Pre-Clinical In Vitro Evaluation of BCL-2 Antisense Compound GenasenseTM (G3139; Genta, Inc.) as a Targeted Pro-Apoptotic Agent for Leukemias with t(4;11)(q21;q23) Translocations.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3360-3360 ◽  
Author(s):  
Blaine W. Robinson ◽  
Kathyrn C. Behling ◽  
Jeffrey S. Barrett ◽  
Manish Gupta ◽  
Jessicca M. Rege ◽  
...  
Keyword(s):  
Response Surface ◽  
Cell Lines ◽  
Caspase 3 ◽  
Single Agent ◽  
Chemotherapy Resistance ◽  
Surface Modeling ◽  
Clinical Activity ◽  
Response Surface Modeling ◽  
Cd34 Cells

Abstract Background: Chemotherapy resistance from reduced apoptosis may contribute to poor outcome in infant leukemias with t(4;11). G3139 has clinical activity against refractory adult leukemias with minimal toxicity. Methods: We quantified anti-apoptotic BCL-2 and pro-apoptotic BAX mRNAs in 56 leukemias cases (55/56 from infants; 41 ALL, 15 AML; 24 t(4;11), 32 non-MLL), the ALL cell lines RS4:11 and SEM-K2, the AML cell line MV4-11, all of which have t(4;11), and normal CD34+ cells. BCL-2 and BAX mRNA levels were normalized to β-actin and examined by the comparative CT method. The in vitro cytotoxicity and pro-apoptotic activity of G3139 was investigated in the cell lines. Cytotoxicity was assessed by MTT after exposing log phase cells for 6 days to single agent G3139, or to fixed-concentration G3139 combined with anti-leukemia cytotoxic drugs over a range of concentrations; transfection reagent was not used for G3139 delivery. Pharmacostatistical response surface modeling was performed to determine synergy. Apoptosis was assessed by flow cytometry analysis of Caspase-3 activation and a FACS TUNEL assay. Results: BCL-2 and BAX mRNAs were abundant in the leukemias and cell lines with t(4;11), whereas CD34+ cells showed lower BCL-2 expression. The difference in normalized expression ratios of BCL-2:BAX relative to CD34+ cells approached significance when t(4;11) leukemias and cell lines were compared to non-MLL leukemias (p=0.07). The approximate IC50 of single agent G3139 was 10 μM in RS4:11 cells, 100 μM in MV4-11, and 180 μM in SEM-K2. Low, biologically achievable G3139 concentrations (5 μM, 1 μM) sensitized RS4:11 cells to the cytotoxicity of doxorubicin, 6-thioguanine, etoposide and cytosine arabinoside, but not methotrexate or dexamethasone. Despite the higher IC50 of G3139 alone in MV4-11 cells, synergy also was suggested when G3139 (50 μM, 10 μM) was combined with etoposide and 6-thioguanine since response surface modeling showed ability to achieve lower effective doses than projected from either single agent. When RS4:11 cells were assayed for proof of principle that the observed cytotoxicity was due to apoptosis, exposure to G3139 and doxorubicin together increased active Caspase-3 and TUNEL staining in a time and dose dependent manner. Conclusions: The imbalanced BCL-2/BAX expression suggests that an anti-apoptotic genotype forms the basis for the chemotherapy resistance in infant leukemias with t(4;11). These in vitro studies indicate that G3139 has pre-clinical activity, that select G3139-cytotoxic agent combinations are synergistic against cell lines with t(4;11), and that the observed activity occurs through apoptosis. Further studies are warranted to determine the pre-clinical in vivo effects of similar combinations against leukemias with t(4;11), with the goal of advancement to the clinic if results are promising.

