Tipifarnib Is Cytotoxic and a Drug Transporter Inhibiting Chemosensitizer in Hodgkin Lymphoma Tumor Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1386-1386
Author(s):  
Boris Boll ◽  
Anna Lang ◽  
Peer Langendorf ◽  
Hinrich P. Hansen ◽  
Elke P. von Strandmann ◽  
...  
Keyword(s):  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Single Agent ◽  
Annexin V ◽  
Hematologic Malignancies ◽  
Clinical Activity ◽  
Drug Transporter ◽  
Toxicity Profile ◽  
Combination Drug ◽  
Index Method

Abstract INTRODUCTION: Patients with Hodgkin lymphoma (HL) still suffer from late toxicities and insufficient treatment of relapses. New therapies should therefore be established. Tipifarnib is a farnesyl transferase inhibitor with an excellent toxicity profile and clinical activity in hematologic malignancies. Recently, tipifarnib has also been shown to potentiate the cytotoxicity of anthracyclines in leukemia cells via the inhibition of the multidrug resistance transporter P-glycoprotein. To date, nothing is known about a functional role of drug resistance transporters or the effects of tipifarnib in HL. METHODS: To test the anti-tumor effects of tipifarnib in HL cell lines, tipifarnib was evaluated in the XTT cytotoxicity assay and the Annexin V binding assay. The combination efficacy of tipifarnib with the anthracycline doxorubicine was monitored using the Chou and Talalay combination index method. Flow cytometry was applied to assess the effects of tipifarnib on drug transporter mediated anthracycline efflux. RESULTS: First, tipifarnib displayed high single agent toxicity in HL cell lines with an average IC50 of < 0.1 μM. Second, the combination of tipifarnib and doxorubicine was highly synergistic at clinically relevant concentrations (1–3 μM and 0.02–0.2 μM, respectively). Third, measurement of residual doxorubicine after incubation with doxorubicine and tipifarnib indicated a strong inhibition of doxorubicine efflux by tipifarnib suggesting a mechanism for the synergy of the two drugs. CONCLUSIONS: Since tipifarnib displays high activity in a panel of HL cell lines, the clinical evaluation of tipifarnib in relapsed/refractory patients as single agent is warranted. Interestingly, tipifarnib exhibits a dual targeting mechanism in HL cells: Potent cytotoxicity as single agent and drug transporter dependent chemosensitization leading to a strong synergy with doxorubicine. The synergistic combinations of tipifarnib with doxorubicine correspond to plasma levels of the two drugs in cancer patients. Doxorubicine is one of the most effective but also most toxic drugs in the standard HL polychemotherapies whereas tipifarnib has a very favourable toxicity profile. Consequently, tipifarnib should be evaluated as a combination drug for HL polychemotherapy to save on doxorubicine dose and to lower acute and long-term toxicities. Tipifarnib might also be examined as a chemosensitizer for the combination therapy of patients with refractory disease.

Upregulated JAK/STAT Signaling Represents a Major Mode of Resistance to HDAC Inhibition In Lymphoma and Provides a Rationale for Novel Combination Therapy

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 434-434 ◽  
Author(s):  
Jason Smith ◽  
Katherine J. Walsh ◽  
Cassandra L Jacobs ◽  
Qingquan Liu ◽  
Siyao Fan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Single Agent ◽  
Acquired Resistance ◽  
Hematologic Malignancies ◽  
Cross Resistance ◽  
Clinical Activity ◽  
Molecular Pathways ◽  
Major Mode ◽  
High Degree

