Keith C. Bible

Serine protease HtrA1 modulates chemotherapy-induced cytotoxicity

Resistance to chemotherapy presents a serious challenge in the successful treatment of various cancers and is mainly responsible for mortality associated with disseminated cancers. Here we show that expression of HtrA1, which is frequently downregulated in ovarian cancer, influences tumor response to chemotherapy by modulating chemotherapy-induced cytotoxicity. Downregulation of HtrA1 attenuated cisplatin- and paclitaxel-induced cytotoxicity, while forced expression of HtrA1 enhanced cisplatin- and paclitaxel-induced cytotoxicity. HtrA1 expression was upregulated by both cisplatin and paclitaxel treatment. This upregulation resulted in limited autoproteolysis and activation of HtrA1. Active HtrA1 induces cell death in a serine protease-dependent manner. The potential role of HtrA1 as a predictive factor of clinical response to chemotherapy was assessed in both ovarian and gastric cancer patients receiving cisplatin-based regimens. Patients with ovarian or gastric tumors expressing higher levels of HtrA1 showed a higher response rate compared with those with lower levels of HtrA1 expression. These findings uncover what we believe to be a novel pathway by which serine protease HtrA1 mediates paclitaxel- and cisplatin-induced cytotoxicity and suggest that loss of HtrA1 in ovarian and gastric cancers may contribute to in vivo chemoresistance.

Characterization of Caspase Processing and Activation in HL-60 Cell Cytosol Under Cell-free Conditions NUCLEOTIDE REQUIREMENT AND INHIBITOR PROFILE

The present studies compared caspase activation under cell-free conditions in vitro and in etoposide-treated HL-60 leukemia cells in situ. Immunoblotting revealed that incubation of HL-60 cytosol at 30u2009°C in the presence of cytochrome c and ATP (or dATP) resulted in activation of procaspases-3, -6, and -7 but not -2 and -8. Although similar selectivity was observed in intact cells, affinity labeling revealed that the active caspase species generated in vitroand in situ differed in charge and abundance. ATP and dATP levels in intact HL-60 cells were higher than required for caspase activation in vitro and did not change before caspase activation in situ. Replacement of ATP with the poorly hydrolyzable analogs 5′-adenylyl methylenediphosphate, 5′-adenylyl imidodiphosphate, or 5′-adenylyl-O-(3-thiotriphos-phate) slowed caspase activation in vitro, suggesting that ATP hydrolysis is required. Caspase activation in vitro was insensitive to phosphatase and kinase inhibitors (okadaic acid, staurosporine, and genistein) but was inhibited by Zn2+, aurintricarboxylic acid, and various protease inhibitors, including 3,4-dichloroisocoumarin,N α-p-tosyl-l-phenylalanine chloromethyl ketone,N α-p-tosyl-l-lysine chloromethyl ketone, andN-(N α-benzyloxycarbonylphenylalanyl)alanine fluoromethyl ketone, each of which inhibited recombinant caspases-3, -6, -7, and -9. Experiments with anti-neoepitope antiserum confirmed that these agents inhibited caspase-9 activation. Collectively, these results suggest that caspase-9 activation requires nucleotide hydrolysis and is inhibited by agents previously thought to affect apoptosis by other means.

Phase 1 Trial of Flavopiridol Combined with Cisplatin or Carboplatin in Patients with Advanced Malignancies with the Assessment of Pharmacokinetic and Pharmacodynamic End Points

