Phase 2 study of imexon, a prooxidant molecule, in relapsed and refractory B-cell non-Hodgkin lymphoma.

Lymphoma cells are subject to higher levels of oxidative stress compared with their normal counterparts and may be vulnerable to manipulations of the cellular redox balance. We therefore designed a phase 2 study of imexon (Amplimexon/NSC-714597), a prooxidant molecule, in patients with relapsed/refractory B-cell non-Hodgkin lymphoma (NHL). Imexon was administered at 1000 mg/m(2) IV daily for 5 days in 21-day cycles. Gene expression analysis performed on pretreatment tumor specimens included 13 transcripts used to generate a redox signature score, previously demonstrated to correlate with lymphoma prognosis. Twenty-two patients were enrolled having follicular (n = 9), diffuse large B-cell (DLBCL) (n = 5), mantle cell (n = 3), transformed follicular (n = 2), small lymphocytic (n = 2), and Burkitt (n = 1) lymphoma. The most common grade 3/4 adverse events were anemia (14%) and neutropenia (9%). The overall response rate was 30%, including responses in follicular lymphoma (4 of 9) and DLBCL (2 of 5). Gene expression analyses revealed CD68 and the redox-related genes, GPX1 and SOD2, as well as a higher redox score to correlate with clinical responses. Therefore, pretreatment markers of oxidative stress may identify patients likely to respond to this therapeutic approach. This trial was registered at www.clinicaltrials.gov as #NCT01314014.

The diaryl oxazole PC-046 is a tubulin-binding agent with experimental anti-tumor efficacy in hematologic cancers.

Microtubule targeting agents are among the most widely used chemotherapeutics for both solid and hematological malignancies. This study characterizes the diaryl-oxazole based anticancer agent PC-046, which was originally identified for development based on selective activity in deleted in pancreas cancer locus 4 (DPC4/SMAD4) deficient tumors. PC-046 has growth inhibitory activity in a variety of tumor types in vitro, and efficacy in SCID mice was shown in human tumor xenografts of MV-4-11 acute myeloid leukemia, MM.1S multiple myeloma, and DU-145 prostate cancer. Pharmacokinetic studies demonstrated relatively high oral bioavailability (71%) with distribution to both plasma and bone marrow. No myelosuppression was seen in non-tumor bearing SCID mice given a single dose just under the acute lethal dose. The COMPARE algorithm in the NCI-60 cell line panel demonstrated that PC-046 closely correlated to other known tubulin destabilizing agents (correlation coefficients ≈0.7 for vincristine and vinblastine). Mechanism of action studies showed cell cycle arrest in metaphase and inhibition of tubulin polymerization. Overall, these studies show that PC-046 is a synthetically-derived, small molecule microtubule destabilizing agent. Advantages over existing microtubule destabilizing agents include ease of synthesis, lack of MDR cross-resistance, good oral bioavailability and the lack of acute myelotoxicity.

Characterization of a membrane-active anti-tumor agent, UA8967.

Deletions or mutations in the tumor suppressor gene DPC4 (deleted in pancreatic carcinoma locus 4) are common in colon and pancreatic cancers. Using the Target-related Affinity Profiling (TRAP) chemical library screening method, a novel agent, UA8967, was selected for further studies because it showed greater potency in DPC4-deleted HCT-116 colon cancer cells. Cytotoxicity studies in six pancreatic cancer cell lines (MiaPaca-2, Panc-1, BxPC3, CF-PAC1, AsPC1, and T3M4), one normal human pancreatic ductal epithelial line (HPDE-6) and the HCT-116 DPC4(+/+) and HCT-116 DPC4(-/-) colon cancer cells showed IC50s ranging from 12-61 µM for exposure times of 72 h. Analysis of schedule dependence showed no advantage for long drug exposure times. There was also no selective inhibition of DNA, RNA or protein synthesis after exposure to UA8967. At 24-48 h, there was an accumulation of cells in G0/G1-phase and a proportionate reduction in S-phase cells. Within 1-6 h of exposure, cells were found to undergo an autophagic response, followed at 24 h by a low level of caspase-independent apoptosis with some necrosis. Because of the relatively non-specific mechanistic effects of UA8967, plasma membrane viability was evaluated using uptake of trypan blue and Sytox® Green dyes, and leakage of LDH. There was a dose dependent increase in Sytox® Green staining, trypan blue uptake and LDH leakage with increasing concentrations of UA8967, suggesting that UA8967 is affecting the plasma membrane. The DPC4(-/-) cells were more sensitive to UA8967 but not to DMSO, suggesting a drug-specific effect on cell membrane integrity.

