Desensitization controls G protein-dependent signaling of chemokine receptors. We investigate the physiologic implication of this process for CXCR4 in a mouse model harboring a heterozygous mutation of the Cxcr4 gene, which engenders a desensitization-resistant receptor. Such anomaly is linked to the warts, hypogammaglobulinemia, infections, myelokathexis (WHIM) syndrome, a human rare combined immunodeficiency. Cxcr4(+/mutant(1013)) mice display leukocytes with enhanced responses to Cxcl12 and exhibit leukopenia as reported in patients. Treatment with CXCL12/CXCR4 antagonists transiently reverses blood anomalies, further demonstrating the causal role of the mutant receptor in the leukopenia. Strikingly, neutropenia occurs in a context of normal bone marrow architecture and granulocyte lineage maturation, indicating a minor role for Cxcr4-dependent signaling in those processes. In contrast, Cxcr4(+/1013) mice show defective thymopoiesis and B-cell development, accounting for circulating lymphopenia. Concomitantly, mature T and B cells are abnormally compartmentalized in the periphery, with a reduction of primary follicles in the spleen and their absence in lymph nodes mirrored by an unfurling of the T-cell zone. These mice provide a model to decipher the role of CXCR4 desensitization in the homeostasis of B and T cells and to investigate which manifestations of patients with WHIM syndrome may be overcome by dampening the gain of CXCR4 function.

We sought to identify miRNAs that can efficiently induce apoptosis in ovarian cancer cells by overcoming BCL-XL and MCL1 anti-apoptotic activity, using combined computational and experimental approaches. We found that miR-491-5p efficiently induces apoptosis in IGROV1-R10 cells by directly inhibiting BCL-XL expression and by inducing BIM accumulation in its dephosphorylated form. This latter effect is due to direct targeting of epidermal growth factor receptor (EGFR) by miR-491-5p and consequent inhibition of downstream AKT and MAPK signalling pathways. Induction of apoptosis by miR-491-5p in this cell line is mimicked by a combination of EGFR inhibition together with a BH3-mimetic molecule. In contrast, SKOV3 cells treated with miR-491-5p maintain AKT and MAPK activity, do not induce BIM and do not undergo cell death despite BCL-XL and EGFR downregulation. In this cell line, sensitivity to miR-491-5p is restored by inhibition of both AKT and MAPK signalling pathways. Altogether, this work highlights the potential of miRNA functional studies to decipher cell signalling pathways or major regulatory hubs involved in cell survival to finally propose the rationale design of new strategies on the basis of pharmacological combinations.

In ovarian carcinomas, recurrence and acquired chemoresistance are the first leading causes of therapeutic failure and are responsible for the poor overall survival rate. Cisplatin exposure of sensitive cells has been previously associated with a down-regulation of Bcl-x L expression and apoptosis, whereas recurrence was systematically observed when Bcl-x L expression was maintained. Bcl-x L down-regulation could thus constitute an interesting chemosensitizing strategy. We showed that a Bclx L targeted RNA interference strategy efficiently sensitized chemoresistant ovarian carcinoma cells to cisplatin, but some of them were still able to re-proliferate. Considering the possible cooperation between Bcl-x L and MCL-1, we investigated the possibility to avoid recurrence in vitro using a multi-targeted RNAi strategy directed against these two anti-apoptotic proteins. We showed that their concomitant inhibition lead to massive apoptosis in absence of cisplatin, this multi-targeted RNAi approach being much more efficient than conventional chemotherapy. We thus demonstrated that Bcl-x L and MCL-1 cooperate to constitute together a strong molecular ''bolt'', which elimination could be sufficient to allow chemoresistant ovarian carcinoma cells apoptosis. Moreover, we demonstrated that in presence of a low concentration of cisplatin, the concomitant down-regulation of Bcl-x L and MCL-1 allowed a complete annihilation of tumour cells population thus avoiding subsequent recurrence in vitro in cell lines highly refractory to any type of conventional chemotherapy. Therefore, Bcl-x L and MCL-1 targeted strategies could constitute an efficient therapeutic tool for the treatment of chemoresistant ovarian carcinoma, in association with conventional chemotherapy.Ovarian cancer is the leading cause of death from gynecological malignancies worldwide and the fifth most common cause of cancer death in women. 1 Early diagnosis is difficult owing to the asymptomatic character of this disease in early stages, and more than 70% of these cancers are diagnosed in an advanced stage (FIGO stages III or IV). Patients with advanced ovarian cancer are treated initially with optimal debulking surgery and standard chemotherapy (platinumdrugs usually associated to taxanes). 2 Despite an initial 70-80% response rate, most patients will relapse within 1-2 years and develop resistance to chemotherapy 3 and the overall 5-year survival is less than 30%. The identification of new drugs or novel therapeutic strategies able to (re)sensitize ovarian carcinoma cells to existing chemotherapy thus appear as a major challenge.Cisplatin, a DNA-damaging agent that forms DNA adducts, is commonly used for the treatment of advanced ovarian cancers. It is widely accepted that these adducts lead to a cell cycle arrest followed by the induction of apoptotic cell death. 4 However, it should be considered that in resistant cells, a strong protection against apoptosis exist when the DNA damages reach their maximum level, to allow the reparation of these injuries and the r...

