Broad Antitumor Activity Research Articles

Abstract 5516: Biodistribution of liposomal docetaxel prodrug MNK-010 in mice bearing A549 human NSCLC xenografts

Abstract The anti-mitotic taxanes belong to one of the most important families of anticancer drugs that have broad anti-tumor activity and are widely used for a variety of human cancer types. However, the taxanes are limited by a number of serious pharmacological and toxicological effects. We are developing a unique delivery system for docetaxel, comprised of a PEGylated liposomal nanoparticle containing a prodrug of docetaxel to improve solubility, tolerability and increase efficacy through improved pharmacokinetics and biodistribution. In this study we evaluated the biodistribution of MNK-010, a liposomal formulation of a docetaxel prodrug in immunodeficient mice bearing A549 Human Non-Small Cell Lung Carcinoma (NSCLC) xenografts. This study tested the hypothesis that MNK-010 can produce greater plasma and tumor accumulation of docetaxel than with delivery of standard of care free docetaxel in tumor-bearing mice. Female nude mice bearing A549 human NSCLC xenografts were given a single intravenous (IV) dose of MNK-010 (40 mg/kg docetaxel molar equivalent) or free docetaxel (50 mg/kg). For each treatment group, three animals were sacrificed at various time points between 1 hr and 504 hr post injection. After euthanasia, blood, tumor, liver, kidney, spleen, lung and skeletal muscle specimens were collected. Tissue and blood samples were assayed to determine docetaxel concentrations. Administration of MNK-010 IV provided a reservoir for the continual slow release of docetaxel in the circulation and in tumors. MNK-010 increased systemic docetaxel exposure (AUC) ten-fold greater than free docetaxel injected at similar doses. MNK-010 produced sustained docetaxel levels in tumor through the 21 day observation period, resulting in four-fold greater docetaxel tumor exposure (AUC) compared to free docetaxel. The increase in MNK-010 plasma exposure as well as the enhanced permeability effect observed with liposomes likely contributed to the increased tumor exposure. In addition to improved accumulating in tumor tissue, docetaxel derived from MNK-010 also accumulated in liver, spleen and kidney. These tissues contain phagocytic cells (macrophages or mesangial) and serve as organs of elimination for chemotherapeutics. Docetaxel derived from MNK-010 was not detectable in skeletal muscle and was only found in lung over the first 24 hours post administration. In contrast, IV injection of free docetaxel produced high initial concentrations of docetaxel in all tissues evaluated, before falling quickly over time. Tumor levels decreased below levels of quantitation after nine days. This study demonstrated that Mallinckrodt Pharmaceuticals’ PEGylated liposomal docetaxel prodrug MNK-010 provided a reservoir for the continual release of docetaxel into the systemic circulation and produced an enhanced accumulation of docetaxel in A549 human NSCLC xenografts in mice compared to the standard of care docetaxel. Citation Format: Richard M. Fitch, James H. Wible, James A. Blackledge, William D. McGhee. Biodistribution of liposomal docetaxel prodrug MNK-010 in mice bearing A549 human NSCLC xenografts. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5516. doi:10.1158/1538-7445.AM2015-5516

Abstract 4472: Plitidepsin shows antitumor activity in patient-derived tumor xenografts and hematologic malignancies

Abstract Plitidepsin is a new marine-derived anti-tumor agent, originally isolated from the tunicate Aplidium albicans that is now fully obtained by total chemical synthesis. The compound is currently being evaluated in a pivotal phase III trial in patients with refractory/relapsed multiple myeloma. Previous in vitro studies in 26 hematological cell lines demonstrated that plitidepsin was more active in multiple myeloma and non-Hodgkin lymphoma compared to CML, AML and ALL derived cell lines as determined in a 4 day proliferation assay in suspension cultures. We have recently shown that plitidepsin interacts selectively with eEF1A2 (eukaryotic translation elongation factor 1 alpha 2) in tumor cells, which results in apoptotic cell death. To identify target tumor types for future clinical studies, the inhibition of colony formation of plitidepsin was assessed in vitro in 116 established patient derived xenografts tumor models from 23 different tumor types, including solid tumors and hematological malignancies such as myeloma, leukemia and lymphoma. The anti-tumor activity of plitidepsin varied depending on the concentration used, achieving a mean IC70 (inhibitory concentration of 70%) value of 3 nM (range between 0.03 nM and >1 μM), which can be also achieved in patients treated using the clinical recommended dose or less. 39 out of 116 tumors showed above average sensitivity (IC70 < 1nM) and IC70 values in these tumors were about 9-fold lower than the mean of all tumors as tested. Sensitive tumor types were seen among different tumor types, such as hematological malignancies, pleuramesothelioma, melanoma, small cell and non-small cell lung cancer (both adeno and large cell), as well as renal cancer. In conclusion plitidepsin showed a broad differential antitumor activity in vitro in hematological malignancies and in solid tumors. Bioinformatic analyses are currently underway to identify biomarkers and/or gene signatures that can predict sensitivity or resistance to plitidepsin for selecting patients being responsive to the drug. Citation Format: Armin Maier, Gerhard Kelter, Vincent Vuaroquaux, Pablo M. Avilés Marin, Carmen Cuevas, Carlos M. Galmarini, Heinz H. Fiebig. Plitidepsin shows antitumor activity in patient-derived tumor xenografts and hematologic malignancies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4472. doi:10.1158/1538-7445.AM2015-4472

