Manipulating the tumor immune microenvironment to improve cancer immunotherapy: IGF1R, a promising target.

Cancer immunotherapy has made impressive advances in improving the outcome of patients affected by malignant diseases. Nonetheless, some limitations still need to be tackled to more efficiently and safely treat patients, in particular for those affected by solid tumors. One of the limitations is related to the immunosuppressive tumor microenvironment (TME), which impairs anti-tumor immunity. Efforts to identify targets able to turn the TME into a milieu more auspicious to current immuno-oncotherapy is a real challenge due to the high redundancy of the mechanisms involved. However, the insulin-like growth factor 1 receptor (IGF1R), an attractive drug target for cancer therapy, is emerging as an important immunomodulator and regulator of key immune cell functions. Here, after briefly summarizing the IGF1R signaling pathway in cancer, we review its role in regulating immune cells function and activity, and discuss IGF1R as a promising target to improve anti-cancer immunotherapy.

Dual IGF1R/IR inhibitors in combination with GD2-CAR T-cells display a potent anti-tumor activity in diffuse midline glioma H3K27M-mutant.

BACKGROUND: Diffuse midline gliomas (DMG) H3K27M-mutant, including diffuse intrinsic pontine glioma (DIPG), are pediatric brain tumors associated with grim prognosis. Although GD2-CAR T-cells demonstrated significant anti-tumor activity against DMG H3K27M-mutant in vivo, a multimodal approach may be needed to more effectively treat patients. We investigated GD2 expression in DMG/DIPG and other pediatric high-grade gliomas (pHGG) and sought to identify chemical compounds that would enhance GD2-CAR T-cell anti-tumor efficacy. METHODS: Immunohistochemistry in tumor tissue samples and immunofluorescence in primary patient-derived cell lines were performed to study GD2 expression. We developed a high-throughput cell-based assay to screen 42 kinase inhibitors in combination with GD2-CAR T-cells. Cell viability, western blots, flow-cytometry, real time PCR experiments, DIPG 3D culture models, and orthotopic xenograft model were applied to investigate the effect of selected compounds on DIPG cell death and CAR T-cell function. RESULTS: GD2 was heterogeneously, but widely, expressed in the tissue tested, while its expression was homogeneous and restricted to DMG/DIPG H3K27M-mutant cell lines. We identified dual IGF1R/IR antagonists, BMS-754807 and linsitinib, able to inhibit tumor cell viability at concentrations that do not affect CAR T-cells. Linsitinib, but not BMS-754807, decreases activation/exhaustion of GD2-CAR T-cells and increases their central memory profile. The enhanced anti-tumor activity of linsitinib/GD2-CAR T-cell combination was confirmed in DIPG models in vitro, ex vivo, and in vivo. CONCLUSION: Our study supports the development of IGF1R/IR inhibitors to be used in combination with GD2-CAR T-cells for treating patients affected by DMG/DIPG and, potentially, by pHGG.

Manipulating the Metabolism to Improve the Efficacy of CAR T-Cell Immunotherapy.

The adoptive transfer of the chimeric antigen receptor (CAR) expressing T-cells has produced unprecedented successful results in the treatment of B-cell malignancies. However, the use of this technology in other malignancies remains less effective. In the setting of solid neoplasms, CAR T-cell metabolic fitness needs to be optimal to reach the tumor and execute their cytolytic function in an environment often hostile. It is now well established that both tumor and T cell metabolisms play critical roles in controlling the immune response by conditioning the tumor microenvironment and the fate and activity of the T cells. In this review, after a brief description of the tumoral and T cell metabolic reprogramming, we summarize the latest advances and new strategies that have been developed to improve the metabolic fitness and efficacy of CAR T-cell products.

Effect of p53 activation through targeting MDM2/MDM4 heterodimer on T regulatory and effector cells in the peripheral blood of Type 1 diabetes patients.

