Teresa Paíno

Inhibition of ATP-sensitive potassium channels increases HSV-tk/GCV bystander effect in U373 human glioma cells by enhancing gap junctional intercellular communication

It is well known that the efficiency of Herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) therapy is improved by the bystander effect, which mainly relies on gap junctional intercellular communication (GJIC). Malignant gliomas communicate poorly through gap junctions, consequently, agents with the ability to increase GJIC are good candidates to improve the efficiency of this therapy. Since we previously showed that the inhibition of ATP-sensitive potassium (KATP) channels promoted by tolbutamide increased GJIC in rat C6 glioma cells, we have investigated whether tolbutamide could increase the bystander effect in HSV-tk/GCV therapy against human glioma cells. We found that tolbutamide increased GJIC in U373 human glioma cells, an effect that was due to the up-regulation of connexin43, a protein that forms gap junctions channels. More interestingly, our results show that tolbutamide increased the efficiency of HSV-tk/GCV in co-cultures containing U373 cells and U373 cells transfected with HSV-tk. This effect was impaired in the presence of carbenoxolone, an inhibitor of GJIC. Furthermore, tolbutamide did not enhance the bystander effect in connexin43-silenced co-cultures. Together our results reveal that the inhibition of KATP channels promoted by tolbutamide enhances the bystander effect in HSV-tk/GCV therapy by increasing connexin43-mediated gap junctional intercellular communication in U373 human glioma cells.

The kinesin spindle protein inhibitor filanesib enhances the activity of pomalidomide and dexamethasone in multiple myeloma

Kinesin spindle protein inhibition is known to be an effective therapeutic approach in several malignancies. Filanesib (ARRY-520), an inhibitor of this protein, has demonstrated activity in heavily pre-treated multiple myeloma patients. The aim of the work herein was to investigate the activity of filanesib in combination with pomalidomide plus dexamethasone backbone, and the mechanisms underlying the potential synergistic effect. The ability of filanesib to enhance the activity of pomalidomide plus dexamethasone was studied in several in vitro and in vivo models. Mechanisms of this synergistic combination were dissected by gene expression profiling, immunostaining, cell cycle and short interfering ribonucleic acid studies. Filanesib showed in vitro, ex vivo, and in vivo synergy with pomalidomide plus dexamethasone treatment. Importantly, the in vivo synergy observed in this combination was more evident in large, highly proliferative tumors, and was shown to be mediated by the impairment of mitosis transcriptional control, an increase in monopolar spindles, cell cycle arrest and the induction of apoptosis in cells in proliferative phases. In addition, the triple combination increased the activation of the proapoptotic protein BAX, which has previously been associated with sensitivity to filanesib, and could potentially be used as a predictive biomarker of response to this combination. Our results provide preclinical evidence for the potential benefit of the combination of filanesib with pomalidomide and dexamethasone, and supported the initiation of a recently activated trial being conducted by the Spanish Myeloma group which is investigating this combination in relapsed myeloma patients.

Synergistic DNA-damaging effect in multiple myeloma with the combination of zalypsis, bortezomib and dexamethasone

Despite new advances in multiple myeloma treatment and the consequent improvement in overall survival, most patients relapse or become refractory to treatment. This suggests that new molecules and combinations that may further inhibit important survival pathways for these tumor cells are needed. In this context, zalypsis is a novel compound, derived from marine organisms, with a powerful preclinical anti-myeloma effect based on the sensitivity of malignant plasma cells to DNA-damage induction; and it has already been tested in a phase I/II clinical trial in multiple myeloma. We hypothesized that the addition of this compound to the combination of bortezomib plus dexamethasone may improve efficacy with acceptable toxicity. The triple combination demonstrated strong synergy and higher efficacy compared with double combinations; not only in vitro, but also ex vivo and, especially, in in vivo experiments. The triple combination triggers cell death, mainly through a synergistic induction of DNA damage and a decrease in the nuclear localization of nuclear factor kappa B. Our findings support the clinical evaluation of this combination for relapsed and refractory myeloma patients.

Amiloride, An Old Diuretic Drug, Is a Potential Therapeutic Agent for Multiple Myeloma

Purpose: The search for new drugs that control the continuous relapses of multiple myeloma is still required. Here, we report for the first time the potent antimyeloma activity of amiloride, an old potassium-sparing diuretic approved for the treatment of hypertension and edema due to heart failure. Experimental Design: Myeloma cell lines and primary samples were used to evaluate cytotoxicity of amiloride. In vivo studies were carried out in a xenograft mouse model. The mechanisms of action were investigated using RNA-Seq experiments, qRT-PCR, immunoblotting, and immunofluorescence assays. Results: Amiloride-induced apoptosis was observed in a broad panel of multiple myeloma cell lines and in a xenograft mouse model. Moreover, amiloride also had a synergistic effect when combined with dexamethasone, melphalan, lenalidomide, and pomalidomide. RNA-Seq experiments showed that amiloride not only significantly altered the level of transcript isoforms and alternative splicing events, but also deregulated the spliceosomal machinery. In addition, disruption of the splicing machinery in immunofluorescence studies was associated with the inhibition of myeloma cell viability after amiloride exposure. Although amiloride was able to induce apoptosis in myeloma cells lacking p53 expression, activation of p53 signaling was observed in wild-type and mutated TP53 cells after amiloride exposure. On the other hand, we did not find a significant systemic toxicity in mice treated with amiloride. Conclusions: Overall, our results demonstrate the antimyeloma activity of amiloride and provide a mechanistic rationale for its use as an alternative treatment option for relapsed multiple myeloma patients, especially those with 17p deletion or TP53 mutations that are resistant to current therapies. Clin Cancer Res; 23(21); 6602–15. ©2017 AACR.