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Molecular Cancer Therapeutics

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Molecular Cancer Therapeutics

Receptor tyrosine kinases (RTK) are transmembrane receptors that regulate signal transduction in cells. As a member of the TAM (Tyro-3, Axl, Mer) RTK subfamily, Axl regulates key processes such as cell growth, migration, aggregation, and apoptosis through several pathways. Its overexpression/overactivation has been underlined in several conditions, especially cancers, and in both chemotherapy and targeted therapy sensitivity loss. In this review, we propose to highlight the therapeutic implication of Axl, starting with the pathways it regulates, validating its interest as a therapeutic target, and defining the tools available to develop strategies for its inhibition. We especially focus on small molecule inhibitors, their structure, inhibition profile, and development stages. Mol Cancer Ther; 13(9); 2141–8. ©2014 AACR.


Choline kinase alpha (ChoK) expression is increasingly being recognized as an important indicator of breast cancer prognosis; however, previous efforts to noninvasively measure ChoK status have been complicated by the spectral limitations of in vivo magnetic resonance spectroscopy (MRS) and the complex network of enzymes involved in choline metabolism. The most effective ChoK inhibitors are symmetric and contain quaternary ammonium groups within heterocyclic head groups connected by an aliphatic spacer. Characterization of these bis-pyridinium and bis-quinolinium compounds has led to phase I clinical trials to assess small-molecule inhibitors of ChoK for solid tumor treatment. We report the development of a novel carbocyanine dye, JAS239, whose bis-indolium structure conforms to the parameters established for ChoK specificity and whose spacer length confers fluorescence in the near-infrared (NIR) window. Fluorimetry and confocal microscopy were used to demonstrate that JAS239 rapidly enters breast cancer cells independent of the choline transporters, with accumulation in the cytosolic space where ChoK is active. Radio-tracing and 1H MRS techniques were used to determine that JAS239 binds and competitively inhibits ChoK intracellularly, preventing choline phosphorylation while inducing cell death in breast cancer cell lines with similar efficacy to known ChoK inhibitors. Fluorescent molecules that report on ChoK status have potential use as companion diagnostics for noninvasive breast tumor staging, because NIR fluorescence allows for detection of real-time probe accumulation in vivo. Furthermore, their ability as novel ChoK inhibitors may prove effective against aggressive, therapy-resistant tumors. Mol Cancer Ther; 13(9); 2149–58. ©2014 AACR.


Recent data suggest that inhibition of the Hedgehog pathway could be a therapeutic target for glioblastoma. Alkaloid cyclopamine inhibits Hedgehog signaling, depleting stem-like cancer cells derived from glioblastoma. However, this compound is toxic for somatic stem cells, preventing its use for clinical applications. In this study, we tested a derivatization product of cyclopamine in the form of cyclopamine glucuronide prodrug (CGP-2). This compound was used in vitro and in vivo toward glioblastoma-initiating cells (GIC). Results obtained in vitro indicate that CGP-2 is active only in the presence of β-glucuronidase, an enzyme detected in high levels in necrotic areas of glioblastomas. CGP-2 decreased proliferation and inhibited the self-renewal of all GIC lines tested. Hedgehog pathway blockade by 10 μmol/L of CGP-2 induced a 99% inhibition of clonogenicity on GICs, similar to cyclopamine treatment. Combination of CGP-2 with radiation decreased clonogenic survival in all GIC lines compared with CGP-2 alone. In a subcutaneous glioblastoma xenograft model, a two-week CGP-2 treatment prevented tumor growth with 75% inhibition at 8 weeks, and this inhibition was still significant after 14 weeks. Unlike cyclopamine, CGP-2 had no detectable toxic effects in intestinal crypts. Our study suggests that inhibition of the Hedgehog pathway with CGP-2 is more effective than conventional temozolomide adjuvant, with much lower concentrations, and seems to be an effective therapeutic strategy for targeting GICs. Mol Cancer Ther; 13(9); 2159–69. ©2014 AACR.


Aurora A kinase orchestrates multiple key activities, allowing cells to transit successfully into and through mitosis. MLN8237 (alisertib) is a selective Aurora A inhibitor that is being evaluated as an anticancer agent in multiple solid tumors and heme-lymphatic malignancies. The antitumor activity of MLN8237 when combined with docetaxel or paclitaxel was evaluated in in vivo models of triple-negative breast cancer grown in immunocompromised mice. Additive and synergistic antitumor activity occurred at multiple doses of MLN8237 and taxanes. Moreover, significant tumor growth delay relative to the single agents was achieved after discontinuing treatment; notably, durable complete responses were observed in some mice. The tumor growth inhibition data generated with multiple dose levels of MLN8237 and paclitaxel were used to generate an exposure–efficacy model. Exposures of MLN8237 and paclitaxel achieved in patients were mapped onto the model after correcting for mouse-to-human variation in plasma protein binding and maximum tolerated exposures. This allowed rank ordering of various combination doses of MLN8237 and paclitaxel to predict which pair would lead to the greatest antitumor activity in clinical studies. The model predicted that 60 and 80 mg/m2 of paclitaxel (every week) in patients lead to similar levels of efficacy, consistent with clinical observations in some cancer indications. The model also supported using the highest dose of MLN8237 that can be achieved, regardless of whether it is combined with 60 or 80 mg/m2 of paciltaxel. The modeling approaches applied in these studies can be used to guide dose-schedule optimization for combination therapies using other therapeutic agents. Mol Cancer Ther; 13(9); 2170–83. ©2014 AACR.


