The Geldanamycin Derivative DMAG Demonstrates Improved Cytotoxicity and Down-Modulation of Hsp90 Client Proteins Relative to 17-AAG in Chronic Lymphocytic Leukemia (CLL) Cells: Justification for Clinical Trials in CLL.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2101-2101
Author(s):  
Amy J. Johnson ◽  
Amy J. Wagner ◽  
Lisa L. Smith ◽  
David M. Lucas ◽  
Michael D. De Lay ◽  
...  
Keyword(s):  
Phase I ◽  
Immunoblot Analysis ◽  
Clinical Development ◽  
Lymphocytic Leukemia ◽  
Pharmacological Properties ◽  
Down Regulation ◽  
Cancer Types ◽  
Client Proteins ◽  
Pharmacodynamic Biomarkers

Abstract The heat shock protein Hsp90 functions to stabilize important cell survival- and proliferation-related kinases such as AKT, IKK, c-Src, Raf-1, and cdk9. Cancer cells have activated Hsp90 as compared to normal cells, making this a relevant therapeutic target to eliminate these kinases. The semi-synthetic geldanamycin derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) binds to and inhibits the activity of Hsp90, and previous work demonstrated the ability of 17-AAG to deplete AKT in several cancer types in vitro. A newer geldanamycin derivative, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (DMAG) has improved pharmacological properties including solubility and oral bioavailability, and was shown to be more effective than 17-AAG in melanoma and pancreatic carcinoma mouse xenograft mouse models. We therefore examined the effects of 17-AAG and DMAG against CD19-positive tumor cells from CLL patients. Cell viability was examined by the MTT assay, and AKT and IKK protein expression was examined by immunoblot analysis. In samples from seven CLL patients, 1.0uM DMAG resulted in 31.5% viability (95% CI: 13.1–50.0%), compared to 61.5% viability (95% CI: 45.0–78.0%) using the same concentration of 17-AAG. In four CLL patient samples treated with 1.0uM DMAG for 24 hours, AKT was decreased an average of 72.5% (95% CI: 57.7–87.3%) relative to the untreated controls. This is in comparison to 1.0uM 17-AAG, which caused a 52.7% decrease in AKT (95%CI: 39.7–65.6%). IKK protein was also decreased at similar levels in all patient samples examined. This data indicates that in CLL cells, DMAG has superior activity both in cytotoxicity and in reduction of relevant Hsp90 client proteins. 17-AAG is currently undergoing Phase I clinical testing in CLL, and DMAG is completing phase I clinical development in solid tumor malignancies. Overall, our data and that of others support clinical development of DMAG in CLL, based on the improved pharmacologic properties, enhanced efficacy relative to 17-AAG, and expected down-regulation of target proteins. In addition, our in vitro observations support using measurement of protein down-regulation of AKT and IKK as pharmacodynamic biomarkers of activity in patients undergoing therapy with these agents.

Phase I study of intranodal direct injection of adenovirus encoding recombinant CD40-ligand (Ad-ISF35) in patients with chronic lymphocytic leukemia

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 3003-3003
Author(s):  
J. E. Castro ◽  
J. D. Sandoval-Sus ◽  
J. Melo-Cardenas ◽  
D. Darrah ◽  
M. Urquiza ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Phase I ◽  
Phase I Study ◽  
Direct Injection ◽  
Leukemia Cell ◽  
Additional Treatment ◽  
Genetically Engineered ◽  
Lymphocytic Leukemia ◽  
Cell Counts