Purpose: Flavopiridol, a cyclin-dependent kinase inhibitor, transcription inhibitor, and DNA-interacting agent, was combined with cisplatin or carboplatin to establish toxicities, evaluate pharmacokinetics, and examine its effects on patient cancers and levels of selected polypeptides in patient peripheral blood mononuclear cells (PBMC). Experimental Design: Therapy was given every 3 weeks. Stage I: cisplatin was fixed at 30 mg/m2 with escalating flavopiridol. Stage II: flavopiridol was fixed at the stage I maximum tolerated dose (MTD) with escalation of cisplatin. Stage III: flavopiridol was fixed at the stage I MTD with escalation of carboplatin. Results: Thirty-nine patients were treated with 136 cycles of chemotherapy. Neutropenia was seen in only 11% of patients. Grade 3 flavopiridol/CDDP toxicities were nausea (30%), vomiting (19%), diarrhea (15%), dehydration (15%), and neutropenia (10%). Flavopiridol combined with carboplatin resulted in unexpectedly high toxicities and one treatment-related death. Stable disease (>3 months) was seen in 34% of treated patients, but there were no objective responses. The stage II MTD was 60 mg/m2 cisplatin and 100 mg/m2/24 hours flavopiridol. As given, CDDP did not alter flavopiridol pharmacokinetics. Flavopiridol induced increased p53 and pSTAT3 levels in patient PBMCs but had no effects on cyclin D1, phosphoRNA polymerase II, or Mcl-1. Conclusions: Flavopiridol and cisplatin can be safely combined in the treatment of cancer patients. Unexpected toxicity in flavopiridol/carboplatin-treated patients attenuates enthusiasm for this alternative combination. Analysis of polypeptide levels in patient PBMCs suggests that flavopiridol may be affecting some, but not all, of its known in vitro molecular targets in vivo.

Flavopiridol disrupts STAT3/DNA interactions, attenuates STAT3-directed transcription, and combines with the Jak kinase inhibitor AG490 to achieve cytotoxic synergy

Up-regulated signal transducers and activators of transcription (STAT)–mediated signaling is believed to contribute to the pathogenesis of a variety of solid and hematologic cancers. Consequently, inhibition of STAT-mediated signaling has recently been proposed as a potential new therapeutic approach to the treatment of cancers. Having shown previously that the pan–cyclin-dependent kinase inhibitor flavopiridol binds to DNA and seems to kill cancer cells via that process in some circumstances, we evaluated the hypothesis that flavopiridol might consequently disrupt STAT3/DNA interactions, attenuate STAT3-directed transcription, and down-regulate STAT3 downstream polypeptides, including the antiapoptotic polypeptide Mcl-1. SDS-PAGE/immunoblotting and reverse transcription-PCR were used to assess RNA and polypeptide levels, respectively. DNA cellulose affinity chromatography and a nuclear elution assay were used to evaluate the ability of flavopiridol to disrupt STAT3/DNA interactions. A STAT3 luciferase reporter assay was used to examine the ability of flavopiridol to attenuate STAT3-directed transcription. Colony-forming assays were used to assess cytotoxic synergy between flavopiridol and AG490. Flavopiridol was found to (a) disrupt STAT3/DNA interactions (DNA cellulose affinity chromatography and nuclear elution assay), (b) attenuate STAT3-directed transcription (STAT3 luciferase reporter assay), and (c) down-regulate the STAT3 downstream antiapoptotic polypeptide Mcl-1 at the transcriptional level (reverse transcription-PCR and SDS-PAGE/immunoblotting). Furthermore, flavopiridol, but not the microtubule inhibitor paclitaxel, could be combined with the STAT3 pathway inhibitor AG490 to achieve cytotoxic synergy in A549 human non–small cell lung cancer cells. Collectively, these data suggest that flavopiridol can attenuate STAT3-directed transcription in a targeted fashion and may therefore be exploitable clinically in the development of chemotherapy regimens combining flavopiridol and other inhibitors of STAT3 signaling pathways. [Mol Cancer Ther 2006;5(1):138–48]

Inhibition of Mitochondrial Respiration as a Source of Adaphostin-induced Reactive Oxygen Species and Cytotoxicity