High-Resolution Epitope Mapping and Affinity Binding Analysis Comparing a New Anti-Human LAG3 Rabbit Antibody Clone to the Commonly Used Mouse 17B4 Clone.

Lymphocyte activation gene 3 (LAG3) is a T cell inhibitory receptor that promotes tumor cell immune escape and is a potential target for cancer diagnostic and immunotherapeutic applications. We used automated capillary electrophoresis (ACE), surface plasmon resonance (SPR), and immunohistochemistry (IHC) to compare the binding characteristics of a new anti-LAG3 rabbit antibody clone, SP464, with the thirty-year old and extensively used anti-LAG3 mouse 17B4 clone. The rabbit SP464 clone exhibited between 20× to 30× greater binding to LAG3 than did the mouse 17B4 clone. Using these tools, we precisely mapped the relative locations of the epitopes of these two antibodies. The SP464 and 17B4 minimal epitopes were localized to separate, but overlapping, sub-fragments within the amino-terminal fifteen acids of the original thirty-mer peptide immunogen used to generate both antibodies. Application of this approach for quantifying the effects of alanine substitutions along the minimal SP464 epitope identified two amino acids essential for binding and four amino acids that likely contribute towards binding. Together, ACE, SPR, and IHC constitute a powerful orthologous approach for comparing antibody-binding characteristics and for fine mapping of linear epitopes within short immunogens. Our results indicate that the rabbit clone SP464 may be useful for assessing LAG3 expression.

A pilot clinical trial of the cytidine deaminase inhibitor tetrahydrouridine combined with decitabine to target DNMT1 in advanced, chemorefractory pancreatic cancer.

DNA methyltransferase 1 (DNMT1) is scientifically validated as a molecular target to treat chemo-resistant pancreatic ductal adenocarcinoma (PDAC). Results of clinical studies of the pyrimidine nucleoside analog decitabine to target DNMT1 in PDAC have, however, disappointed. One reason is high expression in PDAC of the enzyme cytidine deaminase (CDA), which catabolizes decitabine within minutes. We therefore added tetrahydrouridine (THU) to inhibit CDA with decitabine. In this pilot clinical trial, patients with advanced chemorefractory PDAC ingested oral THU ~10 mg/kg/day combined with oral decitabine ~0.2 mg/kg/day, for 5 consecutive days, then 2X/week. We treated 13 patients with extensively metastatic chemo-resistant PDAC, including 8 patients (62%) with ascites: all had received ≥ 1 prior therapies including gemcitabine/nab-paclitaxel in 9 (69%) and FOLFIRINOX in 12 (92%). Median time on THU/decitabine treatment was 35 days (range 4-63). The most frequent treatment-attributable adverse event was anemia (n=5). No deaths were attributed to THU/decitabine. Five patients had clinical progressive disease (PD) prior to week 8. Eight patients had week 8 evaluation scans: 1 had stable disease and 7 PD. Median overall survival was 3.1 months. Decitabine systemic exposure is expected to decrease neutrophil counts; however, neutropenia was unexpectedly mild. To identify reasons for limited systemic decitabine effect, we measured plasma CDA enzyme activity in PDAC patients, and found a > 10-fold increase in those with metastatic vs resectable PDAC. We concluded that CDA activity is increased not just locally but also systemically in metastatic PDAC, suggesting a need for even higher CDA-inhibitor doses than used here.

