Specific targeting of caspase-9/PP2A interaction as potential new anti-cancer therapy.

PURPOSE: PP2A is a serine/threonine phosphatase critical to physiological processes, including apoptosis. Cell penetrating peptides are molecules that can translocate into cells without causing membrane damage. Our goal was to develop cell-penetrating fusion peptides specifically designed to disrupt the caspase-9/PP2A interaction and evaluate their therapeutic potential in vitro and in vivo. EXPERIMENTAL DESIGN: We generated a peptide containing a penetrating sequence associated to the interaction motif between human caspase-9 and PP2A (DPT-C9h), in order to target their association. Using tumour cell lines, primary human cells and primary human breast cancer (BC) xenografts, we investigated the capacity of DPT-C9h to provoke apoptosis in vitro and inhibition of tumour growth (TGI) in vivo. DPT-C9h was intraperitoneally administered at doses from 1 to 25 mg/kg/day for 5 weeks. Relative Tumour Volume (RTV) was calculated. RESULTS: We demonstrated that DPT-C9h specifically target caspase-9/PP2A interaction in vitro and in vivo and induced caspase-9-dependent apoptosis in cancer cell lines. DPT-C9h also induced significant TGI in BC xenografts models. The mouse-specific peptide DPT-C9 also induced TGI in lung (K-Ras model) and breast cancer (PyMT) models. DPT-C9h has a specific effect on transformed B cells isolated from chronic lymphocytic leukemia patients without any effect on primary healthy cells. Finally, neither toxicity nor immunogenic responses were observed. CONCLUSION: Using the cell-penetrating peptides blocking caspase-9/PP2A interactions, we have demonstrated that DPT-C9h had a strong therapeutic effect in vitro and in vivo in mouse models of tumour progression

Progression-associated molecular changes in basal/squamous and sarcomatoid bladder carcinogenesis

International audienceThe aggressive basal/squamous (Ba/Sq) bladder cancer (BLCA) subtype is often diagnosed at the muscle-invasive stage and can progress to the sarcomatoid variant. Identification of molecular changes occurring during progression from non-muscle-invasive BLCA (NMIBC) to Ba/Sq muscle-invasive BLCA (MIBC) is thus challenging in human disease. We used the N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) mouse model of Ba/Sq MIBC to study longitudinally the molecular changes leading to the Ba/Sq phenotype and to the sarcomatoid variant using IHC and microdissection followed by RNA-seq at all stages of progression. A shift to the Ba/Sq phenotype started in early progression stages. Pathway analysis of gene clusters with coordinated expression changes revealed Shh signaling loss and a shift from fatty acid metabolism to glycolysis. An upregulated cluster, appearing early in carcinogenesis, showed relevance to human disease, identifying NMIBC patients at risk of progression. Similar to the human counterpart, sarcomatoid BBN tumors displayed a Ba/Sq phenotype and epithelial–mesenchymal transition (EMT) features. An EGFR/FGFR1 signaling switch occurred with sarcomatoid dedifferentiation and correlated with EMT. BLCA cell lines with high EMT were the most sensitive to FGFR1 knockout and resistant to EGFR knockout. Taken together, these findings provide insights into the underlying biology of Ba/Sq BLCA progression and sarcomatoid dedifferentiation with potential clinical implications

A Small Nucleolar RNA Linked to Diamond-Blackfan Anemia

Résumé de congrè

From a dominant to an oligogenic model of inheritance with environmental modifiers in acute intermittent porphyria

International audienceAcute intermittent porphyria (AIP) is a disease affecting the heme biosynthesis pathway caused by mutations of the hydroxymethylbilane synthase (HMBS) gene. AIP is thought to display autosomal dominant inheritance with incomplete penetrance. We evaluated the prevalence, penetrance and heritability of AIP, in families with the disease from the French reference center for porphyria (CFP) (602 overt patients; 1968 relatives) and the general population, using Exome Variant Server (EVS; 12 990 alleles) data. The pathogenicity of the 42 missense variants identified was assessed in silico, and in vitro, by measuring residual HMBS activity of the recombinant protein. The minimal estimated prevalence of AIP in the general population was 1/1299. Thus, 50 000 subjects would be expected to carry the AIP genetic trait in France. Penetrance was estimated at 22.9% in families with AIP, but at only 0.5-1% in the general population. Intrafamily correlation studies showed correlations to be strong overall and modulated by kinship and the area in which the person was living, demonstrating strong influences of genetic and environmental modifiers on inheritance. Null alleles were associated with a more severe phenotype and a higher penetrance than for other mutant alleles. In conclusion, the striking difference in the penetrance of HMBS mutations between the general population and the French AIP families suggests that AIP inheritance does not follow the classical autosomal dominant model, instead of being modulated by strong environmental and genetic factors independent from HMBS. An oligogenic inheritance model with environmental modifiers might better explain AIP penetrance and heritability

DPT-C9h induces apoptosis in human cell lines.

