Kaufmann, Scott H.

Abstract Human cholangiocarcinomas evade apoptosis by overexpression of Mcl-1. The drug obatoclax (GX15-070) inhibits antiapoptotic members of the Bcl-2 family including Mcl-1. The purpose of this study is to determine if obatoclax sensitizes human cholangiocarcinoma cells to apoptosis. The human cholangiocarcinoma cell lines, KMCH, KMBC, and TFK, were employed for these studies. Protein expression was assessed by immunoblot and protein-protein interactions detected by coprecipitation of the polypeptide of interest with S-tagged Mcl-1. Activation of Bak and Bax was observed by immunocytochemistry with conformation-specific antisera. Obatoclax induced minimal apoptosis alone; however, it increased apoptosis 3- to 13-fold in all three cancer cell lines when combined with Apo2L/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Obatoclax did not alter cellular expression of Bid, Bim, Puma, Noxa, Bak, Bax, Mcl-1, or cFLIP. Mcl-1 binding to Bak was readily identified in untreated cells, and this association was disrupted by treating the cells with obatoclax. Additionally, Bim binding to Mcl-1 was markedly decreased by obatoclax treatment. We also identified alterations in Bak and Bax conformation following treatment with obatoclax plus Apo2L/TRAIL but not with either Apo2L/TRAIL or obatoclax alone. In conclusion, obatoclax releases Bak and Bim from Mcl-1 and sensitizes human cholangiocarcinoma cells to Apo2L/TRAIL-induced apoptosis. Obatoclax is a potentially promising adjunctive agent for the treatment of this cancer. [Mol Cancer Ther 2008;7(8):2339–47]

AbstractOutcomes for older adults with acute myelogenous leukemia (AML) are poor due to both disease and host-related factors. In this phase 2 study, we tested the oral farnesyltransferase inhibitor tipifarnib in 158 older adults with previously untreated, poor-risk AML. The median age was 74 years, and a majority of patients had antecedent myelodysplastic syndrome. Complete remission (CR) was achieved in 22 patients (14%); partial remission or hematologic improvement occurred in 15 patients, for an overall response rate of 23%. The median duration of CR was 7.3 months and the median survival of complete responders was 18 months. Adverse karyotype, age 75 years or older, and poor performance status correlated negatively with survival. Early death in the absence of progressive disease was rare, and drug-related nonhematologic serious adverse events were observed in 74 patients (47%). Inhibition of farnesylation of the surrogate protein HDJ-2 occurred in the large majority of marrow samples tested. Baseline levels of phosphorylated mitogen-activated protein kinase and AKT did not correlate with clinical response. Tipifarnib is active and well tolerated in older adults with poor-risk AML and may impart a survival advantage in those patients who experience a clinical response.

Abstract This study was undertaken to characterize preclinical cytotoxic interactions for human malignancies between the multikinase inhibitor sorafenib (BAY 43-9006) and proteasome inhibitors bortezomib or MG132. Multiple tumor cell lines of varying histiotypes, including A549 (lung adenocarcinoma), 786-O (renal cell carcinoma), HeLa (cervical carcinoma), MDA-MB-231 (breast), K562 (chronic myelogenous leukemia), Jurkat (acute T-cell leukemia), MEC-2 (B-chronic lymphocytic leukemia), and U251 and D37 (glioma), as well as cells derived from primary human glioma tumors that are likely a more clinically relevant model were treated with sorafenib or bortezomib alone or in combination. Sorafenib and bortezomib synergistically induced a marked increase in mitochondrial injury and apoptosis, reflected by cytochrome c release, caspase-3 cleavage, and poly(ADP-ribose) polymerase degradation in a broad range of solid tumor and leukemia cell lines. These findings were accompanied by several biochemical changes, including decreased phosphorylation of vascular endothelial growth factor receptor-2, platelet-derived growth factor receptor-β, and Akt and increased phosphorylation of stress-related c-Jun NH2-terminal kinase (JNK). Inhibition of Akt was required for synergism, as a constitutively active Akt protected cells against apoptosis induced by the combination. Alternatively, the JNK inhibitor SP600125 could also protect cells from apoptosis induced by the combination, indicating that both inhibition of Akt and activation of JNK were required for the synergism. These findings show that sorafenib interacts synergistically with bortezomib to induce apoptosis in a broad spectrum of neoplastic cell lines and show an important role for the Akt and JNK pathways in mediating synergism. Further clinical development of this combination seems warranted. [Mol Cancer Ther 2006;5(9):2378–87]

