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.

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]

Combination of 5-azacytidine (5-AZA) and valproic acid (VPA) in advanced solid cancers: A phase I study

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 3547-3547 ◽  
Author(s):  
A. O. Soriano ◽  
F. Braiteh ◽  
G. Garcia-Manero ◽  
L. H. Camacho ◽  
D. Hong ◽  
...  
Keyword(s):  
Phase I ◽  
Histone Acetylation ◽  
Phase I Study ◽  
Mononuclear Cells ◽  
Neutropenic Fever ◽  
Dna Hypomethylation ◽  
Peripheral Blood Mononuclear ◽  
Western Blots

3547 Background: 5-AZA is a DNA hypomethylating agent. VPA is a histone deacetylase inhibitor (HDACI). The combination of hypomethylating agents and HDACI has synergistic anticancer activity in vitro and in vivo. Based on this, we conducted a phase I study of the combination of 5-AZA and VPA in patients with advanced solid cancers. Methods: 5-AZA was administered SQ daily for 10 days. VPA was given orally daily titrated to 75–100 mcg/ml. Cycles were 28 days long. 5-AZA was started at 20 mg/m2 and escalated using an adaptive algorithm based on toxicity in the prior cohort. Each cohort included 6 patients (pts). Global DNA methylation was estimated with the LINE pyrosequencing assay in peripheral blood mononuclear cells on days 1 and 10 of each cycle. Histone H3 and H4 acetylation was assessed with western blots. Results: Forty seven patients were evaluable. Median age was 60 years (15–77). 5- AZA dose levels included 20, 25, 37.5, 47, 59, 75 and 94 mg/m2. One DLT occurred at 37.5 (Neutropenic fever (NF)). At 75 mg/m2, 1 grade 3 NF and at 94 mg/m2, 3 DLTs (2 NF and 1 thrombocytopenia) were observed. The MTD was 75 mg/m2 SQ daily for 10 days. Other toxicities included drowsiness (N=6), tremor (N=6), hypomagnesemia (N=1), anemia (N=2) and vomiting (N=1). Stable disease (SD) was observed in 16 pts (34%). One pt with rapidly progressive renal cell carcinoma had SD for 6 months (mos). Two pts with leiomyosarcoma of the uterus had SD for 8 and 6 mos. One pt with melanoma and two pts with uveal melanoma had SD for 4 mos. One pt with sinus adenoid cystic carcinoma and 1 with thymic carcinoma had SD for 4 mos. SD was also observed in pts with colon (N=3), sarcoma (N=2), papillary thyroid (N=2) and breast cancer (N=1). Global DNA hypomethylation was demonstrated in all of the doses of 5- AZA. Median LINE methylation pretreatment was 65% (59–70%), it declined to 61% (53–63%) by day 10 (p=0.001) and returned to baseline by day 0 of the next cycle. Histone acetylation was observed in 53% of the pts. Conclusions: The combination of 5-AZA and VPA in patients with advanced solid tumors is safe. The MTD of 5-AZA in combination with VPA was 75 mg/m2 SQ daily for 10 days. DLTs were neutropenic fever and thrombocytopenia. Clinical benefit (SD) was observed in 16 pts (34%). Transient global hypomethylation and histone acetylation were demonstrated. No significant financial relationships to disclose.

Effect of Tight Control of Blood Glucose during Hyper-CVAD Chemotherapy for Acute Lymphocytic Leukemia.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 904-904 ◽  
Author(s):  
Khanh Vu ◽  
Sai-Ching Yeung ◽  
Deborah A. Thomas ◽  
Stefan Faderl ◽  
Marina Konopleva ◽  
...  
Keyword(s):  
Blood Glucose ◽  
Glycemic Control ◽  
Repeated Measures ◽  
Acute Lymphocytic Leukemia ◽  
Fasting Blood Glucose ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Future Research ◽  
High Dose