Ellipticine derivative NSC 338258 represents a potential new antineoplastic agent for the treatment of multiple myeloma.

High-risk multiple myeloma can be correlated with amplification and overexpression of the cell cycle regulator CKS1B. Herein, we used the COMPARE algorithm to correlate high expression of CKS1B mRNA in the NCI-60 cell line panel with the concentration causing 50% growth inhibition (GI(50)) of >40,000 synthetic compounds. This led to the identification of NSC 338258 (EPED3), a highly stable, hydrophilic derivative of the plant alkaloid ellipticine. In vitro, this synthetic anticancer compound exhibits dramatic cytotoxic activity against myeloma cells grown in suspension or in coculture with stromal cells. EPED3-induced cell cycle arrest and an apoptotic progression that appear to be a consequence of the instantaneous effect of the drug on cytoplasmic organelles, particularly mitochondria. Disruption of mitochondria and cytoplasmic distribution of cytochrome c initiated the intracellular proteolytic cascade through the intrinsic apoptotic pathway. EPED3 is able to induce apoptosis in myeloma cells with de novo or acquired resistance to commonly administered antimyeloma agents. Collectively, our data suggest that EPED3 targets mitochondrial function to rapidly deplete chemical energy and initiate apoptosis in myeloma cells at nanomolar concentrations while leaving stromal cells unharmed.

The antitumor agent imexon activates antioxidant gene expression: evidence for an oxidative stress response.

PURPOSE: The aim of this study was to identify biomarkers that may be predictive for the clinical activity of the redox-active antitumor agent imexon. EXPERIMENTAL DESIGN: cDNA microarray and quantitative real-time PCR were used to identify global changes in gene expression in peripheral blood mononuclear cells (PBMC) collected from patients treated with imexon during a phase I trial. Electrophoretic mobility shift assays and Western blot analysis were done using the RPMI8226 myeloma cell line grown in vitro and PBMCs treated ex vivo to investigate the molecular mechanism responsible for these gene changes. RESULTS: Both cDNA microarray and quantitative real-time PCR showed the up-regulation of many antioxidant genes, including thioredoxin reductase-1, glutaredoxin-2, and peroxiredoxin-3 in PBMCs collected from patients treated with imexon. Studies in PBMCs treated ex vivo and RPMI8226 myeloma cells showed that imexon increased binding to the activator protein-1 consensus sequence measured by electrophoretic mobility shift assay. Supershift analysis showed that the majority of the activator protein-1 DNA binding activity was c-Jun, with minor contribution of Jun-D. Nuclear translocation of the nuclear factor (erythroid-derived 1)-like 2 transcription factor and its binding to the antioxidant response element was also increased after imexon treatment, which correlated with an increase in the message levels for nuclear factor (erythroid-derived 1)-like 2/antioxidant response element-regulated antioxidant genes. CONCLUSIONS: Together, these results show that a predominant biological effect of imexon is a change in redox state that can be detected in surrogate normal tissues as increased redox-sensitive transcription factor binding and increased antioxidant gene expression.

Imexon-based combination chemotherapy in A375 human melanoma and RPMI 8226 human myeloma cell lines.

