-regulation of cell proliferation using this treatment. There was a two fold induction of CD 20 with combination treatment, and BIBB515 treatment. The mechanism of cell death in the combination treatment of BIBB515 and fludarabine may be due to the up regulation of cell surface marker CD-20. WAC-3 is another CLL cell line that is sensitive to fludarabine, and resistant to rituximab. BIBB515 sensitizes WAC-3 cells to CD 20 antibody rituximab. There is a 68.7% decrease in cell proliferation with combination treatment of BIBB515 and rituximab. Proliferation of Mec-2 cells were inhibited by 60µM and 30µM terbinafine. Ro-48-8071, showed dose-dependent activity, alone or in combination to fludarabine was seen to induce cell death in Mec-2 cells. Fludarabine alone did not have any effect on these cells. Conclusions: Inhibitors of the mevalonate pathway make resistant CLL cells sensitive to current chemotherapeutic agents. Exploiting this mechanism could alter the current treatment regimens, leading to control of the disease in advanced stages by either inducing the leukemic cells to be static or to regress. This strategy may also limit the toxicities involved with chemotherapy.

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Interferon gamma inhibits apoptotic cell death in B cell chronic lymphocytic leukemia.

Journal of Experimental Medicine ◽

10.1084/jem.177.1.213 ◽

1993 ◽

Vol 177 (1) ◽

pp. 213-218 ◽

Cited By ~ 178

Author(s):

M Buschle ◽

D Campana ◽

S R Carding ◽

C Richard ◽

A V Hoffbrand ◽

...

Keyword(s):

Cell Death ◽

Chronic Lymphocytic Leukemia ◽

Programmed Cell Death ◽

B Cell ◽

Interferon Gamma ◽

Lymphocytic Leukemia ◽

Malignant Cells ◽

Ifn Gamma ◽

Cell Chronic Lymphocytic Leukemia

The malignant, CD5+ B lymphocytes of B cell chronic lymphocytic leukemia (B-CLL) die by apoptosis in vitro. This is in contrast to the prolonged life span of the leukemic cells in vivo and likely reflects the lack of essential growth factors in the tissue culture medium. We found that interferon gamma (IFN-gamma) inhibits programmed cell death and promotes survival of B-CLL cells in culture. This effect may also be important in vivo: increased serum levels of IFN-gamma, ranging from 60 to > 2,200 pg/ml, were found in 7 of 10 B-CLL samples tested, whereas the sera of 10 healthy individuals did not contain detectable levels of this cytokine (< 20 pg/ml). High levels of IFN-gamma message were detected in RNA from T cell-depleted B-CLL peripheral blood samples by Northern blot analysis. Synthesis of IFN-gamma by B-CLL lymphocytes was confirmed by in situ hybridization and flow cytometry. The majority of B-CLL cells (74-82%) expressed detectable levels of IFN-gamma mRNA, and CD19+ B-CLL cells were labeled with anti-IFN-gamma monoclonal antibodies. These results show that IFN-gamma inhibits programmed cell death in B-CLL cells and suggest that the malignant cells are able to synthesize this cytokine. By delaying apoptosis, IFN-gamma may extend the life span of the malignant cells and thereby contribute to their clonal accumulation.

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Clinical Outcome of B-Cell Chronic Lymphocytic Leukemia Following Alemtuzumab Therapy: Retrospective Study within Various Cytogenetic Risk Categories

Blood ◽

10.1182/blood.v112.11.3164.3164 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3164-3164

Author(s):

Michael Fiegl ◽

Martin Erdel ◽

Inge Tinhofer ◽

Georg Hopfinger ◽

Karin Eigenberger ◽

...

