Min H. Kang

Bcl-2 Inhibitors: Targeting Mitochondrial Apoptotic Pathways in Cancer Therapy

Defects in apoptotic pathways can promote cancer cell survival and also confer resistance to antineoplastic drugs. One pathway being targeted for antineoplastic therapy is the anti-apoptotic B-cell lymphoma-2 (Bcl-2) family of proteins (Bcl-2, Bcl-XL, Bcl-w, Mcl-1, Bfl1/A-1, and Bcl-B) that bind to and inactivate BH3-domain pro-apoptotic proteins. Signals transmitted by cellular damage (including antineoplastic drugs) or cytokine deprivation can initiate apoptosis via the intrinsic apoptotic pathway. It is controversial whether some BH3-domain proteins (Bim or tBid) directly activate multidomain pro-apoptotic proteins (e.g., Bax and Bak) or act via inhibition of those anti-apoptotic Bcl-2 proteins (Bcl-2, Bcl-XL, Bcl-w, Mcl-1, Bfl1/A-1, and Bcl-B) that stabilize pro-apoptotic proteins. Overexpression of anti-apoptotic Bcl-2 family members has been associated with chemotherapy resistance in various human cancers, and preclinical studies have shown that agents targeting anti-apoptotic Bcl-2 family members have preclinical activity as single agents and in combination with other antineoplastic agents. Clinical trials of several investigational drugs targeting the Bcl-2 family (oblimersen sodium, AT-101, ABT-263, GX15-070) are ongoing. Here, we review the role of the Bcl-2 family in apoptotic pathways and those agents that are known and/or designed to inhibit the anti-apoptotic Bcl-2 family of proteins.

Initial Testing of the Aurora Kinase A Inhibitor MLN8237 by the Pediatric Preclinical Testing Program (PPTP)

MLN8237 is a small molecule inhibitor of Aurora Kinase A (AURKA) that is currently in early phase clinical testing. AURKA plays a pivotal role in centrosome maturation and spindle formation during mitosis.

Mechanism of Synergy of N-(4-Hydroxyphenyl)Retinamide and ABT-737 in Acute Lymphoblastic Leukemia Cell Lines: Mcl-1 Inactivation

BACKGROUND ABT-737 is a pan-Bcl-2 inhibitor that has a wide range of single-agent activity against acute lymphoblastic leukemia (ALL) cell lines and xenografts. A relationship between expression of myeloid cell leukemia 1 (Mcl-1), an antiapoptotic member of the Bcl-2 family of proteins, and resistance to ABT-737 has been reported for various cancers. The synthetic cytotoxic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) is known to generate reactive oxygen species (ROS), and ROS have been shown to activate c-Jun kinase (JNK), which in turn phosphorylates and inhibits Mcl-1. Thus, we investigated whether 4-HPR-mediated inactivation of Mcl-1 could act synergistically with ABT-737 to promote leukemia cell death. METHODS Cytotoxicity was determined using the fluorescence-based DIMSCAN assay. Synergy was defined as a combination index (CIN) less than 1. The expression of Bcl-2 family messenger RNAs was measured by real-time reverse transcription-polymerase chain reaction, and caspase activity was measured enzymatically. Changes in Bcl-2 family proteins and release of mitochondrial cytochrome c were detected by immunoblotting. ROS, apoptosis, mitochondrial membrane depolarization, and phospho-JNK were measured by flow cytometry. Gene silencing was by small interfering RNA (siRNA). All statistical tests were two-sided. RESULTS ABT-737 decreased Mcl-1 protein expression in ABT-737-sensitive ALL cell lines but not in ABT-737-resistant lines. Using the antioxidant ascorbic acid and siRNA-mediated knockdown of JNK, we showed that 4-HPR decreased Mcl-1 via ROS generation (that phosphorylates JNK) in ABT-737-resistant cell lines. Combining ABT-737 with 4-HPR enhanced the mitochondrial apoptotic cascade (percentage of cells with depolarized mitochondrial membrane at 6 hours, ABT-737 vs ABT-737 plus 4-HPR: 24.5% vs 45.5%, difference = 20.1%, 95% CI = 18.9% to 13.9%; P < .001) and caused caspase-dependent, synergistic multilog cytotoxicity in all seven ALL cell lines examined (mean CIN = 0.57, 95% CI = 0.37 to 0.87), with minimal cytotoxicity for normal lymphocytes. CONCLUSIONS An increase of Mcl-1 protein in response to ABT-737 is one mechanism of ABT-737 resistance that can be overcome by 4-HPR, resulting in synergistic cytotoxicity of ABT-737 combined with 4-HPR in ALL cell lines.

Initial testing of a monoclonal antibody (IMC-A12) against IGF-1R by the pediatric preclinical testing program†

Many childhood malignancies including sarcomas, neuroblastoma, and Wilms tumor show the presence of both, active, type‐1‐insulin‐like growth factor receptor (IGF‐1R), and the autocrine production of its ligands IGF‐1/IGF‐2. IMC‐A12 is a fully human IgG1 antibody that prevents ligand binding to the IGF‐1R.