Well-differentiated/dedifferentiated liposarcomas (WD/DDLPS) are among the most common subtypes of soft tissue sarcomas. Conventional systemic chemotherapy has limited efficacy and novel therapeutic strategies are needed to achieve better outcomes for patients. The cyclin-dependent kinase 4 (CDK4) gene is highly amplified in more than 95% of WD/DDLPS. In this study, we explored the role of CDK4 and the effects of NVP-LEE011 (LEE011), a novel selective inhibitor of CDK4/CDK6, on a panel of human liposarcoma cell lines and primary tumor xenografts. We found that both CDK4 knockdown by siRNA and inhibition by LEE011 diminished retinoblastoma (RB) phosphorylation and dramatically decreased liposarcoma cell growth. Cell-cycle analysis demonstrated arrest at G0–G1. siRNA-mediated knockdown of RB rescued the inhibitory effects of LEE011, demonstrating that LEE011 decreased proliferation through RB. Oral administration of LEE011 to mice bearing human liposarcoma xenografts resulted in approximately 50% reduction in tumor 18F-fluorodeoxyglucose uptake with decreased tumor biomarkers, including RB phosphorylation and bromodeoxyuridine incorporation in vivo. Continued treatment inhibited tumor growth or induced regression without detrimental effects on mouse weight. After prolonged continuous dosing, reestablishment of RB phosphorylation and cell-cycle progression was noted. These findings validate the critical role of CDK4 in maintaining liposarcoma proliferation through its ability to inactivate RB function, and suggest its potential function in the regulation of survival and metabolism of liposarcoma, supporting the rationale for clinical development of LEE011 for the treatment of WD/DDLPS. Mol Cancer Ther; 13(9); 2184–93. ©2014 AACR.


In normal epithelia, the epithelial cell adhesion molecule (EpCAM) expression is relatively low and only present at the basolateral cell surface. In contrast, EpCAM is aberrantly overexpressed in various human carcinomas. Therefore, EpCAM is considered to be a highly promising target for antibody-based cancer immunotherapy. Here, we present a new and fully human cytolytic fusion protein (CFP), designated "anti–EpCAM(scFv)-MAP," that is comprised of an EpCAM-specific antibody fragment (scFv) genetically fused to the microtubule-associated protein tau (MAP). Anti–EpCAM(scFv)-MAP shows potent EpCAM-restricted proapoptotic activity toward rapidly proliferating carcinoma cells. In vitro assays confirmed that treatment with anti–EpCAM(scFv)-MAP resulted in the colocalization and stabilization of microtubules, suggesting that this could be the potential mode of action. Dose-finding experiments indicated that anti–EpCAM(scFv)-MAP is well tolerated in mice. Using noninvasive far-red in vivo imaging in a tumor xenograft mouse model, we further demonstrated that anti–EpCAM(scFv)-MAP inhibited tumor growth in vivo. In conclusion, our data suggest that anti–EpCAM(scFv)-MAP may be of therapeutic value for the targeted elimination of EpCAM+ carcinomas. Mol Cancer Ther; 13(9); 2194–202. ©2014 AACR.


Erlotinib is a tyrosine kinase inhibitor approved for the treatment of patients with advanced non–small cell lung cancer (NSCLC). In these patients, erlotinib prolongs survival but its benefit remains modest because many tumors express wild-type (wt) EGFR or develop a second-site EGFR mutation. To test drug combinations that could improve the efficacy of erlotinib, we combined erlotinib with quinacrine, which inhibits the FACT (facilitates chromatin transcription) complex that is required for NF-B transcriptional activity. In A549 (wtEGFR), H1975 (EGFR-L858R/T790M), and H1993 (MET amplification) NSCLC cells, this drug combination was highly synergistic, as quantified by Chou–Talalay combination indices, and slowed xenograft tumor growth. At a sub-IC50 but more clinically attainable concentration of erlotinib, quinacrine, alone or in combination with erlotinib, significantly inhibited colony formation and induced cell-cycle arrest and apoptosis. Quinacrine decreased the level of active FACT subunit SSRP1 and suppressed NF-B–dependent luciferase activity. Knockdown of SSRP1 decreased cell growth and sensitized cells to erlotinib. Moreover, transcriptomic profiling showed that quinacrine or combination treatment significantly affected cell-cycle–related genes that contain binding sites for transcription factors that regulate SSRP1 target genes. As potential biomarkers of drug combination efficacy, we identified genes that were more strongly suppressed by the combination than by single treatment, and whose increased expression predicted poorer survival in patients with lung adenocarcinoma. This preclinical study shows that quinacrine overcomes erlotinib resistance by inhibiting FACT and cell-cycle progression, and supports a clinical trial testing erlotinib alone versus this combination in advanced NSCLC. Mol Cancer Ther; 13(9); 2203–14. ©2014 AACR.