Keyword(s):

Retrospective Study ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Chemotherapeutic Agents ◽

Lymphocytic Leukemia ◽

Cox Regression Analysis ◽

Trisomy 12 ◽

11Q Deletion ◽

Cell Chronic Lymphocytic Leukemia ◽

13Q Deletion

Abstract B-cell chronic lymphocytic leukemia (CLL) with 17p deletion responds poorly to chemotherapeutic agents. This retrospective study evaluated the benefit of alemtuzumab monotherapy in unselected patients with advanced CLL, categorized by cytogenetic profile. Methods: This is the largest data base with efficacy analysis of alemtuzumab in CLL stratified according to cytogenetics. Detailed data analysis was done in 138 CLL patients, in whom cytogenetic analysis was performed by FISH using the standard CLL analysis categorized according to Doehner et al. (N Engl J Med343, 1910; 2000). Responses were evaluated according to the NCI criteria; progression-free survival (PFS) and overall survival (OS) were also assessed. Results: 73% of the patients were male. At start of alemtuzumab therapy, the median age was 64 years (range, 46–92); 12% were in Rai stage I, 18% in stage II, 20% in stage III, and 50% in stage IV. The median number of two prior therapies was 2 (range, 0–10); of the patients who received prior fludarabine (F) (n=113), 70% were F-refratory, 25% sensitive, and in 5% this was unknown. 30% and 17% of patients had bulky lymphadenopathy (>5 cm) and giant splenomegaly (>20 cm), respectively. Cytogenetic abnormalities were as follows: 13q deletion 14%; trisomy 12, 12%, 11q deletion 20%; 17p deletion, 33%, none of these, 22%. The overall response rate (ORR) was 38% in the total cohort. ORR was 53%, 56%, 21%, and 44% in the subgroup of 13q deletion, trisomy 12, 11q deletion, and 17p deletion, respectively; patients without any of these abnormalities had an ORR of 27%. From start of alemtuzumab, median PFS and OS for the whole cohort was 6.9 months and 30 months, respectively. Notably, PFS and OS in 17p deletion patients was 7.1 months and 19.1 months, respectively, an encouraging outcome when considering the unfavourable risk profile in these patients. In 17p deletion patients, response was remarkable also in disease involved lymph nodes (78%). Patients with F-resistant disease and 17p deletion, an extraordinarily poor prognostic group (n=25), had encouraging ORR, PFS, and OS rates (28%; 7.2 months; and 19.1 months, respectively), which did not differ from those in F-resistant patients with good risk cytogenetics. In a multivariate Cox regression analysis, independent risk factors for shorter OS were anemia (hazard ratio [HR] = 2.48; 95% CI, 1.50–4.11; P <.001), ≥3 of prior lines of therapy (HR = 2.00; 95% CI, 1.24–3.24, P =.005), and poor risk cytogenetics ([17p deletion and 11q deletion], HR = 2.23; 95% CI 1.35–3.69, P =.002). Conclusion: Alemtuzumab was effective in CLL across all cytogenetic categories evaluated. In patients with favorable cytogenetics, we observed that alemtuzumab is a highly effective therapy even when conventional chemotherapy has failed. Patients with 17p deletion achieved quite favorable ORR and OS upon alemtuzumab. Thus, the 17p deletion group can often be shifted to an “intermediate” risk CLL, and responding patients are frequently re-treated with alemtuzumab.

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Acadesine (Acadra‘) for Patients with Resistant/Relapsed Chronic B-Cell Lymphocytic Leukemia (B-CLL): A Multicentre Phase I/II Study.

Blood ◽

10.1182/blood.v114.22.4405.4405 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4405-4405

Author(s):

Eric W Van Den Neste ◽

Eva Gonzalez-Barca ◽

Ann Janssens ◽

Bruno Cazin ◽

J. Pérez de Oteyza ◽

...

