Targeting IKKε in Androgen-Independent Prostate Cancer Causes Phenotypic Senescence and Genomic Instability.

Advanced prostate cancer will often progress to a lethal, castration-resistant state. We previously demonstrated that IKKε expression correlated with the aggressiveness of prostate cancer disease. Here, we address the potential of IKKε as a therapeutic target in prostate cancer. We examined cell fate decisions (proliferation, cell death, and senescence) in IKKε-depleted PC-3 cells, which exhibited delayed cell proliferation and a senescent phenotype, but did not undergo cell death. Using IKKε/TBK1 inhibitors, BX795 and Amlexanox, we measured their effects on cell fate decisions in androgen-sensitive prostate cancer and androgen-independent prostate cancer cell lines. Cell-cycle analyses revealed a G2-M cell-cycle arrest and a higher proportion of cells with 8N DNA content in androgen-independent prostate cancer cells only. Androgen-independent prostate cancer cells also displayed increased senescence-associated (SA)-ß-galactosidase activity; increased γH2AX foci; genomic instability; and altered p15, p16, and p21 expression. In our mouse model, IKKε inhibitors also decreased tumor growth of androgen-independent prostate cancer xenografts but not 22Rv1 androgen-sensitive prostate cancer xenografts. Our study suggests that targeting IKKε with BX795 or Amlexanox in androgen-independent prostate cancer cells induces a senescence phenotype and demonstrates in vivo antitumor activity. These results strengthen the potential of exploiting IKKε as a therapeutic target.

Biomarkers of cardiometabolic complications in survivors of childhood acute lymphoblastic leukemia.

Survivors of childhood acute lymphoblastic leukemia (cALL) are at higher risk of developing cardiometabolic complications. We aimed at exploring the associations between biomarkers of inflammation, oxidative stress, endothelial function, endotoxemia and cardiometabolic risk factors. We conducted a cross-sectional analysis in 246 cALL survivors (mean age, 22.1 ± 6.3 years; mean time since diagnosis, 15.5 ± 5.2 years) and evaluated the associations using a series of logistic regressions. Using structural equation models, we also tested if the relationship between endotoxemia and cardiometabolic complications was mediated by the latent (unobserved) variable inflammation inferred from the observed biomarkers CRP, TNF-α and IL-6. High leptin-adiponectin ratio was associated with obesity [adjusted OR = 15.7; 95% CI (6.2-39.7)], insulin resistance [20.6 (5.2-82.1)] and the metabolic syndrome [11.2 (2.6-48.7)]. Higher levels of plasminogen activator inhibitor-1 and tumor necrosis factor-α were associated with obesity [3.37 (1.6-7.1) and 2.34 (1.3-4.2), respectively] whereas high C-reactive protein levels were associated with insulin resistance [3.3 (1.6-6.8)], dyslipidemia [2.6 (1.4-4.9)] and MetS [6.5 (2.4-17.9)]. Our analyses provided evidence for a directional relationship between lipopolysaccharide binding protein, related to metabolic endotoxemia, inflammation and cardiometabolic outcomes. Identification of biomarkers and biological mechanisms could open new avenues for prevention strategies to minimize the long-term sequelae, improve follow-up and optimize the quality of life of this high-risk population.

Exploiting interconnected synthetic lethal interactions between PARP inhibition and cancer cell reversible senescence.

Senescence is a tumor suppression mechanism defined by stable proliferation arrest. Here we demonstrate that the known synthetic lethal interaction between poly(ADP-ribose) polymerase 1 inhibitors (PARPi) and DNA repair triggers p53-independent ovarian cancer cell senescence defined by senescence-associated phenotypic hallmarks including DNA-SCARS, inflammatory secretome, Bcl-XL-mediated apoptosis resistance, and proliferation restriction via Chk2 and p21 (CDKN1A). The concept of senescence as irreversible remains controversial and here we show that PARPi-senescent cells re-initiate proliferation upon drug withdrawal, potentially explaining the requirement for sustained PARPi therapy in the clinic. Importantly, PARPi-induced senescence renders ovarian and breast cancer cells transiently susceptible to second-phase synthetic lethal approaches targeting the senescence state using senolytic drugs. The combination of PARPi and a senolytic is effective in preclinical models of ovarian and breast cancer suggesting that coupling these synthetic lethalities provides a rational approach to their clinical use and may together be more effective in limiting resistance.