PURPOSE: This study evaluated the cytotoxic effects of imexon (NSC-714597) in tumor cells when combined with a broad panel of chemotherapeutic drugs. METHODS: The sulforhodamine B (SRB) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assays were used to analyze the degree of growth inhibition for the combination studies in the A375 human malignant melanoma and RPMI 8226 human multiple myeloma cell lines, respectively. Cells were continuously exposed to both drugs at a constant molar ratio for 4-5 days. Combination effects were analyzed using the Median Effect method. Statistical significance was inferred if the 95% confidence interval for the combination interaction (C.I.) values for a particular two-drug combination did not include 1.0 (additivity). Synergy was inferred for C.I. values<1.0 and antagonism for CI values>1.0. RESULTS: Imexon was synergistic when combined with DNA-binding agents (cisplatin, dacarbazine, melphalan) and pyrimidine-based antimetabolites (cytarabine, fluorouracil, gemcitabine) in both cell lines. Antagonistic combinations with imexon included methotrexate and the topoisomerase I (TOPO I) and II (TOPO II) inhibitors irinotecan, doxorubicin, mitoxantrone and etoposide. Docetaxel was synergistic with imexon in both cell lines whereas paclitaxel and fludarabine showed a mixed result. Dexamethasone and the proteasome inhibitor bortezomib showed synergy in myeloma cells and additivity in the melanoma cells. The vinca alkaloid, vinorelbine, and the multi-targeted antifol, pemetrexed, were additive with imexon in both cell lines. DISCUSSION: The consistent synergy seen for imexon and alkylating agents may relate to the sulfhydryl-lowering effect of imexon, which would render cells more sensitive to electrophilic species from the alkylators. The marked synergy noted with pyrimidine-based antimetabolites was unexpected and may relate to the induction of cell cycle arrest in S-phase. The strong antagonism noted for imexon with topoisomerase I and II inhibitors may be due to the effect of imexon at increasing oxidant levels which are known to antagonize the cytotoxic effects of topoisomerase poisons. In contrast, the synergy seen with bortezomib in myeloma cells may be related to an increase in reactive oxygen species (ROS) from both drugs. These results suggest that combinations of imexon with alkylating agents and pyrimidine-based antimetabolites are rational to pursue in therapeutic studies in vivo.

Mitochondrial-mediated disregulation of Ca2+ is a critical determinant of Velcade (PS-341/bortezomib) cytotoxicity in myeloma cell lines.

The proteasome inhibitor bortezomib (also known as PS-341/Velcade) is a dipeptidyl boronic acid that has recently been approved for use in patients with multiple myeloma. Bortezomib inhibits the activity of the 26S proteasome and induces cell death in a variety of tumor cells; however, the mechanism of cytotoxicity is not well understood. In this report, oligonucleotide microarray analysis of the 8226 multiple myeloma cell line showed a predominant induction of gene products associated with the endoplasmic reticulum secretory pathway following short-term, high-dose exposure to bortezomib. Examination of mediators of endoplasmic reticulum stress-induced cell death showed specific activation of caspase 12, as well as of caspases 8, 9, 7, and 3, and cleavage of bid. Treatment of myeloma cells with bortezomib also showed disregulation of intracellular Ca2+ as a mechanism of caspase activation. Cotreatment with a panel of Ca2+-modulating agents identified the mitochondrial uniporter as a critical regulatory factor in bortezomib cytotoxicity. The uniporter inhibitors ruthenium red and Ru360 prevented caspase activation and bid cleavage, and almost entirely inhibited bortezomib-induced cell death, but had no effect on any other chemotherapeutic drug examined. Additional Ca2+-modulating agents, including 2-amino-ethoxydiphenylborate, 1,2-bis (o-aminophenoxy) ethane-tretraacetic acid (acetoxymethyl) ester, and dantrolene, did not alter bortezomib cytotoxicity. Analysis of intracellular Ca2+ showed that the ruthenium-containing compounds inhibited Ca2+ store loading and abrogated the desensitized capacitative calcium influx associated with bortezomib treatment. These data support the hypothesis that intracellular Ca2+ disregulation is a critical determinant of bortezomib cytotoxicity.

Correlates of imexon sensitivity in human multiple myeloma cell lines.

