Genome-Protective Topoisomerase 2a-Dependent G2 Arrest Requires p53 in hTERT-Positive Cancer Cells.

Topoisomerase 2a (Topo2a)-dependent G2 arrest engenders faithful segregation of sister chromatids, yet in certain tumor cell lines where this arrest is dysfunctional, a PKCε-dependent failsafe pathway can be triggered. Here we elaborate on recent advances in understanding the underlying mechanisms associated with this G2 arrest by determining that p53-p21 signaling is essential for efficient arrest in cell lines, in patient-derived cells, and in colorectal cancer organoids. Regulation of this p53 axis required the SMC5/6 complex, which is distinct from the p53 pathways observed in the DNA damage response. Topo2a inhibition specifically during S phase did not trigger G2 arrest despite affecting completion of DNA replication. Moreover, in cancer cells reliant upon the alternative lengthening of telomeres (ALT) mechanism, a distinct form of Topo2a-dependent, p53-independent G2 arrest was found to be mediated by BLM and Chk1. Importantly, the previously described PKCε-dependent mitotic failsafe was engaged in hTERT-positive cells when Topo2a-dependent G2 arrest was dysfunctional and where p53 was absent, but not in cells dependent on the ALT mechanism. In PKCε knockout mice, p53 deletion elicited tumors were less aggressive than in PKCε-replete animals and exhibited a distinct pattern of chromosomal rearrangements. This evidence suggests the potential of exploiting synthetic lethality in arrest-defective hTERT-positive tumors through PKCε-directed therapeutic intervention. SIGNIFICANCE: The identification of a requirement for p53 in stringent Topo2a-dependent G2 arrest and engagement of PKCε failsafe pathways in arrest-defective hTERT-positive cells provides a therapeutic opportunity to induce selective synthetic lethality.

A genome-wide RNAi screen identifies the SMC5/6 complex as a non-redundant regulator of a Topo2a-dependent G2 arrest.

The Topo2a-dependent arrest is associated with faithful segregation of sister chromatids and has been identified as dysfunctional in numerous tumour cell lines. This genome-protecting pathway is poorly understood and its characterization is of significant interest, potentially offering interventional opportunities in relation to synthetic lethal behaviours in arrest-defective tumours. Using the catalytic Topo2a inhibitor ICRF193, we have performed a genome-wide siRNA screen in arrest-competent, non-transformed cells, to identify genes essential for this arrest mechanism. In addition, we have counter-screened several DNA-damaging agents and demonstrate that the Topo2a-dependent arrest is genetically distinct from DNA damage checkpoints. We identify the components of the SMC5/6 complex, including the activity of the E3 SUMO ligase NSE2, as non-redundant players that control the timing of the Topo2a-dependent arrest in G2. We have independently verified the NSE2 requirement in fibroblasts from patients with germline mutations that cause severely reduced levels of NSE2. Through imaging Topo2a-dependent G2 arrested cells, an increased interaction between Topo2a and NSE2 is observed at PML bodies, which are known SUMOylation hotspots. We demonstrate that Topo2a is SUMOylated in an ICRF193-dependent manner by NSE2 at a novel non-canonical site (K1520) and that K1520 sumoylation is required for chromosome segregation but not the G2 arrest.

Cardiac RKIP induces a beneficial ß-adrenoceptor-dependent positive inotropy.

In heart failure therapy, it is generally assumed that attempts to produce a long-term increase in cardiac contractile force are almost always accompanied by structural and functional damage. Here we show that modest overexpression of the Raf kinase inhibitor protein (RKIP), encoded by Pebp1 in mice, produces a well-tolerated, persistent increase in cardiac contractility that is mediated by the ß1-adrenoceptor (ß1AR). This result is unexpected, as ß1AR activation, a major driver of cardiac contractility, usually has long-term adverse effects. RKIP overexpression achieves this tolerance via simultaneous activation of the ß2AR subtype. Analogously, RKIP deficiency exaggerates pressure overload-induced cardiac failure. We find that RKIP expression is upregulated in mouse and human heart failure, indicative of an adaptive role for RKIP. Pebp1 gene transfer in a mouse model of heart failure has beneficial effects, suggesting a new therapeutic strategy for heart failure therapy.

RKIP: a governor of intracellular signaling.