Abstract Purpose: The serine/threonine kinase inhibitor flavopiridol targets multiple cyclin-dependent kinases, induces checkpoint arrest, and interrupts transcriptional elongation. We designed a phase I clinical trial using a timed sequential therapy approach where flavopiridol was given for the dual purpose of initial cytoreduction and enhancing cell cycle progression of the remaining leukemia cell cohort followed by cycle-dependent drugs 1-β-d-arabinofuranosylcytosine (ara-C) and mitoxantrone. Experimental Design: Flavopiridol was given by 1-hour infusion daily for 3 days beginning day 1 followed by 2 g/m2/72 h ara-C beginning day 6 and 40 mg/m2 mitoxantrone beginning day 9. In vivo correlates included pharmacokinetics, modulation of blast cycle regulators, and serum and marrow supernatant vascular endothelial growth factor levels. Results: Of 34 adults receiving induction therapy, 16 (47%) evinced direct leukemia cytotoxicity with ≥50% drop in peripheral blast counts and tumor lysis in 9 (26%). Four (12%) died during therapy (two fungal infections and two sudden death). Dose-limiting toxicity occurred at 60 mg/m2/d with profound neutropenia >40 days duration, and maximal tolerated dose was 50 mg/m2/d. Overall response rate was 31% in 26 acute myelogenous leukemia and 12.5% in acute lymphoblastic leukemia. Pharmacokinetics showed that a linear two-compartment model with first-order elimination provided the best fit of the observed concentration versus time data. Flavopiridol down-regulated one or more target proteins in marrow blasts in vivo. Vascular endothelial growth factor was detected in sera and marrow supernatant pretreatment, and sera obtained on day 3 inhibited bovine aortic endothelial cell proliferation by a mean of 32% (range, 10-80%). Conclusions: Our data suggest that flavopiridol is cytotoxic to leukemic cells and, when followed by ara-C and mitoxantrone, exerts biological and clinical effects in patients with relapsed and refractory acute leukemias. These findings warrant continuing development of flavopiridol at 50 mg/m2/d × 3 days in combination with cytotoxic and biological agents for acute leukemias.

Abstract The ability of cancers to evade immune surveillance and resist immunotherapy raises a fundamental question of how tumor cells survive in the presence of a competent immune system. Studies to address this question have primarily focused on mechanisms by which tumor cells avoid recognition by or induce tolerance in the immune system. However, little is known about whether cancer cells also acquire an intrinsic ability to resist killing by immune effectors. We find that cancer cells enhance their ability to withstand an attack by cytotoxic immune effector cells via acquisition of specific genetic alterations that interfere with the shared mitochondrial death signaling pathway entrained by granzyme B, IFN-γ, and Apo2 ligand/tumor necrosis factor–related apoptosis inducing ligand (Apo2L/TRAIL), three key mediators of immunologic cell–mediated cytotoxicity. We show that the coexistence of specific mitochondrial signaling defects (either deletion of Bax, overexpression of Bcl-xL, or deletion of Smac) with expression of X-linked inhibitor of apoptosis protein decreases the sensitivity of cancer cells to IFN-γ/Apo2L/TRAIL– or granzyme B–induced apoptosis, lymphocyte-mediated cytotoxicity in vitro, and adoptive cellular immunotherapy in vivo. Conversely, negating X-linked inhibitor of apoptosis protein expression or function in tumor cells with defective mitochondrial signaling enables direct activation of caspase-3/-7 by granzyme B or Apo2L/TRAIL, and restores their susceptibility to immunologic cytotoxicity. These findings identify an important mechanism by which cancers evade elimination by immune effector cells and suggest that cancer immunotherapy might be improved by concurrent strategies to alleviate or circumvent the intrinsic mitochondrial death signaling defects that help cancer cells resist immunologic cytotoxicity. (Cancer Res 2006; 66(3): 1730-9)