3003 Background: Transduction of chronic lymphocytic leukemia (CLL) cells with replication-defective adenovirus (Ad) encoding a genetically engineered, membrane-stablized CD154 (ISF35) converts transduced, and “bystander” non-transduced, CLL cells into proficient antigen presenting cells that can induce immunity against autologous leukemia cells. Preclinical studies demonstrated that direct injection of Ad-ISF35 into lymphoma nodules can induce potent anti-lymphoma immune responses in test animals, capable of eradicating lethal tumors at distal sites and protect against recurrent disease upon subsequent re-challenge with syngeneic tumor. Methods: We conducted a phase I study on 15 patients to evaluate the safety of intranodal direct injection (IDI) of Ad-ISF35. Pts, ages 45–71 yrs, with rapidly progressive disease (median CLL doubling time of 3.7 months) each received a single ultrasound guided IDI of 1 to 30 x 1010 Ad-ISF35 viral particles in 4 different dose cohorts. Results: IDI of Ad-ISF35 was well-tolerated and effective in inducing systemic responses. Some pts had grade ≤ 2 injection-site erythema, pain and/or swelling, or flu-like symptoms. Some pts in the highest-dose cohorts had transient, asymptomatic grade 3/4 hypophosphatemia. No long-term (≥ 6 wk) adverse effects were observed. Although there was no evidence for dissemination of Ad-ISF35 beyond the injected lymph node, IDI of Ad-ISF35 induced blood CLL cells to express death receptors, pro-apoptotic proteins, and immune co-stimulatory molecules similar to those induced on “bystander” CLL cells co-cultured with Ad-ISF35 transduced cells in vitro. Importantly, IDI of Ad-ISF35 resulted in significant reductions in blood leukemia cell counts and a median reduction of 53.2% (range 25–75.4%) in the size of lymph nodes and/or spleen, which was durable (≥ 4 months) in 9 pts. Despite aggressive disease prior to treatment, the median treatment-free survival was 5.3 months and 3 pts have yet to require additional treatment after 1-year follow-up. Conclusions: Single IDI of Ad-ISF35 was safe and effective in inducing systemic biologic and clinical responses in pts with CLL. IDI of Ad-ISF35 might be effective in the treatment of CLL and related lymphomas. [Table: see text]

Results of a phase I trial of the proteasome inhibitor carfilzomib in patients with relapsed or refractory chronic lymphocytic leukemia (CLL) and small lymphocytic leukemia (SLL).

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 7077-7077
Author(s):  
Jennifer Ann Woyach ◽  
Joseph M. Flynn ◽  
Jeffrey Alan Jones ◽  
Leslie A. Andritsos ◽  
Margaret Lucas ◽  
...  
Keyword(s):  
Phase I ◽  
Dose Level ◽  
Phase I Trial ◽  
Dose Range ◽  
Plasma Concentrations ◽  
Area Under The Curve ◽  
Lymphocytic Leukemia ◽  
Maximum Plasma ◽  
Maximal Dose

7077 Background: CLL is an incurable malignancy, and survival for patients (pts) with relapsed disease is limited. Carfilzomib (CFZ) has shown efficacy in multiple myeloma, and our group has shown significant in vitro activity in primary CLL cells. Therefore, we have undertaken a phase I trial of this agent in CLL. Methods: This is a single institution phase I trial of CFZ in pts with relapsed or refractory CLL. Primary endpoints were to determine maximal tolerated dose (MTD) and describe toxicity. Pts with CLL relapsed after at least one therapy were enrolled using a 3x3 design. CFZ was administered on the standard myeloma schedule. The first two doses were administered at 20 mg/m2 with remainder given at doses starting at 27 mg/m2 for dose level 1 with escalation to 56 mg/m2. Results: 17 pts received at least 1 dose of CFZ. 12 pts completed at least 1 cycle of therapy, with the remaining 5 experiencing PD during cycle 1. The MTD was not reached, with 3 pts accrued to each dose level to the maximal dose tested without dose limiting toxicity. Most adverse events (AE) were grade (G) 1 or 2. G3/4 AE were quickly reversible and included G3 neutropenia (4 pts), G4 neutropenia (2), G3 febrile neutropenia (1), and G3 thrombocytopenia (3). G1/2 toxicities observed in ≥ 20% of pts included anemia (10), thrombocytopenia (7), and hypocalcemia (8). Median number of cycles was 3, with 9 pts achieving stable disease after 2 cycles. Of 3 pts enrolled at maximal dose level, 2 remain on therapy after 5 and 7 months, with 1 achieving a clinical partial response. Of 5 evaluable pts, at least 50% proteasome inhibition was seen in all at 1 hour, with minimal recovery at 24 hours. PK was best characterized by a two-compartment model. Maximum plasma concentrations across all dose levels ranged from 0.81 to 8.1 uM. Across the evaluated dose range, area under the curve increased in an apparent dose-proportional manner. Conclusions: Despite relatively limited efficacy in this study, CFZ has acceptable toxicity in CLL, with no MTD identified up to 56 mg/m2. This suggests that CFZ may be better studied in CLL using a different schedule or in combination with other active agents. Clinical trial information: NCT01212380.