Synthesis and potential antitumor activity of 7-(4-substituted piperazin-1-yl)-4-oxoquinolines based on ciprofloxacin and norfloxacin scaffolds: in silico studies

The potential antitumor activities of a series of 7-(4-substituted piperazin-1-yl)fluoroquinolone derivatives (1–14a,b) using ciprofloxacin and norfloxacin as scaffolds are described. These compounds exhibit potent and broad spectrum antitumor activities using 60 human cell lines in addition to the inherent antibacterial activity. Compounds 1a, 2a, 3b, 6b and 7a were found to be the most potent, while 2b, 5b, and 6a were found to have an average activity. The results of this study demonstrated that compounds 1a, 2a, 3b, 6b and 7a (mean GI50; 2.63–3.09 µM) are nearly 7-fold more potent compared with the positive control 5-fluorouracil (mean GI50; 22.60 µM). More interestingly, compounds 1a, 2a, 3b, 6b and 7a have an almost antitumor activity similar to gefitinib (mean GI50; 3.24 µM) and are nearly 2-fold more potent compared to erlotinib (mean GI50; 7.29 µM). In silico study and ADME-Tox prediction methodology were used to study the antitumor activity of the most active compounds and to identify the structural features required for antitumor activity.

Broad Anti-tumor Activity of a Small Molecule that Selectively Targets the Warburg Effect and Lipogenesis

Malignant cells exhibit aerobic glycolysis (the Warburg effect) and become dependent on de novo lipogenesis, which sustains rapid proliferation and resistance to cellular stress. The nuclear receptor liver-X-receptor (LXR) directly regulates expression of key glycolytic and lipogenic genes. To disrupt these oncogenic metabolism pathways, we designed an LXR inverse agonist SR9243 that induces LXR-corepressor interaction. Incancer cells, SR9243 significantly inhibited the Warburg effect and lipogenesis by reducing glycolytic and lipogenic gene expression. SR9243 induced apoptosis in tumors without inducing weight loss, hepatotoxicity, or inflammation. Our results suggest that LXR inverse agonists may be an effective cancer treatment approach.

Shikonin inhibits TNF-α-induced growth and invasion of rat aortic vascular smooth muscle cells.

Shikonin is a naphthoquinone compound extracted from the Chinese herb purple gromwell. Shikonin has broad antibacterial, anti-inflammatory, and antitumor activities. The tumor necrosis factor-α (TNF-α)-induced proliferation and invasion of vascular smooth muscle cells (VSMCs) is an important factor that contributes to atherosclerosis. The effects of shikonin on the proliferation and apoptosis of VSMCs have been reported; however, the function of shikonin on TNF-α-mediated growth and invasion of VSMCs during atherosclerosis remains unclear. In this study, we used Western blot, flow cytometry, real-time quantitative PCR, and enzyme-linked immunosorbent assay to investigate the effect of shikonin on the TNF-α-induced growth and invasion of VSMCs and to determine the underlying mechanism. Our results showed that shikonin inhibits the TNF-α-mediated growth and invasion. Further study revealed that shikonin regulates the activation of nuclear factor kappa B and phosphatidyl inositol 3-kinase signaling pathways; modulates the expression of cyclin D1, cyclin E, B-cell lymphoma 2, and Bax; activates caspase-3 and caspase-9; induces cell cycle arrest; and promotes the apoptosis of VSMCs. Together, our results indicate that shikonin may become a promising agent for the treatment of atherosclerosis and they also establish foundation for the development of anti-atherosclerosis drugs.

A phase I, dose-escalation, multicenter study of ACT-PFK-158, 2HCl in patients with advanced solid malignancies explores a first-in-human inhibitor of glycolysis.

