AATF/Che-1 acts as a phosphorylation-dependent molecular modulator to repress p53-driven apoptosis.

Following genotoxic stress, cells activate a complex signalling network to arrest the cell cycle and initiate DNA repair or apoptosis. The tumour suppressor p53 lies at the heart of this DNA damage response. However, it remains incompletely understood, which signalling molecules dictate the choice between these different cellular outcomes. Here, we identify the transcriptional regulator apoptosis-antagonizing transcription factor (AATF)/Che-1 as a critical regulator of the cellular outcome of the p53 response. Upon genotoxic stress, AATF is phosphorylated by the checkpoint kinase MK2. Phosphorylation results in the release of AATF from cytoplasmic MRLC3 and subsequent nuclear translocation where AATF binds to the PUMA, BAX and BAK promoter regions to repress p53-driven expression of these pro-apoptotic genes. In xenograft experiments, mice exhibit a dramatically enhanced response of AATF-depleted tumours following genotoxic chemotherapy with adriamycin. The exogenous expression of a phospho-mimicking AATF point mutant results in marked adriamycin resistance in vivo. Nuclear AATF enrichment appears to be selected for in p53-proficient endometrial cancers. Furthermore, focal copy number gains at the AATF locus in neuroblastoma, which is known to be almost exclusively p53-proficient, correlate with an adverse prognosis and reduced overall survival. These data identify the p38/MK2/AATF signalling module as a critical repressor of p53-driven apoptosis and commend this pathway as a target for DNA damage-sensitizing therapeutic regimens.

Splice-switching of the insulin receptor pre-mRNA alleviates tumorigenic hallmarks in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is an aggressive pediatric tumor with a poor prognosis for metastasis and recurrent disease. Large-scale sequencing endeavors demonstrate that Rhabdomyosarcomas have a dearth of precisely targetable driver mutations. However, IGF-2 signaling is known to be grossly altered in RMS. The insulin receptor (IR) exists in two alternatively spliced isoforms, IR-A and IR-B. The IGF-2 signaling molecule binds both its innate IGF-1 receptor as well as the insulin receptor variant A (IR-A) with high affinity. Mitogenic and proliferative signaling via the canonical IGF-2 pathway is, therefore, augmented by IR-A. This study shows that RMS patients express increased IR-A levels compared to control tissues that predominantly express the IR-B isoform. We also found that Hif-1α is significantly increased in RMS tumors, portraying their hypoxic phenotype. Concordantly, the alternative splicing of IR adapts to produce more IR-A in response to hypoxic stress. Upon examining the pre-mRNA structure of the gene, we identified a potential hypoxia-responsive element, which is also the binding site for the RNA-binding protein CUG-BP1 (CELF1). We designed Splice Switching Oligonucleotides (SSO) against this binding site to decrease IR-A levels in RMS cell lines and, consequently, rescue the IR-B expression levels. SSO treatment resulted in a significant reduction in cell proliferation, migration, and angiogenesis. Our data shows promising insight into how impeding the IGF-2 pathway by reducing IR-A expression mitigates tumor growth. It is evident that Rhabdomyosarcomas use IR alternative splicing as yet another survival strategy that can be exploited as a therapeutic intervention in conjunction with already established anti-IGF-1 receptor therapies.

SRSF2 Regulation of MDM2 Reveals Splicing as a Therapeutic Vulnerability of the p53 Pathway.

MDM2 is an oncogene and critical negative regulator of tumor suppressor p53. Genotoxic stress causes alternative splicing of MDM2 transcripts, which leads to alterations in p53 activity and contributes to tumorigenesis. MDM2-ALT1 is one of the alternatively spliced transcripts predominantly produced in response to genotoxic stress, and is comprised of terminal coding exons 3 and 12. Previously, we found that SRSF1 induces MDM2-ALT1 by promoting MDM2 exon 11 skipping. Here we report that splicing regulator SRSF2 antagonizes the regulation of SRSF1 by facilitating the inclusion of exon 11 through binding at two conserved exonic splicing enhancers. Overexpression of SRSF2 reduced the generation of MDM2-ALT1 under genotoxic stress, whereas SRSF2 knockdown induced the expression of MDM2-ALT1 in the absence of genotoxic stress. Blocking the exon 11 SRSF2-binding sites using oligonucleotides promoted MDM2-ALT1 splicing and induced p53 protein expression, and apoptosis in p53 wild-type cells. The regulation of MDM2 splicing by SRSF2 is also conserved in mice, as mutation of one SRSF2-binding site in Mdm2 exon 11, using CRISPR-Cas9, increased the expression of the MDM2-ALT1 homolog Mdm2-MS2. IMPLICATIONS: Taken together, the data indicate that modulating MDM2 splicing may be a useful tool for fine-tuning p53 activity in response to genotoxic stress.