The Novel Histone Deacetylase Inhibitor OSU-HDAC42 Has Class I and II Histone Deacetylase (HDAC) Inhibitory Activity and Represents a Novel Therapy for Chronic Lymphocytic Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2807-2807
Author(s):  
Derek A. West ◽  
David M. Lucas ◽  
Melanie E. Davis ◽  
Michael D. De Lay ◽  
Amy J. Johnson ◽  
...  
Keyword(s):  
Cell Death ◽  
Chronic Lymphocytic Leukemia ◽  
Histone Deacetylase ◽  
Inhibitory Activity ◽  
Hdac Inhibitors ◽  
Clinical Development ◽  
Lymphocytic Leukemia ◽  
In Vivo Efficacy

Abstract Inhibitors of histone deacetylase (HDAC) have generated major interest for the treatment of multiple cancers including B-cell Chronic Lymphocytic Leukemia (CLL). To date, HDAC inhibitors introduced for clinical development in CLL have been associated either with suboptimal activity relative to concentrations required to mediate cytotoxicity in vitro (Valproic Acid, MS-275, SAHA), or demonstrate unacceptable acute or long-term toxicities (depsipeptide) that limit their clinical potential. Fortunately, several alternative HDAC inhibitors are in pre-clinical or early clinical development. One such agent currently undergoing pre-clinical testing by the National Cancer Institute-sponsored RAID program is OSU-HDAC42 (s-HDAC-42), a novel, orally bioavailable phenylbutyrate-derived HDAC inhibitor with both in vitro and in vivo efficacy against prostate cancer cells. We therefore tested OSU-HDAC42 against CD19-positive cells obtained from patients with CLL to determine its potential in this disease. The LC50 of OSU-HDAC42 in CLL cells was 0.46 uM at 48 hours of continuous incubation by MTT assay, which was corroborated by annexin V-FITC/propidium iodide flow cytometry. To determine the minimum amount of time that OSU-HDAC42 must be present to induce cell death, cells were incubated for various times, washed, resuspended in fresh media without drug, then assessed by MTT at a total of 48 hours incubation. The effects of OSU-HDAC42 were eliminated in CLL cells when drug was removed after 4 or 6 hours. However, there was a gradual increase in effect over time, and by 16 hours, approximately 60% of the cytotoxicity achieved with continuous incubation was retained. OSU-HDAC42 induced acetylation of histone proteins H3 and H4 as early as 4 hours that was dose and time dependent. LC/MS interrogation of OSU-HDAC42-treated CLL cells is currently underway to determine specific post-translational modification changes of all histone proteins and variants. OSU-HDAC42 also was able to sensitize CLL cells to TNF-Related Apoptosis Inducing Ligand (TRAIL) at 24 hours in a dose-dependent manner, supporting its class I HDAC inhibitory activity as recently reported by Inoue and colleagues (Cancer Res.2006; 66:6785). Evidence of class II HDAC inhibitory activity was also observed with OSU-HDAC42 at 12 hours with acetylation of tubulin. Unlike depsipeptide, OSU-HDAC42 activated both caspase-8 and -9 followed by PARP processing. Cell death induced by OSU-HDAC42 was completely inhibited with pre-treatment by the pan-caspase inhibitor Z-VAD-FMK. In vivo experiments are underway to examine the efficacy of OSU-HDAC42 in several murine models of leukemia to confirm in vivo efficacy as well as influence on murine effector cells. Our data strongly support continued investigation of OSU-HDAC42 in CLL and related B-cell malignancies.