The Raf kinase inhibitor protein (RKIP) increasingly evolves as an important regulator of intracellular signaling networks and thus participates in diverse physiological functions ranging from growth and differentiation processes to muscle contraction. Several molecular events contribute to the ability of RKIP to tightly coordinate kinase signaling. The elucidation of the molecular mechanisms leading to substrate specificity of RKIP and substrate binding efficacy is of great interest for a better understanding of the overall role of RKIP in the organism but also for the design of specific and potent kinase inhibitors. In this work, we will review mechanistic details of the regulation of RKIP as inhibitor of Raf-1 and G protein-coupled receptor kinase 2 (GRK2) that enable RKIP to coordinate the cell's balance between inhibition and potentiation of mitogenic ERK1/2 signaling--a prominent example of RKIP's function as a regulator of intracellular signaling.

Interference with ERK(Thr188) phosphorylation impairs pathological but not physiological cardiac hypertrophy.

Extracellular signal-regulated kinases 1 and 2 (ERK1/2) are central mediators of cardiac hypertrophy and are discussed as potential therapeutic targets. However, direct inhibition of ERK1/2 leads to exacerbated cardiomyocyte death and impaired heart function. We have previously identified ERK(Thr188) autophosphorylation as a regulatory phosphorylation of ERK1/2 that is a key factor in cardiac hypertrophy. Here, we investigated whether interference with ERK(Thr188) phosphorylation permits the impairment of ERK1/2-mediated cardiac hypertrophy without increasing cardiomyocyte death. The impact of ERK(Thr188) phosphorylation on cardiomyocyte hypertrophy and cell survival was analyzed in isolated cells and in mice using the mutant ERK2(T188A), which is dominant-negative for ERK(Thr188) signaling. ERK2(T188A) efficiently attenuated cardiomyocyte hypertrophic responses to phenylephrine and to chronic pressure overload, but it affected neither antiapoptotic ERK1/2 signaling nor overall physiological cardiac function. In contrast to its inhibition of pathological hypertrophy, ERK2(T188A) did not interfere with physiological cardiac growth occurring with age or upon voluntary exercise. A preferential role of ERK(Thr188) phosphorylation in pathological types of hypertrophy was also seen in patients with aortic valve stenosis: ERK(Thr188) phosphorylation was increased 8.5 ± 1.3-fold in high-gradient, rapidly progressing cases (≥40 mmHg gradient), whereas in low-gradient, slowly progressing cases, the increase was not significant. Because interference with ERK(Thr188) phosphorylation (i) inhibits pathological hypertrophy and (ii) does not impair antiapoptotic ERK1/2 signaling and because ERK(Thr188) phosphorylation shows strong prevalence for aortic stenosis patients with rapidly progressing course, we conclude that interference with ERK(Thr188) phosphorylation offers the possibility to selectively address pathological types of cardiac hypertrophy.

Raf kinase inhibitor protein (RKIP) dimer formation controls its target switch from Raf1 to G protein-coupled receptor kinase (GRK) 2.

Proteins controlling cellular networks have evolved distinct mechanisms to ensure specificity in protein-protein interactions. Raf kinase inhibitor protein (RKIP) is a multifaceted kinase modulator, but it is not well understood how this small protein (21 kDa) can coordinate its diverse signaling functions. Raf1 and G protein-coupled receptor kinase (GRK) 2 are direct interaction partners of RKIP and thus provide the possibility to untangle the mechanism of its target specificity. Here, we identify RKIP dimer formation as an important mechanistic feature in the target switch from Raf1 to GRK2. Co-immunoprecipitation and cross-linking experiments revealed RKIP dimerization upon phosphorylation of RKIP at serine 153 utilizing purified proteins as well as in cells overexpressing RKIP. A functional phosphomimetic RKIP mutant had a high propensity for dimerization and reproduced the switch from Raf1 to GRK2. RKIP dimerization and GRK2 binding, but not Raf1 interaction, were prevented by a peptide comprising amino acids 127-146 of RKIP, which suggests that this region is critical for dimer formation. Furthermore, a dimeric RKIP mutant displayed a higher affinity to GRK2, but a lower affinity to Raf1. Functional analyses of phosphomimetic as well as dimeric RKIP demonstrated that enhanced dimerization of RKIP translates into decreased Raf1 and increased GRK2 inhibition. The detection of RKIP dimers in a complex with GRK2 in murine hearts implies their physiological relevance. These findings represent a novel mechanistic feature how RKIP can discriminate between its different interaction partners and thus advances our understanding how specific inhibition of kinases can be achieved.