Abstract Abstract 434 Background Histone deacetylase inhibitors (HDACis) have demonstrated significant clinical activity in hematologic malignancies; however, single agent response rates have ranged between 20–50% with the duration of response often measured in months, suggesting that drug resistance is a major mode of failure. The pathways through which these agents work and the means by which tumors develop resistance to them are poorly understood. Combination therapy targeting multiple oncogenic pathways holds the promise to improve upon both the depth and durability of these responses. We investigated the mechanisms of inherent and acquired resistance to HDACis in a broad range of lymphomas. By detailing the molecular pathways implicated in resistance to HDACi, we sought to identify novel combinations of compounds that could overcome potential mechanisms that confer resistance. Methods and Results We tested two separate HDACis, LBH589 and SAHA in 51 cell lines representing a wide range of lymphomas including Burkitt lymphoma, diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma, and Hodgkin lymphoma. Gene expression array data was generated for all these cell lines. We then identified genes that were significantly associated with resistance to both LBH589 and SAHA (p<.01) and applied hierarchical clustering to identify the functional significance of these genes. Histology was not predictive of sensitivity to either HDACi. These data were then analyzed using gene set enrichment to identify known molecular pathways associated with resistance. Activation of JAK/STAT signaling was found to be a major determinant of resistance among the cell lines that were relatively resistant to HDACi. (P<0.001, FDR <.25). To determine whether these genes that we found to be associated with resistance reflected potential mechanisms of acquired resistance to HDACi therapy, we separately engineered resistance to LBH589 and SAHA in three DLBCL cell lines (LY3, BJAB, Farage) through incremental dose escalation over a period of up to 6 months. Each of these three cell lines demonstrated sustained growth at drug concentrations that were at or above their original IC50. Each of these cell lines were then exposed to the other HDACi and tested for cross resistance. In each case, the cell lines demonstrated complete cross-resistance to the other drug. We then profiled the gene expression of these cell lines that had acquired resistance. Similar to our previous results, these cell lines demonstrated increased signaling through the JAK/STAT pathway, suggesting that mechanisms of inherent and acquired resistance are similar. We therefore reasoned that combining HDAC and JAK inhibition may overcome both inherent and acquired resistance. To investigate this hypothesis, we tested LBH589 and INCB018424, a JAK1/2 inhibitor, alone and in combination in the LY3, TMD-8, U2932, and BJAB cell lines. While INCB018424 demonstrated no single agent cytotoxicity, it yielded a high degree of synergy when combined with LBH589 with the combination index computed by the Chou-Talalay method ranging from .19 to .9. Conclusion HDACis show single agent activity in the treatment of a number of hematologic malignancies, however most patients develop resistance to these drugs after relatively short-lived remissions. Thus, the greatest promise of these drugs may lie in combination with other agents that target molecular pathways that underlie resistance to these drugs. Using gene expression profiling of a broad range of tumor types and sensitivity to HDACis we were able to identify activation of the JAK/STAT pathway as a common feature of inherent and acquired resistance to HDACis. We combined the JAK1/2 inhibitor INCB018424 with LBH589 and demonstrated a high degree of synergy. As the number of small molecule inhibitors with clinical activity increases, the need to identify rational preclinical combinations becomes greater. Pairing gene expression profiling and resistant cell lines is a promising approach to the selection of combinations likely to maximize clinical benefit while limiting toxicity. Disclosures: No relevant conflicts of interest to declare.

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 2193

2017 ◽  
Vol 1 (S1) ◽  
pp. 58-59
Author(s):  
Houda Alachkar ◽  
Martin Mutonga ◽  
Amanda de Albuquerque ◽  
Rucha Deo ◽  
Gregory Malnassy ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Leucine Zipper ◽  
Lymphoblastic Leukemia ◽  
Annexin V ◽  
Hematologic Malignancies ◽  
Cytotoxic Activities ◽  
Knock Down ◽  
Protein Levels