The Para-Isomer of Nitric Oxide Donating Acetylsalicylic Acid (p-NO-ASA) Induces Apoptosis in Chronic Lymphocytic Leukemia (CLL) in Vitro and In Vivo without Gross Systemic Toxicities.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3783-3783
Author(s):  
Regina Razavi ◽  
Iris Gehrke ◽  
Rajesh Kumar Gandhirajan ◽  
Simon Jonas Poll-Wolbeck ◽  
Julian Paesler ◽  
...  
Keyword(s):  
B Cells ◽  
Tumor Volume ◽  
Annexin V ◽  
Immunoblot Analysis ◽  
Vehicle Control ◽  
Lymphocytic Leukemia ◽  
Atp Content ◽  
Meta Isomer

Abstract Abstract 3783 Poster Board III-719 Chronic lymphocytic leukemia (CLL) is characterized by an accumulation of mature, non functional B cells. WNT/β-catenin(CTNNB1)/TCF/Lef-1 signalling appears to be constitutively and aberrantly activated in these cells. Furthermore, several compounds related to the non-steroidal anti-inflammatory drugs (NSAIDs) are reported to inhibit β-catenin stability and/or function in WNT active cancers in vitro. However, so far clinical studies with such substances generated disappointing results which is likely to the fact that therapeutic plasma concentrations could not be reached without producing significant toxicities; hence, the required high concentrations limit their clinical use. Recently, nitric oxide-donating acetylsalicylic acid (NO-ASA) has been shown to achieve high plasma levels in doses not leading to any relevant side effects in humans. In addition, NO-ASA could disrupt complexation of β-catenin and TCF-4 in vitro. Because the general structure of NO-ASA enables more variants, the aim of our study was to evaluate the effect of the para- (p-NO-ASA) and meta-isomer (m-NO-ASA) in CLL in vitro and in vivo. Primary CLL cells as well as healthy peripheral blood monocytes (PBMCs) and healthy B cells were treated with varying concentrations of p- and m-NO-ASA. Cytotoxicity was assessed by microscopic cell viability testing and measurement of the reduction of the ATP content. Induction of apoptosis was investigated by Annexin V-FITC/propidiumiodide staining and immunoblotting of the caspases-9, -3 as well as PARP. Further, the β-catenin protein amount and the expression of WNT effector proteins like cyclin D1 (CCND1), C-MYC and LEF-1 was evaluated by immunoblot analysis. In vivo activity of NO-ASA was evaluated by treating irradiated CD1 nu/nu female mice, containing a JVM-3 cell line xenograft, with 100 mg/kg/day of p- and m-NO-ASA or vehicle control p.o. for 3 weeks continuously. We found a significant concentration dependent reduction of the ATP content in CLL cells after treatment with p-NO-ASA, whereas the meta-isomer showed no effect on CLL cells. While healthy B cells and healthy PBMCs were not significantly affected by any of the isomers the mean lethal concentration (LC50) was 4.64 μM in CLL cells. Annexin V-FITC/PI staining revealed that reduced cell survival occurs in a time and concentration dependent manner and is mediated by apoptosis. Treatment with 10 μM of p-NO-ASA for 24 hours reduced survival to 46.3 ± 10.1%. This effect was achieved as early as 6 hours after treatment. Immunoblot analysis showed that only p-NO-ASA but not m-NO-ASA activates caspases-9, -3 and cleaves PARP at the same concentrations, which lead to induction of cell death. β-catenin protein levels and WNT pathway target genes are down regulated between 1 to 10 μM also only by the para-isomer. In vivo results revealed that exclusively p-NO-ASA show a strong antitumor efficacy with an IRmax value of 83.1%. After 9 days of treatment p-NO-ASA lead to a significantly reduced tumor volume compared to vehicle control (126.4 ± 22.3 mm3 for p-NO-ASA vs. 290.0 ± 65.9 mm3 for the vehicle control, p=0.0303). Tumor volume of vehicle treated controls increased up to 775.4 ± 219.6 mm3 whereas the tumor volume of p-NO-ASA treated group remained stable at 128.7 ± 27.6 mm3 (p=0.0091) over a treatment period of 21 days. The meta-isomer exhibited no significant antineoplastic effect. Our findings show that the para- but not the meta-isomer of NO-ASA selectively induces caspase mediated apoptosis in CLL cells. The mechanism of action might include inhibition of β-catenin/Lef-1 signaling since we observed downregulation of specific target gene expression. Due to our promising in vivo results, discovering a strong inhibition of tumor growth without producing gross side effects, p-NO-ASA might be a valuable compound for the treatment of CLL. More investigations of the mechanism of action and the specific difference between the positional isomers are needed. Disclosures: Hallek: Roche: Consultancy, Honoraria, Research Funding.

