Alisertib demonstrates significant antitumor activity in bevacizumab resistant, patient derived orthotopic models of glioblastoma.

Aurora A kinase (AURKA), a member of the serine/threonine kinase family, plays a critical role in cell division, and it is widely overexpressed in a variety of tumors including glioblastoma (GBM). Alisertib (MLN8237) is an orally administered selective AURKA inhibitor with potent antiproliferative activity, currently undergoing clinical testing in different tumor types. In vitro evaluation of alisertib against the primary GBM lines, GBM6, GBM10, GBM12 and GBM39 showed significant antitumor activity with IC50s ranging between 30 and 95 nM. Orthotopic xenografts of GBM10 and the bevacizumab resistant lines GBM6 and GBM39 were established by implantating 3 × 105 cells in the caudate nucleus of nude mice; animals were randomized to treatment with either alisertib 30 mg/kg/day or vehicle. In all three models, treatment with alisertib resulted in a statistically significant prolongation of survival (p < 0.0001). In addition, alisertib administration in these mice decreased phosphorylated aurora-A, induced mitotic arrest and significantly decreased histone H3 phosphorylation in tumors. In conclusion, alisertib displays significant antitumor activity against primary GBM lines and xenografts, including patient derived GBM lines resistant to bevacizumab; these data support clinical translation in GBM.

Inhibition of the Aurora A kinase augments the anti-tumor efficacy of oncolytic measles virotherapy.

Oncolytic measles virus (MV) strains have demonstrated broad spectrum preclinical anti-tumor efficacy, including breast cancer. Aurora A kinase controls mitotic spindle formation and has a critical role in malignant transformation. We hypothesized that the Aurora A kinase inhibitor MLN8237 (alisertib) can increase MV oncolytic effect and efficacy by causing mitotic arrest. Alisertib enhanced MV oncolysis in vitro and significantly improved outcome in vivo against breast cancer xenografts. In a disseminated MDA-231-lu-P4 lung metastatic model, the MV/alisertib combination treatment markedly increased median survival to 82.5 days with 20% of the animals being long-term survivors versus 48 days median survival for the control animals. Similarly, in a pleural effusion model of advanced breast cancer, the MV/alisertib combination significantly improved outcome with a 74.5 day median survival versus the single agent groups (57 and 40 days, respectively). Increased viral gene expression and IL-24 upregulation were demonstrated, representing possible mechanisms for the observed increase in anti-tumor effect. Inhibiting Aurora A kinase with alisertib represents a novel approach to enhance MV-mediated oncolysis and antitumor effect. Both oncolytic MV strains and alisertib are currently tested in clinical trials, this study therefore provides the basis for translational applications of this combinatorial strategy in the treatment of patients with advanced breast cancer.

The mitotic kinase Aurora--a promotes distant metastases by inducing epithelial-to-mesenchymal transition in ERα(+) breast cancer cells.

In this study, we demonstrate that constitutive activation of Raf-1 oncogenic signaling induces stabilization and accumulation of Aurora-A mitotic kinase that ultimately drives the transition from an epithelial to a highly invasive mesenchymal phenotype in estrogen receptor α-positive (ERα(+)) breast cancer cells. The transition from an epithelial- to a mesenchymal-like phenotype was characterized by reduced expression of ERα, HER-2/Neu overexpression and loss of CD24 surface receptor (CD24(-/low)). Importantly, expression of key epithelial-to-mesenchymal transition (EMT) markers and upregulation of the stemness gene SOX2 was linked to acquisition of stem cell-like properties such as the ability to form mammospheres in vitro and tumor self-renewal in vivo. Moreover, aberrant Aurora-A kinase activity induced phosphorylation and nuclear translocation of SMAD5, indicating a novel interplay between Aurora-A and SMAD5 signaling pathways in the development of EMT, stemness and ultimately tumor progression. Importantly, pharmacological and molecular inhibition of Aurora-A kinase activity restored a CD24(+) epithelial phenotype that was coupled to ERα expression, downregulation of HER-2/Neu, inhibition of EMT and impaired self-renewal ability, resulting in the suppression of distant metastases. Taken together, our findings show for the first time the causal role of Aurora-A kinase in the activation of EMT pathway responsible for the development of distant metastases in ERα(+) breast cancer cells. Moreover, this study has important translational implications because it highlights the mitotic kinase Aurora-A as a novel promising therapeutic target to selectively eliminate highly invasive cancer cells and improve the disease-free and overall survival of ERα(+) breast cancer patients resistant to conventional endocrine therapy.