Abstract Weiser et al discovered that patients (pts) with acute lymphocytic leukemia (ALL) who developed hyperglycemia (HG) during induction chemotherapy with hyper-CVAD (fractionated cyclophosphamide, vincristine, doxorubicin, dexamethasone alternating with methotrexate and high-dose cytarabine) had shorter median overall survival (OS) as well as shorter complete remission duration (CRD). We hypothesized that normalization of HG would reverse this adverse prognosis. Therefore, a prospective study was performed to determine if intensive management of HG with insulin during hyper-CVAD consolidation therapy would improve outcome. Between May 2004 and July 2008, 52 pts with newly diagnosed ALL, Burkitt’s lymphoma, or lymphoblastic lymphoma treated with hyper-CVAD and who developed HG (glucose level of > 180 mg/dL on at least two random measurements) were enrolled. Pts were randomized to the standard care arm or the intervention care arm of HG control. Pts randomized to the standard arm were treated according to conventional care; whereas those in the intervention arm received insulin therapy with insulin glargine and aspart which were titrated to keep fasting blood glucose (Glc) levels <120 mg/dL and postprandial Glc levels <180 mg/dL without hypoglycemia. An interim analysis for both superiority and futility was planned after one half of the total number of expected deaths were observed. In the standard and intervention arms, the median age was 46.5 yrs (range 17–70) and 57.5 yrs (range 18–85) , respectively (p=0.053). Median follow-up among all patients was 20 mos (range 2 – 47). Based on a repeated measures model with terms for treatment arm, cycle, and the interaction between the two, mean serum Glc levels were significantly lower in the intervention arm as compared to the standard arm (p = 0.009). Eighteen pts died, 8 in the standard arm and 10 in the intensive arm. OS was not statistically different between the two study arms (p = 0.386). OS at one yr was 80.8% (CI 67–97.4%) and 63.5% (CI 47–85.8%) for the standard and intervention arms, respectively. Five of the deaths in the intervention arm were in pts aged 70 years or older. When adjusted for age, the intervention arm was associated with a 13% reduction in the hazard of death compared to the standard arm (hazard ratio = 0.87, 95% CI 0.32, 2.39, p = 0.79). Achievement of CR, number of pts with an ICU admission, number of hospitalizations, and number of infections were not statistically different between the two arms. CRD was not statistically different between the two arms (p = 0.503). In conclusion, intensive insulin management of glucocorticoid-induced HG as practiced did not improve OS or CRD. Since glycemic control improved without impact on survival, either the improvement was not sufficient enough to be clinically significant or insulin had a deleterious effect on the malignancy that negates the benefits of glycemic control. Future research will address the latter possibility as we have preliminary in vitro data demonstrating that elevated insulin concentrations stimulate cell growth and resistance to doxorubicin-induced apoptosis in leukemia cell lines and primary ALL samples.

Lenalidomide Inhibits Proliferation of Chronic Lymphocytic Leukemia Cells in Vitro

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1775-1775
Author(s):  
Jessie-Farah Fecteau ◽  
Diahnn Futalan ◽  
Ila Bharati ◽  
Emanuela M. Ghia ◽  
Laura G Corral ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Research Funding ◽  
Single Agent ◽  
Leukemia Cell ◽  
S Phase ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Cell Expression

Abstract Abstract 1775 Promising clinical responses have been observed in chronic lymphocytic leukemia (CLL) patients treated with lenalidomide as a single agent or in combination with other agents, The mechanisms of action of lenalidomide are under study; unlike most other anti-leukemia drugs, lenalidomide has no direct cytoxic effects in vitro on primary CLL cells, which typically are in G0/1 phase of the cell cycle. We examined the activity of lenalidomide on CLL cells that were induced to proliferate in vitro. To induce proliferation, CLL cells were cultured in media containing human interleukin (IL)-4 and IL-10 and with stromal cells (HeLa) made to express CD154. Labeling CLL cells with carboxyfluorescein succinimidyl ester (CFSE), allowed us to monitor for several rounds of induced CLL-cell division via flow cytometry. We found that addition of 0.33–10 micro M lenalidomide to such cultures resulted in a dose-dependent reduction in the number of leukemia cells induced to undergo cell-division. Moreover, we found that lenalidomide could significantly reduce the number of dividing CLL cells in each patient sample tested (n=4) by an average of 1.7 fold, reducing the fraction of dividing cells from 77% ± 27% to 44% ± 22% after 6 days of culture (mean +/− SD, P < 0.05). Evaluation of the DNA content of CLL cells using propidium iodide (PI) and flow cytometry revealed that lenalidomide significantly decreased the percentage of CLL cells in the G2/M phase of the cell cycle from 9% ± 2.7% to 5.1% ± 2.1% (mean +/− SD, n=4; P< 0.05) in control versus lenalidomide-treated cultures, respectively. Furthermore, lenalidomide appeared also to reduce the percentages of CLL cells in S phase from 12% ± 8% to 6.2% ± 3.1%. We found that the capacity of lenalidomide to inhibit CLL cell-division was associated with lenalidomide-induced leukemia-cell expression of p21/WAF/Cip, which can directly inhibit the activity of cyclin-dependent kinases required for progression from G1 into the S phase of the cell cycle. Gene expression analysis of CLL cells (n=10) revealed that lenalidomide induced increased leukemia-cell expression of p21/WAF/Cip at 6h and at 24h, an effect that also was noted for leukemia cells in the blood of patients treated with single-agent lenalidomide. Lenalidomide-induced expression of p21/WAF/Cip was associated with induced expression of the pro-apoptotic protein Bim, a downstream target of of p21/WAF/Cip. However, lenalidomide did not appear to induce leukemia-cell expression of TP53, which can induce p21/WAF/Cip, suggesting that lenalidomide induces p21/WAF/Cip via a TP53-independent mechanism. These results suggest that lenalidomide has effects on CLL cells that are distinct from those induced by CD40-ligation. Moreover, these studies reveal a potential mechanism for the anti-leukemia activity of lenalidomide, which might inhibit factors that potentially drive leukemia-cell proliferation in vivo. Disclosures: Corral: Celgene: Employment. Kipps:Igenica: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding. Messmer:Celgene: Research Funding.