Selective Bcl-2 Inhibition With ABT-199 Is Highly Active Against Chronic Lymphocytic Leukemia (CLL) Irrespective Of TP53 Mutation Or Dysfunction

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1304-1304 ◽  
Author(s):  
Mary Ann Anderson ◽  
Constantine C.S. Tam ◽  
John F. Seymour ◽  
Anthony Bell ◽  
David A Westerman ◽  
...  
Keyword(s):  
Phase I ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Phase I Clinical Trials ◽  
Advisory Committees ◽  
Tp53 Mutations ◽  
Heavily Pretreated ◽  
Clinical Responses ◽  
Tp53 Pathway

Abstract Aim CLL is characterized by universal overexpression of the anti-apoptotic protein Bcl-2 which promotes inappropriate survival and chemotherapy resistance. ABT-199 is a Bcl-2 selective BH3-mimetic molecule currently in phase I clinical trials for the treatment of CLL. Del(17p) is a key negative prognostic indicator in CLL, associated with reduced response rates, progression-free and overall survival with standard therapy. Reduction or loss of TP53 function is considered the biological basis for its impact. Consistent with this, TP53 mutations in CLL lacking del(17p) also confer similar negative prognostic effects. BH3-mimetics are small molecule inhibitors that theoretically act downstream of TP53. We therefore hypothesized that ABT-199 will be equally effective in patients (pts) with CLL irrespective of TP53 function. We aimed to: (i) examine the incidence of TP53 aberrations in a heavily pretreated group of patients referred to two centers for ABT-199 therapy; (ii) determine the impact of TP53 aberrations on in vitro and clinical responses of CLL to ABT-199; and (iii) determine if CLL cells deficient in TP53 pathway function are as sensitive to ABT-199 as CLL with intact TP53 function. Methods This work was conducted in parallel with phase I clinical trials of ABT-199 monotherapy (NCT01328626; n = 25) or monotherapy followed by combination with rituximab (NCT01682616; n = 5) in pts with relapsed and refractory (r/r) CLL, after written, informed consent. Pts were tested for potential TP53 aberration using (i) FISH for del(17p); (ii) bone marrow (BM) immunohistochemistry (IHC) for elevated TP53 expression (>20% CLL cells staining positive on BM trephine); (iii) a 72hour in vitro nutlin-3a cytotoxicity assay for functional assessment of the TP53 pathway; and (iv) targeted next generation sequencing of the TP53 gene (with confirmatory Sanger sequencing) to detect deleterious mutations. Del(17p) and TP53 status of CLL samples at screening was determined and correlated with in vitro responses to ABT-199 after 24 hours and objective clinical responses to therapy with ABT-199 as determined within the trial protocol using iwCLL criteria. Results In this r/r group of CLL pts, 13/30 (43%) had evidence of del(17p) by FISH (range 9 - 90% of cells); 15/28 (54%) had evidence of TP53 mutation by sequencing; and 7/29 (24%) had TP53 overexpression by IHC, all of whom had TP53 mutations. For 27 pts with data for all three assays, ten had no evidence of TP53 abnormality. In the 10 samples with >20% del(17p) by FISH, deleterious TP53 mutations were detected in all. For the purposes of comparing responses to ABT-199 by TP53 status, three categories were identified: negative for both del(17p) and p53 mutation, positive for one only, and positive for both (table 1). CLL cells in all groups were similarly highly sensitive in vitro to ABT-199 (ANOVA p>0.05). The overall response rate was at least 87.5% in all groups. Given that CLL is characterized by heterogeneity for TP53 aberrations within individual patients, we selected four samples with high level del(17p) and TP53 mutations for assessment of TP53 function. All showed resistance to nutlin 3a-induced cytotoxicity (median IC50 12mM [95%CI 9 – 41mM]) when compared with CLL cells without del(17p) or TP53 mutations which were 15 – 30-fold more sensitive. No differences in vitro sensitivity to ABT-199 were detected when these nutlin-3a resistant samples were compared with cells lacking TP53 mutations or del(17p). Conclusions Defects in the TP53 pathway are present in the majority of patients with heavily pretreated CLL; novel treatment for such patients must include activity against TP53 aberrant clones. CLL cells lacking TP53 pathway function have similar sensitivity to ABT-199 in vitro as do TP53 pathway intact cells, and clinical responses were observed in ≥87.5% of patients. These data suggest that ABT-199 should be evaluated alone and in combination in pts with TP53-aberrant lymphoid neoplasms. Disclosures: Anderson: Abbvie: Research Funding. Off Label Use: ABT-199 is an unlicensed drug currently in phase I and II studies in patients with chronic lymphocytic leukemia. Seymour:Roche: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Huang:Genentech: Employee of Walter and Eliza Hall Institute which recieves commercial income related to ABT-199. Other, Research Funding; Abbvie: Research Funding. Roberts:Abbvie: Research Funding; Genentech: Employee Walter and Eliza Hall Institute which recieves commercial income related to ABT-199. Other, Research Funding.