Various immunotherapies for the treatment of type 1 diabetes are currently under investigation. Some of these aim to rescue the remaining beta cells from autoimmune attack caused by the disease. Among the strategies employed, p53 has been envisaged as a possible target for immunomodulation. We studied the possible effect of p53 activation on Treg subsets and Treg/Teff balance in type 1 diabetes patients' PBMC. Upon p53 activation, we observed an increase in CD8+ Treg and activated CD8+ Teff whilst CD8+ Teff cells significantly decreased in healthy PBMC when stimulated with anti-CD3/CD28. No effect was detected on percentages of CD4+ Treg, while a reduction was seen in CD4+ Teff cells and an increase in activated CD4+ Teff cells. In patients' PBMC, upon p53 activation followed by 6 days of anti-CD3/CD28 stimulation, CD8+ Treg and activated CD8+ Teff were increased while CD8+ Teff were decreased. No differences were detected in the CD4+ counterparts. CD8+ Teff PD1+, CD8+ Teff PD1low were increased upon p53 activation in type 1 diabetics compared to controls while CD8+ Teff PD1high were increased in both groups. The same increased percentages were detected for CD4+ counterparts. CD4+ Treg PD1high cells were decreased in diabetics upon p53 activation at day 6 of anti-CD3/CD28 stimulation. In conclusion, a Teff dysregulation is observed upon p53 activation suggesting that molecules promoting p53 cannot be used for therapy in type 1 diabetics.

Estrogens Counteract Platinum-Chemosensitivity by Modifying the Subcellular Localization of MDM4.

Estrogen activity towards cancer-related pathways can impact therapeutic intervention. Recent omics data suggest possible crosstalk between estrogens/gender and MDM4, a key regulator of p53. Since MDM4 can either promote cell transformation or enhance DNA damage-sensitivity, we analysed in vivo impact of estrogens on both MDM4 activities. In Mdm4 transgenic mouse, Mdm4 accelerates the formation of fibrosarcoma and increases tumor sensitivity to cisplatin as well, thus confirming in vivo Mdm4 dual mode of action. Noteworthy, Mdm4 enhances chemo- and radio-sensitivity in male but not in female animals, whereas its tumor-promoting activity is not affected by mouse gender. Combination therapy of transgenic females with cisplatin and fulvestrant, a selective estrogen receptor degrader, was able to recover tumor cisplatin-sensitivity, demonstrating the relevance of estrogens in the observed sexual dimorphism. Molecularly, estrogen receptor-α alters intracellular localization of MDM4 by increasing its nuclear fraction correlated to decreased cell death, in a p53-independent manner. Importantly, MDM4 nuclear localization and intra-tumor estrogen availability correlate with decreased platinum-sensitivity and apoptosis and predicts poor disease-free survival in high-grade serous ovarian carcinoma. These data demonstrate estrogen ability to modulate chemo-sensitivity of MDM4-expressing tumors and to impinge on intracellular trafficking. They support potential usefulness of combination therapy involving anti-estrogenic drugs.

Identification and functional characterization of CD8+ T regulatory cells in type 1 diabetes patients.

Type 1 diabetes is an autoimmune disease where autoreactive T lymphocytes destroy pancreatic beta cells. We previously reported a defect in CD4+ Tregs cell proliferation and reduced CD4+ Tregs PD-1 expression in patients. Another 'memory-like' regulatory subset, CD8+ Tregs, evaluated as CD8+CD25+FOXP3+, has recently raised interest for their effective suppressive activity. Different CD8+ T cell populations, their proliferation capacity and expression of PD-1 molecule were evaluated by flow-cytometer analysis in newly diagnosed, long-term Type 1 diabetes patients compared to healthy normal donors. Under basal conditions, CD8+ Tregs and CD8+ Teffs were seemingly represented among study groups while there was evidence of diminished expression of PD-1 in Teff subsets of long-term patients. After 3 days of PMA/ionomycin stimulation, patients CD8+ Tregs showed decreased percentage in respect to control group. CD8+ Teffs were instead increased in long-term diabetics versus controls. PD-1+CD8+ Tregs were represented at a much lower percentage in long-term diabetic patients, in respect to controls. Importantly, patients CD8+ Tregs and CD8+ Teffs presented a significant proliferation defect in respect to the control group. In conclusion, our study indicates that a defect of CD8+ Tregs is observed in diabetics. This subset could thus represent a novel target of immunotherapy in patients.

Exploiting novel tailored immunotherapies of type 1 diabetes: Short interfering RNA delivered by cationic liposomes enables efficient down-regulation of variant PTPN22 gene in T lymphocytes.