OBJECTIVES/SPECIFIC AIMS: Unlike the high cure rates (90%) of children with acute lymphoblastic leukemia (ALL), that of adults is still lagging behind and better therapies are needed. Maternal embryonic leucine-zipper kinase (MELK) is aberrantly upregulated in cancer, and implicated in cancer stem cell survival. A recent study has identified FOXM1, a MELK substrate, as a therapeutic target in B cell ALL (B-ALL). Thus, we hypothesized that MELK may act as a therapeutic target in ALL via targeting FOXM1 activity. METHODS/STUDY POPULATION: Western blot and qPCR were used to assess MELK expression in 14 ALL cell lines. Knock-down and kinase inhibition approaches targeting MELK expression and function, followed by CCK-8 and Annexin V (flow cytometry) assays to measure cell viability and apoptosis, respectively. RESULTS/ANTICIPATED RESULTS: MELK was significantly upregulated in patients with ALL (oncomine data analysis). MELK was also significantly higher in B-ALL and T-ALL cell lines compared with that in blood cells of healthy donors. MELK knock-down significantly decreased cell viability (40%–70%, p<0.05, Fig. 1) in ALL cells, and induced apoptosis (~40%). OTS167, a potent MELK inhibitor exhibited cytotoxic activities in both B and T-ALL cells. The IC50 of OTS167 ranged from 20 to 60 nM; we also found a significant increase in apoptosis (p<0.05). Mechanistically, MELK inhibition resulted in decrease of FOXM1 protein levels 3 hours post-treatment. DISCUSSION/SIGNIFICANCE OF IMPACT: MELK is highly expressed in ALL and represents a novel therapeutic target likely via modulating FOXM1 activity. Functional and mechanistic studies will complement and ensure the success of the undergoing Phase I/II clinical trial of OTS167 in patients with refractory or relapsed AML, ALL, and other advanced hematologic malignancies.

scholarly journals MicroRNA High Throughput Loss-of-Function Screening Reveals an Oncogenic Role for miR-21-5p in Hodgkin Lymphoma

2018 ◽  
Vol 49 (1) ◽  
pp. 144-159 ◽  
Author(s):  
Ye Yuan ◽  
Fubiao Niu ◽  
Ilja M. Nolte ◽  
Jasper Koerts ◽  
Debora de Jong ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Growth ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Western Blotting ◽  
High Throughput ◽  
Annexin V ◽  
Loss Of Function ◽  
Empty Vector ◽  
Competition Assays

Background/Aims: Classical Hodgkin lymphoma (cHL) is among the most frequent lymphoma subtypes. The tumor cells originate from crippled germinal center (GC)-B cells that escaped from apoptosis. MicroRNAs (miRNAs) play important roles in B-cell maturation and aberrant expression of miRNAs contributes to the pathogenesis of cHL. Our aim was to identify oncogenic miRNAs relevant for growth of cHL using a high-throughput screening approach. Methods: A lentiviral pool of 63 miRNA inhibition constructs was used to identify miRNAs essential to cell growth in three cHL cell lines in duplicate. As a negative control we also infected cHL cell lines with a lentiviral barcoded empty vector pool consisting of 222 constructs. The abundance of individual constructs was followed over time by a next generation sequencing approach. The effect on growth was confirmed using individual GFP competition assays and on apoptosis using Annexin-V staining. Our previously published Argonaute 2 (Ago2) immunoprecipitation (IP) data were used to identify target genes relevant for cell growth / apoptosis. Luciferase assays and western blotting were performed to confirm targeting by miRNAs. Results: Four miRNA inhibition constructs, i.e. miR-449a-5p, miR-625-5p, let-7f-2-3p and miR-21-5p, showed a significant decrease in abundance in at least 4 of 6 infections. In contrast, none of the empty vector constructs showed a significant decrease in abundance in 3 or more of the 6 infections. The most abundantly expressed miRNA, i.e. miR-21-5p, showed significantly higher expression levels in cHL compared to GC-B cells. GFP competition assays confirmed the negative effect of miR-21-5p inhibition on HL cell growth. Annexin-V staining of cells infected with miR-21-5p inhibitor indicated a significant increase in apoptosis at day 7 and 9 after viral infection, consistent with the decrease in growth. Four miR-21-5p cell growth- and apoptosis-associated targets were AGO2-IP enriched in cHL cell lines and showed a significant decrease in expression in cHL cell lines in comparison to normal GC-B cells. For the two most abundantly expressed, i.e. BTG2 and PELI1, we confirmed targeting by miR-21-5p using luciferase assays and for PELI1 we also confirmed this at the protein level by western blotting. Conclusion: Using a miRNA loss-of-function high-throughput screen we identified four miRNAs with oncogenic effects in cHL and validated the results for the in cHL abundantly expressed miR-21-5p. MiR-21-5p is upregulated in cHL compared to GC-B cells and protects cHL cells from apoptosis possibly via targeting BTG2 and PELI1.

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.