TPS494 Background: In human cancers, loss of PTEN, stabilization of hypoxia inducible factor-1α, and activation of Ras and AKT converge to increase the activity of a regulator of glycolysis, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3). This enzyme synthesizes fructose-2,6-bisphosphate (F2,6BP), which is an activator of 6-phosphofructo-1-kinase, a crucial step of glycolysis that is tightly controlled by multiple metabolic feedback mechanisms and dictates the rate of glycolytic flow. The vast majority of pancreatic ductal adenocarcinomas and approximately 50% of colon adenocarcinomas harbor activating mutations in Ras and these tumors have been reported to be highly glycolytic. PFK158 is a potent small molecule inhibitor of PFKFB3 that is selectively cytotoxic to Ras-transformed epithelial cells and displays broad anti-tumor activity causing ~80% growth inhibition in several mouse models of human-derived tumors and syngeneic murine models of colon cancer. Importantly, IND-enabling safety and toxicity studies have demonstrated that PFK158 is well tolerated in rats and dogs with an expected good therapeutic index, lending support for a phase 1 trial that is now underway. Methods: The primary objective of the study is to describe the dose limiting toxicity and to determine either the maximum tolerated dose or biological effective dose of PFK-158 in a “3+3” cohort-based dose escalation design that follows a modified Fibonacci scheme. Multiple secondary endpoints have been incorporated to assess the effects of PFK-158 on peripheral blood mononuclear cell F2,6BP activity and on glucose uptake using FDG-PET imaging. This trial is currently enrolling at two US sites; Cohort 1 has been completed without dose-limiting toxicity and Cohort 2 is enrolling with two subjects under treatment as of September 2014. In conclusion, PFK158 is the first-in-man and first-in-class PFKFB3 inhibitor to be examined in a phase I trial and may have significant clinical utility either as a monotherapy or when combined with other targeted agents. Clinical trial information: NCT02044861.

Broad Anti-tumor Activity of a Small Molecule that Selectively Targets the Warburg Effect and Lipogenesis

Broad Anti-tumor Activity of a Small Molecule that Selectively Targets the Warburg Effect and Lipogenesis

SYK and STAT3 Are Active in Diffuse Large B-Cell Lymphoma: Activity of Cerdulatinib, a Dual SYK/JAK Inhibitor

Non-Hodgkin Lymphoma (NHL) represents about 5 percent of all cancers diagnosed in the United States. While incidence of NHL has increased slightly over the past decade, death rates have been declining steadily. These declines in mortality can be attributed to improvements in treatment that are based on an increased understanding of the biology of the disease. Diffuse large B-cell lymphoma (DLBCL) accounts for ~30% of NHLs and greater than 80% of aggressive NHLs. Recent studies including large-scale genetic analyses have demonstrated the critical roles of the B-cell receptor’s (BCR) and JAK/STAT pathways in DLBCL. Herein, we investigated the anti-lymphoma activity of cerdulatinib (aka PRT062070), a novel compound that dually targets both SYK and JAK/STAT signaling pathways.To determine whether targeting both SYK and JAK/STAT is relevant in DLBCL, we examined the expression of p-SYK (pY525/526) and p-STAT3 (pY705) on a tissue microarray of 62 DLBCL primary tumors, including 41 GCB and 21 non-GCB cases. p-SYK expression was detected in 29 (47%) cases with a characteristic peri-membrane staining pattern. Of those 29 p-SYK positive cases, 17 were GCB type (17/41, 41%) and 12 were non-GCB type (12/21, 57%). p-STAT3 exhibits a characteristic nuclear staining pattern in DLBCL cases. A total of 26 (42%) stained positive for p-STAT3; 16 were GCB type (16/41, 39%) and 10 were non-GCB type (10/21, 48%). Interestingly, there are 19 cases (31%) with reactivity for both p-SYK and p-STAT3, among which, 11 were GCB type (27%) and 8 were non-GCB type (38%).SYK and STAT3 are also phosphorylated in a panel of nine DLBCL cell lines. Immunoblotting analyses showed that ABC and GCB subtypes of DLBCL cells appear to exhibit different JAK/STAT and BCR signaling profiles. For instance, p-AKT was highly expressed in GCB cells, whereas p-STAT3 was more strongly expressed in ABC cells. Overall, the DLBCL cells are more sensitive to the dual inhibitor than to the SYK-specific inhibitor alone. In both GCB and ABC cell lines, cerdulatinib induced apoptosis via down-regulation of MCL1 protein and PARP cleavage. The compound also blocked G1/S transition and caused cell cycle arrest through inhibition of RB phosphorylation and down-regulation of cyclin E. Further analyses of the cell signaling activities showed that STAT3 phosphorylation was sensitive to inhibition by cerdulatinib in ABC cell lines while phosphorylation of SYK, PLCg2, AKT and ERK was sensitive to inhibition by cerdulatinib in GCB cell lines. Importantly, JAK/STAT and BCR signaling can be blocked by cerdulatinib in GCB and non-GCB primary human DLBCL cells, which led to cell death of these cells. Our work provided mechanistic insights into the actions of SYK/JAK dual inhibitor cerdulatinib, suggesting that the drug may be a potent treatment of DLBCL with a broader anti-tumor activity in both ABC and GCB subtypes of the lymphoma. DisclosuresPandey:Portola Pharmaceuticals: Employment. Conley:Portola Pharmaceuticals: Employment. Coffey:Portola Pharmaceuticals: Employment.