The WHIM syndrome, which features high susceptibility to human papillomavirus (HPV) infection, is a rare immunodeficiency associated with autosomal dominant heterozygous mutations of the CXCR4 chemokine receptor. CXCL12 and its receptors, CXCR4 and CXCR7, are linked to tumorigenesis, and we reported that abnormal expression of CXCL12 in epidermal keratinocytes correlates with HPV infection. However, the HPV-related pathologies observed in WHIM patients remain mechanistically unexplained. We show that keratinocytes immortalized by oncogenic HPV16 or HPV18 upregulate CXCL12 and its receptors in a manner dependent upon expression of the viral proteins E6 and E7. Autocrine signaling activated by CXCL12-engagement of its receptors controls motility and survival of the infected cells. Strikingly, expression of a WHIM syndrome-related gain-of-function CXCR4 mutant confers transforming capacity to HPV18-immortalized keratinocytes. These results establish a pivotal role for CXCL12 signaling in HPV-mediated transformation and provide a mechanistic basis for understanding HPV pathogenesis in WHIM syndrome.

Malignant pleural mesothelioma (MPM) is an aggressive tumor with poor prognosis and limited response to platinum-based chemotherapy. Several lines of evidence support a role for the anti-apoptotic protein Bcl-x(L) in MPM chemoresistance. Since it has been recently suggested that Mcl-1 cooperates with Bcl-x(L) for protection against cell death, we investigated the response of mesothelioma cell lines to the downregulation of Bcl-x(L) (alone or in combination with cisplatin) and the potential interest of its concomitant inhibition with that of Mcl-1. Using RNA interference, we showed that Bcl-x(L) depletion sensitized two highly chemoresistant mesothelioma cell lines to cisplatin and that under this treatment, one cell line, MSTO-211H, displayed an apoptotic type of cell death, whereas the other, NCI-H28, evidenced mainly necrotic-type cell death. Otherwise, the inhibition of Mcl-1 by cisplatin may contribute to this induction of cell death observed after Bcl-x(L) downregulation. Strikingly, we observed that the simultaneous inhibition of Bcl-x(L) and Mcl-1 using small interfering RNA (siRNA) induced a massive cell death in the absence of chemotherapy and was sufficient to avoid escape to treatment in MSTO-211H cells. In NCI-H28, the addition of a low cisplatin concentration allowed to impede the long-term recovery observed after treatment by the siRNA combination. Together, these findings provide a strong molecular basis for the clinical evaluation of therapies targeting both Bcl-x(L) and Mcl-1, alone or in combination with conventional chemotherapy, for the treatment of MPM.