Stage 2 combination testing of rapamycin with cytotoxic agents by the pediatric preclinical testing program

Rapamycin demonstrated broad-spectrum tumor growth inhibition activity against the in vivo panels of childhood tumors used in the Pediatric Preclinical Testing Program (PPTP). Here we have evaluated rapamycin combined with agents used frequently in the treatment of childhood malignancies. Rapamycin was tested in vitro against 23 cell lines alone or in combination with melphalan, cisplatin, vincristine, or dexamethasone (leukemic models only). In vivo, the impact of combining rapamycin with a cytotoxic agent was evaluated using two measures: 1) the therapeutic enhancement measure, and 2) a linear regression model for time-to-event to formally evaluate for sub- and supraadditivity for the combination compared to the agents used alone. Combining rapamycin with cytotoxic agents in vitro gave predominantly subadditive or additive effects, except for dexamethasone in leukemia models for which supra-additive activity was observed. In vivo testing demonstrated that therapeutic enhancement was common for rapamycin in combination with cyclophosphamide and occurred for 4 of 11 evaluable xenografts for the rapamycin and vincristine combination. The combinations of rapamycin with either cyclophosphamide or vincristine were significantly more effective than the respective standard agents used alone at their maximum tolerated doses (MTD) for most evaluable xenografts. The combination of rapamycin and cisplatin produced excessive toxicity requiring cisplatin dose reductions, and therapeutic enhancement was not observed for this combination. Addition of rapamycin to either cyclophosphamide or vincristine at their respective MTDs appears promising, as these combinations are relatively well tolerated and as many of the pediatric preclinical models evaluated demonstrated therapeutic enhancement for these combinations. Mol Cancer Ther; 9(1); 101–12

Phase I Study of Infusional Paclitaxel in Combination With the P-Glycoprotein Antagonist PSC 833

PURPOSE PSC 833 (valspodar) is a second-generation P-glycoprotein (Pgp) antagonist developed to reverse multidrug resistance. We conducted a phase I study of a 7-day oral administration of PSC 833 in combination with paclitaxel, administered as a 96-hour continuous infusion. PATIENTS AND METHODS Fifty patients with advanced cancer were enrolled onto the trial. PSC 833 was administered orally for 7 days, beginning 72 hours before the start of the paclitaxel infusion. Paclitaxel dose reductions were planned because of the pharmacokinetic interactions known to occur with PSC 833. RESULTS In combination with PSC 833, maximum-tolerated doses were defined as paclitaxel 13.1 mg/m(2)/d continuous intravenous infusion (CIVI) for 4 days without filgrastim, and paclitaxel 17.5 mg/m(2)/d CIVI for 4 days with filgrastim support. Dose-limiting toxicity for the combination was neutropenia. Statistical analysis of cohorts revealed similar mean steady-state concentrations (C(pss)) and areas under the concentration-versus-time curve (AUCs) when patients received paclitaxel doses of 13.1 or 17.5 mg/m(2)/d for 4 days with PSC 833, as when they received a paclitaxel dose of 35 mg/m(2)/d for 4 days without PSC 833. However, the effect of PSC 833 on paclitaxel pharmacokinetics varied greatly among individual patients, although a surrogate assay using CD56+ cells suggested inhibition of Pgp was complete or nearly complete at low concentrations of PSC 833. Responses occurred in three of four patients with non-small-cell lung cancer, and clinical benefit occurred in five of 10 patients with ovarian carcinoma. CONCLUSION PSC 833 in combination with paclitaxel can be administered safely to patients provided the paclitaxel dose is reduced to compensate for the pharmacokinetic interaction. Surrogate studies with CD56+ cells indicate that the maximum-tolerated dose for PSC 833 gives serum levels much higher than those required to block Pgp. The variability in paclitaxel pharmacokinetics, despite complete inhibition of Pgp in the surrogate assay, suggests that other mechanisms, most likely related to P450, contribute to the pharmacokinetic interaction. Future development of combinations such as this should include strategies to predict pharmacokinetics of the chemotherapeutic agent. This in turn will facilitate dosing to achieve comparable CPss and AUCs.

Initial testing (stage 1) of AZD6244 (ARRY‐142886) by the pediatric preclinical testing program

AZD6244 (ARRY‐142886) is a potent small molecule inhibitor of MEK1/2 that is in phase 2 clinical development.

National Cancer Institute Pediatric Preclinical Testing Program: Model Description for In Vitro Cytotoxicity Testing

The National Cancer Institute (NCI) has established the Pediatric Preclinical Testing Program (PPTP) for testing drugs against in vitro and in vivo childhood cancer models to aid in the prioritization of drugs considered for early phase pediatric clinical trials.

Initial testing (stage 1) of LCL161, a SMAC mimetic, by the Pediatric Preclinical Testing Program.

LCL161, a SMAC mimetic, was tested against the PPTP in vitro panel (1.0 nM to 10.0 µM) and the PPTP in vivo panels (30 or 75 mg/kg [solid tumors] or 100 mg/kg [ALL]) administered orally twice in a week. LCL161 showed a median relative IC50 value of >10 µM, being more potent against several leukemia and lymphoma lines. In vivo LCL161 induced significant differences in EFS distribution in approximately one‐third of solid tumor xenografts (osteosarcoma and glioblastoma), but not in ALL xenografts. No objective tumor responses were observed. In vivo LCL161 demonstrated limited single agent activity against the pediatric preclinical models studied. Pediatr Blood Cancer 2012; 58: 636–639.

Initial testing (stage 1) of the IGF-1 receptor inhibitor BMS-754807 by the pediatric preclinical testing program†

BMS‐754807 is a small molecule ATP‐competitive inhibitor of the type‐1 insulin‐like growth factor receptor currently in phase 1 clinical trials.