Antitumor evaluation and molecular docking study of substituted 2-benzylidenebutane-1,3-dione, 2-hydrazonobutane-1,3-dione and trifluoromethyl-1H-pyrazole analogues

A series of 2-(arylidene)-1-(4-chlorophenyl)-4,4,4-trifluorobutane-1,3-diones (2–4), 4-(arylidene)-3-(4-chlorophenyl)-5-(trifluoromethyl)-4H-pyrazoles (5–7), 1-(4-chlorophenyl)-4,4,4-trifluoro-2-(2-(aryl)hydrazono)butane-1,3-diones (8, 9), 3-(4-chlorophenyl)-4-(2-(aryl)hydrazono)-5-(trifluoromethyl)-4H-pyrazoles (10, 11), 2-((3-(4-chlorophenyl)-1-phenyl-5-(trifluoromethyl)-1H-pyrazol-4-yl)methylene)malononitrile (13), 2-((5-(4-chlorophenyl)-1-phenyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)methylene)cycloalkan-1-ones (14, 15) and 1-(aryl)-3-(5-(4-chlorophenyl)-1-phenyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)prop-2-en-1-ones (16, 17) were designed, synthesized and evaluated for their in vitro antitumor activity. 1-(4-Chlorophenyl)-4,4,4-trifluoro-2-(2-(4-methoxyphenyl)hydrazono)butane-1,3-dione (8) showed potential and broad spectrum antitumor activity compared to the known drug 5-FU with GI50, (6.61 and 22.60 µM), TGI (42.66 and <100 µM) and LC50 (93.33 and <100 µM) values, respectively. On the other hand, compound 8 yielded selective activities toward melanoma, colon, non-small lung and breast cancer cell lines compared with erlotinib and gefitinib. Molecular docking methodology was performed for compound 8 into binding site of B-RAFV600E and EGFR kinases which showed similar binding mode to vemurafenib (PLX4032) and erlotinib, respectively.

Carfilzomib demonstrates broad anti-tumor activity in pre-clinical non-small cell and small cell lung cancer models.

BackgroundCarfilzomib (CFZ) is a proteasome inhibitor that selectively and irreversibly binds to its target and has been approved in the US for treatment of relapsed and refractory multiple myeloma. Phase 1B studies of CFZ reported signals of clinical activity in solid tumors, including small cell lung cancer (SCLC). The aim of this study was to investigate the activity of CFZ in lung cancer models.MethodsA diverse panel of human lung cancer cell lines and a SHP77 small cell lung cancer xenograft model were used to investigate the anti-tumor activity of CFZ.ResultsCFZ treatment inhibited both the constitutive proteasome and the immunoproteasome in lung cancer cell lines. CFZ had marked anti-proliferative activity in A549, H1993, H520, H460, and H1299 non-small cell lung cancer (NSCLC) cell lines, with IC50 values after 96 hour exposure from <1.0 nM to 36 nM. CFZ had more variable effects in the SHP77 and DMS114 SCLC cell lines, with IC50 values at 96 hours from <1 nM to 203 nM. Western blot analysis of CFZ-treated H1993 and SHP77 cells showed cleavage of poly ADP ribose polymerase (PARP) and caspase-3, indicative of apoptosis, and induction of microtubule-associated protein-1 light chain-3B (LC3B), indicative of autophagy. In SHP77 flank xenograft tumors, CFZ monotherapy inhibited tumor growth and prolonged survival, while no additive or synergistic anti-tumor efficacy was observed for CFZ + cisplatin (CDDP).ConclusionsCFZ demonstrated anti-proliferative activity in lung cancer cell lines in vitro and resulted in a significant survival advantage in mice with SHP77 SCLC xenografts, supporting further pre-clinical and clinical investigations of CFZ in NSCLC and SCLC.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-014-0111-8) contains supplementary material, which is available to authorized users.

Activating and propagating polyclonal gamma delta T cells with broad specificity for malignancies.

To activate and propagate populations of γδ T cells expressing polyclonal repertoire of γ and δ T-cell receptor (TCR) chains for adoptive immunotherapy of cancer, which has yet to be achieved. Clinical-grade artificial antigen-presenting cells (aAPC) derived from K562 tumor cells were used as irradiated feeders to activate and expand human γδ T cells to clinical scale. These cells were tested for proliferation, TCR expression, memory phenotype, cytokine secretion, and tumor killing. γδ T-cell proliferation was dependent upon CD137L expression on aAPC and addition of exogenous IL2 and IL21. Propagated γδ T cells were polyclonal as they expressed TRDV1, TRDV2-2, TRDV3, TRDV5, TRDV7, and TRDV8 with TRGV2, TRGV3F, TRGV7, TRGV8, TRGV9*A1, TRGV10*A1, and TRGV11 TCR chains. IFNγ production by Vδ1, Vδ2, and Vδ1(neg)Vδ2(neg) subsets was inhibited by pan-TCRγδ antibody when added to cocultures of polyclonal γδ T cells and tumor cell lines. Polyclonal γδ T cells killed acute and chronic leukemia, colon, pancreatic, and ovarian cancer cell lines, but not healthy autologous or allogeneic normal B cells. Blocking antibodies demonstrated that polyclonal γδ T cells mediated tumor cell lysis through combination of DNAM1, NKG2D, and TCRγδ. The adoptive transfer of activated and propagated γδ T cells expressing polyclonal versus defined Vδ TCR chains imparted a hierarchy (polyclonal>Vδ1>Vδ1(neg)Vδ2(neg)>Vδ2) of survival of mice with ovarian cancer xenografts. Polyclonal γδ T cells can be activated and propagated with clinical-grade aAPCs and demonstrate broad antitumor activities, which will facilitate the implementation of γδ T-cell cancer immunotherapies in humans.