Ovarian cancer is the leading cause of death from gynecological cancer. The anti-apoptotic protein Bcl-x(L) is frequently overexpressed in ovarian carcinoma which correlates with chemotherapy resistance. It has been demonstrated that Bcl-x(L) cooperates with another anti-apoptotic protein, Mcl-1, to protect ovarian cancer cells against apoptosis, and that their concomitant inhibition induces massive cell death. Here, we examined the interest of ABT-737, a potent BH3-mimetic molecule targeting Bcl-x(L), both alone and in combination with Mcl-1 modulators, in ovarian cancer cell lines. As a single agent, ABT-737 was ineffective at promoting cell death in the four cell lines we tested in vitro. However, the specific inhibition of Mcl-1 by siRNA dramatically increased the sensitivity of chemoresistant cells to ABT-737. Platinum compounds also sensitize to ABT-737 by dose-dependently decreasing Mcl-1 expression or by increasing the expression of pro-apoptotic BH3-only proteins Noxa and, to a lower extent, Bim. Furthermore, we demonstrated that Noxa accumulation was involved in apoptosis occurring in response to the combination of ABT-737 and platinum compounds, since cells were protected from apoptosis by its silencing. Moreover, the combination was also highly cytotoxic ex vivo in sliced SKOV3 tumor nodes. However we observed in these slices a strong basal expression of Noxa and apoptotic cell death in response to ABT-737 alone. Therefore, we have revealed that the modulation of the Mcl-1/Noxa axis by platinum compounds results in a strong sensitization of chemoresistant ovarian carcinoma cells to ABT-737, which could constitute a promising therapeutic in these cancers.

We previously showed that Bcl-xL and Mcl-1 cooperatively protect platinum-resistant ovarian cancer cells from apoptosis. Here we assessed the anticancer potential of combining ABT-737-induced inhibition of Bcl-xL with Mcl-1 inhibition via PI3K/Akt/mTOR pathway disruption using NVP-BEZ235. NVP-BEZ235 inhibited cell proliferation without inducing apoptosis. It strongly repressed Mcl-1 expression and induced Puma expression in both cell lines tested while differentially modulating Bim between the two. Interestingly, NVP-BEZ235 efficiently sensitized ovarian carcinoma cells to ABT-737, provided that Bim expression was induced. Moreover, inhibiting the ERK1/2 pathway restored Bim expression and sensitized low Bim-expressing cancer cells to the BEZ235/ABT-737 treatment.

Chemoresistance of ovarian carcinoma has been associated previously to the absence of Bcl-x L expression downregulation in response to cisplatin. Among BH3-mimetic molecules constituting promising anticancer agents able to inhibit the activity of antiapoptotic Bcl-2 family proteins, we evaluated the effect of one of them, HA14-1, on various ovarian carcinoma cell lines. In response to HA14-1, the cisplatin-resistant IGROV1-R10 cell line underwent massive cell death, whereas other cell lines presented a partial response (IGROV1, SKOV3, and A2780) or did not respond to this molecule (OAW42 and OAW42-R). However, the expression of HA14-1 targets (Bcl-2 and Bcl-x L ) did not correlate to these different responses. In contrast, cell death was associated with the disappearance of Mcl-1 after exposure to HA14-1. We showed that, in the HA14-1 nonresponsive cell lines (SKOV3 and OAW42), small interfering RNA-mediated Mcl-1 downregulation allowed HA14-1-induced massive apoptosis in the absence of chemotherapy. Furthermore, cisplatininduced Mcl-1 downregulation was also able to sensitize highly chemoresistant SKOV3 cells to HA14-1. Taken together, these results show that Bcl-x L and Mcl-1 are able to cooperate to protect ovarian carcinoma cells against oncogenic stress or chemotherapy-induced apoptosis and suggest that the development of multitargeted strategies directed against these two antiapoptotic proteins may constitute a major challenge for the therapeutic care of chemoresistant ovarian carcinomas. BH3-mimetic compounds represent promising tools for this purpose either on their own (direct or indirect pan-inhibitors) or in combination with new drugs aiming to inactivate