Adaphostin is a dihydroquinone derivative that is undergoing extensive preclinical testing as a potential anticancer drug. Previous studies have suggested that the generation of reactive oxygen species (ROS) plays a critical role in the cytotoxicity of this agent. In this study, we investigated the source of these ROS. Consistent with the known chemical properties of dihydroquinones, adaphostin simultaneously underwent oxidation to the corresponding quinone and generated ROS under aqueous conditions. Interestingly, however, this quinone was not detected in intact cells. Instead, high performance liquid chromatography demonstrated that adaphostin was concentrated by up to 300-fold in cells relative to the extracellular medium and that the highest concentration of adaphostin (3000-fold over extracellular concentrations) was detected in mitochondria. Consistent with a mitochondrial site for adaphostin action, adaphostin-induced ROS production was diminished by >75% in MOLT-4 rho0 cells, which lack mitochondrial electron transport, relative to parental MOLT-4 cells. In addition, inhibition of oxygen consumption was observed when intact cells were treated with adaphostin. Loading of isolated mitochondria to equivalent adaphostin concentrations caused inhibition of uncoupled oxygen consumption in mitochondria incubated with the complex I substrates pyruvate and malate or the complex II substrate succinate. Further analysis demonstrated that adaphostin had no effect on pyruvate or succinate dehydrogenase activity. Instead, adaphostin inhibited reduced decylubiquinone-induced cytochrome c reduction, identifying complex III as the site of inhibition by this agent. Moreover, adaphostin enhanced the production of ROS by succinate-charged mitochondria. Collectively, these observations demonstrate that mitochondrial respiration rather than direct redox cycling of the hydroquinone moiety is a source of adaphostin-induced ROS and identify complex III as a potential target for antineoplastic agents.

Annatto constituent cis-bixin has selective antimyeloma effects mediated by oxidative stress and associated with inhibition of thioredoxin and thioredoxin reductase

In pursuit of the anticancer effects of seeds of the rain forest plant Bixa orellana (annatto), we found that its constituent cis-bixin induced cytotoxicity in a wide variety of tumor cell lines (IC(50) values from 10 to 50 microM, 24-h exposures) and, importantly, also selectively killed freshly collected patient multiple myeloma cells and highly drug-resistant multiple myeloma cell lines. Mechanistic studies indicated that cis-bixin-induced cytotoxicity was greatly attenuated by co-treatment with glutathione or N-acetylcysteine (NAC); whereas fluorescence-activated cell sorting (FACS) assays using the cell-permeable dyes 5-(and-6) chloromethyl-2,7-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H(2)DCFDA), or dihydroethidium demonstrated that cis-bixin rapidly induced cellular reactive oxygen species (ROS) in dose- and time-dependent fashions, collectively implicating ROS as contributory to cis-bixin-induced cytotoxicity. In pursuit of potential contributors to ROS imposition by cis-bixin, we found that cis-bixin inhibited both thioredoxin (Trx) and thioredoxin reductase (TrxR1) activities at concentrations comparable to those required for cytotoxicity, implicating the inhibition of these redox enzymes as potentially contributing to its ability to impose cellular ROS and to kill cancer cells. Collectively, our studies indicate that the annatto constituent cis-bixin has intriguing selective antimyeloma activity that appears to be mediated through effects on redox signaling.

Effect of P-glycoprotein on flavopiridol sensitivity

Flavopiridol is the first potent inhibitor of cyclin-dependent kinases (CDKs) to enter clinical trials. Little is known about mechanisms of resistance to this agent. In order to determine whether P-glycoprotein (Pgp) might play a role in flavopiridol resistance, we examined flavopiridol sensitivity in a pair of Chinese hamster ovary cell lines differing with respect to level of Pgp expression. The IC50s of flavopiridol in parental AuxB1 (lower Pgp) and colchicine-selected CHRC5 (higher Pgp) cells were 90.2 ± 6.6 nM and 117 ± 2.3 nM, respectively (P< 0.01), suggesting that Pgp might have a modest effect on flavopiridol action. Consistent with this hypothesis, pretreatment with either quinidine or verapamil (inhibitors of Pgp-mediated transport) sensitized CHRC5 cells to the antiproliferative effects of flavopiridol. Because of concern that colony forming assays might not accurately reflect cytotoxicity, we also examined flavopiridol-treated cells by trypan blue staining and flow cytometry. These assays confirmed that flavopiridol was less toxic to cells expressing higher levels of Pgp. Further experiments revealed that flavopiridol inhibited the binding of [3H]-azidopine to Pgp in isolated membrane vesicles, but only at high concentrations. Collectively, these results identify flavopiridol as a weak substrate for Pgp.