Imexon (NSC-714597) is an aziridine-containing imminopyrolidone in Phase I clinical trials. The current studies compared biological indices of cytotoxicity in 7 human multiple myeloma (MM) cell lines to develop a correlative model for imexon sensitivity. In the MM cell lines there was a wide range of sensitivity to imexon measured by standard cytotoxicity assays (MTT) and by viability/apoptosis/necrosis (Annexin-V-FITC/PI) measurements. The following sensitivity pattern was observed in order of decreasing sensitivity: IM-9 > 8226/S > MM.1S, ARH-77, H929 > 8226/I > U266. The same descending rank order was seen for loss of mitochondrial membrane potential (MMP), generation of reactive oxygen species (ROS) and, at high drug concentrations, thiol depletion. Cell cycle analysis showed imexon sensitive cells accumulate at the G2/M interphase. Although there was a positive correlation between increasing CuZnSOD levels and imexon resistance, no relationship was found for catalase, Bcl-2, mitochondrial thioredoxin or MnSOD levels. These findings suggest consistent phenotypes for imexon sensitivity and resistance in human MM cell lines exposed to drug for 48 h, with a combination of apoptosis and necrosis. Resistance is correlated with CuZnSOD expression, reduced drug accumulation, lack of ROS generation and maintenance of MMP. Oxidation of cellular thiols occurs only at high (supra-cytotoxic) drug levels and is, therefore, weakly correlated with cytotoxicity. This unique mechanism involving oxidation and the previously reported absence of myelosuppression suggests that imexon may be rationally combined with existing cytotoxic agents to improve therapeutic activity in MM.

Magnetic Resonance Imaging Identifies Differential Response to Pro-Oxidant Chemotherapy in a Xenograft Model.

Induction of oxidative stress is a key component of cancer therapy. Pro-oxidant drugs have been demonstrated to enhance the efficacy of radiotherapy and chemotherapy. An emerging concept is that therapeutic outcomes are dictated by the differential redox buffering reserve in subpopulations of malignant cells, indicating the need for noninvasive biomarkers of tumor redox that can be used for dose identification and response assessment in a longitudinal setting. Magnetic resonance imaging (MRI) enhanced with the thiol-binding contrast agent Gd-LC6-SH, and hemodynamic response imaging (HRI) in combination with hypercapnia and hyperoxia were investigated as biomarkers of the pharmacodynamics of the small molecule pro-oxidant imexon (IMX). Human multiple myeloma cell lines 8226/S and an IMX-resistant variant, 8226/IM10, were established as contralateral tumors in SCID mice. T1slope, an MRI measure of the washout rate of Gd-LC6-SH, was significantly lower post-IMX therapy in 8226/S tumors compared with vehicle controls, indicating treatment-related oxidization of the tumor microenvironment, which was confirmed by analysis of tumor tissue for thiols. T1slope and ex vivo assays for thiols both indicated a more reduced microenvironment in 8226/IM10 tumors following IMX therapy. HRI with hypercapnia challenge revealed IMX inhibition of vascular dilation in 8226/S tumors but not 8226/IM10 tumors, consistent with decreased immunohistochemical staining for smooth muscle actin in treated 8226/S tumors. MRI enhanced with Gd-LC6-SH, and HRI coupled with a hypercapnic challenge provide noninvasive biomarkers of tumor response to the redox modulator imexon.

Cell adhesion-mediated drug resistance (CAM-DR) is associated with activation of NF-kappa B (RelB/p50) in myeloma cells.

The microenvironment has been shown to influence tumor cell phenotype with respect to growth, metastasis, and response to chemotherapy. We have utilized oligonucleotide microarray analysis to identify signal transduction pathways and gene products altered by the interaction of myeloma tumor cells with the extracellular matrix component fibronectin that may contribute to the antiapoptotic phenotype conferred by the microenvironment. Genes with altered expression associated with fibronectin cell adhesion, either induced or repressed, were numerically ranked by fold change. FN adhesion repressed the expression of 469 gene products, while 53 genes with known coding sequences were induced by twofold or more. Of these 53 genes with two fold, or greater increase in expression, 11 have been reported to be regulated by the nuclear factor-kappa B (NF-kappa B) family of transcription factors. EMSA analysis demonstrated NF-kappa B binding activity significantly increased in cells adhered to fibronectin compared to cells in suspension. This DNA binding activity consisted primarily of RelB-p50 heterodimers, which was distinct from the NF-kappa B activation of TNF alpha. These data demonstrate the selectivity of signal transduction from the microenvironment that may contribute to tumor cell resistance to programmed cell death.

