Summary Ischemia-associated oxidative damage leading to necrosis is a major cause of catastrophic tissue loss in human health. Elucidating its signaling mechanism is of paramount importance. p53 is a central stress sensor responding to multiple insults including oxidative stress to orchestrate apoptotic and autophagic types of cell death. Whether p53 can also activate oxidative stress-induced necrosis is unknown. Here we uncover a role of p53 in activating necrosis. In response to oxidative stress, p53 accumulates in the mitochondrial matrix and triggers mitochondrial permeability transition pore (PTP) opening and necrosis by physical interaction with the critical PTP regulator Cyclophilin D (CypD). Intriguingly, a robust p53-CypD complex forms during brain ischemia/reperfusion injury. In contrast, reduction of p53 levels or Cyclosporine A-pretreatment of mice prevents this complex and is associated with effective stroke protection. Our study identifies the mitochondrial p53-CypD axis as an important contributor to oxidative stress-induced necrosis and implicates this axis in stroke pathology.

p53 is one of the most mutated tumor suppressors in human cancers and as such has been intensively studied for a long time. p53 is a major orchestrator of the cellular response to a broad array of stress types by regulating apoptosis, cell cycle arrest, senescence, DNA repair and genetic stability. For a long time it was thought that these functions of p53 solely rely on its function as a transcription factor, and numerous p53 target genes have been identified [1]. In the last 8 years however, a novel transcription-independent proapoptotic function mediated by the cytoplasmic pool of p53 has been revealed. p53 participates directly in the intrinsic apoptosis pathway by interacting with the multidomain members of the Bcl-2 family to induce mitochondrial outer membrane permeabilization. Our review will discuss these studies, focusing on recent advances in the field.

Although human cancers have complex genotypes and are genomically unstable, they often remain dependent on the continued presence of single-driver mutations-a phenomenon dubbed ''oncogene addiction.'' Such dependencies have been demonstrated in mouse models, where conditional expression systems have revealed that oncogenes able to initiate cancer are often required for tumor maintenance and progression, thus validating the pathways they control as therapeutic targets. Here, we implement an integrative approach that combines genetically defined mouse models, transcriptional profiling, and a novel inducible RNAi platform to characterize cellular programs that underlie addiction to MLL-AF9-a fusion oncoprotein involved in aggressive forms of acute myeloid leukemia (AML). We show that MLL-AF9 contributes to leukemia maintenance by enforcing a Mybcoordinated program of aberrant self-renewal involving genes linked to leukemia stem cell potential and poor prognosis in human AML. Accordingly, partial and transient Myb suppression precisely phenocopies MLL-AF9 withdrawal and eradicates aggressive AML in vivo without preventing normal myelopoiesis, indicating that strategies to inhibit Myb-dependent aberrant self-renewal programs hold promise as effective and cancer-specific therapeutics. Together, our results identify Myb as a critical mediator of oncogene addiction in AML, delineate relevant Myb target genes that are amenable to pharmacologic inhibition, and establish a general approach for dissecting oncogene addiction in vivo.

The p53 family member p73 is essential for brain development, but its precise role and scope remain unclear. Global p73 deficiency determines an overt and highly penetrant brain phenotype marked by cortical hypoplasia with ensuing hydrocephalus and hippocampal dysgenesis. The ΔNp73 isoform is known to function as a prosurvival factor of mature postmitotic neurons. In this study, we define a novel essential role of p73 in the regulation of the neural stem cell compartment. In both embryonic and adult neurogenesis, p73 has a critical role in maintaining an adequate neurogenic pool by promoting self-renewal and proliferation and inhibiting premature senescence of neural stem and early progenitor cells. Thus, products of the p73 gene locus are essential maintenance factors in the central nervous system, whose broad action stretches across the entire differentiation arch from stem cells to mature postmitotic neurons.