355 Altiratinib (DCC-2701): a balanced inhibitor of MET, TIE2, and VEGFR2 kinases that exhibits broad anti-tumor and anti-angiogenic activities

355 Altiratinib (DCC-2701): a balanced inhibitor of MET, TIE2, and VEGFR2 kinases that exhibits broad anti-tumor and anti-angiogenic activities

Abstract CT325: First in human study of JNJ-42756493, a potent pan fibroblast growth factor receptor (FGFR) inhibitor in patients with advanced solid tumors

Abstract Background: JNJ-42756493 is an FGFR 1, 2, 3, and 4 inhibitor with nanomolar affinity, orally bioavailable, and has demonstrated a broad spectrum antitumor activity in cell line, xenograft and patient-derived explant models with abnormality in FGFR signaling pathway such as FGFR gene amplification, mutation and translocation. Methods: A multipart phase 1 first in human study of JNJ-42756493 was initiated in advanced solid tumor patients (NCT01962532) including, Part 1 dose-escalation to determine the recommended phase 2 dose (RP2D) based on safety, pharmacokinetic, and pharmacodynamic data; Part 2 biopsy cohort to confirm the RP2D and Part 3 expansion phase to evaluate anti-tumor activity in NSCLC, SCLC, Breast cancer and other solid tumor patients with FGFR gene amplification, mutation or translocation at RP2D. Multiple biomarkers from tissue (including phospho-FGFR, phospho-Erk and phospho-S6) and serum (phosphate, calcium, Vitamin D, PTH and FGF23) were also assessed in the study. Results: At data cut-off (22 October 2013), total of 28 patients had been treated at 5 dose levels (0.5, 2, 4, 6 and 9 mg daily continuously) in Part 1 of the study. JNJ-42756493 appeared safe, did not generate any dose-limiting toxicities or any drug related severe adverse events. One patient from 4 mg cohort died after receiving 6 doses in Cycle 1, autopsy indicated a serious adverse event (SAE) of pulmonary edema which not related to the study drug. The most common adverse events (AEs) were hyperphosphatemia (57%), asthenia (46%), dry mouth (32%), abdominal pain (29%), diarrhea (25%), vomiting (25%), decreased appetite (21%) and constipation (21%). Grade 1 or 2 hyperphosphatemia was managed by co-administration of phosphate lowering therapy and by treatment interruption, other AEs were generally mild to moderate. No clinically significant cardiac safety findings were observed. Pharmacokinetics was linear and predictable with a half-life of ∼50 hours. Dose levels ≥ 6mg achieved plasma concentrations predicted to be efficacious from preclinical experiments. Dose dependent changes in biomarkers including increases in phosphate, FGF23 and calcium and decreases in PTH were observed in blood, evaluation of biomarker response in tissue is ongoing. A bladder cancer patient with lung metastasis harboring a FGFR3-TACC3 translocation treated at 9 mg had a confirmed PR. 9 mg was declared as the first RP2D, but safety evaluation of JNJ-42756493 at higher doses is ongoing. Conclusions: JNJ-42756493 has excellent pharmaceutical properties and appeared safe with manageable side effects at dose levels that elicit anti-tumor activity. Citation Format: Rodrigo Dienstmann, Rastilav Bahleda, Barbara Adamo, Jordi Rodon, Andrea Varga, Anas Gazzah, Suso Platero, Hans Smit, Timothy Perera, Bob Zhong, Kim Stuyckens, Yusri Elsayed, Chris Takimoto, Vijay Peddareddigari, Josep Tabernero, Feng Roger Luo, Jean-Charles Soria. First in human study of JNJ-42756493, a potent pan fibroblast growth factor receptor (FGFR) inhibitor in patients with advanced solid tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT325. doi:10.1158/1538-7445.AM2014-CT325

Abstract 815: CBL0137 demonstrates significant antitumor activity against hepatocellular carcinoma alone and in combination with sorafenib