Role of the tumor microenvironment in mediating de novo resistance to drugs and physiological mediators of cell death.

The emergence of clinical drug resistance continues to be an obstacle for the successful treatment of cancer. Our current understanding of mechanisms associated with drug resistance has been ascertained by investigating drug-resistant models created by exposing a parental population to increasing concentrations of a cytotoxic. These unicellular drug-resistant models have been critical in elucidating drug-resistant mechanism and in some cases have aided in the identification of drug targets. However, these models do not address resistance mechanisms that contribute to de novo drug resistance. We propose that specific niches within the tumor microenvironment may provide a sanctuary for subpopulations of tumors cells that affords a survival advantage following initial drug exposure and may facilitate the acquisition of acquired drug resistance. More specifically, we propose that the bone marrow microenvironment is a sanctuary for hema-topoietic cancers. This review will focus on the bone marrow microenvironment and its role in conferring resistance to cytotoxics and physiological mediators of cell death.

Inhibition of Bcr-Abl kinase activity by PD180970 blocks constitutive activation of Stat5 and growth of CML cells.

Chronic myelogenous leukemia (CML) is a myeloproliferative disease characterized by the BCR-ABL genetic translocation and constitutive activation of the Abl tyrosine kinase. Among members of the Signal Transducers and Activators of Transcription (STAT) family of transcription factors, Stat5 is activated by the Bcr-Abl kinase and is implicated in the pathogenesis of CML. We recently identified PD180970 as a new and highly potent inhibitor of Bcr-Abl kinase. In this study, we show that blocking Bcr-Abl kinase activity using PD180970 in the human K562 CML cell line resulted in inhibition of Stat5 DNA-binding activity with an IC(50) of 5 nM. Furthermore, abrogation of Abl kinase-mediated Stat5 activation suppressed cell proliferation and induced apoptosis in K562 cells, but not in the Bcr-Abl-negative myeloid cell lines, HEL 92.1.7 and HL-60. Dominant-negative Stat5 protein expressed from a vaccinia virus vector also induced apoptosis of K562 cells, consistent with earlier studies that demonstrated an essential role of Stat5 signaling in growth and survival of CML cells. RNA and protein analyses revealed several candidate target genes of Stat5, including Bcl-x, Mcl-1, c-Myc and cyclin D2, which were down-regulated after treatment with PD180970. In addition, PD180970 inhibited Stat5 DNA-binding activity in cultured primary leukemic cells derived from CML patients. To detect activated Stat5 in CML patient specimens, we developed an immunocytochemical assay that can be used as a molecular end-point assay to monitor inhibition of Bcr-Abl signaling. Moreover, PD180970 blocked Stat5 signaling and induced apoptosis of STI-571 (Gleevec, Imatinib)-resistant Bcr-Abl-positive cells. Together, these results suggest that the mechanism of action of PD180970 involves inhibition of Bcr-Abl-mediated Stat5 signaling and provide further evidence that compounds in this structural class may represent potential therapeutic agents for CML.

Induction of apoptosis and cell cycle arrest by imexon in human pancreatic cancer cell lines.