Profound Hair and Skin Hypopigmentation in an African American Woman Treated With the Multi-Targeted Tyrosine Kinase Inhibitor Pazopanib

A 40-year-old African American woman with advanced, progressive radioiodine-refractory Hürthle cell carcinoma of the thyroid involving lymph nodes, lungs, and bone was treated with the investigational multitargeted tyrosine kinase inhibitor (TKI) pazopanib (starting dose, 800 mg daily orally, continuously) in accord with an independent review board– and National Cancer Institute– approved phase II clinical trial in patients with advanced, rapidly progressive,radioiodine-refractorydifferentiatedthyroidcancers.Thepatient experienced declining thyroglobulin tumor marker levels (Fig 1) and developed a prompt Response Evaluation Criteria in Solid Tumors (RECIST) partial response (see Fig 1 for a summary of RECIST tumor measurements and Fig 2 for representative computed tomography images). In parallel, she also developed adverse effects, including hypertension requiring medical therapy; anorexia due to altered taste perception; therapy-related nausea, vomiting, and diarrhea; moderate asymptomatic hyperbilirubinemia; and mild thrombocytopenia and leucopenia, ultimately prompting dose reduction to 600 mg per day after the fourth 28-day cycle of pazopanib therapy. After the first 2 months of pazopanib therapy, the patient began to develop progressive hair and skin hypopigmentation and hair thinning. Her skin pigmentation continued to lighten while on pazopanib through the first 12 months of therapy. The patient is shown (left images in Fig 3) after 11 cycles of pazopanib therapy in comparison to her daughter (right images in Fig 3), who was of similar skin pigmentation to the patient before initiation of pazopanib therapy (note that effects on hair pigmentation of the patient are obscured by a hair prosthesis). The patient incurred a sunburn (grade 3) for the first time in her life while on a Caribbean cruise during her eleventh month of therapy, presumably related to her skin depigmentation, despite no similar problems with sunburn in her lifetime previously even in response to more intense sun exposures. Moreover, the patient has at times perceived that members of her community have been treating her differently than when she was previously more highly pigmented, presumably as a consequence of her now lighter skin pigmentation. The use of TKIs as cancer therapeutics is becoming increasingly prevalent, with agents such as imatinib, sunitinib and sorafenib already approved for clinical use—and several other TKIs presently under investigational use. Hair hypopigmentation has been commonly reported in patients receiving the c-Kit inhibitors. In contrast, skin hypopigmentation has been rarely seen in response to TKI therapy. We report here for the first time, to the best of our knowledge, the development of profound hair and diffuse skin hypopigmentation in an African American patient treated with the orally bioavailable vascular endothelial growth factor, platelet-derived

Development of a Multidisciplinary, Multicampus Subspecialty Practice in Endocrine Cancers

