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The transcriptional co-activator YAP1 oncogene is the downstream effector of the Hippo pathway, which regulates tissue homeostasis, organ size, regeneration, and tumorigenesis. Multiple cancers are dependent on sustained expression of YAP1 for cell proliferation, survival, and tumorigenesis, but the molecular basis of this oncogene dependency is not well understood. To identify genes that can functionally substitute for YAP1, we performed a genome-scale genetic rescue screen in YAP1-dependent colon cancer cells expressing an inducible YAP1-specific shRNA. We found that the transcription factor PRDM14 rescued cell proliferation and tumorigenesis upon YAP1 suppression in YAP1-dependent cells, xenografts, and colon cancer organoids. YAP1 and PRDM14 individually activated the transcription of calmodulin 2 (CALM2) and a glucose transporter SLC2A1 upon YAP1 suppression, and CALM2 or SLC2A1 expression was required for the rescue of YAP1 suppression. Together, these findings implicate PRDM14-mediated transcriptional upregulation of CALM2 and SLC2A1 as key components of oncogenic YAP1 signaling and dependency.

SUMMARY Metastasis is a complex and poorly understood process. In pancreatic cancer, loss of the transforming growth factor (TGF)-β/BMP effector SMAD4 is correlated with changes in altered histopathological transitions, metastatic disease, and poor prognosis. In this study, we use isogenic cancer cell lines to identify SMAD4 regulated genes that contribute to the development of metastatic colonization. We perform an in vivo screen identifying FOSL1 as both a SMAD4 target and sufficient to drive colonization to the lung. The targeting of these genes early in treatment may provide a therapeutic benefit., Graphical abstract, In brief Loss of SMAD4 is associated with poor outcome in pancreatic cancer. Using an in vivo, isogenic metastatic colonization assay, Dai et al. identified SMAD4-regulated genes that affect metastasis but not primary tumor growth. FOSL1 is a SMAD4-regulated gene that is necessary and sufficient to drive metastatic colonization.

Activated T-cells express Programmed cell Death protein 1 (PD-1), a key immune checkpoint receptor. PD-1 functions primarily in peripheral tissues, where T cells may encounter tumor-derived immunosuppressive ligands. Monoclonal antibodies that disrupt the interaction between T-cell derived PD-1 and immunosuppressive ligands, such as PD-L1, have revolutionized approaches to cancer therapy. For instance, Nivolumab is a monoclonal Ab that targets human PD-1 and has played an important role in immune checkpoint therapy. Herein we report the purification and initial characterization of a ~27 kDa single chain variable fragment (scFv) of Nivolumab that targets human PD-1 and blocks binding by PD-L1. The possibility that the anti-PD-1 scFv can serve as both an anti-tumor agent and as an anti-viral agent is discussed. Importance The clinical significance of anti-PD-1 antibodies for treatment of a range of solid tumors is well documented (reviewed in [1–4]). In this report, we describe the results of studies that establish that an anti-PD-1 scFv purified from E. coli binds tightly to human PD-1. Furthermore, we demonstrate that upon binding, the anti-PD-1 scFv disrupts the interaction between PD-1 and PD-L1. Thus, the properties of this scFv, including its small size, stability and affinity for human PD-1, suggest that it has the potential to be a useful reagent in subsequent immunotherapeutic, diagnostic and anti-viral applications., Highlights • Computer based design of a single chain variable fragment (scFv) of Nivolumab. • An E.coli based method for the purification of the anti-PD-1 scFv. • Demonstration that when bound to PD-1, the anti-PD-1 scFv blocks binding by PD-L1.

Merkel cell polyomavirus (MCV) has been recently described as the cause for most human Merkel cell carcinomas. MCV is similar to simian virus 40 (SV40) and encodes a nuclear large T (LT) oncoprotein that is usually mutated to eliminate viral replication among tumor-derived MCV. We identified the hVam6p cytoplasmic protein involved in lysosomal processing as a novel interactor with MCV LT but not SV40 LT. hVam6p binds through its clathrin heavy chain homology domain to a unique region of MCV LT adjacent to the retinoblastoma binding site. MCV LT translocates hVam6p to the nucleus, sequestering it from involvement in lysosomal trafficking. A naturally occurring, tumor-derived mutant LT (MCV350) lacking a nuclear localization signal binds hVam6p but fails to inhibit hVam6p-induced lysosomal clustering. MCV has evolved a novel mechanism to target hVam6p that may contribute to viral uncoating or egress through lysosomal processing during virus replication.

The p21-activated kinases (PAKs) contain an N-terminal Cdc42/Rac interactive binding domain, which in the group 1 PAKs (PAK1, 2, and 3) regulates the activity of an adjacent conserved autoinhibitory domain. In contrast, the group 2 PAKs (PAK4, 5, and 6) lack this autoinhibitory domain and are not activated by Cdc42/Rac binding, and the mechanisms that regulate their kinase activity have been unclear. This study found that basal PAK6 kinase activity was repressed by a p38 mitogen-activated protein (MAP) kinase antagonist and could be strongly stimulated by constitutively active MAP kinase kinase 6 (MKK6), an upstream activator of p38 MAP kinases. Mutation of a consensus p38 MAP kinase target site at serine 165 decreased PAK6 kinase activity. Moreover, PAK6 was directly activated by MKK6, and mutation of tyrosine 566 in a consensus MKK6 site (threonine-proline-tyrosine, TPY) in the activation loop of the PAK6 kinase domain prevented activation by MKK6. PAK6 activation by MKK6 was also blocked by mutation of an autophosphorylated serine (serine 560) in the PAK6 activation loop, indicating that phosphorylation of this site is necessary for MKK6-mediated activation. PAK4 and PAK5 were similarly activated by MKK6, consistent with a conserved TPY motif in their activation domains. The activation of PAK6 by both p38 MAP kinase and MKK6 suggests that PAK6 plays a role in the cellular response to stress-related signals.

Recent studies have demonstrated that introduction of hTERT in combination with SV40 large T antigen (LT), small t antigen (st), and H-rasV12 suffices to transform many primary human cells. In human mammary epithelial cells (HMECs) expressing elevated c-Myc, activated H-Ras is dispensable for anchorage-independent growth. Using this system, we show that st activates the PI3K pathway and that constitutive PI3K signaling substitutes for st in transformation. Moreover, using constitutively active versions of Akt1 and Rac1, we show that these downstream pathways of PI3K synergize to achieve anchorage-independent growth. At lower levels of c-myc expression, activated PI3K also replaces st to complement H-rasV12 and LT and confers both soft agar growth and tumorigenicity. However, elevated c-myc expression cannot replace H-rasV12 for tumorigenesis. These observations begin to define the pathways perturbed during the transformation of HMECs.

The large tumor antigen of simian virus 40 (SVLT) is a potent oncogene. Although inactivation of the p53 and pRb tumor suppressors has been causally linked to the transforming properties of SVLT, its exact mechanism of action remains undefined. Previous data indicated that Ras is activated in SVLT-expressing cells. In this report we show that SVLT also increases Raf kinase activity in both insect and mammalian cells, thus identifying the Raf kinase as an additional target of SVLT. Our results further show that SVLT was still able to activate Raf in cells where Ras levels had been drastically reduced through expression of an antisense construct, indicating that SVLT may activate Raf at least partly by a mechanism that is independent of its stimulatory effect on Ras.