Pan-BCL-2 Family Small Molecule Inhibitor GX015-070 (GeminX, Inc.) Exhibits Single Agent Cytotoxicity, Is Synergistic with Select Anti-Leukemia Cytotoxic Agents, and Induces Apoptosis in Cell Lines with t(4;11)(q21;q23) Translocations.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3368-3368 ◽  
Author(s):  
Jessicca M. Rege ◽  
Blaine W. Robinson ◽  
Manish Gupta ◽  
Jeffrey S. Barrett ◽  
Peter C. Adamson ◽  
...  
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Family Member ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Caspase 3 ◽  
Single Agent ◽  
Cytotoxic Agents ◽  
Response Surface Modeling

Abstract Background: Leukemias with MLL translocations, especially t(4;11), often are resistant to common chemotherapeutic agents, which may be due to abnormal apoptosis regulation. Pro- and anti-apoptotic BCL-2 family member interactions govern initiation of the intrinsic apoptosis pathway. GX015-070, which currently is in Phase I/IIA clinical trials, mimics the BH3 domain on pro-apoptotic BCL-2 family proteins and can bind the BH3 binding pocket of anti-apoptotic BCL-2 family members and modulate apoptosis. We performed comprehensive protein expression profiling of BCL-2 family member proteins and evaluated in vitro activity and mechanism of action of GX015-070 in cell lines with t(4;11). Methods: Baseline expression of BCL-2 family proteins was determined by Western blot analysis. Cytotoxicity was assessed by MTT after a 3 day exposure of RS4:11, SEM-K2 and MV4-11 cells in log phase growth to single agent GX015-070 at concentrations from 5 nM to 7.5 μM. Combined effects of fixed-concentration GX015-070 with cytotoxic agents over a range of concentrations were assayed by MTT, and the results were analyzed by pharmacostatistical response surface modeling. Disruption of specific pro- and anti-apoptotic BCL-2 family member interactions was investigated by co-immunoprecipitation/Western blot analysis. Flow cytometry and/or Western blot analysis of Caspase-3 activation, and a FACS TUNEL assay, were used to assess apoptosis in GX015-070 treated and untreated cells. Results: The three cell lines had similar baseline levels of expression of BCL-2 family proteins. BCL-2 and BAX were most abundant followed by PUMA, BAK, BCL-XL, BIM-EL, MCL-1, BIK and NOXA. Results of assays of GX015-070 activity and mechanism of action are in shown in the table. Conclusions: These data indicate that GX015-070 has potent cytotoxic activity in cell lines with t(4;11) as a single agent and that the cytotoxicity results from apoptosis. Response surface modeling in RS4:11 cells suggested ability to achieve effective doses with GX015-070 combined with cytosine arabinoside (Ara-C), dexamethasone (Dex) or doxorubicin (ADR) that are lower than projected from the single agents, but synergy was not suggested when GX015-070 was combined with etoposide, methotrexate or 6-thioguanine. The co-IP experiments give proof of principle that GX015-070 disrupts pro- and anti-apoptotic BCL-2 family protein interactions in cell lines with t(4;11). Additional pre-clinical experiments directed at overcoming drug resistance from abnormal cell death regulation in leukemias with t(4;11) using GX015-070 are in progress. These studies provide a framework to understand the cell death/survival machinery in primary leukemias with t(4;11) translocations more completely and manipulate that machinery to achieve better treatments. GX015-070 Activity and Mechanism Cell Line Single Agent Activity Synergy Inhibition Caspase-3 Activation TUNEL RS4:11 IC50=43.5 nM Ara-C, Dex, ADR Mcl1:Bak; Bcl2:Bak + + SEM-K2 IC50=156 nM In progress Mcl1:Bak; Bcl2:Bak + In Progress MV4-11 IC50=123 nM In progress Mcl1:Bak In progress +

Combination of Sorafenib, Idarubicin, and Cytarabine Has a High Response Rate in Patients with Newly Diagnosed Acute Myeloid Leukemia (AML) Younger Than 65 Years

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 768-768 ◽  
Author(s):  
Farhad Ravandi ◽  
Jorge Cortes ◽  
Stefan Faderl ◽  
Guillermo Garcia-Manero ◽  
Susan O’Brien ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Phase I ◽  
Cell Lines ◽  
Phase Ii ◽  
Kinase Inhibitor ◽  
Single Agent ◽  
High Response Rate ◽  
Clinical Activity ◽  
Mutation Burden