scholarly journals In Vitro Characterization of the Pharmacological Properties of the Anti-Cancer Chelator, Bp4eT, and Its Phase I Metabolites

PLoS ONE ◽  
2015 ◽  
Vol 10 (10) ◽  
pp. e0139929 ◽  
Author(s):  
Eliška Potůčková ◽  
Jaroslav Roh ◽  
Miloslav Macháček ◽  
Sumit Sahni ◽  
Ján Stariat ◽  
...  
Keyword(s):  
Phase I ◽  
Pharmacological Properties ◽  
In Vitro Characterization ◽  
Anti Cancer

Expression of MicroRNA (miR) miR-15a/miR-16-1 Downregulates Expression of BCL-2 Protein in Chronic Lymphocytic Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2796-2796 ◽  
Author(s):  
Liguang Chen ◽  
Li Tang ◽  
George Calin ◽  
Carlo M. Croce ◽  
Thomas J. Kipps
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
Human Cancer ◽  
Human Tumor ◽  
Immunoblot Analysis ◽  
Lymphocytic Leukemia ◽  
Control Vector ◽  
Rt Pcr ◽  
Target Mrna

Abstract MicroRNAs (miRNAs) comprise a family of small RNA, each member of which can potentially regulate post-transcription gene expression in a temporal and tissue-specific manner. Each miRNA encodes a transcript of about 22 nucleotides that can modulate expression of specific target mRNA. We identified that the chronic lymphocytic leukemia (CLL) cells of a large proportion of patients have aberrant, low-level expression of two clustered miRNAs located at 13q14, designated miR-15a and miR-16–1, providing the first example of dysregulated miRNA expression in a human cancer (PNAS USA99:15524, 2002). These miRNAs each have sequences that potentially could target mRNA encoding the anti-apoptotic protein bcl-2, which is expressed at high levels in CLL. Transfection of a lymphoma B cell line lacking expression of miR-15a and miR-16–1 with an expression vector encoding miR-15a and miR-16–1, designated pmiR-15/16, attenuated the expression-level of bcl-2 protein and enhanced the susceptibility of such lymphoma B cells to apoptosis in vitro. However, it was not certain whether miR-15a and miR-16–1 could modulate expression of bcl-2 in primary leukemia cells that were not adapted for propagation in vitro. We selected primary CLL cell samples (n = 3) that harbored deletions at 13q14 and that lacked expression of miR-15a and miR-16–1, as assessed via microRNA array and quantitative RT-PCR analyses. We transfected these cells with pmiR-15/16 or a control vector via electroporation and, in parallel studies, also transfected these CLL cells with sense and antisense control oligo-RNAs via transmessenger transfection. Before and after such manipulations we monitored for expression of miR-15a and miR-16–1 by RT-PCR, for relative expression of BCL-2-family member transcripts using a multiplex ligation-dependent probe amplification (MLPA) assay, and for expression of bcl-2 protein using immunoblot analysis and flow cytometry. We found that CLL cells transfected with either pmiR15/16 or with miR-15a and miR-16–1 sense oligo-RNAs had increased expression-levels of miR-15a and miR-16–1 within 24 hours after transfection, whereas CLL transfected with the control vector or antisense oligo-RNA did not. Depite high-level expression of miR-15a and miR-16–1, the relative levels of BCL-2 transcript did not change over the 48 hours after transfection that we examined. However, in this time period we observed that CLL cells made to express miR-15a and miR-16–1, experienced significant reductions in the levels of bcl-2 protein, which were not observed in control transfected CLL cells. This is the first demonstration that miRNAs can effect post-transcriptional regulation of protein expression in a primary human tumor and suggest that miRNAs may have potential therapeutic utility in the modulation of pathogenic gene-expression in CLL and other cancers.