Abstract Primary hepatocellular carcinoma (HCC) is the 6th most common malignancy diagnosed worldwide with an incidence that continues to rise. Even with the use of the current standard of care drug sorafenib, survival of patients remains at ∼10-11 months and sorafenib toxicity results in dose interruptions and delays. Therefore, tolerable, effective therapies against HCC are greatly needed. CBL0137 represents a novel class of small molecules that simultaneously activate p53 and inhibit cancer-associated stress response pathways, such as NF-κB and HSF-1, and has demonstrated broad range antitumor activity against pre-clinical tumor models. The effects of CBL0137 are mediated by the inhibition of the chromatin remodelling factor FACT (FAcilitates Chromatin Transcription) comprised of SSRP1 and SPT16 subunits. FACT is expressed in undifferentiated progenitors and stem cells of adult tissues, but is almost undetectable in differentiated cells. It is expressed in many tumor types, including HCC, and is correlated with high grade and worse overall survival. Thus, FACT represents a potentially important target for cancer therapy. We investigated the effect of CBL0137 on HCC using HepG2 and Hep3B xenograft models. Treatment of tumor bearing mice intravenously with 60-70 mg/kg CBL0137 every 4th day resulted in ∼88-94% tumor growth inhibition in both models, including tumor regression in 60-80% of tumors during the course of treatment. Furthermore, combination of suboptimal doses of CBL0137 with sorafenib had greater efficacy compared to either drug alone (tumor growth inhibition: HepG2- 87.9% combination vs 73.6% CBL0137 only (P=0.0014), 75.7% sorafenib only (P=0.0006); Hep3B- 79.6% combination vs 38.2% CBL0137 only (P=0.003), 51.4% sorafenib only (P=0.032)). Although the mechanism resulting in the combination effect is unknown, in vitro analysis of markers of CBL0137 and sorafenib activity revealed that both drugs caused a marked decrease in the basal and induced expression of NF-κB target genes (IL-8 and TNF). Furthermore, the combination of CBL0137 and sorafenib almost completely abolished the expression of these genes in both HCC cell lines, suggesting that the combination effect may be in part mediated through inhibition of the NF-κB pathway. Together, these data indicate that CBL0137 may provide a clinical benefit for the treatment of HCC both alone and in combination with sorafenib. Citation Format: Catherine Burkhart, Rachael Kohrn, Brittany Walker, Katerina Gurova, Andrei Purmal, Andrei Gudkov. CBL0137 demonstrates significant antitumor activity against hepatocellular carcinoma alone and in combination with sorafenib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 815. doi:10.1158/1538-7445.AM2014-815

Abstract 607: Targeting PIM1 kinase enhances 1,25-dihydroxyvitamin D3-mediated anti-tumor activity in bladder cancer cells

Abstract Bladder cancer is the 5th most common in the United States. 30% of cases are advanced stage requiring multimodality therapy at diagnosis and many which are first diagnosed when superficial will progress to more advanced disease. High recurrence and progression rate and drug resistance highlight the need for new therapeutic strategies to improve clinical outcomes. 1,25-dihydroxyvitamin D3 (1,25D3), the most active vitamin D metabolite, has demonstrated broad spectrum antitumor activities in vitro and in vivo. In addition, 1,25D3 potentiates the antitumor effects of common chemotherapeutic agents in several cancer cell types including bladder cancer. 24-hydroxylase, encoded by the CYP24A1 gene, plays a key role in degrading 1,25D3. Increased expression of CYP24A1 has been found in several human tumors. Several studies reveal that the protein kinase signaling pathways including PIM1 are involved in the regulation of CYP24A1 expression. The PIM kinases are a family of serine/threonine kinases (PIM-1, PIM-2, PIM-3) that have been associated with tumorigenesis and drug resistance. PIM1 has been extensively studied in lymphoma and upregulation of PIM kinase expression has been reported in many malignancies. We analyzed mRNA expression of CYP24A1 and PIM1 in 17 bladder cancer cell lines by qRT-PCR. We found that CYP24A1 expression is increased in 12 out of 17 bladder cancer cell lines and PIM1 expression is increased in 8 out of 17 bladder cancer cell lines compared to expression in a benign bladder cancer cell line. Therefore, we hypothesized that inhibition of PIM kinase activity would suppress CYP24A1 expression and thus enhancing 1,25D3-mediated anti-tumor activity. Using the PIM1 inhibitor, JP11646, we found that inhibition of PIM1 kinase activity reduces CYP24A1 expression at transcriptional level in bladder cancer cells RT112 and RT112D21 which express high level of PIM1 and CYP24A1. We further evaluated the efficacy of the PIM1 inhibitor alone and in combination with 1,25D3. Inhibition of PIM1 kinase activity by JP11646 reduces cell growth in a panel of bladder cancer cell lines in a dose-dependent manner as measured by MTT assays. MTT assays detected a significant inhibition of cell proliferation by 50% compared to the control when cells were treated with 128 nM of JP11646 for 72 h in 6 of bladder cancer cell lines tested. Furthermore, inhibition of PIM1 kinase activity enhances 1,25D3-mediated inhibitory effect on cell growth in bladder cancer cells RT112 and RT112D21. This study provides the first evidence that inhibition of PIM kinase activity may be an attractive therapeutic strategy to facilitate the optimization of antitumor therapy of vitamin D in bladder cancer. This study was supported by NIH/NCI grants 5R01CA067267 and 5R01CA095045 Citation Format: Wei Luo, Carmen M. Baldino, Justin Caserta, Yingyu Ma, Candace Johnson, Donald L. Trump. Targeting PIM1 kinase enhances 1,25-dihydroxyvitamin D3-mediated anti-tumor activity in bladder cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 607. doi:10.1158/1538-7445.AM2014-607