Abstract Background: Sorafenib is an orally active multi-kinase inhibitor with potent activity against the Raf/ERK/MEK pathway, VEGFR, PDGFR-β, and c-KIT. In vitro, it has growth-inhibitory effects in several AML cell lines with or without constitutive activation of ERK signaling. Sorafenib selectively induces cell growth arrest and apoptosis in FLT3-mutant human AML cell lines at nM concentrations. In a phase I study of single agent sorafenib in patients (pts) with AML escalating doses were well tolerated with no myelosuppression and with significant clinical activity predominantly (but not exclusively) in FLT3 mutated pts. Methods: This study was conducted to determine the tolerability and efficacy of combination of sorafenib with cytarabine 1.5 g/m2 iv over 24 hours daily × 4 (× 3 for pts over 60) and idarubicin 12 mg/m2 iv daily × 3. In the phase I portion of study, pts with relapsed AML were treated with escalating doses of sorafenib po (400 mg qod, 400 mg daily and 400 mg bid) for 7 days during induction, and 400 mg bid was established as a safe dose for phase II evaluation. Pts achieving CR receive up to 5 courses of consolidation with idarubicin 8 mg/m2 iv daily × 2 and cytarabine 0.75 g/m2 iv daily × 3 in addition to continuous sorafenib 400 mg po bid for up to 28 days per cycle. Maintenance with sorafenib 400 mg bid would continue for up to a year after consolidation. Results: Ten pts (median age 34 years, range 21–58) with relapsed AML (median prior therapy 2, range 1–6) were treated on the phase I portion. Seven had FLT3-ITD mutation (5 with high mutation burden, 2 with low), and 3 were negative. Four achieved CR, and 6 failed. In the phase II portion, 30 pts (including 8 with FLT3-ITD and 2 with FLT3-TKD) have been treated. Median age is 53 years (range 18 – 65) Cytogenetics were diploid in 13, +8 in 3, −5/−7 in 3, t(9;11) in 1, miscellaneous in 6, and unavailable in 4. The median presentation WBC was 4.6 × 109/L (range 1.5 –122.7 × 109/L). FLT3 mutation burden was low in blasts from 4 pts, and high in 6). Five pts were FLT3-ITD+/NPM1-. Among 25 evaluable pts, 22 (88%) have achieved CR (n=19), or CRi (n=3); 1 achieved PR, 1 died at induction from pneumonia, 1 was resistant; 5 pts are too early. The regimen is well tolerated and grade 3 adverse events thought to be possibly related to the study combination have included elevation of transaminases (3), hyperbilirubinemia (4), small bowel obstruction (1), diarrhea (2), rash (2), pericarditis (1), elevated creatinine (1), and atrial fibrillation (1). Median follow-up is 8 weeks (range, 1 – 28) with the probability of survival at 6 months of 87%; 2 pts have relapsed with CR durations of 2 and 3 months. Samples from 8 pts were studied prior to and 24–48 hours post sorafenib administration, and prior to chemotherapy. In six pts (75%), sorafenib alone induced apoptosis in peripheral blood blasts and in CD33/CD34 positive leukemia progenitor cells as determined by flow cytometry. Expression of phospho-ERK (pERK) was detectable by flow cytometry in 5 out of 7 samples tested at baseline; 24-hour exposure to sorafenib resulted in >50% downregulation of pERK in 3 of the 5 samples. Plasma inhibitory assay was performed using day 7 samples from 10 pts; mutant FLT3 was suppressed by all samples with 5-fold more potent suppression against mutant versus wildtype FLT3. Conclusions: Combination of sorafenib with idarubicin and cytarabine is safe and has a high CR rate in frontline therapy of younger pts with AML. Correlative studies confirm potent activity of sorafenib against ERK and FLT3 signaling.

scholarly journals Comparison of Fractional Inhibitory Concentration Index with Response Surface Modeling for Characterization of In Vitro Interaction of Antifungals against Itraconazole-Susceptible and -Resistant Aspergillus fumigatus Isolates