A Selective PIM Kinase Inhibitor Is Highly Active In Multiple Myeloma: Mechanism of Action and Signal Transduction Studies

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4084-4084 ◽  
Author(s):  
Veerendra Munugalavadla ◽  
Leanne Berry ◽  
Yung-Hsiang Chen ◽  
Gauri Deshmukh ◽  
Jake Drummond ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Combination Treatment ◽  
Kinase Inhibitor ◽  
Single Agent ◽  
Myeloma Cell ◽  
Negative Regulator ◽  
Combination Drug ◽  
Employment Equity ◽  
Equity Ownership

Abstract Abstract 4084 Related work from our group has shown the therapeutic utility of PIM inhibition in multiple myeloma cell lines, xenografts, and primary patient samples (Ebens A. et al., ASH 2010 submitted abstr.). In this study we provide detailed mechanistic findings to show that PIM kinase inhibition co-regulates several important elements of the PI3K/AKT/mTOR pathway, resulting in significant synergy for combination drug treatments. The PIM kinases are a family of 3 ser/thr growth factor- & cytokine-induced proteins hypothesized to have redundant survival and growth functions. GNE-652 is a pan-PIM kinase inhibitor with picomolar biochemical potencies and an excellent kinase selectivity profile. Myeloma cell lines exhibit sensitivity to single agent PIM inhibition and a striking synergy in combination with the PI3K inhibitor GDC-0941. Cells respond to this combination with cell cycle arrest and marked apoptosis in vitro. We tested a panel of selective PI3K/AKT/mTOR inhibitors and found PI3K and AKT inhibitors showed the greatest extent of synergy with GNE-652, whereas mTOR inhibitors were synergistic to a lesser extent. These results suggest that PIM signaling converges on both TORC1 and AKT to generate these differential synergies. BAD is a negative regulator of both Bcl-2 and Bcl-XL, and we were able to confirm previous reports that AKT and PIM cooperate to inactivate BAD (Datt et al., 1997; Yan et al., 2003). Pim has been shown to potentially inactivate PRAS40, a negative regulator of TORC1 (Zhang et al., 2009). We demonstrate that PIM or PI3K inhibition caused a loss of phosphorylation on PRAS40 and results in a physical association of PRAS40 and TORC1 and a decrease in phosphorylated p70S6K and S6RP. These reductions were apparent in 7 of 7 cell lines assayed and enhanced by the combination of PI3K and PIM inhibition in these cell lines. Consistent with prior reports (Hammerman et al., 2005), we show that a second node of convergence between PIM and TORC1 is 4E-BP1. Both GDC-0941 and GNE-652 treatments reduced phosphorylation of 4E-BP1 in 7 of 7 myeloma cell lines. Since dephosphorylated 4E-BP1 competes with eIF4G for the mRNA cap binding factor eIF4E, we assayed immunoprecipitates of eIF4E for the presence of eIF4G and 4E-BP1 and observed increased BP1 and decreased 4G. The combination treatment significantly enhanced the loss of 4G relative to either single agent, and importantly, even at 5× the IC50 concentrations for single agents, combination drug treatment achieved greater extent of effect than single agent treatment. Thus PI3K and PIM pathways are redundant at the level of cap-dependent translational initiation mediated by eIF4E. It has been hypothesized a subset of mRNAs are particularly sensitive to inhibition of cap-dependent translation, and that this includes a number of oncogenes such as cyclin D1. We assayed global protein synthesis in MM1.s cells using 35S-methionine and as expected we observed only a modest ≂∼f20% decrease caused by either GNE-652 or GDC-0941 and this decrease was not enhanced by combination treatment. However, we noted across 7 different myeloma cell lines, strong decreases in levels of cyclin D1 that were enhanced by combination treatment. In summary, we have identified several points at which PIM and PI3K/AKT/mTOR converge to provide synergistic apoptosis in multiple myeloma cell lines. These results provide the rationale for further preclinical development of PIM inhibitors and provide the basis for a possible clinical development plan in multiple myeloma. Disclosures: Munugalavadla: Genentech: Employment, Equity Ownership. Berry:Genentech: Employment, Equity Ownership. Chen:Genentech: Employment, Equity Ownership. Deshmukh:Genentech: Employment, Equity Ownership. Drummond:Genentech: Employment, Equity Ownership. Du:Genentech: Employment, Equity Ownership. Eby:Genentech: Employment, Equity Ownership. Fitzgerald:Genentech: Employment, Equity Ownership. S.Friedman:Genentech: Employment, Equity Ownership. E.Gould:Genentech: Employment, Equity Ownership. Kenny:Genentech: Employment, Equity Ownership. Maecker:Genentech: Employment, Equity Ownership. Moffat:Genentech: Employment, Equity Ownership. Moskalenko:Genentech: Employment, Equity Ownership. Pacheco:Genentech: Employment, Equity Ownership. Saadat:Genentech: Employment, Equity Ownership. Slaga:Genentech: Employment, Equity Ownership. Sun:Genentech: Employment, Equity Ownership. Wang:Genentech: Employment, Equity Ownership. Yang:Genentech: Employment, Equity Ownership. Ebens:Genentech Inc: Employment, Equity Ownership.