In autoimmune diseases as Type 1 diabetes, the actual treatment that provides the missing hormones is not able, however, to interrupt the underlining immunological mechanism. Importantly, novel immunotherapies are exploited to protect and rescue the remaining hormone producing cells. Among probable targets of immunotherapy, the C1858T mutation in the PTPN22 gene, which encodes for the lymphoid tyrosine phosphatase (Lyp) variant R620W, reveals an autoimmunity related pathophysiological role. Our scope was to establish new C1858T PTPN22 siRNA duplexes delivered by liposomal carriers (lipoplexes) to patients' PBMC. Following lipoplexes treatment, CD3+ and CD3- immunotypes were efficiently transfected; cell integrity and viability were preserved. Specific target mRNA down-modulation was observed. After T cell receptor stimulation, in lipoplexes-treated PBMC Lyp function was restored by increased release of IL-2 in cultures. Results set-up the stage for ultimate trials in the treatment of autoimmunity based on the specific inhibitory targeting of C1858T PTPN22 by lipoplexes.

A Novel Homozygous Mutation of the AIRE Gene in an APECED Patient From Pakistan: Case Report and Review of the Literature.

Autoimmune-poly-endocrinopathy-candidiasis-ectodermal-dystrophy syndrome (APECED) is a rare monogenic recessive disorder caused by mutations in the autoimmune regulator (AIRE) gene. Criteria for the diagnosis of APECED are the presence of two of the following disorders: chronic mucocutaneous candidiasis (CMC), chronic hypoparathyroidism (CHP), and Addison's disease. APECED develops at high incidence in Finns, Sardinians, and Iranian Jews and presents with a wide range of clinical phenotypes and genotypes. In this manuscript, we report the clinical, endocrinological, and molecular features of a 16-year-old female patient from Pakistan living in Italy and presenting the major APECED clinical manifestations CMC, CHP, and primary adrenal insufficiency. Premature ovarian failure, chronic bronchopneumopathy, vitiligo, Hashimoto's thyroiditis emerged as associated diseases. In our patient, AIRE gene screening revealed the novel c.396G>C (p.Arg132Ser; p.R132S) mutation in homozygosity thus confirming APECED diagnosis. This is the first reported mutation within the nuclear localization signal (NLS) that is associated with APECED. The NLS mutation affects the nuclear import of classical transcription factors through nuclear pore by recognition of nuclear import receptors, the importin α molecules. By displaying crystal structures of the peptide containing the KRK basic residue cluster bound to α importins, we show that p.R132S replacement in 131-KRK-133 does not reproduce these interactions. Thus, we propose that the novel mutation exerts its pathogenetic effect by impairing the nuclear import of the Aire protein. The present case report is added to a limited series of Pakistani APECED patients who we reviewed from the scientific literature, mostly diagnosed on clinical findings.

APECED in Turkey: A case report and insights on genetic and phenotypic variability.

APECED is a rare monogenic recessive disorder caused by mutations in the AIRE gene. In this manuscript, we report a male Turkish patient with APECED syndrome who presented with chronic mucocutaneous candidiasis associated with other autoimmune manifestations developed over the years. The presence of the homozygous R257X mutation of the AIRE gene confirmed the diagnosis of APECED syndrome. We further performed literature review in 23 published Turkish APECED patients and noted that Finnish major mutation R257X is common in Turks. In particular, we assessed retrospectively how often the Ferre/Lionakis criteria would have resulted in earlier diagnosis in Finns, Sardinians and Turks in respect to the classic criteria. Since an earlier diagnosis could have been possible in 18.8% of Turkish, in 23.8% of Sardinian and 38.55% of Finnish patients we reviewed from literature, Ferre/Lionakis criteria could indeed allow in future earlier initiation of immunomodulatory treatments, if found effective in future studies.

Identification of GAD65 AA 114-122 reactive 'memory-like' NK cells in newly diagnosed Type 1 diabetic patients by HLA-class I pentamers.