scholarly journals The Glutaminase Activity of L-Asparaginase Mediates Suppression of Asns Upregulation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3959-3959 ◽  
Author(s):  
Wai Kin Chan ◽  
Lin Tan ◽  
Karine Harutyunyan ◽  
Di Du ◽  
Leona Martin ◽  
...  
Keyword(s):  
Cell Death ◽  
Amino Acid ◽  
Anticancer Activity ◽  
Single Agent ◽  
Leukemia Cell ◽  
Cell Types ◽  
Leukemia Cells ◽  
E Coli ◽  
Glutaminase Activity

Abstract L-asparaginase (ASNase) is a standard component of treatment regimens used for acute lymphoblastic leukemia and is being tested against other cancer types, including acute myeloid leukemia, lymphoma, and pancreatic cancer. We and others have reported that the anticancer activity of ASNase requires the enzyme's glutaminase activity, but the underlying glutaminase-mediated mechanism(s) that lead to leukemia cell death are unknown. Glutamine, the most abundant amino acid in the blood, is known for pleiotropic roles in numerous biological pathways, including energy metabolism, redox metabolism, nucleotide anabolism, and amino acid anabolism. Many cancer cells have been found to reprogram their metabolic pathways to become highly dependent on glutamine for survival and proliferation. Glutaminase (GLS/GLS2)-mediated conversion of glutamine to glutamic acid provides the latter as a substrate for conversion to α‐ketoglutarate by transaminases or glutamate dehydrogenases (GLUD1/GLUD2) to fuel the TCA cycle. Consequently, targeting glutamine metabolism has become an attractive strategy for anticancer therapy. The enzyme asparagine synthetase (ASNS) mediates resistance to ASNase through synthesis of asparagine. ASNS is expressed in most cell types, and its expression is upregulated in response to a wide variety of cell stresses, including amino acid limitation and endoplasmic reticulum stress. We and others have shown that ASNS-positive leukemia cells capable of synthesizing asparagine de novo are less responsive than ASNS-negative leukemia cells to ASNase therapy (Chan et al., Blood, 2014). Moreover, ASNase resistance has been associated with elevated ASNS expression. In fact, we have shown that ASNS expression is a predictive marker of the in vitro response of leukemia cell lines and some solid tumor cell types to ASNase. The expression of ASNS in most cells in the body poses a serious challenge for successful therapy with ASNase; for example, production of asparagine by the liver and cells (e.g., mesenchymal stem cells and adipocytes) of the tumor microenvironment may contribute significantly to ASNase resistance in vivo. Here we used the high-glutaminase E. chrysanthemi ASNase (Erwinaze®), wild-type E. coli ASNase (ASNaseWT), and the glutaminase-deficient E. coli mutant, ASNaseQ59L, as models of high, medium-, and low-glutaminase, respectively, to explore ASNase glutaminase activity-mediated mechanisms of leukemia cell death. Unexpectedly, we found that increasing glutaminase activity caused an increase in the suppression of ASNS upregulation in vitro (Figure 1A). In NSG mice injected with luciferase-labeled Sup-B15 cells, single-agent ASNaseWT yielded a durable response approximating cure, whereas glutaminase-deficient ASNaseQ59L yielded a complete response but with recurrence. Together, the results suggest that ASNase glutaminase activity is associated with suppression of ASNS upregulation, making durable, single-agent anticancer activity easier to achieve. Overall, the results provide new insight into the mechanism of action of ASNase. Disclosures Konopleva: Stemline Therapeutics: Research Funding. Weinstein:NIH: Patents & Royalties: L-asparaginase. Lorenzi:Erytech Pharma: Consultancy; NIH: Patents & Royalties.