CAL-101, a Potent Selective Inhibitor of the p110δ Isoform of Phosphatidylinositol 3-kinase, Attenuates Pathway Signaling, Induces Apoptosis, and Overcomes Signals From the Microenvironment In Cellular Models of Hodgkin Lymphoma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3926-3926 ◽  
Author(s):  
Sarah A Meadows ◽  
Adam Kashishian ◽  
Dave Johnson ◽  
Volker Diehl ◽  
Brian Lannutti
Keyword(s):  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Stromal Cells ◽  
Specific Inhibitor ◽  
Lymphocytic Leukemia ◽  
Class I ◽  
Clinical Activity ◽  
Dependent Manner ◽  
Cellular Models ◽  
Dose Dependent

Abstract Abstract 3926 Phosphatidylinositide 3-kinases (PI3Ks) are a family of lipid kinases that are involved in signaling events which control a diverse number of cellular processes. The class I kinases contain 4 isoforms designated p110α, β, δ, γ, and are activated by cell surface receptors. Aberrant regulation of the PI3K signaling pathway is frequently observed in human malignancies including those of hematological origin. CAL-101 is an oral p110δ-specific inhibitor which has shown preclinical and clinical activity in non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL). This compound is a potent p110δ inhibitor (EC50 of 65 nM in a whole-blood assay) with >200-fold selectivity over the other class I PI3K isoforms and no activity against Class II and III PI3K family members or other PI3K-related proteins, including mTOR and DNA-PK. Prior in vitro NHL studies revealed that CAL-101 induces caspase-dependent apoptosis, and inhibits CD40L-, BAFF-, CXCL12- and CXCL13-derived survival signals in cellular models (Lannutti BJ, et al., Blood 2010). To investigate the potential role of p110δ in Hodgkin lymphoma (HL) we screened a number of HL cell lines for p110δ isoform expression and constitutive PI3K pathway activation. We report high levels of p110δ protein and activated Akt in 5 of 5 HL cell lines evaluated (L428, L540, L591, L1236, KM-H2). Inhibition of p110δ with CAL-101 treatment of cell lines resulted in a reduction of Akt phosphorylation and a decrease in cellular viability. Because previous studies have established the importance of signals from the microenvironment for the survival and proliferation of malignant cells as well as for their resistance to standard therapies, we investigated the effect of p110δ inhibition by CAL-101 in HL cell line-stroma cocultures. In this setting, CAL-101 overcame tumor cell growth induced by coculture of HL cells with bone marrow stromal cells. In addition, CAL-101 induced dose-dependent apoptosis of HL cells at 48 hours. Furthermore, stromal cell coculture resulted in increased CCL5, CCL17, and CCL22 levels; productions of these chemokines by HL cells cultured in the presence of stromal cells were reduced by CAL-101 in a dose-dependent manner. These results indicate that specific inhibition of p110δ may disrupt signals between HL cells and their microenvironment, thereby providing the preclinical rationale for clinical evaluation of CAL-101 as a novel therapeutic approach in patients with Hodgkin lymphoma. Disclosures: Meadows: Calistoga Pharmaceuticals: Employment. Kashishian:Calistoga Pharmaceuticals: Employment. Johnson:Calistoga Pharmaceuticals: Employment. Lannutti:Calistoga Pharmaceutical Inc.: Employment.