Type 1 diabetes is an autoimmune disease, in which pancreatic ß cells are destroyed by autoreactive T cells in genetically predisposed individuals. Serum beta cell autoantibody specificities have represented the mainstay for classifying diabetes as autoimmune-mediated and for stratifying risk in first-degree relatives. In recent years, approaches were attempted to solve the difficult issue of detecting rare antigen-specific autoreactive T cells and their significance to etiopathogenesis such as the use of the MHC multimer technology. This tool allowed the specific detection of increased percentages of GAD65 autoreactive T cells by means of HLA A*02:01 GAD65 AA 114-122 pentamers in newly diagnosed diabetics. Here we provide evidence that GAD65 AA 114-122 pentamers can depict a GAD65 AA114-122 peptide expandable population of functionally and phenotypically skewed, preliminary characterized CD3-CD8dullCD56+ 'memory-like' NK cells in PBMC of newly diagnosed diabetics. Our data suggest that the NK cell subset could bind the HLA class I GAD65 AA 114-122 pentamer through ILT2 inhibitory receptor. CD107a expression revealed increased degranulation of CD3-CD8dullCD56+ NK cells in GAD65 AA 114-122 and FLU peptide expanded peripheral blood mononuclear cells of diabetics following GAD65 AA 114-122 peptide HLA A*02:01 presentation in respect to the unpulsed condition. CD107a expression was enriched in ILT2 positive NK cells. As opposite to basal conditions where similar percentages of CD3-CD56+ILT2+ cells were detected in diabetics and controls, CD3-CD56+CD107a+ and CD3-CD56+ILT2+CD107a+ cells were significantly increased in T1D PBMC either GAD65 AA 114-122 or FLU peptides stimulated after co-culture with GAD65 AA 114-122 pulsed APCs. As control, healthy donor NK cells showed similar degranulation against both GAD65 AA 114-122 pulsed and unpulsed APCs. The pathogenetic significance of the CD3-CD8dullCD56+ 'memory-like NK cell subset' with increased response upon secondary challenge in diabetics remains to be elucidated.

Use of short interfering RNA delivered by cationic liposomes to enable efficient down-regulation of PTPN22 gene in human T lymphocytes.

Type 1 diabetes and thyroid disease are T cell-dependent autoimmune endocrinopathies. The standard substitutive administration of the deficient hormones does not halt the autoimmune process; therefore, development of immunotherapies aiming to preserve the residual hormonal cells, is of crucial importance. PTPN22 C1858T mutation encoding for the R620W lymphoid tyrosine phosphatase variant, plays a potential pathophysiological role in autoimmunity. The PTPN22 encoded protein Lyp is a negative regulator of T cell antigen receptor signaling; R620W variant, leading to a gain of function with paradoxical reduced T cell activation, may represent a valid therapeutic target. We aimed to develop novel wild type PTPN22 short interfering RNA duplexes (siRNA) and optimize their delivery into Jurkat T cells and PBMC by using liposomal carriers. Conformational stability, size and polydispersion of siRNA in lipoplexes was measured by CD spectroscopy and DLS. Lipoplexes internalization and toxicity evaluation was assessed by confocal microscopy and flow cytometry analysis. Their effect on Lyp expression was evaluated by means of Western Blot and confocal microscopy. Functional assays through engagement of TCR signaling were established to evaluate biological consequences of down-modulation. Both Jurkat T cells and PBMC were efficiently transfected by stable custom lipoplexes. Jurkat T cell morphology and proliferation was not affected. Lipoplexes incorporation was visualized in CD3+ but also in CD3- peripheral blood immunotypes without signs of toxicity, damage or apoptosis. Efficacy in affecting Lyp protein expression was demonstrated in both transfected Jurkat T cells and PBMC. Moreover, impairment of Lyp inhibitory activity was revealed by increase of IL-2 secretion in culture supernatants of PBMC following anti-CD3/CD28 T cell receptor-driven stimulation. The results of our study open the pathway to future trials for the treatment of autoimmune diseases based on the selective inhibition of variant PTPN22 allele using lipoplexes of siRNA antisense oligomers.

MDM4 actively restrains cytoplasmic mTORC1 by sensing nutrient availability.

BACKGROUND: Many tumor-related factors have shown the ability to affect metabolic pathways by paving the way for cancer-specific metabolic features. Here, we investigate the regulation of mTORC1 by MDM4, a p53-inhibitor with oncogenic or anti-survival activities depending on cell growth conditions. METHOD: MDM4-mTOR relationship was analysed through experiments of overexpression or silencing of endogenous proteins in cell culture and using purified proteins in vitro. Data were further confirmed in vivo using a transgenic mouse model overexpressing MDM4. Additionally, the Cancer Genome Atlas (TCGA) database (N = 356) was adopted to analyze the correlation between MDM4 and mTOR levels and 3D cultures were used to analyse the p53-independent activity of MDM4. RESULTS: Following nutrient deprivation, MDM4 impairs mTORC1 activity by binding and inhibiting the kinase mTOR, and contributing to maintain the cytosolic inactive pool of mTORC1. This function is independent of p53. Inhibition of mTORC1 by MDM4 results in reduced phosphorylation of the mTOR downstream target p70S6K1 both in vitro and in vivo in a MDM4-transgenic mouse. Consistently, MDM4 reduces cell size and proliferation, two features controlled by p70S6K1, and, importantly, inhibits mTORC1-mediated mammosphere formation. Noteworthy, MDM4 transcript levels are significantly reduced in breast tumors characterized by high mTOR levels. CONCLUSION: Overall, these data identify MDM4 as a nutrient-sensor able to inhibit mTORC1 and highlight its metabolism-related tumor-suppressing function.