Imexon is an aziridine-containing small molecule currently in Phase I clinical trials. This agent has been shown to bind to thiols and increase intracellular oxidants, inducing apoptosis in hematologic cancer cells. Pancreatic cancers are known to be sensitive to oxidation, suggesting this disease may be an appropriate target for this agent. The current report examines the activity of imexon in pancreatic cells. Imexon induced concentration-dependent and time-dependent apoptosis in a panel of six human pancreatic carcinoma cell (PCC) lines. The mean IC50 (SD) for growth inhibition by the SRB assay was 200 (101) microM for a 48 h exposure with a range of 64-358 microM. Cell killing was schedule-dependent, favoring exposure times > or =48 h. Imexon-treated MiaPaCa-2 cells underwent non-lethal growth arrest following exposure to concentrations < or =200 microM for 48 h. When concentrations were increased to 300 microM for > or =48 h, the MiaPaCa-2 cells arrested in G2 phase and activated caspases 3, 8, and 9 were detected. After a 72 h exposure to the IC80 concentration of imexon, cells exhibited a loss of mitochondrial membrane potential detected by CMXRos staining. However, there was no loss of reduced cellular thiols unless very high concentrations of > or =400 microM were used. In contrast, reactive oxygen species (ROS) were elevated in a dose-dependent fashion, starting at very low imexon concentrations. Imexon also significantly inhibited MiaPaCa-2 tumor growth in SCID mice at 100 mg/kg/d for 9 d. The tumor growth inhibition (% T/C) was 27% of control, and the tumor growth delay was 21 d, indicating an active agent by NCI standards. The levels of imexon that are cytotoxic in human PCC's are achievable based on the preliminary results of the ongoing Phase I trial. Imexon appears to be active against PCCs in vitro and has an entirely novel mechanism of action involving G2 arrest, accumulation of ROS, and the induction of apoptosis.

Gemcitabine resistant pancreatic cancer cell lines acquire an invasive phenotype with collateral hypersensitivity to histone deacetylase inhibitors.

Gemcitabine based treatment is currently a standard first line treatment for patients with advanced pancreatic cancer, however overall survival remains poor, and few options are available for patients that fail gemcitabine based therapy. To identify potential molecular targets in gemcitabine refractory pancreatic cancer, we developed a series of gemcitabine resistant (GR) cell lines. Initial drug exposure selected for an early resistant phenotype that was independent of drug metabolic pathways. Prolonged drug selection pressure after 16 weeks, led to an induction of cytidine deaminase (CDA) and enhanced drug detoxification. Cross resistance profiles demonstrate approximately 100-fold cross resistance to the pyrimidine nucleoside cytarabine, but no resistance to the same in class agents, azacytidine and decitabine. GR cell lines demonstrated a dose dependent collateral hypersensitivity to class I and II histone deacetylase (HDAC) inhibitors and decreased expression of 3 different global heterochromatin marks, as detected by H4K20me3, H3K9me3 and H3K27me3. Cell morphology of the drug resistant cell lines demonstrated a fibroblastic type appearance with loss of cell-cell junctions and an altered microarray expression pattern, using Gene Ontology (GO) annotation, consistent with progression to an invasive phenotype. Of particular note, the gemcitabine resistant cell lines displayed up to a 15 fold increase in invasive potential that directly correlates with the level of gemcitabine resistance. These findings suggest a mechanistic relationship between chemoresistance and metastatic potential in pancreatic carcinoma and provide evidence for molecular pathways that may be exploited to develop therapeutic strategies for refractory pancreatic cancer.

Combined micro CT and histopathology for evaluation of skeletal metastasis in live animals.

Bone is a favored site for solid tumor metastasis, especially among patients with breast, lung or prostate carcinomas. Micro CT is a powerful and inexpensive tool that can be used to investigate tumor progression in xenograft models of human disease. Many previous studies have relied on terminal analysis of harvested bones to document metastatic tumor activity. The current protocol uses live animals and combines sequential micro CT evaluation of lesion development with matched histopathology at the end of the study. The approach allows for both rapid detection and evaluation of bone lesion progression in live animals. Bone resident tumors are established either by direct (intraosseous) or arterial (intracardiac) injection, and lesion development is evaluated for up to eight weeks. This protocol provides a clinically relevant method for investigating bone metastasis progression and the development of osteotropic therapeutic strategies for the treatment of bone metastases.

Targeting the bone marrow microenvironment in hematologic malignancies.