Assessing Functional Roles of the Senescence-Associated Secretory Phenotype (SASP).

Cellular senescence is linked to many normal biological processes, including tumor suppression, development, and wound healing, but it is also associated with age-related pathologies such as cancer progression. Numerous functions of senescent cells depend on their ability to secrete bioactive molecules, a characteristic termed the senescence-associated secretory phenotype (SASP). Although the SASP is generally described as proinflammatory, its true microenvironmental impact and composition may vary according to cell types (i.e., fibroblasts/epithelial, normal/cancerous) and senescence-triggering stimuli (i.e., replicative senescence, DNA damage-induced senescence, oncogene-induced senescence). The SASP reinforces autocrine cell-autonomous functions such as the senescence-associated proliferation arrest, but also mediates potent paracrine, non-cell-autonomous effects. In a paracrine manner, senescent cells influence the remodeling of surrounding tissues and the biology of adjacent cells, including modulation of proliferation and migration/invasion, reinforcement/induction of peripheral senescence, and immune cell activity or recruitment. Overall, the complexity of the context-dependent SASP composition and varied microenvironmental impact demonstrate the importance of properly assessing SASP functions directly on target cells. In this chapter, we focus on experimental approaches to evaluate the impact of SASP on the proliferation and migration/invasion capacities of target cancer cells. These techniques, with combined supplemental notes, will facilitate the assessment of novel functions of senescent cells on their microenvironment, and can be easily adapted beyond the use of the presented SASP-cancer scenario.

Comparison of early and recent influenza A(H1N1)pdm09 isolates harboring or not the H275Y neuraminidase mutation, in vitro and in animal models.

After 6 years of circulation in humans, a novel antigenic variant of influenza A(H1N1)pdm09 (i.e., A/Michigan/45/2015) emerged in 2015-16 and has predominated thereafter worldwide. Herein, we compared in vitro and in vivo properties of 2016 wild-type (WT) A/Michigan/45/15-like isolate and its H275Y neuraminidase (NA) variant to the original A/California/07/09-like counterparts. The H275Y mutation induced comparable levels of resistance to oseltamivir and peramivir without altering zanamivir susceptibility in both 2009 and 2016 isolates. In vitro, the two WT isolates had comparable replicative properties. The 2016-H275Y isolate had lower titers at 36 h post-inoculation (PI) (P < 0.05) while the 2009-H275Y titers were lower at both 24 h (P < 0.01) and 36 h PI (P < 0.001) vs the respective WTs. In mice, the 2016-WT isolate caused less weight losses (P < 0.001) and lower lung viral titers (LVTs) (P < 0.01) vs the 2009-WT. The LVTs of 2016-WT and 2016-H275Y groups were comparable whereas the 2009-H275Y LVTs were lower vs the respective WT (P < 0.01). Ferrets infected with the 2016-WT isolate and their contacts had higher nasal viral titers (NVTs) at early time points vs the 2009-WT group (P < 0.01). Also, NVTs of 2016-H275Y animals were lower vs the 2016-WT group at early time points in both infected (P < 0.01) and contact animals (P < 0.001). In conclusion, while the H275Y mutation similarly impacts the A/California/07/2009- and A/Michigan/45/2015-like A(H1N1)pdm09 NAs, the fitness of these isolates differs according to animal models with the 2016 virus being less virulent in mice but slightly more virulent in ferrets, potentially reflecting a period of cumulative changes in surface and internal genes.