Estrogens enhance myoblast differentiation in facioscapulohumeral muscular dystrophy by antagonizing DUX4 activity.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder that is characterized by extreme variability in symptoms, with females being less severely affected than males and presenting a higher proportion of asymptomatic carriers. The sex-related factors involved in the disease are not known. Here, we have utilized myoblasts isolated from FSHD patients (FSHD myoblasts) to investigate the effect of estrogens on muscle properties. Our results demonstrated that estrogens counteract the differentiation impairment of FSHD myoblasts without affecting cell proliferation or survival. Estrogen effects are mediated by estrogen receptor ß (ERß), which reduces chromatin occupancy and transcriptional activity of double homeobox 4 (DUX4), a protein whose aberrant expression has been implicated in FSHD pathogenesis. During myoblast differentiation, we observed that the levels and activity of DUX4 increased progressively and were associated with its enhanced recruitment in the nucleus. ERß interfered with this recruitment by relocalizing DUX4 in the cytoplasm. This work identifies estrogens as a potential disease modifier that underlie sex-related differences in FSHD by protecting against myoblast differentiation impairments in this disease.

Association of TrkA and APP Is Promoted by NGF and Reduced by Cell Death-Promoting Agents.

The amyloid precursor protein (APP) interacts with the tropomyosin receptor kinase A (TrkA) in normal rat, mouse, and human brain tissue but not in Alzheimer's disease (AD) brain tissue. However, it has not been reported whether the two proteins interact directly, and if so, which domains are involved. Clarifying these points will increase our understanding of the role and regulation of the TrkA/APP interaction in normal brain functioning as well as in AD. Here we addressed these questions using bimolecular fluorescence complementation (BiFC) and the proximity ligation assay (PLA). We demonstrated that exogenously expressed APP and TrkA associate through their juxtamembrane/transmembrane domains, to form a complex that localizes mainly to the plasma membrane, endoplasmic reticulum (ER) and Golgi. Formation of the complex was inhibited by p75NTR, ShcC and Mint-2. Importantly, we demonstrated that the association between endogenous APP and TrkA in primary septal neurons were modified by NGF, or by drugs that either inhibit ER-to-Golgi transport or perturb microtubules and microfilaments. Interestingly, several agents that induce cell death [amyloid ß (Aß)-peptide, staurosporine and rapamycin], albeit via different mechanisms, all caused dissociation of APP/TrkA complexes and increased production of C-terminal fragment (ß-CTF) APP fragment. These findings open new perspectives for investigating the interplay between these proteins during neurodegeneration and AD.

The Double Role of p53 in Cancer and Autoimmunity and Its Potential as Therapeutic Target.

p53 is a sequence-specific short-lived transcription factor expressed at low concentrations in various tissues while it is upregulated in damaged, tumoral or inflamed tissue. In normally proliferating cells, p53 protein levels and function are tightly controlled by main regulators, i.e., MDM2 (mouse double minute 2) and MDM4 proteins. p53 plays an important role due to its ability to mediate tumor suppression. In addition to its importance as a tumor suppressor, p53 coordinates diverse cellular responses to stress and damage and plays an emerging role in various physiological processes, including fertility, cell metabolism, mitochondrial respiration, autophagy, cell adhesion, stem cell maintenance and development. Interestingly, it has been recently implicated in the suppression of autoimmune and inflammatory diseases in both mice and humans. In this review based on current knowledge on the functional properties of p53 and its regulatory pathways, we discuss the potential utility of p53 reactivation from a therapeutic perspective in oncology and chronic inflammatory disorders leading to autoimmunity.

Peptides and peptidomimetics in the p53/MDM2/MDM4 circuitry - a patent review.

INTRODUCTION: Restoration of the p53 tumor suppressor function is an attractive anticancer strategy. Despite the development of several therapeutics targeting the two main p53 negative regulators, MDM2 and MDM4, no one has yet reached clinical application. In the past, several efforts have been employed to develop more specific and efficient compounds that can improve and/or overcome some of the features related to small molecule compounds (SMC). Peptides and peptidomimetics are emerging as attractive molecules given their increased selectivity, reduced toxicity and reduced tendency to develop tumor-resistance compared to SMC. Area covered: This article reviews publications and patents (publicly available up to April 2016) for peptides and derivatives aimed to reactivate the oncosuppressive function of p53, with a particular focus on inhibitors of MDM2/MDM4. Emphasis is placed on the efficacy of these compounds compared to the p53-reactivating small molecules developed so far. Expert opinion: A number of promising peptides for p53 reactivation in cancer therapy have been developed. These compounds appear to possess improved features compared to SMC, especially for their ability to simultaneously target the MDM2/MDM4 inhibitors, and their increased specificity.