Abstract 2405: Rad18 regulates epistatic relationship between FA-BRCA and homologous recombination pathways to repair camptothecin induced DSB

Abstract DNA topoisomerase 1 (Top1) inhibiting anticancer agents such as camptothecin (CPT) show broad spectrum antitumor activity. The therapeutic activities of CPT and its analogues are attributed to prolonged stabilization of Top1-DNA covalent complexes and converting them into replication mediated double strand breaks (DSB). In spite of their therapeutic success, acquired tumor resistance poses major challenges for their therapeutic efficacy. Combination therapies using DNA repair inhibitors can increase the cytotoxic effects of these drugs and aid in overcoming such problems. Homologous recombination (HR) is the predominant DNA repair pathway involved in the repair of CPT induced DSB. Therefore, inhibition of either HR or tumors that are defective in HR could be effectively killed by Top1 inhibitors. In the same way, cells deficient in HR (ex. Rad51) or its associated proteins (such as Rad18, Fanconi anemia (FA) and breast cancer associated (BRCA1, BRCA2) proteins) are known to exhibit hypersensitivity to CPT. Specifically, FA proteins are known to associate with replication forks and implicated in repair of fork stalling lesions by HR in association with BRCA proteins. Thus, an epistatic relationship between FA and BRCA pathway has been suggested in repair of fork stalling lesions, but molecular basis for this cross-talk is not known. We previously shown that Rad18 regulates FANCD2 monoubiquitination (a key step in FA pathway) to promote HR mediated repair of CPT-induced DSB and cell survival. To further characterize the molecular interactions between Rad18 and FA-BRCA pathway in regulating HR, we have assessed their role in HR by DR-GFP reporter assays by downregulating Rad18 and FANCD2 proteins by siRNAs either alone or in combination. Consistent with survival data, cells deficient in Rad18 or FANCD2 exhibited reduced level of HR. Likewise, cells deficient in both the genes also exhibited similar results of single gene deficient cells, suggesting these two proteins work in a common pathway that promotes HR. Further biochemical and biophysical studies in Rad18 and FANCD2 deficient cells revealed dramatic decrease in nuclear localization of Rad51 and its CPT induced foci formation. Since, FANCD2 is required for BRCA2 recruitment to the sites of DSB, and BRCA2 known to associate with Rad51 and stabilize it on single stranded DNA, we further analyzed BRCA2 foci formation and its colocalization with Rad51 in response to CPT. Interestingly, our studies revealed reduced BRCA2 and Rad51 foci and their colocalization in response to CPT in Rad18 and FANCD2 deficient cells. Consistent with their role in HR, cells deficient in either of these genes exhibited chromosomal aberrations, implicating their role in accurate repair of replication coupled DSB. Together these data support that Rad18 regulates epistatic relationship between FA-BRCA and HR pathways in response to replication coupled DSB induced by CPT. Citation Format: Kaushlendra Tripathi, Chinnadurai Mani, David Clark, Reagan Barnett, Komaraiah Palle. Rad18 regulates epistatic relationship between FA-BRCA and homologous recombination pathways to repair camptothecin induced DSB. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2405. doi:10.1158/1538-7445.AM2014-2405

Abstract 962: Identification of a PFKFB3 inhibitor suitable for phase I trial testing that synergizes with the B-Raf inhibitor vemurafenib

Abstract In human cancers, loss of PTEN, stabilization of hypoxia inducible factor-1α, and activation of Ras and AKT converge to increase the activity of a regulator of glycolysis, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3). This enzyme synthesizes fructose-2,6-bisphosphate, which is an activator of 6-phosphofructo-1-kinase, a key step of glycolysis that is tightly controlled by multiple metabolic feedback mechanisms. We recently identified the first competitive small molecule inhibitor of PFKFB3, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), and demonstrated that this agent is selectively cytotoxic to Ras-transformed epithelial cells and displays broad anti-tumor activity. We now report the discovery of a novel 3PO-derivative, PFK158, that is markedly more potent than 3PO, has excellent pharmacokinetic properties and causes ∼80% growth inhibition in several mouse models of human-derived tumors. Importantly, IND-enabling safety and toxicity studies have demonstrated that PFK158 is well tolerated in rats and dogs, providing support for a phase 1 trial in cancer patients planned for early 2014. Once the maximum tolerated dose is identified, we intend to conduct multiple phase 1/2 trials of PFK158 in combination with targeted agents given the ability of PFK158 to suppress glucose metabolism and multiple survival mechanisms. One such targeted agent, vemurafenib, is a mutant B-Raf inhibitor that was recently FDA-approved for BRAFV600E-positive melanoma patients. Unfortunately, a multitude of resistance mechanisms have resulted in disease progression in less than one year for the majority of melanoma patients treated with vemurafenib. We hypothesized that PFK158 would sensitize vemurafenib-resistant melanoma cells and found that the addition of PFK158 to vemurafenib caused a marked increase in the apoptotic death of several melanoma cell lines in vitro. These data provide rationale for the conduct of pre-clinical studies of PFK158 combined with vemurafenib in transgenic BRAFV600E melanoma mouse models which are in turn expected to justify a phase I/II trial of the combination in BRAFV600E-positive melanoma patients. In conclusion, PFK158 is the first PFKFB3 inhibitor to be examined in a phase I trial and may have significant clinical utility when combined with agents that target driver oncogenes. Citation Format: Julie O'Neal, Gilles Tapolsky, Brian Clem, Sucheta Telang, Jason Chesney. Identification of a PFKFB3 inhibitor suitable for phase I trial testing that synergizes with the B-Raf inhibitor vemurafenib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 962. doi:10.1158/1538-7445.AM2014-962