Phase I study of intravenous Seneca Valley virus (NTX-010), a replication competent oncolytic virus, in patients with neuroendocrine (NE) cancers

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 4629-4629 ◽  
Author(s):  
C. M. Rudin ◽  
N. Senzer ◽  
J. Stephenson ◽  
D. Loesch ◽  
K. Burroughs ◽  
...  
Keyword(s):  
Phase I ◽  
Phase I Study ◽  
Single Agent ◽  
Neutralizing Antibody ◽  
Response Assessment ◽  
Toxicity Assessment ◽  
Serum Markers ◽  
Small Cell ◽  
Viral Kinetics

4629 Background: NTX-010 is a naturally occurring replication competent picornavirus with potent and selective tropism for human NE tumors, including small cell cancers and carcinoid. NTX-010 elicts rapid cytolysis in vitro and durable responses following IV dosing in multiple xenograft models. Methods: A first-in-human phase I study of IV NTX-010 was conducted across 5 log-increment dose cohorts from 107 vp/kg to 1011 vp/kg, in patients with NE cancers. Study endpoints included toxicity assessment, response assessment, evaluation of viral titers and clearance in blood, sputum, nasal swabs, urine, and stool, and assessment of neutralizing antibody (Ab) development. Results: 30 patients were treated (6 small cell, 24 carcinoid-type). All small cell patients were heavily pretreated (> third line) and received 107 vp/kg. In these patients, median PFS was 1.2 months and median OS was 4.1 months, including 1 long term (16 month +) survivor with prolonged SD after progressing through prior therapies. Carcinoid patients in cohorts 1–4 have 70% SD rate and median PFS of 5.4 months (95% CI 3.6 to NE); median OS has not been reached. Cohort 5, a 12 patient expansion cohort at 1011 vp/kg restricted to carcinoid, is still being monitored and shows promising antitumor activity including improvement in carcinoid syndrome symptoms, decline in 5HIAA and other serum markers, minor responses by CT scan, and an objective PET response (>50% decrease in SUV). There were no DLTs in any cohort. Evidence of intratumoral viral replication includes delayed kinetics in serum viral titer, post-infusion serum titers greater then the dose administered and positive immunohistochemistry and/or RT-PCR signal for viral antigens in tumor mass despite Ab production. Viral clearance was documented in all subjects and correlated temporally with development of antiviral Ab. Conclusions: NTX-010 is the first picornavirus to be evaluated as an anticancer therapeutic. A single IV dose of 1011 vp/kg of NTX-010 is safe, has predictable viral kinetics, and shows promising activity against NE tumors. Phase II testing of this novel agent either as a single agent or in combination with standard cytotoxic therapies is warranted. [Table: see text]

scholarly journals Expression of Apoptosis-Regulating Proteins in Chronic Lymphocytic Leukemia: Correlations With In Vitro and In Vivo Chemoresponses

Blood ◽  
1998 ◽  
Vol 91 (9) ◽  
pp. 3379-3389 ◽  
Author(s):  
Shinichi Kitada ◽  
Janet Andersen ◽  
Sophie Akar ◽  
Juan M. Zapata ◽  
Shinichi Takayama ◽  
...  
Keyword(s):  
Cell Death ◽  
Chronic Lymphocytic Leukemia ◽  
Caspase 3 ◽  
Single Agent ◽  
White Blood Cells ◽  
Lymphocytic Leukemia ◽  
In Vitro Chemosensitivity ◽  
Apoptotic Protein