scholarly journals Substantial Susceptibility of Chronic Lymphocytic Leukemia to BCL2 Inhibition: Results of a Phase I Study of Navitoclax in Patients With Relapsed or Refractory Disease

2012 ◽  
Vol 30 (5) ◽  
pp. 488-496 ◽  
Author(s):  
Andrew W. Roberts ◽  
John F. Seymour ◽  
Jennifer R. Brown ◽  
William G. Wierda ◽  
Thomas J. Kipps ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Phase I ◽  
Progression Free Survival ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Refractory Disease ◽  
Phase Ii Studies ◽  
Continual Reassessment ◽  
Related Proteins

Purpose BCL2 overexpression is a hallmark of chronic lymphocytic leukemia (CLL). The novel BH3 mimetic navitoclax (ABT-263) specifically inhibits BCL2 and related proteins BCL-xl and BCL-w, potently inducing apoptosis of CLL cells in vitro. A phase I trial in patients with CLL was conducted to evaluate the safety, pharmacokinetics, and biologic activity of oral navitoclax. Patients and Methods Twenty-nine patients with relapsed or refractory CLL received daily navitoclax for 14 days (10, 110, 200, or 250 mg/d; n = 15) or 21 days (125, 200, 250, or 300 mg/d; n = 14) of each 21-day cycle. Dose escalation decisions were informed by continual reassessment methodology. Results Lymphocytosis was reduced by more than 50% in 19 of 21 patients with baseline lymphocytosis. Among 26 patients treated with navitoclax ≥ 110 mg/d, nine (35%) achieved a partial response and seven maintained stable disease for more than 6 months. Median treatment duration was 7 months (range, 1 to ≥ 29 months). Median progression-free survival was 25 months. Activity was observed in patients with fludarabine-refractory disease, bulky adenopathy, and del(17p) CLL. Thrombocytopenia due to BCL-xl inhibition was the major dose-limiting toxicity and was dose-related. Low MCL1 expression and high BIM:MCL1 or BIM:BCL2 ratios in leukemic cells correlated with response. We determined that the navitoclax dose of 250 mg/d in a continuous dosing schedule was optimal for phase II studies. Conclusion BCL2 is a valid therapeutic target in CLL, and its inhibition by navitoclax warrants further evaluation as monotherapy and in combination in this disease.

Depsipeptide (FR901228) induces histone acetylation and inhibition of histone deacetylase in chronic lymphocytic leukemia cells concurrent with activation of caspase 8–mediated apoptosis and down-regulation of c-FLIP protein

Blood ◽  
2003 ◽  
Vol 102 (2) ◽  
pp. 652-658 ◽  
Author(s):  
Jennifer L. Aron ◽  
Mark R. Parthun ◽  
Guido Marcucci ◽  
Shinichi Kitada ◽  
Andrew P. Mone ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Chronic Lymphocytic Leukemia ◽  
Histone Acetylation ◽  
Histone Deacetylase ◽  
Histone H3 ◽  
Lymphocytic Leukemia ◽  
Caspase 8 ◽  
Tnf Receptor ◽  
Down Regulation

AbstractDepsipeptide is in clinical trials for chronic lymphocytic leukemia (CLL) on the basis of earlier observations demonstrating selective in vitro activity in CLL. We sought to determine the relationship of histone H3 and H4 acetylation, inhibition of histone deacetylase, and apoptosis observed in CLL cells to justify a pharmacodynamic end point in these clinical trials. We demonstrate that in vitro depsipeptide induces histone H3 and H4 acetylation and histone deacetylase enzyme inhibition at concentrations corresponding to the LC50 (concentration producing 50% cell death) for cultured CLL cells (0.038 μM depsipeptide). The changes in histone acetylation are lysine specific, involving H4 K5, H4 K12, and H3 K9, and to a lesser extent H4 K8, but not H4 K16 or H3 K14. Depsipeptide-induced apoptosis is caspase dependent, selectively involving the tumor necrosis factor (TNF) receptor (extrinsic pathway) initiating caspase 8 and effector caspase 3. Activation of caspase 8 was accompanied by the down-regulation of cellular FLICE-inhibitory protein (c-FLIP, I-FLICE) without evidence of Fas (CD95) up-regulation. Changes in other apoptotic proteins, including Bcl-2, Bax, Mcl-1, and X-linked inhibitor of apoptosis (XIAP), were not observed. Our results demonstrate a relationship between target enzyme inhibition of histone deacetylase, histone H3 and H4 acetylation, and apoptosis involving the TNF-receptor pathway of apoptosis that is not used by other therapeutic agents in CLL. These data suggest use of histone H3 and H4 acetylation, inhibition of histone deacetylase, and down-regulation of FLIP as pharmacodynamic end points for further evaluation of this drug in patients.