<p><b>A</b>) Daudi, Jurkat, and HeLa cell lines were cultured in the presence of DPT-C9h, DPT-Sh1, C9h, or C9 peptides for 20 h at 100 µM and apoptosis was estimated by Annexin-V staining. <b>B</b>) Mouse lung cancer cell lines LKR10 and LKR13 were cultured in the presence of DPT-C9h, DPT-C9, or DPT-Sh1 at 100 µM. After 24 h of incubation, apoptosis was estimated by Annexin staining. The basal level of apoptosis of control non-treated cells is shown. P values are also shown (*<0.05; **<0.001; ***<0.0001). <b>C</b>) Breast, uveal melanoma and lung cancer cell lines isolated from primary human xenografs were cultured in the presence or absence of DPT-C9h peptide (100 µM) for 24h and apoptosis was estimated by Annexin V-FITC. Basal level of apoptosis without peptide addition is shown (grey colour) p values are shown. <b>D</b>). Breast cancer cell lines derived from the primary human xenografts, were incubated with C9h in culture medium at 150 µM and apoptosis induction was estimated at different times. <b>E</b>) Breast cancer cell lines isolated from primary human xenografts BCx-3 and Bcx-12 were cultured in the presence or absence (control) of the peptide DPT-C9h for 24 h and apoptosis was estimated.</p

Apoptotic effect of DPT-C9h peptide on primary and tumour cells.

<p><b>A</b>) Peripheral blood mononuclear cells (PBMC) from healthy donors or CLL patients were cultured in the presence of DPT-C9h (150 µM) for 3 h, then washed, transferred to complete medium and apoptosis was estimated 6h later. Selection of B cells was done by anti-CD19 antibody before Annexin V-FITC staining. Non-treated cells were used as control. <b>B</b>) Cells isolated from bone marrow of CLL patients and healthy donors were treated as in A and analyzed for apoptosis. P values are shown.</p

Effect of DPT-C9h on caspase-9 activation, mitochondrial membrane depolarization, cytochrome <i>c</i> release and cell cycle.

<p>A) HBCx-3 cells were cultured for 3 or 6 h with medium (control), 100 µM of DPT-C9h or 10 µM of the caspase inhibitor Z-VAD (pre incubation of 1h) and 100 µM of DPT-C9h. Caspase-9 activity was estimated using a luminogenic substrate. Results are represented relative to control non-treated cells as arbitrary units. P values are shown. B) HBCx-3 cells were cultured for 24 h with medium (control), DPT-Sh1 (100 µM), DPT-C9h (100 µM) or Z-VAD (10 µM, pre incubation of 1h) and DPT-C9h (100 µM). Apoptosis was estimated by Annexin-V-FITC binding. C) HBCx-3 cells were treated for different periods of time with DPT-C9h (100 µM) and then incubated for 30 min at 37°C protected from the light with the fluorescent probe JC-10. Green and red fluorescence were measured. Data are represented relative to the control non-treated cells. P values are shown. D) HBCx-12A and HBCx-3 cell lines were treated for 24 h with 100 µM of DPT-C9 h. Mitochondrial fraction was separated from whole cell lysates and immunoblotted for cytochrome <i>c</i>. The WB was also hybridized with the mitochondrial marker Tim23 as internal control of protein loading. E) HBCx-3 cells were non-treated (control) or treated with 10 or 25 µM of DPT-C9h for 24 or 48 h and the cell cycle was analyzed by FACS.</p

<i>In vivo</i> antibody responses and toxicity induced by DPT-C9h.

<p><b>A</b><b> </b>) Serum antibodies taken from nude mice treated for different periods of time were detected by ELISA at two different concentrations of DPT-C9h peptide (10 and 50 µM). <b>B</b>) Serum antibodies from wild type mice treated for different periods for time were tested by ELISA against DPT-C9h and DPT-Sh1 (50 µM). <b>C</b>) DPT-C9h was intraperitoneally administered in mice bearing tumors HBCx-12A at 1, 5, or 25 mg/kg once daily for 5 weeks; the median weight of mice for each experimental group is represented at different times. A total of 10 mice were included per group. Similarly, DPT-C9h was intraperitoneally administered in mice bearing tumours HBCx-8 at 10 mg/kg twice daily for 4 weeks. DPT-C9 was IP administrated at mice model PyMT model at dose of 5 mg/kg. The median weight of mice for each experimental group is represented at different times. Ten mice were included per group.</p

Recurring mutations in RPL15 are linked to hydrops fetalis and treatment independence in diamond-blackfan anemia

Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure disorder linked predominantly to ribosomal protein gene mutations. Here the European DBA consortium reports novel mutations identified in the RPL15 gene in 6 unrelated individuals diagnosed with DBA. Although point mutations have not been previously reported for RPL15, we identified 4 individuals with truncating mutations p.Tyr81* (in 3 of 4) and p.Gln29*, and 2 with missense variants p.Leu10Pro and p.Lys153Thr. Notably, 75% (3 of 4) of truncating mutation carriers manifested with severe hydrops fetalis and required intrauterine transfusions. Even more remarkable is the observation that the 3 carriers of p.Tyr81* mutation became treatment-independent between four and 16 months of life and maintained normal blood counts until their last follow up. Genetic reversion at the DNA level as a potential mechanism of remission was not observed in our patients. In vitro studies revealed that cells carrying RPL15 mutations have pre-rRNA processing defects, reduced 60S ribosomal subunit formation, and severe proliferation defects. Red cell culture assays of RPL15-mutated primary erythroblast cells also showed a severe reduction in cell proliferation, delayed erythroid differentiation, elevated TP53 activity, and increased apoptosis. This study identifies a novel subgroup of DBA with mutations in the RPL15 gene with an unexpected high rate of hydrops fetalis and spontaneous, long-lasting remission