The Combination Of Hypomethylating Agents and Histone Deacetylase Inhibitors(HDACi) Are Synergistically Cytotoxic and Reverse The Malignant Phenotype In Preclinical Models Of T-Cell Lymphoma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 646-646 ◽  
Author(s):  
Owen A. O'Connor ◽  
Enrica Marchi ◽  
Kelly Zullo ◽  
Luigi Scotto ◽  
Jennifer E. Amengual ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Board Of Directors ◽  
Cell Lines ◽  
Single Agent ◽  
Hematologic Malignancies ◽  
Advisory Committees ◽  
Methylation Array ◽  
Simultaneous Exposure ◽  
The Mean

Abstract Both HDAC inhibitors (HDACIs) and DNA methyltransferase inhibitors (DNMTIs) are known to influence global expression patterns in hematologic malignancies. Little is known about the combination of these two drug classes in lymphoid malignancies. HDACIs have marked single agent activity in the T- cell lymphomas (TCL), although the mechanism of action is not well defined. DNMTIs affect cytosine methylation of genomic DNA and have activity mainly restricted to the myeloid derived hematologic malignancies. The single agent efficacy and synergistic interaction of a panel of HDACIs (panobinostat, belinostat, romidepsin and vorinostat) and DNMTIs (decitabine (DEC), 5-azacytadine (5-AZA)) was evaluated in models of TCL. The molecular basis for the synergistic effect of HDACIs and DNMTIs was evaluated by gene expression profiling (GEP) and CpG methylation CTCL. Single agent concentration and time effect relationships were generated for 2 CTCL (HH, H9) and 2 T-ALL (P12, PF382) cell lines. Romidepsin and belinostat were the most potent HDACIs with the mean 48 hour IC50 of 8.8 nM (range 1.7-2.7 nM) and 85 nM (range 36-136 nM), respectively. Cell viability was not affected by treatment with DEC or 5-AZA at 24 and 48 hours at concentrations as high as 20 μM. Reduction in viability was first demonstrated after 72 hours of exposure to DEC, with the mean IC50 of 14.8 μM (range 0.4 μM- >20uM). Simultaneous exposure of combinations of DEC plus romidepsin or DEC plus belinostat at their IC10, IC20, and IC50 produced marked synergy in all TCL derived cell lines. Simultaneous exposure of DEC plus romidepsin demonstrated the deepest synergy at 72 hours with synergy coefficients in the range of 0.3. Cells treated with the combination of DEC plus romidepsin also demonstrated significant induction of apoptosis as evaluated by annexinV/propridium iodide via FACS analysis and an increase in acetylated histone 3 by immunoblot. The in vivo activity of the combination of DEC plus belinostat was investigated in a xenograft model of CTCL using HH, the most resistant TCL derived cell line. Mice were treated with DEC 1.5 mg/kg (day 29, 33, 35, 37, 39, 41, 43) and/or belinostat 100 mg/kg (day 29-day 47). The combination mouse cohort demonstrated statistically significant tumor growth delay compared to DEC alone (p=0.002) and belinostat alone (p=0.001). The interaction of DEC and romidepsin was analyzed by GEP and methylation array. Interestingly, the baseline malignant phenotype seen in the CTCL cell-lines was reversed. A significant down-regulation of genes involved in biosynthetic pathways including protein and lipid synthesis, and a significant up-regulation of genes responsible for cell cycle arrest were seen. The vast majority (114/138; 92%) of genes modulated by the single agents were similarly modulated by the combination. However, the latter induced a further significant change in the transcriptome, affecting an additional 390 genes. Similarly, methylation array data was analyzed following treatment of these drugs alone and in combination. DEC induced de-methylation of 190 different gene regions corresponding to 175 genes and an additional 335 loci. Interestingly, when combined with romidepsin the number of demethylated gene regions decreased to 85 corresponding to 79 genes, 78 of which were common with DEC and 148 additional loci. The comparison of gene expression and methylation demonstrated a significant inverse relationship (R2 = 0.657) with genes found to be differentially expressed in GEP and methylation analysis. (Figure 1)Figure 1Summary of gene expression and methylation analysis.Figure 1. Summary of gene expression and methylation analysis. These data support the observation that DNMTIs in combination with HDACIs produces significant synergistic activity in models of TCL. Further evaluation of the mechanism of action with DNMTIs in combination with HDACIs is ongoing, and a clinical trial of the combination is now open. Disclosures: O'Connor: Celgene Pharmaceuticals: Consultancy; Spectrum Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees; Allos Therapeutics: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Off Label Use: Hypomethylating Agents in T-cell lymphoma. Amengual:Acetylon Pharmacueticals, INC: Membership on an entity’s Board of Directors or advisory committees, Research Funding.