Targeting the MDM2/MDM4 interaction interface as a promising approach for p53 reactivation therapy.

Restoration of wild-type p53 tumor suppressor function has emerged as an attractive anticancer strategy. Therapeutics targeting the two p53-negative regulators, MDM2 and MDM4, have been developed, but most agents selectively target the ability of only one of these molecules to interact with p53, leaving the other free to operate. Therefore, we developed a method that targets the activity of MDM2 and MDM4 simultaneously based on recent studies indicating that formation of MDM2/MDM4 heterodimer complexes are required for efficient inactivation of p53 function. Using computational and mutagenesis analyses of the heterodimer binding interface, we identified a peptide that mimics the MDM4 C-terminus, competes with endogenous MDM4 for MDM2 binding, and activates p53 function. This peptide induces p53-dependent apoptosis in vitro and reduces tumor growth in vivo. Interestingly, interfering with the MDM2/MDM4 heterodimer specifically activates a p53-dependent oxidative stress response. Consistently, distinct subcellular pools of MDM2/MDM4 complexes were differentially sensitive to the peptide; nuclear MDM2/MDM4 complexes were particularly highly susceptible to the peptide-displacement activity. Taken together, these data identify the MDM2/MDM4 interaction interface as a valuable molecular target for therapeutic reactivation of p53 oncosuppressive function.

IGF-1R/MDM2 relationship confers enhanced sensitivity to RITA in Ewing sarcoma cells.

Ewing sarcoma is one of the most frequent bone cancers in adolescence. Although multidisciplinary therapy has improved the survival rate for localized tumors, a critical step is the development of new drugs to improve the long-term outcome of recurrent and metastatic disease and to reduce side effects of conventional therapy. Here, we show that the small molecule reactivation of p53 and induction of tumor cell apoptosis (RITA, NSC652287) is highly effective in reducing growth and tumorigenic potential of Ewing sarcoma cell lines. These effects occur both in the presence of wt-p53 as well as of mutant or truncated forms of p53, or in its absence, suggesting the presence of additional targets in this tumor histotype. Further experiments provided evidence that RITA modulates an important oncogenic mark of these cell lines, insulin-like growth factor receptor 1 (IGF-1R). Particularly, RITA causes downregulation of IGF-1R protein levels. MDM2 degradative activity is involved in this phenomenon. Indeed, inhibition of MDM2 function by genetic or pharmacologic approaches reduces RITA sensitivity of Ewing sarcoma cell lines. Overall, these data suggest that in the cell context of Ewing sarcoma, RITA may adopt additional mechanism of action besides targeting p53, expanding its field of application. Noteworthy, these results envisage the promising utilization of RITA or its derivative as a potential treatment for Ewing sarcomas.

MDM4 (MDMX) localizes at the mitochondria and facilitates the p53-mediated intrinsic-apoptotic pathway.

MDM4 is a key regulator of p53, whose biological activities depend on both transcriptional activity and transcription-independent mitochondrial functions. MDM4 binds to p53 and blocks its transcriptional activity; however, the main cytoplasmic localization of MDM4 might also imply a regulation of p53-mitochondrial function. Here, we show that MDM4 stably localizes at the mitochondria, in which it (i) binds BCL2, (ii) facilitates mitochondrial localization of p53 phosphorylated at Ser46 (p53Ser46(P)) and (iii) promotes binding between p53Ser46(P) and BCL2, release of cytochrome C and apoptosis. In agreement with these observations, MDM4 reduction by RNA interference increases resistance to DNA-damage-induced apoptosis in a p53-dependent manner and independently of transcription. Consistent with these findings, a significant downregulation of MDM4 expression associates with cisplatin resistance in human ovarian cancers, and MDM4 modulation affects cisplatin sensitivity of ovarian cancer cells. These data define a new localization and function of MDM4 that, by acting as a docking site for p53Ser46(P) to BCL2, facilitates the p53-mediated intrinsic-apoptotic pathway. Overall, our results point to MDM4 as a double-faced regulator of p53.