CBP501 is an anticancer drug candidate that was investigated in two randomized phase II clinical trials for patients with nonsquamous non–small cell lung cancer (NSCLC) and malignant pleural mesothelioma (MPM). CBP501 has been shown to have two mechanisms of action, namely calmodulin modulation and G2 checkpoint abrogation. Here, we searched for a biomarker to predict sensitivity to CBP501. Twenty-eight NSCLC cell lines were classified into two subgroups, CBP501-sensitive and -insensitive, by quantitatively analyzing the cis-diamminedichloro-platinum (II) (CDDP)–enhancing activity of CBP501 through treatments with short-term (1 hour) coexposure to CDDP and CBP501 or to either alone. Microarray analysis was performed on these cell lines to identify gene expression patterns that correlated with CBP501 sensitivity. We found that multiple nuclear factor erythroid-2–related factor 2 (Nrf2) target genes showed high expression in CBP501-insensitive cell lines. Western blot and immunocytochemical analysis for Nrf2 in NSCLC cell lines also indicated higher protein level in CBP501-insensitive cell lines. Moreover, CBP501 sensitivity is modulated by silencing or sulforaphane-induced overexpression of Nrf2. These results indicate that Nrf2 transcription factor is a potential candidate as a biomarker for resistance to CBP501. This study might help to identify those subpopulations of patients who would respond well to the CBP501 and CDDP combination treatment of NSCLC. Mol Cancer Ther; 13(9); 2215–25. ©2014 AACR.


Identifying molecular markers for tumor recurrence is critical in successfully selecting patients with colon cancer who are more likely to benefit from adjuvant chemotherapy. We investigated the effect of single-nucleotide polymorphisms (SNP) within genes involved in oxaliplatin and fluoropyrimidines metabolism, DNA repair mechanisms, drug transport, or angiogenesis pathways on outcome for patients with stage II and III colon cancer treated with adjuvant chemotherapy. Genomic DNA was extracted from formalin-fixed paraffin-embedded samples of 202 patients with stage II and III colon cancer receiving oxaliplatin-based adjuvant chemotherapy from January 2004 to December 2009. Genotyping was performed for 67 SNPs in 32 genes using the MassARRAY (SEQUENOM) technology. Our results were validated in an independent cohort of 177 patients treated with the same chemotherapy regimens. The combination of the selectin E (SELE) rs3917412 G>A G/G and the methylentetrahydrofolate reductase (MTHFR) rs1801133 T/T genotypes was associated with a significantly increased risk for recurrence in both the training [RR = 4.103; 95% confidence interval (CI), 1.803–9.334; P = 0.001] and the validation cohorts (RR = 3.567; 95% CI, 1.253–10.151; P = 0.017) in the multiple regression analysis considering the stage, lymphovascular invasion, and bowel perforation as covariates. The combined analysis of these polymorphisms was also significantly associated with overall survival in both cohorts (RR = 3.388; 95% CI, 0.988–11.623; P = 0.052, and RR = 3.929; 95% CI, 1.144–13.485; P = 0.020, respectively). Our findings suggest that the SELE rs3917412 and MTHFR rs1801133 SNPs could serve as pharmacogenetic predictors of tumor recurrence in patients with early-stage colon cancer treated with oxaliplatin-based adjuvant chemotherapy, thus allowing personalized selection of treatment to optimize clinical outcomes. Mol Cancer Ther; 13(9); 2226–37. ©2014 AACR.


The number of approved antiangiogenic drugs is constantly growing and emphasizes the need for predictive biomarkers. The aim of this study was to analyze the predictive value of epidermal growth factor–like domain 7 (EGFL7) and microRNA-126 (miR126) to first-line chemotherapy combined with bevacizumab, in patients with metastatic colorectal cancer (mCRC). A total of 158 patients from two different, but comparable, cohorts were included. Analyses were performed on tumor tissue from the primary tumor either based on a whole-tumor resection or an endoscopic biopsy. EGFL7 was analyzed by immunohistochemistry (IHC) and miR126 by in situ hybridization (ISH). Both biomarkers were quantified by image-guided analyses. Endpoints were response rate (RR) and progression-free survival (PFS). The EGFL7 vessel area (VA) in tumor resections was closely related to treatment response with a median EGFL7 VA in responding patients of 4 [95% confidence interval (CI), 4–6] compared with 8.5 (95% CI, 7–11) in nonresponders, P = 0.0008. This difference translated into a borderline significant difference in PFS (P = 0.06). Furthermore, a significant relationship between high EGFL7 VA and KRAS mutation was detected (P = 0.049). The results showed no significant relationship between the miR126 VA and the clinical endpoints. Our study suggests a predictive value of EGFL7 in regard to first-line chemotherapy and bevacizumab in patients with mCRC and supports the mechanism of a dual blocking of the vascular endothelial growth factor-A and EGFL7 axis in this setting. Mol Cancer Ther; 13(9); 2238–45. ©2014 AACR.