Unicellular drug-resistant models have been critical in elucidating intrinsic drug-resistant mechanisms; however, these models do not consider resistance mechanisms that may be elicited by extrinsic influences such as the tumor microenvironment. We propose that specific niches within the tumor microenvironment may provide a sanctuary for subpopulations of tumor cells to evade or circumvent drug-induced death and that this may represent a form of de novo drug resistance. We have found that elements of the bone marrow microenvironment, including extracellular matrices and normal stromal elements, protect malignant cells, including leukemia and myeloma cells, from drug-induced cell death. This extrinsic form of drug resistance may allow cells to survive initial drug treatment and thereby acquire a more complex, intrinsic drug-resistant phenotype. Focusing on this form of de novo drug resistance may ultimately prevent the emergence of acquired drug resistance and enhance drug therapy for hematologic malignancies.

Targeting integrin α6 stimulates curative-type bone metastasis lesions in a xenograft model.

Laminin-binding integrin receptors are key mediators of epithelial cell migration and tumor metastasis. Recent studies have demonstrated a role for the α6 integrin (ITGA6/CD49f) in maintaining stem cell compartments within normal bone marrow and in residency of tumors metastatic to bone. In this study, we tested a function-blocking antibody specific for ITGA6, called J8H, to determine if preexisting cancer lesions in bone could be slowed and/or animal survival improved. Human prostate tumors were established by intracardiac injection into male SCID mice and treatment with J8H antibody was initiated after 1 week. Tumor progression was monitored by micro-computed tomography (CT) imaging of skeletal lesions. Animals that received weekly injections of the anti-ITGA6 antibody showed radiographic progression in only 40% of osseous tumors (femur or tibia), compared with control animals, where 80% of the lesions (femur or tibia) showed progression at 5 weeks. Kaplan-Meier survival analysis demonstrated a significant survival advantage for J8H-treated animals. Unexpectedly, CT image analysis revealed an increased proportion of bone lesions displaying a sclerotic rim of new bone formation, encapsulating the arrested lytic lesions in animals that received the anti-ITGA6 antibody treatment. Histopathology of the sclerotic lesions demonstrated well-circumscribed tumor within bone, surrounded by fibrosis. These data suggest that systemic targeting of the ITGA6-dependent function of established tumors in bone may offer a noncytotoxic approach to arrest the osteolytic progression of metastatic prostate cancer, thereby providing a new therapeutic strategy for advanced disease.

Preventing the autophagic survival response by inhibition of calpain enhances the cytotoxic activity of bortezomib in vitro and in vivo.

PURPOSE: Bortezomib, a first-generation proteasome inhibitor, induces an endoplasmic reticulum (ER) stress response, which ultimately leads to dysregulation of intracellular Ca(2+) and apoptotic cell death. This study investigated the role of the Ca(2+)-dependent enzyme, calpain, in bortezomib cytotoxicity. A novel therapeutic combination was evaluated in which HIV protease inhibitors were used to block calpain activity and enhance bortezomib cytotoxicity in myeloma cells in vitro and in vivo. METHODS: Bortezomib-mediated cell death was examined using assays for apoptosis (Annexin V staining), total cell death (trypan blue exclusion), and growth inhibition (MTT). The effects of calpain on bortezomib-induced cytotoxicity were investigated using siRNA knockdown or pharmaceutical inhibitors. Enzyme activity assays and immunofluorescence analysis were used to identify mechanistic effects. RESULTS: Inhibition of the Ca(2+)-dependent cysteine protease calpain, either by pharmacologic or genetic means, enhances or accelerates bortezomib-induced myeloma cell death. The increase in cell death is not associated with an increase in caspase activity, nor is there evidence of greater inhibition of proteasome activity, suggesting an alternate, calpain-regulated mechanism of bortezomib-induced cell death. Bortezomib initiates an autophagic response in myeloma cells associated with cell survival. Inhibition of calpain subverts the cytoprotective function of autophagy leading to increased bortezomib-mediated cell death. Combination therapy with bortezomib and the calpain-blocking HIV protease inhibitor, nelfinavir, reversed bortezomib resistance and induced near-complete tumor regressions in an SCID mouse xenograft model of myeloma.