Vitamin D and miRNAs in cancer.

Vitamin D is a steroid hormone that regulates mineral homeostasis, bone metabolism and many other physiological processes. The active metabolite of vitamin D, 1α, 25-dihydroxyvitamin D (1,25D(3)), has broad spectrum antitumor activities and potentiates the effects of a number of chemotherapeutic agents. 1,25D(3) exerts its anti-tumor effects mainly through genomic mechanisms involving the regulation of gene transcription through vitamin D response elements (VDREs). More recently, miRNAs have been shown to be regulated by 1,25D(3). miRNAs are short non-coding RNAs that post-transcriptionally modulate the expression of a wide range of genes. Therefore, they have important regulatory roles in the development and progression of many diseases including cancer. This review focuses on the regulation of miRNA expression by 1,25D(3) in cancer model systems and the contribution of the regulated miRNAs to the anti-tumor effect of 1,25D(3). In addition, the impact of miRNAs on 1,25D(3) signaling is discussed.

Synthesis, biological evaluation and molecular modeling study of 2-(1,3,4-thiadiazolyl-thio and 4-methyl-thiazolyl-thio)-quinazolin-4-ones as a new class of DHFR inhibitors

A new series of 2-(1,3,4-thiadiazolyl- or 4-methyl-thiazolyl)thio-6-substituted-quinazolin-4-one analogs was designed, synthesized, and evaluated for their in vitro DHFR inhibition, antimicrobial, and antitumor activities. Compounds 29, 34, and 39 proved to be the most active DHFR inhibitors with IC50 values range of 0.1–0.6μM. Compounds 28, 31 and 33 showed remarkable broad-spectrum antimicrobial activity comparable to the known antibiotic Gentamicin. Compounds 26, 33, 39, 43, 44, 50, 55 and 63 showed broad spectrum antitumor activity with GI values range of 10.1–100%. Molecular modeling study concluded that recognition with key amino acid Glu30, Phe31 and Phe34 is essential for binding. ADMET properties prediction of the active compounds suggested that compounds 29 and 34 could be orally absorbed with diminished toxicity.

Comparative toxicity and efficacy of engineered anthrax lethal toxin variants with broad anti-tumor activities

We have previously designed and characterized versions of anthrax lethal toxin that are selectively cytotoxic in the tumor microenvironment and which display broad and potent anti-tumor activities in vivo. Here, we have performed the first direct comparison of the safety and efficacy of three engineered anthrax lethal toxin variants requiring activation by either matrix-metalloproteinases (MMPs), urokinase plasminogen activator (uPA) or co-localized MMP/uPA activities. C57BL/6J mice were challenged with six doses of engineered toxins via intraperitoneal (I.P.) or intravenous (I.V.) dose routes to determine the maximum tolerated dose for six administrations (MTD6) and dose-limiting toxicities. Efficacy was evaluated using the B16-BL6 syngraft model of melanoma; mice bearing established tumors were treated with six I.P. doses of toxin and tumor measurements and immunohistochemistry, paired with terminal blood work, were used to elaborate upon the anti-tumor mechanism and relative efficacy of each variant. We found that MMP-, uPA- and dual MMP/uPA-activated anthrax lethal toxins exhibited the same dose-limiting toxicity; dose-dependent GI toxicity. In terms of efficacy, all three toxins significantly reduced primary B16-BL6 tumor burden, ranging from 32% to 87% reduction, and they also delayed disease progression as evidenced by dose-dependent normalization of blood work values. While target organ toxicity and effective doses were similar amongst the variants, the dual MMP/uPA-activated anthrax lethal toxin exhibited the highest I.P. MTD6 and was 1.5–3-fold better tolerated than the single MMP- and uPA-activated toxins. Overall, we demonstrate that this dual MMP/uPA-activated anthrax lethal toxin can be administered safely and is highly effective in a preclinical model of melanoma. This modified bacterial cytotoxin is thus a promising candidate for further clinical development and evaluation for use in treating human cancers.