Keyword(s):

Cell Death ◽

Chronic Lymphocytic Leukemia ◽

Phase I ◽

B Cell ◽

Dose Escalation ◽

Safety Profile ◽

Whole Blood ◽

Lymphocytic Leukemia ◽

Pharmacokinetic Data ◽

Cell Chronic Lymphocytic Leukemia

Abstract Abstract 4405 Acadesine (Acadra‘) is a nucleoside that induces cell death in B-cell chronic lymphocytic leukemia (B-CLL) cells ex vivo in a dose-dependent manner over the concentration range 50 mM to 1 mM. Acadesine enters B-cells and is phosphorylated to its active metabolite, ZMP, which induces apoptosis independently of ATM or p53. It is active against B-CLL cells from patients who have not responded to prior treatment with fludarabine and/or chlorambucil (Campas et al, 2003). Based on this pre-clinical data, a Phase I/II clinical study was designed to determine the safety and tolerability of acadesine given intravenously as a 4-hour infusion to patients with B-CLL. This is an open-label dose escalation study of acadesine with two parts. In Part I, the dose escalation part of the study, patients receive a single dose of acadesine on Day 1 and are followed up to Day 22. In Part II, patients will receive up to 5 doses of acadesine at the maximum tolerated dose (MTD) identified in Part I over a period of up to 20 days starting on Day 1. The patient population includes B-CLL patients with refractory or relapsed disease who have received one or more (≥ 1) prior lines of treatment which must have included either a fludarabine or an alkylator based regimen. Primary endpoints of the study evaluate the safety and tolerability of acadesine. Secondary endpoints evaluate the pharmacokinetics of acadesine in plasma, its metabolite ZMP in whole blood, and changes in peripheral blood B-cell and T-cell counts as efficacy biomarkers. Optimal Biological Dose (OBD) is defined as the minimal acadesine dose that gives a maximum Cmax for ZMP in whole blood with a good safety profile. Twelve patients (6 males and 6 females) have been administered acadesine to date in 3 cohorts in Part I. Acadesine has an excellent safety profile to date. The principal safety finding was asymptomatic acadesine-related hyperuricaemia of short duration in four patients. Prophylactic allopurinol has significantly reduced the incidence of hyperuricaemia. Pharmacokinetic data showed acadesine rapidly disappears from plasma to be converted into ZMP inside blood cells. The OBD has not been reached yet and maximum dose administered to date is 139.5 mg/kg. The next cohort will receive 210 mg/kg. Additional safety, pharmacokinetics and efficacy data will be presented at the meeting. Disclosures: Off Label Use: Acadesine (AcadraTM): nucleoside that induces cell death in B-cell chronic lymphocytic leukemia.. Auton:BTG Limited: Consultancy; Advancell Technologies Inc.: Consultancy. Saunders:BTG limited: Consultancy; Advancell Technologies: Consultancy. Campas:Advancecell Technologies Inc: Employment, Equity Ownership.

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Evidence for distinct pathomechanisms in B-cell chronic lymphocytic leukemia and mantle cell lymphoma by quantitative expression analysis of cell cycle and apoptosis-associated genes

Blood ◽

10.1182/blood.v99.12.4554 ◽

2002 ◽

Vol 99 (12) ◽

pp. 4554-4561 ◽

Cited By ~ 94

Author(s):

Christian Korz ◽

Armin Pscherer ◽

Axel Benner ◽

Daniel Mertens ◽

Claudia Schaffner ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Cell Lymphoma ◽

Lymphocytic Leukemia ◽

Expression Data ◽

Mantle Cell ◽

Chromosome Bands ◽

Cell Chronic Lymphocytic Leukemia

The B-cell lymphoproliferative malignancies B-cell chronic lymphocytic leukemia (B-CLL) and mantle cell lymphoma (MCL) share characteristics, including overlapping chromosomal aberrations with deletions on chromosome bands 13q14, 11q23, 17p13, and 6q21 and gains on chromosome bands 3q26, 12q13, and 8q24. To elucidate the biochemical processes involved in the pathogenesis of B-CLL and MCL, we analyzed the expression level of a set of genes that play central roles in apoptotic or cell proliferation pathways and of candidate genes from frequently altered genomic regions, namely ATM, BAX, BCL2, CCND1, CCND3, CDK2,CDK4, CDKN1A, CDKN1B,E2F1, ETV5, MYC, RB1,SELL, TFDP2, TNFSF10, andTP53. Performing real-time quantitative reverse transcription polymerase chain reaction in a panel of patients with MCL and B-CLL and control samples, significant overexpression and underexpression was observed for most of these genes. Statistical analysis of the expression data revealed the combination of CCND1 and CDK4 as the best classifier concerning separation of both lymphoma types. Overexpression in these malignancies suggests ETV5 as a new candidate for a pathogenic factor in B-cell lymphomas. Characteristic deregulation of multiple genes analyzed in this study could be combined in a comprehensive picture of 2 distinctive pathomechanisms in B-CLL and MCL. In B-CLL, the expression parameters are in strong favor of protection of the malignant cells from apoptosis but did not provide evidence for promoting cell cycle. In contrast, in MCL the impairment of apoptosis induction seems to play a minor role, whereas most expression data indicate an enhancement of cell proliferation.