Inhibition of Rho-associated coiled-coil-forming kinase increases efficacy of measles virotherapy.

RhoA and its downstream effector Rho-associated coiled-coil-forming kinase (ROCK) are known regulators of the formation of actin cytoskeleton in cells. Actin cytoskeleton is involved in paramyxovirus infection; we, therefore, examined the effect of ROCK inhibition on measles virus (MV) cytopathic effect and replication. Treatment with the ROCK inhibitor, Y27632, significantly increased syncytia size in tumor cell lines following MV infection, associated with cytoskeleton disruption as demonstrated by actin staining. Treatment of prostate cancer, breast cancer and glioblastoma tumor cell lines with Y27632 following MV infection resulted in increased cytopathic effect, as assessed by trypan blue exclusion assays. In addition, there was a significant increase in viral proliferation by at least one log or more as tested in one-step viral growth curves. Increased viral replication was also observed in athymic nude mice bearing MDA-MB-231 xenografts following combination treatment with MV and Y27632. In summary, inhibition of the ROCK kinase by Y27632 enhanced the oncolytic effect of MV and viral proliferation; this approach merits further translational investigation.

Attenuated oncolytic measles virus strains as cancer therapeutics.

Attenuated measles virus vaccine strains have emerged as a promising oncolytic vector platform, having shown significant anti-tumor activity against a broad range of malignant neoplasms. Measles virus strains derived from the attenuated Edmonston-B (MV-Edm) vaccine lineage have been shown to selectively infect, replicate in and lyse cancer cells while causing minimal cytopathic effect on normal tissues. This review summarizes the preclinical data that led to the rapid clinical translation of oncolytic measles vaccine strains and provides an overview of early clinical data using this oncolytic platform. Furthermore, novel approaches currently under development to further enhance the oncolytic efficacy of MV-Edm strains, including strategies to circumvent immunity or modulate immune system responses, combinatorial approaches with standard treatment modalities, virus retargeting as well as strategies for in vivo monitoring of viral replication are discussed.

Heat shock protein inhibitors increase the efficacy of measles virotherapy.