Impact of R152K and R368K neuraminidase catalytic substitutions on in vitro properties and virulence of recombinant A(H1N1)pdm09 viruses.

Neuraminidase (NA) mutations conferring resistance to NA inhibitors (NAIs) are expected to occur at framework or catalytic residues of the NA enzyme. Numerous clinical and in vitro reports already described NAI-resistant A(H1N1)pdm09 variants harboring various framework NA substitutions. By contrast, variants with NA catalytic changes remain poorly documented. Herein, we investigated the effect of R152K and R368K NA catalytic mutations on the NA enzyme properties, in vitro replicative capacity and virulence of A(H1N1)pdm09 recombinant viruses. In NA inhibition assays, the R152K and R368K substitutions resulted in reduced inhibition [10- to 100-fold increases in IC50 vs the wild-type (WT)] or highly reduced inhibition (>100-fold increases in IC50) to at least 3 approved NAIs (oseltamivir, zanamivir, peramivir and laninamivir). Such resistance phenotype correlated with a significant reduction of affinity observed for the mutants in enzyme kinetics experiments [increased Km from 20 ±â€¯1.77 for the WT to 200.8 ±â€¯10.54 and 565.2 ±â€¯135 µM (P < 0.01) for the R152K and R368K mutants, respectively]. The R152K and R368K variants grew at comparable or even higher titers than the WT in both MDCK and ST6GalI-MDCK cells. In experimentally-infected C57BL/6 mice, the recombinant WT and the R152K and R368K variants induced important signs of infection (weight loss) and resulted in mortality rates of 87.5%, 37.5% and 100%, respectively. The lung viral titers were comparable between the three infected groups. While the NA mutations were stable, an N154I substitution was detected in the HA2 protein of the R152K and R368K variants after in vitro passages as well as in lungs of infected mice. Due to the multi-drug resistance phenotypes and conserved fitness, the emergence of NA catalytic mutations accompanied with potential compensatory HA changes should be carefully monitored in A(H1N1)pdm09 viruses.

Peramivir susceptibilities of recombinant influenza A and B variants selected with various neuraminidase inhibitors.

BACKGROUND: Peramivir is a parenteral neuraminidase inhibitor (NAI) approved for treating influenza infections in a few countries. We determined peramivir susceptibilities of several uncharacterized influenza A and B neuraminidase (NA) and haemagglutinin (HA) mutants selected with different NAIs. METHODS: Recombinant wild-type (WT) and mutant NA proteins were expressed in 293T cells and susceptibility to peramivir, oseltamivir and zanamivir was determined by NA inhibition assay using the MUNANA substrate. Recombinant/reassortant influenza A(H1N1), A(H3N2) and B HA mutants were rescued by reverse genetics and assessed by plaque size or viral yield assays for drug susceptibility. RESULTS: Recombinant R152K, I222K/T, G248R+I266V, Q312R+I427T and R371K (A[H1N1]pdm09); E41G, 1222L/V, Q226H and S247P (A[H3N2]) and D198Y, A246D/S/T and G402S (B) mutant NA proteins (N2 numbering) were analysed. Peramivir exhibited the lowest IC50 values against both influenza A and B WT NAs. Peramivir and oseltamivir generally shared similar phenotypes. Of note, peramivir retained activity against I222K/T (A[H1N1]pdm09), I222L/V (A[H3N2]) and A246T (B) mutants, which had reduced inhibition (RI) or highly RI (HRI) against oseltamivir. Cross-RI/HRI against the three NAIs was observed for R152K, R371K and Q312R+I427T (A[H1N1]pdm09); S247P (A[H3N2]) and D198Y (B) mutants. All tested recombinant/reassortant R208K (A/Puerto Rico/8/34 [H1N1]); A28T, R124M and K189E (A/Victoria/3/75 [H3N2]) and T139N (B/Phuket/3073/13) HA mutants were susceptible to peramivir in cell culture experiments. CONCLUSIONS: Peramivir is highly active against seasonal influenza subtypes. Although peramivir and oseltamivir generally share similar phenotypes, peramivir still possesses activity against some variants with RI/HRI against oseltamivir. Finally, NAI-induced HA substitutions alone did not significantly impact NAI susceptibility.