SUMMARY Our recent ERK1/2 inhibitor analyses in pancreatic ductal adenocarcinoma (PDAC) indicated ERK1/2-independent mechanisms maintaining MYC protein stability. To identify these mechanisms, we determined the signaling networks by which mutant KRAS regulates MYC. Acute KRAS suppression caused rapid proteasome-dependent loss of MYC protein, through both ERK1/2-dependent and -independent mechanisms. Surprisingly, MYC degradation was independent of PI3K-AKT-GSK3β signaling and the E3 ligase FBWX7. We then established and applied a high-throughput screen for MYC protein degradation and performed a kinome-wide proteomics screen. We identified an ERK1/2-inhibition-induced feed-forward mechanism dependent on EGFR and SRC, leading to ERK5 activation and phosphorylation of MYC at S62, preventing degradation. Concurrent inhibition of ERK1/2 and ERK5 disrupted this mechanism, synergistically causing loss of MYC and suppressing PDAC growth.

p53 can induce apoptosis through mitochondrial membrane permeabilization by interaction of its DNA binding region with the anti-apoptotic proteins BclxL and Bcl2. However, little is known about the action of p53 at the mitochondria in molecular detail. By using NMR spectroscopy and fluorescence polarization we characterized the binding of wildtype and mutant p53 DNA binding domains to BclxL and show that the wild-type p53 DNA binding domain leads to structural changes in the BH3 binding region of BclxL, whereas mutants fail to induce such effects due to reduced affinity. This was probed by induced chemical shift and residual dipolar coupling data. These data imply that p53 partly achieves its pro-apoptotic function at the mitochondria by facilitating interaction between BclxL and BH3-only proteins in an allosteric mode of action. Furthermore, we characterize for the first time the binding behavior of Pifithrin-, a specific small molecule inhibitor of the p53-BclxL interaction, and present a structural model of the protein-ligand complex. A rather unusual behavior is revealed whereby Pifithrin-binds to both sides of the protein-protein complex. These data should facilitate the rational design of more potent specific BclxL-p53 inhibitors.Due to its central role in the induction of apoptosis and cellcycle arrest, p53 is one of the most important checkpoint proteins of cell survival and genomic integrity induced by a variety of different damage stimuli like DNA damage, oncogenic or hypoxic stress (1). p53 can induce apoptosis via transcriptiondependent and transcription-independent functions (2). In particular, p53 mediates its pro-apoptotic response via transcriptional activation of pro-apoptotic target genes, such as BAX and PUMA, and trans-repression of pro-survival proteins (for review, see Ref.3). In addition, death signals are mediated through the transcription-independent p53 death pathway, which acts via p53 protein-mediated direct mitochondrial outer membrane permeabilization (MOMP), 2 leading to rapid cytochrome c release.MOMP is believed to depend on the opening of the dynamic Bax/Bak lipid pore and is triggered by BH3-only proteins (4, 5). In response to a death stimulus, a fraction of stress-stabilized p53 rapidly translocates to mitochondria in primary, immortal, and transformed cells (6). The primary targets of p53 at the mitochondria are pro-survival Bcl2 proteins. Members of the Bcl2 family can be grouped into pro-and anti-apoptotic subfamilies, based on their modular content of BH1, BH2, BH3, and BH4 (Bcl2 homology) domains. The BH1234 domain proteins BclxL and Bcl2 are prototype anti-apoptotic members that stabilize the outer mitochondrial membrane by binding and inhibiting pro-apoptotic members via their hydrophobic BH123 binding pocket (also called the BH3 peptide binding pocket) (7). In contrast, BH123 domain proteins Bax and Bak act as ultimate pro-apoptotic effector proteins. A third class, the BH3-only proteins, like Bad, tBid, or Puma, act as key transducers of death signals and fall into t...