Abstract The insulin-like growth factor receptor (IGF-IR) and insulin receptor are either overactivated and/or overexpressed in a wide range of tumor types and contribute to tumorigenicity, proliferation, metastasis, and drug resistance. Here, we show that BMS-554417, a novel small molecule developed as an inhibitor of IGF-IR, inhibits IGF-IR and insulin receptor kinase activity and proliferation in vitro, and reduces tumor xenograft size in vivo. In a series of carcinoma cell lines, the IC50 for proliferation ranged from 120 nmol/L (Colo205) to >8.5 μmol/L (OV202). The addition of stimulatory ligands was unnecessary for the antiproliferative effect in MCF-7 and OV202 cells. BMS-554417 treatment inhibited IGF-IR and insulin receptor signaling through extracellular signal-related kinase as well as the phosphoinositide 3-kinase/Akt pathway, as evidenced by decreased Akt phosphorylation at Ser473. At doses that inhibited proliferation, the compound also caused a G0-G1 arrest and prevented nuclear accumulation of cyclin D1 in response to LR3 IGF-I. In Jurkat T-cell leukemia cells, this agent triggered apoptotic cell death via the mitochondrial pathway. BMS-554417 was orally bioavailable and significantly inhibited the growth of IGF1R-Sal tumor xenografts in vivo. BMS-554417 is a member of a novel class of IGF-IR/insulin receptor inhibitors that have potential clinical applications because of their antiproliferative and proapoptotic activity in vitro and in vivo. (Cancer Res 2006; 66(1): 362-71)

Abstract Purpose: According to some studies, susceptibility of cells to anticancer drug-induced apoptosis is markedly inhibited by targeted deletion of genes encoding apoptotic protease activating factor 1 (Apaf-1) or certain caspases. Information about levels of these polypeptides in common cancer cell types and any possible correlation with drug sensitivity in the absence of gene deletion is currently fragmentary. Experimental Design: Immunoblotting was used to estimate levels of Apaf-1 as well as procaspase-2, -3, -6, -7, -8, and -9 in the 60-cell-line panel used for drug screening by the National Cancer Institute. Sensitivity of the same lines to >80,000 compounds was determined with 48-hour sulforhodamine B binding assays. Additional 6-day assays were performed for selected agents. Results: Levels of Apaf-1 and procaspases varied widely. Apaf-1 and procaspase-9, which are implicated in caspase activation after treatment of cells with various anticancer drugs, were detectable in all of the cell lines, with levels of Apaf-1 ranging from ∼1 × 105 to 2 × 106 molecules per cell and procaspase-9 from ∼5 × 103 to ∼1.6 × 105 molecules per cell. Procaspase-8 levels ranged from 1.7 × 105 to 8 × 106 molecules per cell. Procaspase-3, a major effector caspase, varied from undetectable to ∼1.6 × 106 molecules per cell. Correlations between levels of these polypeptides and sensitivity to any of a variety of experimental or conventional antineoplastic agents in either 2-day or 6-day cytotoxicity assays were weak at best. Conclusions: With the exception of caspase-3, all of the components of the core cell-death machinery are expressed in all of the cell lines examined. Despite variations in expression, levels of any one component are not a major determinant of drug sensitivity in these cells in vitro.

Abstract Purpose: To assess the maximum tolerated dose, toxicities, pharmacokinetics, and antileukemic activity of topotecan and carboplatin in adults with recurrent or refractory acute leukemias. Experimental Design: Patients received topotecan and carboplatin by 5-day continuous infusion at nine dose levels. Patients achieving a complete remission received up to two additional courses for consolidation. Plasma topotecan and ultrafilterable platinum were assayed on days 1 to 5. In addition, pretreatment levels of various polypeptides in leukemic cells were examined by immunoblotting to assess possible correlations with response. Results: Fifty-one patients received a total of 69 courses of therapy. Dose-limiting toxicity consisted of grade 4/5 typhlitis and grade 3/4 mucositis after one course of therapy or grade 4 neutropenia and thrombocytopenia lasting >50 days when a second course was administered on day 21. Among 45 evaluable patients, there were 7 complete remissions, 2 partial remissions, 1 incomplete complete remission, and 1 reversion to chronic-phase chronic myelogenous leukemia. Topotecan steady-state plasma concentrations increased with dose. No accumulation of topotecan or ultrafilterable platinum occurred between days 1 and 5 of therapy. Leukemic cell levels of topoisomerase I, checkpoint kinase 1, checkpoint kinase 2, and Mcl-1 correlated with proliferating cell nuclear antigen but not with response. In contrast, low Bcl-2 expression correlated with response (P = 0.014, Mann-Whitney U test). Conclusions: The maximum tolerated dose was 1.6 mg/m2/d topotecan plus 150 mg/m2/d carboplatin. The complete remission rate in a heavily pretreated population was 16% (33% at the highest three dose levels). Responses seem to correlate with low pretreatment blast cell Bcl-2 expression.