2002 ◽  
Vol 46 (3) ◽  
pp. 702-707 ◽  
Author(s):  
D. T. A. Te Dorsthorst ◽  
P. E. Verweij ◽  
J. F. G. M. Meis ◽  
N. C. Punt ◽  
J. W. Mouton
Keyword(s):  
Aspergillus Fumigatus ◽  
Response Surface ◽  
Filamentous Fungi ◽  
Inhibitory Concentration ◽  
Surface Modeling ◽  
Drug Combinations ◽  
Surface Model ◽  
Response Surface Modeling ◽  
Fractional Inhibitory Concentration

ABSTRACT Although the fractional inhibitory concentration (FIC) index is most frequently used to define or to describe drug interactions, it has some important disadvantages when used for drugs against filamentous fungi. This includes observer bias in the determination of the MIC and no agreement on the endpoints (MIC-0, MIC-1, or MIC-2 [≥95, ≥75, and ≥50% growth inhibition, respectively]) when studying drug combinations. Furthermore, statistical analysis and comparisons are troublesome. The use of a spectrophotometric method to determine the effect of drug combinations yields quantitative data and permits the use of model fits to the whole response surface. We applied the response surface model described by Greco et al. (W. R. Greco, G. Bravo, and J. C. Parsons, Pharmacol. Rev. 47:331-385, 1995) to determine the interaction coefficient alpha (ICα) using a program developed for that purpose and compared the results with FIC indices. The susceptibilities of amphotericin B (AM), itraconazole (IT), and terbinafine (TB) were tested either alone or in combination against 10 IT-susceptible (IT-S) and 5 IT-resistant (IT-R) clinical strains of Aspergillus fumigatus using a modified checkerboard microdilution method that employs the dye MTT [3-(4,5-dimethyl-2-thiazyl)2,5-diphenyl-2H-tetrazolium bromide]. Growth in each well was determined by a spectrophotometer. FIC indices were determined and ICα values were estimated for each organism strain combination, and the latter included error estimates. Depending on the MIC endpoint used, the FIC index ranged from 1.016 to 2.077 for AM-IT, from 0.544 to 1.767 for AM-TB, and from 0.656 to 0.740 for IT-TB for the IT-S strains. For the IT-R strains the FIC index ranged from 0.308 to 1.767 for AM-IT, from 0.512 to 1.646 for AM-TB, and from 0.403 to 0.497 for IT-TB. The results indicate that the degree of interaction is not only determined by the agents themselves but also by the choice of the endpoint. Estimates of the ICα values showed more consistent results. Although the absolute FIC indices were difficult to interpret, there was a good correlation with the results obtained using the ICα values. The combination of AM with either IT or TB was antagonistic in vitro, whereas the combination of IT and TB was synergistic in vitro for both IT-S and IT-R strains. The use of response surface modeling to determine the interaction of drugs against filamentous fungi is promising, and more consistent results are obtained by this method than by using FIC indices.

The Hsp90 Inhibitor SNX7081 Restores the Fludarabine Sensitivity of Chronic Lymphocytic Leukemia (CLL) Cells Harbouring Mutations of ATM or TP53

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2473-2473
Author(s):  
O. Giles Best ◽  
Stephen P Mulligan
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Cell Lines ◽  
Caspase 3 ◽  
Mononuclear Cells ◽  
Single Agent ◽  
Treatment Strategies ◽  
Lymphocytic Leukemia ◽  
Functional Assay ◽  
Simultaneous Exposure