scholarly journals SAR650984 (SAR) Directly Promotes Homotypic Adhesion-Related Multiple Myeloma (MM) Cell Death and SAR-Induced Anti-MM Activities Are Enhanced By Pomalidomide, More Potently Than Lenalidomide

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2124-2124 ◽  
Author(s):  
Hua Jiang ◽  
Gang An ◽  
Chirag Acharya ◽  
Mike Y Zhong ◽  
Ti Cai ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Caspase 3 ◽  
Single Agent ◽  
Annexin V ◽  
Cell Killing ◽  
Individual Agent ◽  
Direct Cell ◽  
Direct Killing

Abstract SAR650984 (SAR) is a naked humanized IgG1 monoclonal antibody (mAb) selectively targeting the membrane protein CD38 in early clinical development to treat multiple myeloma (MM) and other CD38+ hematological malignancies. SAR has demonstrated encouraging single agent activity in relapsed/refractory (R/R) MM patients (ASCO abstract #8532) and even better efficacy when combined with Dexamethasone and Lenalidomide (Len), without reaching a maximum tolerated dose in patients with heavily pretreated MM (ASCO Abstract #8512). It functions through multiple mechanisms including antibody dependent cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), and direct killing against CD38-positive tumor cells including MM. Although SAR induces lysis of all CD38-expressing MM cell lines via ADCC, it only significantly induces direct killing of MOLP8 cells that express the highest CD38 surface density (~580,000/cell) among > 17 MM cell lines. We first sought to determine whether direct cell death induced by SAR depends on CD38 levels on MM cell membrane by generating RPMI8226 cells overexpressing CD38 (R-CD38) (Abstract #67338). R-CD38 cells express > 6-fold higher CD38 mRNA and surface protein levels than parental RPMI8226 cells (577,304/cell vs. 128,713/cell). Direct MM cell killing by SAR was determined using caspase 3/7 activity and CellTiter-Glo luminescent cell viability assays without goat anti-human IgG crosslinking, in the presence or absence of IL-6 or bone marrow stromal cells (BMSCs). Following overnight incubation, SAR significantly induced homotypic aggregation (HA) of R-CD38, but not control RPMI8226 cells, associated with dose-dependent activation of pro-apoptotic caspase 3/7 in R-CD38, but not control cells. Importantly, SAR decreased the viability of R-CD38, but not control cells, regardless of the presence of IL-6 or BMSCs. Direct cell death induced by SAR depends on SAR-induced HA in MM cells since SAR only blocked survival of R-CD38 and MOLP8 MM cells that show significant HA. Thus, direct apoptosis induced by SAR depends on the level of CD38 surface expression, which may contribute to clinical responses in R/R MM expressing higher CD38 levels. Next, we evaluated the combination effect of Len or Pomalidomide (Pom) with SAR on MM cells. BM mononuclear cells from MM patients were incubated with SAR (10 mg/ml) with or without 10 mM of Len or Pom overnight, followed by flow cytometric analysis to determine % Annexin V/PI staining of CD138+/BCMA+ MM cells. As expected, Pom alone induced slightly higher % of Annexin V+/PI+ MM cells than Len (41 + 1.8 % vs 49 + 1.5 %). Either combination further increased the % of double positive MM patient cells when compared with individual agent alone (from 40 + 2.1% to 70 + 3.1% combined with Len; from 40 + 2.1% to 86 + 3.4% combined with Pom). In addition, PBMC effectors from normal donors (n=4) were pretreated with Len or Pom (5 mM) for 3-7 days and used for SAR-mediated ADCC assays against MM cells (MM1S, MM1R, RPMI8226, R-38, MOLP8), with or without HS-5 or BMSCs from patients. Pom, more potently than Len, further increased SAR-induced MM cell lysis regardless of the presence of BMSCs. Moreover, additional pretreatment of MM cells with Pom overnight further enhanced SAR-induced ADCC by Pom-pretreated PBMC effectors. Both MOLP8 and R-CD38 are relative resistant to direct cytotoxicities induced by Len or Pom. Significantly, Pom, also more potently than Len, augmented direct toxicities induced by SAR in MOLP8 and R-CD38 MM cells. Taken together, we here demonstrate that SAR directly induces apoptosis of MM cells with higher CD38 levels; and that Pom, more effectively than Len, increases SAR-induced MM cell killing via apoptosis and ADCC. These data strongly support SAR as a monotherapy or in combination treatment to improve the outcome of MM patients. Disclosures Cai: Sanofi: Employment. Song:Sanofi: Employment. Yang:Sanofi: Employment. Adrian:Sanofi: Employment. Munshi:Celgene: Consultancy; Onyx: Consultancy; Janssen: Consultancy; Sanofi-Aventis: Consultancy; Ocopep: Consultancy, Equity Ownership, Patents & Royalties. Anderson:Celgene: Consultancy; Onyx: Consultancy; Gilead Sciences: Consultancy; Sanofi-Aventis US: Consultancy; Acetylon: Scientific Founder Other; Oncoprep: Scientific Founder Other.