OBJECTIVESnRelative to more abundant neoplasms, endocrine cancers have been historically neglected, yet their incidence is increasing. We therefore sought to build interest in endocrine cancers, improve physician experience, and develop innovative approaches to treating patients with these neoplasms.nnnMETHODSnBetween 2005 and 2010, we developed a multidisciplinary Endocrine Malignancies Disease Oriented Group involving all 3 Mayo Clinic campuses (Rochester, Minnesota; Jacksonville, Florida; and Scottsdale, Arizona). In response to higher demand at the Rochester campus, we sought to develop a Subspecialty Tumor Group and an Endocrine Malignancies Tumor Clinic within the Division of Medical Oncology.nnnRESULTSnThe intended groups were successfully formed. We experienced difficulty in integration of the Mayo Scottsdale campus resulting from local uncertainty as to whether patient volumes would be sufficient to sustain the effort at that campus and difficulty in developing enthusiasm among clinicians otherwise engaged in a busy clinical practice. But these obstacles were ultimately overcome. In addition, with respect to the newly formed medical oncology subspecialty endocrine malignancies group, appointment volumes quadrupled within the first year and increased 7 times within 2 years. The number of active therapeutic endocrine malignancies clinical trials also increased from 1 in 2005 to 5 in 2009, with all 3 Mayo campuses participating.nnnCONCLUSIONSnThe development of subspecialty tumor groups for uncommon malignancies represents an effective approach to building experience, increasing patient volumes and referrals, and fostering development of increased therapeutic options and clinical trials for patients afflicted with otherwise historically neglected cancers.PURPOSEnRelative to more abundant neoplasms, endocrine cancers have been historically neglected, yet their incidence is increasing. We therefore sought to build interest in endocrine cancers, improve physician experience, and develop innovative approaches to treating patients with these neoplasms.nnnMETHODSnBetween 2005 and 2010, we developed a multidisciplinary Endocrine Malignancies Disease Oriented Group involving all three Mayo Clinic campuses (Rochester, MN; Jacksonville, FL; and Scottsdale, AZ). In response to higher demand at the Rochester campus, we sought to develop a Subspecialty Tumor Group and an Endocrine Malignancies Tumor Clinic within the Division of Medical Oncology.nnnRESULTSnThe intended groups were successfully formed. We experienced difficulty in integration of the Mayo Scottsdale campus resulting from local uncertainty as to whether patient volumes would be sufficient to sustain the effort at that campus and difficulty in developing enthusiasm among clinicians otherwise engaged in a busy clinical practice. But these obstacles were ultimately overcome. In addition, with respect to the newly formed medical oncology subspecialty endocrine malignancies group, appointment volumes quadrupled within the first year and increased seven times within two years. The number of active therapeutic endocrine malignancies clinical trials also increased from one in 2005 to five in 2009, with all three Mayo campuses participating.nnnCONCLUSIONnThe development of subspecialty tumor groups for uncommon malignancies represents an effective approach to building experience, increasing patient volumes and referrals, and fostering development of increased therapeutic options and clinical trials for patients afflicted with otherwise historically neglected cancers.

Systemic therapeutic approaches to advanced thyroid cancers.

Until only recently, few effective systemic therapies were available to treat patients with metastatic thyroid cancers. Recent advances in better understanding the pathogenesis and altered signaling pathways-especially in medullary and differentiated thyroid cancers (MTCs and DTCs)-have begun to change this situation substantially. Vandetanib, an orally bioavailable inhibitor of the RET kinase that is constitutively activated in MTC, has now been approved by the U.S. Food and Drug Administration (FDA) for use in progressive and symptomatic metastatic MTC; it has been shown to delay time to progression relative to placebo in a randomized phase III trial. Further, vascular endothelial growth factor receptor (VEGF-R) inhibitory agents including sorafenib, sunitinib, pazopanib, and axitinib that are already approved in the United States for use in advanced renal cell carcinoma have shown high response rates in treating advanced DTCs in multiple phase II trials, and have become commonly used in progressive radioiodine-refractory metastatic DTC. Yet additional agents are now in development, with several including XL184 (cabozantinib) also showing promise in DTC and MTC. In anaplastic thyroid cancer (ATC), progress has been slower, with the greatest apparent gains resulting more from the application of systemic therapies earlier in the disease course, especially when used in conjunction with initial surgical and radiation therapies. Despite recent progress, additional effective systemic therapeutic approaches remain sorely needed for treating metastatic MTC, DTC, and ATC.