Strategies to induce p53 activation in tumors that retain wild-type p53 are promising for cancer therapy. Nutlin is a potent and selective pharmacological MDM2 inhibitor that competitively binds to its p53-binding pocket, thereby leading to non-genotoxic p53 stabilization and activation of growth arrest and apoptosis pathways. Nutlin-induced apoptosis is thought to occur via p53’s transcriptional program. Here we report that the transcription-independent mitochondrial p53 program plays an important role in Nutlin-induced p53-mediated tumor cell death. Aside from nuclear stabilization, Nutlin causes cytoplasmic p53 accumulation and translocation to mitochondria. Monoubiquitinated p53, originating from a distinct cytoplasmic pool, is the preferred p53 species that translocates to mitochondria in response to stress. Nutlin does not interfere with MDM2’s ability to monoubiquitinate p53, due to the fact that MDM2-p53 complexes are only partially disrupted and that Nutlin-stabilized MDM2 retains its E3 ubiquitin ligase activity. Nutlin-induced mitochondrial p53 translocation is rapid and associated with cytochrome C release that precedes induction of p53 target genes. Specific inhibition of mitochondrial p53 translocation by Pifithrin μ reduces the apoptotic Nutlin response by 2.5-fold, underlining the significance of p53’s mitochondrial program in Nutlin-induced apoptosis. Surprisingly, blocking the transcriptional arm of p53, either via α-Amanitin or the p53-specific transcriptional inhibitor Pifithrin α, not only fails to inhibit, but greatly potentiates Nutlin-induced apoptosis. In sum, the direct mitochondrial program is a major mechanism in Nutlin-induced p53-mediated apoptosis. Moreover, at least in some tumors the transcriptional p53 activities in net balance not only are dispensable for the apoptotic Nutlin response, but appear to actively block its therapeutic effect.

Purpose: Epidermal growth factor receptor (EGFR) family members (e.g., EGFR, HER2, HER3, and HER4) are commonly overexpressed in pancreatic cancer. We investigated the effects of inhibition of EGFR/HER2 signaling on pancreatic cancer to elucidate the role(s) of EGFR/HER2 in radiosensitization and to provide evidence in support of further clinical investigations.Experimental Design: Expression of EGFR family members in pancreatic cancer lines was assessed by quantitative reverse transcription-PCR. Cell growth inhibition was determined by MTS assay. The effects of inhibition of EGFR family receptors and downstream signaling pathways on in vitro radiosensitivity were evaluated using clonogenic assays. Growth delay was used to evaluate the effects of nelfinavir on in vivo tumor radiosensitivity.Results: Lapatinib inhibited cell growth in four pancreatic cancer cell lines, but radiosensitized only wild-type K-ras-expressing T3M4 cells. Akt activation was blocked in a wild-type K-ras cell line, whereas constitutive phosphorylation of Akt and extracellular signal-regulated kinase (ERK) was seen in lines expressing mutant K-ras. Overexpression of constitutively active K-ras (G12V) abrogated lapatinib-mediated inhibition of both Akt phosphorylation and radiosensitization. Inhibition of MAP/ERK kinase/ERK signaling with U0126 had no effect on radiosensitization, whereas inhibition of activated Akt with LY294002 (enhancement ratio, 1.2-1.8) or nelfinavir (enhancement ratio, 1.2-1.4) radiosensitized cells regardless of K-ras mutation status. Oral nelfinavir administration to mice bearing mutant K-rascontaining Capan-2 xenografts resulted in a greater than additive increase in radiation-mediated tumor growth delay (synergy assessment ratio of 1.5).Conclusions: Inhibition of EGFR/HER2 enhances radiosensitivity in wild-type K-ras pancreatic cancer. Nelfinavir, and other phosphoinositide 3-kinase/Akt inhibitors, are effective pancreatic radiosensitizers regardless of K-ras mutation status. Clin Cancer Res; 16(3); 912-23. ©2010 AACR.Pancreatic cancer, with ∼33,000 cases diagnosed annually, is the fourth leading cause of cancer deaths in the United States (1). Improvements in understanding the molecular aberrations underlying pancreatic cancer (reviewed in ref.2) have led to the approval of drugs targeting these abnormalities (3). Some of these agents target the members of the epidermal growth factor receptor family (EGFR/ErbB-1/HER1, ErbB-2/HER2/neu, ErbB-3/ HER3, and ErbB-4/HER4).Ligand activation of EGFR family proteins (EGFR is a member of the receptor tyrosine kinase superfamily of transmembrane proteins) results in perturbation of a variety of downstream signaling cascades. The clinical efficacy of drugs targeting the EGFR family of proteins was hypothesized due to the observed overexpression of EGFR in 40% to 70% of pancreatic cancers (4, 5), along with the overexpression of HER2 in a smaller subset of cases (6-8). The use of EGFR family inhibitors has been supported by data showing that blockade of EGFR or HER2 in...