Abstract Abstract 2473 Introduction: Resistance to fludarabine-based treatment represents a challenge in the clinical management of Chronic Lymphocytic Leukemia (CLL). Despite the unprecedented response rates seen with the fludarabine (F), cyclophosphamide (C), Rituximab (R) regimen novel treatment strategies are required that do not rely on an intact p53 signaling pathway. We recently described the activity of a novel, synthetic inhibitor of the molecular chaperone, heat-shock protein 90 (Hsp90) named SNX7081 (Serenex, now Pfizer) against CLL cells in vitro (Best et al., 2010 BJH). Here we explored the effect of this inhibitor on the fludarabine sensitivity of 3 haematological cell lines and 12 patient samples with mutations of ATM or TP53. Methods: Mononuclear cells were isolated by density centrifugation from CLL patients following informed consent. The 13 patient samples selected for study were determined to have mutations of either ATM or TP53 using a functional assay described in detail elsewhere (Best et al., 2008). The Mec1 (CLL), Mec2 (CLL) and U266 (B-ALL) cell lines were maintained under standard conditions in RPMI-1640 with 2mM L-glut and 1% pen/strep. Sensitivity to fludarabine, with and without SNX7081, was assessed using the MTT (3–4, 5-dimethylthiazol-2,5-diphenyl tetrazolium bromide) assay. Synergy between the agents, activation of caspase-3 and the induction of double stranded DNA (dsDNA) breaks following treatment were all assessed by flow cytometry using the mitochondrial membrane potential dye DilC1 (5) and propidium iodide (PI) or appropriate antibodies. Results: The IC50 for fludarabine was significantly higher in the 3 cell lines and 13 patient samples with ATM/TP53 lesions than in 4 cell lines or 10 patient samples defined as ATM/TP53 wild-type. Simultaneous exposure to a combination of fludarabine and SNX7081 at a ratio based on the IC50 of the compounds as single agent significantly reduced the IC50 for fludarabine (P<0.01); in 11 patient samples the IC50 for fludarabine was reduced to within a clinically achievable range (<5μM). Synergy between fludarabine and SNX7081 was evident as an effect on the distribution of the cell lines in the cell cycle and as a marked effect on the proportion of apoptotic cells (DilC (1)5 negative/PI negative) in cultures of both the cell lines and patient samples. Furthermore, we show that the combination of the compounds has a greater than additive effect on the activation of caspase-3 and on the formation of dsDNA breaks, as evidenced by the phosphorylation of g-H2Ax. Conclusions: Our studies suggest that inhibition of Hsp90 may overcome fludarabine resistance conferred by mutations of ATM or TP53. The mechanism of the synergy between these compounds appears to be via augmentation of fludarabine-induced dsDNA breaks and is concomitant with an increase in caspase-3 signaling. The data suggest that this combination may represent a promising regimen in the treatment of fludarabine-refractory CLL. Disclosures: Mulligan: Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer Schering, now Genzyme: Honoraria.

scholarly journals SL-401, a Targeted Therapy Directed to the Interleukin-3 Receptor (CD123), and SL-801, a Reversible Inhibitor of Exportin-1 (XPO1), Display Synergistic Anti-Tumor Activity Against Hematologic Malignancies in Vitro

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4724-4724 ◽  
Author(s):  
John Gionco ◽  
Janice Chen ◽  
Ross Lindsay ◽  
Vince Macri ◽  
Christopher L. Brooks
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Single Agent ◽  
Hematologic Malignancies ◽  
Clinical Activity ◽  
Employment Equity ◽  
Equity Ownership ◽  
Rpmi 8226 ◽  
Anti Tumor Activity