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B-Cell Chronic Lymphocytic Leukemia Cells Exposed to the Non-Genotoxic p53 Activator Nutlin-3 Are Characterized by a Specific Gene Expression Signature.

Blood ◽

10.1182/blood.v114.22.4374.4374 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4374-4374

Author(s):

Michele Dal-Bo ◽

Paola Secchiero ◽

Massimo Degan ◽

Riccardo Bomben ◽

Dania Benedetti ◽

...

Keyword(s):

Gene Expression ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Differentially Expressed Genes ◽

Chemotherapeutic Agents ◽

Lymphocytic Leukemia ◽

Differentially Expressed ◽

Specific Gene ◽

P53 Mutations ◽

Cell Chronic Lymphocytic Leukemia

Abstract Abstract 4374 Introduction p53 plays a key role in determining the clinical features of B cell chronic lymphocytic leukemia (CLL). Disruption of p53 by point mutations, deletion at 17p13, or both, occurs in a fraction of cases at diagnosis and predicts poor survival and chemorefractoriness. In cells with functional p53, p53 activity is inhibited through interaction with MDM2. In fact, p53 can be activated upon exposure of cells to inhibitors of p53/MDM2 interaction, like Nutlins. Exposure of CLL cells to Nutlin-3 is effective in raising the levels of p53 protein with subsequent induction of cell cycle arrest and/or apoptosis, independently of the most relevant prognostic markers. The aim of the present study was to analyze the gene expression profile (GEP) induced by Nutlin-3 exposure in primary CLL cells from p53wt and p53del/mut cases. Patients and methods purified cells from 24 PB CLL samples, all characterized for IGHV mutational status, CD38 and ZAP-70 and p53 mutations (16 p53wt CLL, 8 p53del/mut CLL of which 6 with del17p13 and p53 mutations, 1 with del17p13 alone, and 1 with p53 mutations alone), were exposed to 10 mM Nutlin-3 for 24 hours. GEP was performed using a dual labelling strategy; the differential expression of the below reported genes were validated by quantitative real-time PCR. Results i) signature of Nutlin-3 exposure in p53wt CLL: 144 differentially expressed genes (143 up-regulated, 1 down-regulated) were correlated with response to Nutlin-3. Among the over-expressed genes, several genes were related to apoptosis (e.g. BAX, BBC3, E124, IKIP, FAS, LRDD, FLJ11259, TRIAP1, GADD45, TP53INP1, ISG20L1, ZMAT3, TNFRS10C, TNFRSF10B/TRAIL-R2), while other genes (e.g. MDM2, CDKN1A, PCNA) were up-regulated by Nutlin-3 as a part of a negative feed-back mechanism. Of note, this signature was not shared by 3/16 p53wt cases (identified as “non-responder” p53wt CLL) and 7/8 p53del/mut cases (identified as “non-responder” p53del/mut CLL); consistently, cells from these cases were also significantly resistant to the in-vitro cytotoxic effects of Nutlin-3; ii) signature of Nutlin-3 “non-responder” p53wt CLL: by comparing the constitutive GEP of 13 “responder” versus 3 “non-responder” p53wt CLL, we obtained 278 differentially expressed genes, 149 up-regulated and 129 down-regulated in “non-responder” p53wt CLL. Among up-regulated genes, we focused on MDM4/MDMX, a gene whose product was known to have an inhibitor activity of p53-dependent transcription and to form Nutlin-3 resistant complexes with p53. Among down-regulated genes, validations were made for BIRC4BP, whose product is known to act as an antagonist of the anti-apoptotic protein XIAP; iii) signature of Nutlin-3 “non-responder” p53del/mut CLL: by comparing the constitutive GEP of 13 “responder” versus 7 “non-responder” p53del/mut cases, we obtained 72 differentially expressed genes, 26 up-regulated and 46 down-regulated (31/46 located at the 17p segment) in “non-responder” p53del/mut CLL. Validations were made for several genes whose products display pro-apoptotic activities (e.g. PSMB6, RPL26 and ZBTB4, located at 17p segment, and GNAZ located at chromosome 22) among down-regulated genes, and ARHGDIA, whose gene product displays anti-apoptotic activities and mediates cellular resistance to chemotherapeutic agents, among up-regulated genes. Notably, CLL cells (n=43) displayed constitutively higher levels of MDM4/MDMX (p<0.0001) and ARHGDIA (p=0.0002) transcripts than purified normal B cells (n=15), irrespectively to the major biologic prognosticators. Conclusions specific gene-sets and GEP were documented to be associated with response or resistance to Nutlin-3 exposure in p53wt or p53del/mut CLL. These findings may help to identify novel molecular targets for CLL therapy. Disclosures: No relevant conflicts of interest to declare.