AATF suppresses apoptosis, promotes proliferation and is critical for Kras-driven lung cancer.

A fundamental principle in malignant tranformation is the ability of cancer cells to escape the naturally occurring cell-intrinsic responses to DNA damage. Tumors progress despite the accumulation of DNA lesions. However, the underlying mechanisms of this tolerance to genotoxic stress are still poorly characterized. Here, we show that replication stress occurs in Kras-driven murine lung adenocarcinomas, as well as in proliferating murine embryonic and adult tissues. We identify the transcriptional regulator AATF/CHE-1 as a key molecule to sustain proliferative tissues and tumor progression in parts by inhibiting p53-driven apoptosis in vivo. In an autochthonous Kras-driven lung adenocarcinoma model, deletion of Aatf delayed lung cancer formation predominantly in a p53-dependent manner. Moreover, targeting Aatf in existing tumors through a dual recombinase strategy caused a halt in tumor progression. Taken together, these data suggest that AATF may serve as a drug target to treat KRAS-driven malignancies.

Discovery of Stromal Regulatory Networks that Suppress Ras-Sensitized Epithelial Cell Proliferation.

Mesodermal cells signal to neighboring epithelial cells to modulate their proliferation in both normal and disease states. We adapted a Caenorhabditis elegans organogenesis model to enable a genome-wide mesodermal-specific RNAi screen and discovered 39 factors in mesodermal cells that suppress the proliferation of adjacent Ras pathway-sensitized epithelial cells. These candidates encode components of protein complexes and signaling pathways that converge on the control of chromatin dynamics, cytoplasmic polyadenylation, and translation. Stromal fibroblast-specific deletion of mouse orthologs of several candidates resulted in the hyper-proliferation of mammary gland epithelium. Furthermore, a 33-gene signature of human orthologs was selectively enriched in the tumor stroma of breast cancer patients, and depletion of these factors from normal human breast fibroblasts increased proliferation of co-cultured breast cancer cells. This cross-species approach identified unanticipated regulatory networks in mesodermal cells with growth-suppressive function, exposing the conserved and selective nature of mesodermal-epithelial communication in development and cancer.

Putting the brakes on p53-driven apoptosis.

Following genotoxic stress, cells activate a complex, kinase-based signaling network to arrest the cell cycle and initiate DNA repair or apoptosis. The tumor suppressor p53 lies at the heart of this DNA damage response. p53 mediates the transactivation of both cell cycle-regulating and pro-apoptotic clusters of target genes. However, it remains incompletely understood which signaling molecules dictate the choice between these two opposing p53-dependent cellular outcomes. Over recent years, numerous regulatory mechanisms impacting on the cellular outcome of p53 signaling have been described. However, no single dominant mechanism has thus far been identified to regulate the cellular choice between p53-driven apoptosis or senescence. The transcriptional regulator AATF has recently emerged as a novel factor impacting on the cellular outcome of the p53 response. Upon genotoxic stress, cytoplasmic pools of MRLC-bound AATF are phosphorylated through the p38MAPK/MK2 checkpoint kinase complex. This AATF phosphorylation results in the disruption of cytoplasmic MRLC3:AATF complexes followed by rapid nuclear localization of AATF. Once in the nucleus, AATF binds to the PUMA, BAX and BAK promoters to repress the DNA damage-induced expression of these pro-apoptotic p53 target genes. Depletion of AATF in tumor cells results in a dramatically enhanced response to DNA-damaging chemotherapeutics, both in vitro and in vivo. Furthermore, focal copy number gains at the AATF locus in neuroblastoma correlate with adverse prognosis and reduced overall survival in this typically p53-proficient malignancy. These data identify the p38/MK2/AATF signaling pathway as a critical repressor of p53-driven apoptosis in tumor cells and implicate this signaling cascade as a novel target for chemotherapy-sensitizing therapeutic efforts.