Abstract Background: Novel combination therapies have shown success in combating tumor heterogeneity and drug resistance. SL-401 is a targeted therapy directed to the interleukin-3 receptor (CD123), which is overexpressed on numerous hematologic malignancies. SL-401 has demonstrated high single agent response rates in an ongoing Phase 2 trial of blastic plasmacytoid dendritic cell neoplasm (BPDCN) and is also being evaluated in the clinic for additional cancers, including acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPNs) as a single agent, and multiple myeloma (MM) in combination with other agents. While SL-401 has demonstrated robust single agent clinical activity in patients with BPDCN, its unique mechanism of action and non-overlapping side effect profile with other agents may lend itself to combination therapy as well. Another class of drugs that has demonstrated clinical activity against several hematologic and solid malignancies is Exportin-1 (XPO1) inhibitors. SL-801 is a novel oral small molecule that reversibly inhibits XPO1 and has shown potent in vitro and in vivo anti-tumor activity against a broad range of hematologic and solid malignancies. SL-801 is currently being evaluated in a Phase 1 trial of patients with advanced solid tumors, and a Phase 1 trial in advanced hematologic cancers is planned. Here, we investigated the in vitro effect of combination treatment of SL-401 and SL-801 against cell lines of chronic myeloid leukemia (CML), AML, MM, and Hodgkin's lymphoma (HL). Methods: The human K562 CML cell line, MV4-11 AML cell line, RPMI-8226 MM cell line, and L-428 HL cell line were treated with varying concentrations of SL-401 and SL-801 alone or in combination for 48 hours. Cell viability was assessed by the CellTiter Glo in vitro cytotoxicity assay. Combination index (CI) values were calculated using CompuSyn software by the method of Chou and Talalay, and treatment was considered to be synergistic when CI < 1. Caspase activation was measured using the Caspase-Glo 3/7 assay, and lactate dehydrogenase (LDH) release was measured using the CytoTox 96 Non-Radioactive Cytotoxicity Assay. Results: As single agents, SL-401 and SL-801 demonstrated anti-tumor activity in all four cell lines tested. MV4-11 cells were the most sensitive to both drugs, with an IC50 of 34 pM for SL-401 and 21 nM for SL-801. In the other cell lines, the IC50s for SL-401 were 17 nM in K562 cells, 25 nM in RPMI-8226 cells, and 100 nM in L-428 cells, and the IC50s for SL-801 were 99 nM in K562 cells, 51 nM in RPMI-8226 cells, and 494 nM in L-428 cells. When combined with each other, SL-401 and SL-801 potently inhibited cell growth in all cell lines, and CI calculations indicated that the interaction between the two drugs was synergistic at most dose combinations. Notably, CI values < 0.3 were observed in MV4-11 and L-428 cells, indicative of strong synergy. Consistent with these observations, the combination of SL-401 and SL-801 also induced higher levels of caspase activation and LDH release in MV4-11 and L-428 cells than either drug alone. Conclusion: These findings demonstrate that SL-401 and SL-801, when combined, act synergistically in their in vitro anti-tumor activity against CML, AML, MM, and HL cells. Investigations into the molecular mechanisms underlying the observed synergy are in progress. These promising results provide rationale for further development of SL-401 and SL-801 combination therapy in the treatment of a broad range of hematologic malignancies. Disclosures Gionco: Stemline Therapeutics, Inc.: Employment. Chen:Stemline Therapeutics, Inc.: Employment, Equity Ownership. Lindsay:Stemline Therapeutics, Inc.: Employment, Equity Ownership. Macri:Stemline Therapeutics, Inc.: Employment, Equity Ownership. Brooks:Stemline Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties.

scholarly journals In Vitro Synergistic Interaction between Amphotericin B and Pentamidine against Scedosporium prolificans

2002 ◽  
Vol 46 (10) ◽  
pp. 3323-3326 ◽  
Author(s):  
Javier Afeltra ◽  
Eric Dannaoui ◽  
Jacques F. G. M. Meis ◽  
Juan L. Rodriguez-Tudela ◽  
Paul E. Verweij
Keyword(s):  
Response Surface ◽  
Amphotericin B ◽  
Clinical Laboratory ◽  
Geometric Mean ◽  
Surface Modeling ◽  
National Committee ◽  
Response Surface Modeling ◽  
Microdilution Method ◽  
Scedosporium Prolificans

ABSTRACT To develop new approaches for the treatment of invasive infections caused by Scedosporium prolificans, the in vitro interaction between amphotericin B and pentamidine against 30 clinical isolates was evaluated using a checkerboard microdilution method based on the National Committee for Clinical Laboratory Standards M38-P guidelines. The interaction between the drugs was analyzed using fractional inhibitory concentration index (FICI) analysis and response surface modeling. Amphotericin B alone was inactive against all the isolates. The geometric mean MIC for pentamidine was 57 μg/ml (range, 8 to 256 μg/ml; MIC at which 50% of the isolates tested were inhibited [MIC50], 64 μg/ml; MIC90, 128 μg/ml). The combination was synergistic against 28 of 30 isolates (93.3%) by FICI analysis and 30 of 30 (100%) by response surface modeling analysis. Antagonism was not observed.

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