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Characterization of Interleukin-10 Receptor Expression on B-Cell Chronic Lymphocytic Leukemia Cells

Blood ◽

10.1182/blood.v89.11.4146 ◽

1997 ◽

Vol 89 (11) ◽

pp. 4146-4152 ◽

Cited By ~ 52

Author(s):

Jesper Jurlander ◽

Chun-Fai Lai ◽

Jimmy Tan ◽

Chuan-Chu Chou ◽

Christian H. Geisler ◽

...

Keyword(s):

Cell Death ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Interleukin 10 ◽

Receptor Activation ◽

Lymphocytic Leukemia ◽

Receptor Expression ◽

Interferon Α ◽

Ifn Γ ◽

Cell Chronic Lymphocytic Leukemia

Abstract B-cell chronic lymphocytic leukemia (B-CLL) cells accumulate in vivo in the G0/G1 phase of the cell cycle, suggesting that their malignant expansion is due, at least in part, to a delay in cell death. However, the cellular or molecular factors responsible for a delay in B-CLL cell death are unknown. B-CLL cells do express receptors for interferon-α (IFN-α) and IFN-γ, and activation of both has been shown to promote B-CLL survival in vitro by preventing apoptosis. The interleukin-10 (IL-10) receptor is another member of the IFN receptor family, but its ligand, IL-10, has been reported to induce apoptosis in B-CLL cells. In the current study, we undertook a biochemical analysis of IL-10 receptor expression on freshly isolated B-CLL cells and characterized the functional responsiveness of IL-10 binding to its constitutively expressed receptor. We show that B-CLL cells bind IL-10 with significant specificity and express between 47 and 127 IL-10 receptor sites per cell, with a dissociation constant in the range of 168 to 426 × 10−12 mol/L. Ligand binding and activation of the IL-10 receptor expressed on B-CLL cells results in the phosphorylation of signal transducer and activator of transcription 1 (STAT1) and STAT3 proteins. This pattern of STAT protein phosphorylation is identical to IL-10 receptor activation on normal cells and similar to IFN-α (STAT1 and STAT3) and IFN-γ (STAT1) receptor activation in CLL. Further, in consecutive samples of fresh blood obtained from patients with B-CLL cells, the addition of IL-10 inhibited B-CLL proliferation, enhanced B-CLL differentiation, but did not induce apoptosis. Indeed, IL-10, like IFN-γ, was able to significantly reduce the amount of B-CLL cell death caused by hydrocortisone-induced apoptosis. We conclude that cytokines, which signal through the interferon family of receptors, have comparable functional effects on B-CLL cells.

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