Phase I Study of 5-aza-2′-Deoxycitidine, Alone or in Combination with Hyper-CVAD, in Relapsed or Refractory Acute Lymphocytic Leukemia (ALL).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2826-2826 ◽  
Author(s):  
Guillermo Garcia-Manero ◽  
Deborah Thomas ◽  
Michael Rytting ◽  
Patrick Zweidler-McKay ◽  
Zeev Estrov ◽  
...  
Keyword(s):  
Cell Death ◽  
Phase I ◽  
Phase I Study ◽  
Acute Lymphocytic Leukemia ◽  
Single Agent ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Dna Hypomethylation ◽  
Sequential Phase

Abstract Acute lymphocytic leukemia (ALL) is characterized by concomitant aberrant methylation of multiple promoter associated CpG islands. Specific DNA methylation patterns predict for poor prognosis. Furthermore, reactivation of epigenetically repressed molecular pathways results in in-vitro selective induction of leukemia cell death (Kuang et al. Oncogene 2007). In vitro modeling in ALL indicates that more frequent dosing of 5-aza-2′-deoxycitidine (DAC) results in increased control of leukemia cell proliferation and induction of cell death (Yang H. Leuk Res 2005). Based on all this data, we designed a phase I clinical trial of DAC for patients (pts) with relapsed or refractory ALL. Pts of any age are eligible for this study. There are no requirements in terms of performance status or prior therapies. DAC is infused over 1 hour daily × 5 every other week. One cycle of therapy is considered as 4 weeks. Pts that do not respond or progress after single agent DAC can participate in a sequential phase I study of DAC in combination with standard hyperCVAD therapy. In that phase of the study, DAC is administered daily × 5 concomitantly with hyperCVAD once per course. DAC is escalated using a conventional “3+3” design in both portions of the study. Thirteen pts have been treated so far. Nine of these pts have also been treated on the sequential phase I portion of the combination of DAC with hyperCVAD. Their median age is 33 years (range 8–66); number of prior therapies 2 (range 1–8); 2 pts had diploid cytogenetics;1 t(4;11) and the rest had complex alterations. No pts was BCR/ABL positive. Pretreatment median WBC was 5 (range 0.5–97). Single agent DAC has been escalated up to doses of 60 mg/m2 IV daily × 5 every other week (total per course= 600 mg/m2). No dose limiting toxicities have been observed. When used in combination with hyperCVAD, DAC doses of 15 mg/m2 daily × 5 (total: 75 mg/m2) have not been associated with significant drug-related toxicities either. Four pts (30%) achieved a complete marrow response (no morphological evidence of leukemia in the marrow) with single agent DAC. Hematological control with single agent DAC was also achieved in pts with rapidly proliferating disease with or without steroid support. Three pts of 9 (30%) achieved a complete remission with DAC + HCVAD. One of these pts had previously achieved a marrow response with single agent DAC. The effect of DAC on DNA hypomethylation induction was measured using the LINE bisulfite pyrosequencing assay. Of importance, no plateau in terms of DNA hypomethylation induction has been detected at doses up to 60 mg/m2 IV × 5 every other week. Based on the lack of toxicity and continuous pharmacodynamic effect, dose escalation continues for both the single agent DAC portion and the DAC + hyperCVAD combination. These results indicate that DAC administered every other week, or in combination with hyperCVAD, is safe and associated with clinical activity in pts with advanced relapsed/refractory ALL. These data also suggest that the optimal dose of hypomethylating agent is disease dependent.

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