The I427T neuraminidase (NA) substitution, located outside the NA active site of an influenza A(H1N1)pdm09 variant with reduced susceptibility to NA inhibitors, alters NA properties and impairs viral fitness.

Emergence of pan neuraminidase inhibitor (NAI)-resistant variants constitutes a serious clinical concern. An influenza A(H1N1)pdm09 variant containing the I427T/Q313R neuraminidase (NA) substitutions was previously identified in a surveillance study. Although these changes are not part of the NA active site, the variant showed reduced susceptibility to many NAIs. In this study, we investigated the mechanism of resistance for the I427T/Q313R substitution and its impact on the NA enzyme and viral fitness. Recombinant wild-type (WT), I427T/Q313R and I427T A(H1N1)pdm09 viruses were generated by reverse genetics and tested for their drug susceptibilities, enzymatic properties and replication kinetics in vitro as well as their virulence in mice. Molecular dynamics (MD) simulations were performed for NA structural analysis. The I427T substitution, which was responsible for the resistance phenotype observed in the double (I427T/Q313R) mutant, induced 17-, 56-, 7-, and 14-fold increases in IC50 values against oseltamivir, zanamivir, peramivir and laninamivir, respectively. The I427T substitution alone or combined to Q313R significantly reduced NA affinity. The I427T/Q313R and to a lesser extent I427T recombinant viruses displayed reduced viral titers vs WT in vitro. In experimentally-infected mice, the mortality rates were 62.5%, 0% and 14.3% for the WT, I417T/Q313R and I427T viruses, respectively. There were about 2.5- and 2-Log reductions in mean lung viral titers on day 5 post-infection for the I427T/Q313R and I427T mutants, respectively, compared to WT. Results from simulations revealed that the I427T change indirectly altered the stability of the catalytic R368 residue of the NA enzyme causing its reduced binding to the substrate/inhibitor. This study demonstrates that the I427T/Q313R mutant, not only alters NAI susceptibility but also compromises NA properties and viral fitness, which could explain its infrequent detection in clinic.

Blockade of the acute activation of mTOR complex 1 decreases hypertrophy development in rats with severe aortic valve regurgitation.

BACKGROUND: Hypertrophy (H) is an adaptive response of the heart to a hemodynamic overload. Severe left ventricular (LV) volume overload (VO) from valve regurgitations (aortic (AR) or mitral regurgitation) leads to eccentric LVH. Increased protein turnover is a major event during development of LVH and the mechanistic target of rapamycin (mTOR) is a key molecule for its control. The role of mTOR inhibition in the development of LVH using rapamycin for relatively short periods of time (days to a few weeks) has been studied in the past in pressure overload models but not in VO models. We investigated if mTOR pathway was activated during LVH development in a model of severe VO (AR) in rats and if a rapamycin treatment can slow heart remodeling in this situation. METHODS AND RESULTS: Male rats with severe AR were studied acutely at 2 days, at 8 weeks (compensated phase) and 6 months (late phase) after VO induction. mTOR complex (mTORC) 1 (ribosomal S6 protein phosphorylation) was activated early after AR induction but not later in the disease whereas mTORC2 activity levels (Akt phosphorylation at Ser473) remained stable. We observed that a moderate dose of rapamycin (2 mg/kg/day; orally) for 8 weeks prevented severe LVH caused by AR (-46 %: p < 0.001). Rapamycin treatment specifically inhibited LV mTORC1 without altering mTORC2 activity at 8 weeks. Rapamycin also prevented cardiac myocyte hypertrophy caused by AR. CONCLUSION: Rapamycin slows hypertrophy in LV VO by inhibiting early activation of mTORC1 without modulating mTORC2.