Effect of obatoclax (GX15-070) on cell survival, apoptosis, and prosurvival autophagy in head and neck squamous cell carcinoma (HNSCC).

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e13587-e13587
Author(s):  
Victor Y. Yazbeck ◽  
Changyou Li ◽  
Daniel E Johnson
Keyword(s):  
Head And Neck ◽  
Cell Lines ◽  
Performance Status ◽  
Single Agent ◽  
Annexin V ◽  
In Vivo Studies ◽  
Dependent Manner ◽  
Autophagy Inhibitor ◽  
Dismal Prognosis ◽  
Hnscc Cell

e13587 Background: Approximately 500,000 new cases of head and neck cancer are diagnosed annually worldwide, with 300,000 deaths per year. Patients with recurrent and metastatic disease have a dismal prognosis with a median survival of less than one year. Current standard of care involves a platinum-based regimen for patients with good performance status, but is usually difficult to tolerate. Novel therapeutic approaches are in great need for this patient population. The antiapoptotic BCL-2 family proteins, including BCL-2, BCL-XL, and MCL-1 are involved in oncogenesis and chemoresistance and are overexpressed in HNSCC. Obatoclax is a pan-BCL2 inhibitor capable of inhibiting MCL-1, in addition to BCL-2 and BCL-XL. Methods: We determined the activity of obatoclax in 4 well-characterized HNSCC cell lines (UMSCC-1, Cal-33, 1483, UMSCC-22A). Cell viability was determined by MTT assay and apoptosis by Annexin-V binding, FACS analysis, and immunoblotting. Autophagy was assessed by immunofluorescence and immunoblotting. Results: All 4 HNSCC cell lines were highly sensitive to single-agent obatoclax with IC50’s ranging from 50-200 nM. Obatoclax induced apoptosis in all cell lines as evidenced by increased Annexin-V binding at 48 hours in a dose-dependent manner from less than 10% in vehicle treated cells to 30-60% in cells treated with obatoclax. Processing/activation of caspase-3 and cleavage of PARP protein was also observed. In addition, obatoclax induced prosurvival autophagy in all cell lines and the addition of the autophagy inhibitor chloroquine enhanced obatoclax cytotoxicity. In vivo studies of obatoclax using human HNSCC xenograft tumors is currently underway. Conclusions: This preclinical study suggests that obatoclax might have therapeutic value in HNSCC, as a single agent or in combination with an autophagy inhibitor.

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