Abstract B-cell chronic lymphocytic leukemia (B-CLL) represents a neoplastic disorder caused primarily by defective programmed cell death (PCD), as opposed to increased cell proliferation. Defects in the PCD pathway also contribute to chemoresistance. The expression of several apoptosis-regulating proteins, including the Bcl-2 family proteins Bcl-2, Bcl-XL, Mcl-1, Bax, Bak, and BAD; the Bcl-2–binding protein BAG-1; and the cell death protease Caspase-3 (CPP32), was evaluated by immunoblotting using 58 peripheral blood B-CLL specimens from previously untreated patients. Expression of Bcl-2, Mcl-1, BAG-1, Bax, Bak, and Caspase-3 was commonly found in circulating B-CLL cells, whereas the Bcl-XL and BAD proteins were not present. Higher levels of the anti-apoptotic protein Mcl-1 were strongly correlated with failure to achieve complete remission (CR) after single-agent therapy (fludarabine or chlorambucil) (P = .001), but the presence of only seven CRs among the 42 patients for whom follow-up data were available necessitates cautious interpretation of these observations. Higher levels of the anti-apoptotic protein BAG-1 were also marginally associated with failure to achieve CR (P = .04). Apoptosis-regulating proteins were not associated with patient age, sex, Rai stage, platelet count, hemoglobin (Hb) concentration, or lymph node involvement, although higher levels of Bcl-2 and a high Bcl-2:Bax ratio were correlated with high numbers (>105/μL) of white blood cells (WBC) (P = .01; .007) and higher levels of Bak were weakly associated with loss of allelic heterozygosity at 13q14 (P = .04). On the basis of measurements of apoptosis induction by fludarabine using cultured B-CLL specimens, in vitro chemosensitivity data failed to correlate with in vivo clinical response rates (n = 42) and expression of the various apoptosis-regulating proteins. Although larger prospective studies are required before firm conclusions can be reached, these studies show the expression in B-CLLs of multiple apoptosis-regulating proteins and suggest that the relative levels of some of these, such as Mcl-1, may provide information about in vivo responses to chemotherapy. In vitro chemosensitivity data, however, do not appear to be particularly useful in predicting responses in B-CLL.

Adaphostin-Induced Apoptosis in CLL B-Cells Is Associated with Induction of Oxidative Stress and Exhibits Synergy with Fludarabine.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3482-3482 ◽  
Author(s):  
Tait D. Shanafelt ◽  
Y. Kit Lee ◽  
Nancy D. Bone ◽  
Ann K. Strege ◽  
Scott H. Kaufmann ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
B Cells ◽  
Alkylating Agents ◽  
Single Agent ◽  
Lymphocytic Leukemia ◽  
Parp Cleavage ◽  
Ros Generation ◽  
Mutation Status

Abstract Background : B-Chronic Lymphocytic leukemia (CLL) is the most common leukemia in North America. The standard current treatments use purine nucleoside analogues as single agent therapy or in combination with rituximab, steroids, and alkylating agents. Overall response rates with these treatments in previously untreated patients may reach 90 % with CR rates of 50–60%, however, most patients will relapse. Thus, despite refinements in therapy, CLL remains an incurable malignancy and there remains significant and urgent need to identify and develop new agents with novel mechanisms of action for the treatment of CLL. In line with this we have explored the mechanism and in vitro activity for a tyrphostin, adaphostin. Methods: We evaluated the in vitro efficacy of adaphostin to induce apoptosis in CLL B-cells. Peripheral blood was collected from patients with CLL (n=57). Highly purified CLL B-cells ((minimum 80% CD19+, mean 92.2% CD19+ and 94.4% CD19+/CD5+ positive)) were cultured with freshly prepared adaphostin for 24 – 120 hours. Cell death was analyzed by flow cytometry using Anexin V/Propidium iodide (PI). PARP cleavage and anti-apoptotic protein levels were measured by immunoblot techniques. The effect of combination treatment on CLL B cells with adaphostin and fludarabine on CLL B-cells was also assessed. Results: Analysis by Annexin V/PI staining revealed that the mean IC50 for adaphostin at 24 h was 4.2 uM (range 1.10 uM-11.25 uM; median = 4.25; n = 29) for CLL isolates and >10 uM for B and T-cells isolated from normal donors. Median IC50 levels for Adaphostin were not significantly different based on IgVH mutation status, level of CD38 expression, or cytogenetic abnormalities by FISH testing (Table 1). Immunoblots demonstrated adaphostin-induced PARP cleavage and cleavage of caspase 3 substrates, suggesting that adaphostin induces cell death through apoptosis. Adaphostin increased the intra-cellular level of reactive oxygen species (ROS) in CLL B-cells; and the antioxidant N-acetylcysteine blocked both adaphostin-induced ROS generation and apoptosis (mean reduction cell death=60%; range 23–99% reduction) suggesting generation of ROS is critical to adaphostin’s induction of apoptosis. Adaphostin also caused a decrease in the level of the anti-apoptotic proteins Bcl-2 in a majority of patients on both flow cytometry and immunoblots. When adaphostin was combined with fludarabine (F-ARA-ATP), a synergistic effect on cell death was observed in all 10 CLL samples when analyzed by mathematical modeling software. Summary: These findings indicate that adaphostin induces selective apoptosis in CLL B-cells from all risk categories through a mechanism that involves ROS generation. Importantly, we also demonstrate its ability to augment the effects of fludarabine. We continue to explore the preclinical development of adaphostin as a novel agent for the treatment of CLL. IC50 Adaphostin Dose Levels by Prognostic Groups FACTOR N Median IC 50 Adaphostin Range p-Value     Rai Stage Group Low/Int (0–II) 19 4.7 1.8 – 11.25 0.018 High (III – IV) 10 3.05 1.1 – 5.4 - IgVH Mutation Status Mutated 11 4.45 1.1 – 11.25 0.72 Nonmutated 10 3.93 1.8 – 5.8 - CD38 Status Negative 20 4.55 1.1 – 11.25 0.74 Positive 9 4.25 1.8 – 6.6 -     FISH defects 13q- 8 4.70 1.75 – 11.25 Normal 6 3.53 1.1 – 5.8 0.83 12+ 4 3.93 2.25 – 6.6 17p/11q 5 4.20 2.0 – 5.4