Oncolytic measles virus strains have activity against multiple tumor types and are currently in phase I clinical testing. Induction of the heat shock protein 70 (HSP70) constitutes one of the earliest changes in cellular gene expression following infection with RNA viruses including measles virus, and HSP70 upregulation induced by heat shock has been shown to result in increased measles virus cytotoxicity. HSP90 inhibitors such as geldanamycin (GA) or 17-allylaminogeldanamycin result in pharmacologic upregulation of HSP70 and they are currently in clinical testing as cancer therapeutics. We therefore investigated the hypothesis that heat shock protein inhibitors could augment the measles virus-induced cytopathic effect. We tested the combination of a measles virus derivative expressing soluble human carcinoembryonic antigen (MV-CEA) and GA in MDA-MB-231 (breast), SKOV3.IP (ovarian) and TE671 (rhabdomyosarcoma) cancer cell lines. Optimal synergy was accomplished when GA treatment was initiated 6-24 h following MV infection. Western immunoblotting confirmed HSP70 upregulation in combination-treated cells. Combination treatment resulted in statistically significant increase in syncytia formation as compared to MV-CEA infection alone. Clonogenic assays demonstrated significant decrease in tumor colony formation in MV-CEA/GA combination-treated cells. In addition there was increase in apoptosis by 4,6-diamidino-2-phenylindole staining. Western immunoblotting for caspase-9, caspase-8, caspase-3 and poly(ADP-ribose) polymerase (PARP) demonstrated increase in cleaved caspase-8 and PARP. The pan-caspase inhibitor Z-VAD-FMK and caspase-8 inhibitor Z-IETD-FMK, but not the caspase-9 inhibitor Z-IEHD-FMK, protected tumor cells from MV-CEA/GA-induced PARP activation, indicating that apoptosis in combination-treated cells occurs mainly via the extrinsic caspase pathway. Treatment of normal cells, such as normal human fibroblasts, however, with the MV-CEA/GA combination, did not result in cytopathic effect, indicating that GA did not alter the MV-CEA specificity for tumor cells. One-step viral growth curves, western immunoblotting for MV-N protein expression, QRT-PCR quantitation of MV-genome copy number and CEA levels showed comparable proliferation of MV-CEA in GA-treated vs -untreated tumor cells. Rho activation assays and western blot for total RhoA, a GTPase associated with the actin cytoskeleton, demonstrated decrease in RhoA activation in combination-treated cells, a change previously shown to be associated with increase in paramyxovirus-induced cell-cell fusion. The enhanced cytopathic effect resulting from measles virus/GA combination supports the translational potential of this approach in the treatment of cancer.

Cross-reaction between the genus-specific lipopolysaccharide antigen of Chlamydia spp. and the lipopolysaccharides of Porphyromonas gingivalis, Escherichia coli O119 and Salmonella newington: implications for diagnosis.

Seven hybridoma clones, secreting monoclonal antibodies (MAbs) against the genus-specific chlamydial lipopolysaccharide (LPS) antigen were obtained after immunization of BALB/c mice with formalin killed Chlamydia psittaci. The antigen-binding properties of the MAbs were characterized in different immunologic reactions with purified chlamydial elementary bodies and LPS antigens from S- and R-forms of Gram-negative bacteria. Four MAbs reacted with the heterologous LPS antigens of Salmonella R-mutants, Escherichia coli Re chemotype and Acinetobacter calcoaceticus. Two MAbs demonstrated in addition a significant reactivity with Porphyromonas gingivalis, E. coli O119 and Salmonella newington LPS in ELISA, dot-ELISA and passive hemolysis assay (for clone 204G9). One MAb cross-reacted only with Salmonella minnesota Re LPS in ELISA. In indirect immunofluorescent assay six MAbs produced bright green fluorescence with all tested chlamydial strains and five of them reacted with the Re and Rb2 chemotypes of S. minnesota. The results demonstrate a wide cross-reactivity of the produced MAbs with LPS antigens of various Gram-negative bacteria, posing the question for careful consideration and interpretation of serology results for Chlamydia spp.

Monoclonal antibodies of IgA isotype specific for lipopolysaccharide of Salmonella enteritidis: production, purification, characterization and application as serotyping reagents.

Hybridomas secreting immunoglobulin A (IgA) monoclonal antibodies (MAbs) against Salmonella enteritidis lipopolysaccharide (LPS) were generated after mucosal immunization of BALB/c mice with heat killed bacteria. Antigen binding properties and specificity of the produced MAbs were studied in ELISA and immunoblotting with purified LPS. Two IgA MAbs agglutinated all Salmonella OD1 strains and all S. enteritidis clinical isolates. MAb 178H11 recognized O:9 antigen of subserogroup OD1 LPS. MAb 177E6/A9 reacted also with OD3 LPS antigen and agglutinated OD3 strains. These data suggest the existence of different O:9 antigen subspecificities, one presented in subgroup OD1 and the other common for OD1 and OD3. Thus the produced IgA MAbs prove to be useful reagents, which could differentiate OD1 and OD3 from OD2 strains.