scholarly journals Expression of Apoptosis-Regulating Proteins in Chronic Lymphocytic Leukemia: Correlations With In Vitro and In Vivo Chemoresponses

Blood ◽  
1998 ◽  
Vol 91 (9) ◽  
pp. 3379-3389 ◽  
Author(s):  
Shinichi Kitada ◽  
Janet Andersen ◽  
Sophie Akar ◽  
Juan M. Zapata ◽  
Shinichi Takayama ◽  
...  
Keyword(s):  
Cell Death ◽  
Chronic Lymphocytic Leukemia ◽  
Caspase 3 ◽  
Single Agent ◽  
White Blood Cells ◽  
Lymphocytic Leukemia ◽  
In Vitro Chemosensitivity ◽  
Apoptotic Protein

B-cell chronic lymphocytic leukemia (B-CLL) represents a neoplastic disorder caused primarily by defective programmed cell death (PCD), as opposed to increased cell proliferation. Defects in the PCD pathway also contribute to chemoresistance. The expression of several apoptosis-regulating proteins, including the Bcl-2 family proteins Bcl-2, Bcl-XL, Mcl-1, Bax, Bak, and BAD; the Bcl-2–binding protein BAG-1; and the cell death protease Caspase-3 (CPP32), was evaluated by immunoblotting using 58 peripheral blood B-CLL specimens from previously untreated patients. Expression of Bcl-2, Mcl-1, BAG-1, Bax, Bak, and Caspase-3 was commonly found in circulating B-CLL cells, whereas the Bcl-XL and BAD proteins were not present. Higher levels of the anti-apoptotic protein Mcl-1 were strongly correlated with failure to achieve complete remission (CR) after single-agent therapy (fludarabine or chlorambucil) (P = .001), but the presence of only seven CRs among the 42 patients for whom follow-up data were available necessitates cautious interpretation of these observations. Higher levels of the anti-apoptotic protein BAG-1 were also marginally associated with failure to achieve CR (P = .04). Apoptosis-regulating proteins were not associated with patient age, sex, Rai stage, platelet count, hemoglobin (Hb) concentration, or lymph node involvement, although higher levels of Bcl-2 and a high Bcl-2:Bax ratio were correlated with high numbers (>105/μL) of white blood cells (WBC) (P = .01; .007) and higher levels of Bak were weakly associated with loss of allelic heterozygosity at 13q14 (P = .04). On the basis of measurements of apoptosis induction by fludarabine using cultured B-CLL specimens, in vitro chemosensitivity data failed to correlate with in vivo clinical response rates (n = 42) and expression of the various apoptosis-regulating proteins. Although larger prospective studies are required before firm conclusions can be reached, these studies show the expression in B-CLLs of multiple apoptosis-regulating proteins and suggest that the relative levels of some of these, such as Mcl-1, may provide information about in vivo responses to chemotherapy. In vitro chemosensitivity data, however, do not appear to be particularly useful in predicting responses in B-CLL.

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