Summary Mutations in p53 and RAS potently cooperate in oncogenic transformation and correspondingly these genetic alterations frequently coexist in pancreatic ductal adenocarcinoma (PDA) and other human cancers. Previously we identified a set of genes synergistically activated by combined RAS and p53 mutations as frequent downstream mediators of tumorigenesis. Here, we show that the synergistically activated gene Plac8 is critical for pancreatic cancer growth. Silencing of Plac8 in cell lines suppresses tumor formation by blocking autophagy, a process essential for maintaining metabolic homeostasis in PDA, and genetic inactivation in an engineered mouse model inhibits PDA progression. We show that Plac8 is a critical regulator of the autophagic machinery, localizing to the lysosomal compartment and facilitating lysosome-autophagosome fusion. Plac8 thus provides a mechanistic link between primary oncogenic mutations and the induction of autophagy, a central mechanism of metabolic reprogramming, during PDA progression.

In searching for small-molecule compounds that inhibit proliferation and survival of diffuse large B-cell lymphoma (DLBCL) cells and may, therefore, be exploited as potential therapeutic agents for this disease, we identified the commonly used and well-tolerated antibiotic doxycycline as a strong candidate. Here, we demonstrate that doxycycline inhibits the growth of DLBCL cells both in vitro and in mouse xenograft models. In addition, we show that doxycycline accumulates in DLBCL cells to high concentrations and affects multiple signaling pathways that are crucial for lymphomagenesis. Our data reveal the deneddylating activity of COP-9 signalosome (CSN) as a novel target of doxycycline and suggest that doxycycline may exert its effects in DLBCL cells in part through a CSN5-HSP90 pathway. Consistently, knockdown of CSN5 exhibited similar effects as doxycycline treatment on DLBCL cell survival and HSP90 chaperone function. In addition to DLBCL cells, doxycycline inhibited growth of several other types of non-Hodgkin lymphoma cells in vitro. Together, our results suggest that doxycycline may represent a promising therapeutic agent for DLBCL and other non-Hodgkin lymphomas subtypes.

Highlights d A network of non-mutated genes is critical to the malignant state d TopNet can accurately model cellular responses to genetic perturbations d TopNet is capable of pinpointing key architectural features of cancer cells

Malignant cell transformation and the underlying genomic scale reprogramming of gene expression require cooperation of multiple oncogenic mutations. Notably, this cooperation is reflected in the synergistic regulation of downstream genes, so-called cooperation response genes (CRGs). CRGs impact diverse hallmark features of cancer cells and are not known to be functionally connected. Yet, they act as critical mediators of the cancer phenotype at an unexpectedly high frequency of >50%, as indicated by genetic perturbations. Here we demonstrate that CRGs function within a network of strong genetic interdependencies that are critical to the robustness of the malignant state. Our approach, termed TopNet, utilizes attractor-based ternary network modeling that takes the novel approach of incorporating uncertainty in the underlying gene perturbation data and is capable of identifying non-linear gene interactions. TopNet reveals topological gene network architecture that effectively predicts previously unknown, functionally relevant epistatic gene interactions, and thus, among a broad range of applications, has utility for identification of non-mutant targets for cancer intervention.

A) Pancreatic ductal adenocarcinoma (PDA) depends on a marked reprogramming of metabolic pathways, including the acquisition of autophagy dependence, for survival and growth. How common mutations in PDA cause autophagy dependence as well as the timing of autophagy activation in the course of cancer progression, have not been established. Here we show how Plac8, a gene synergistically up-regulated in response to the common cooperating oncogenic mutations found in PDA (RAS activation and functional loss of p53), is critical to the growth of PDA by sustaining autophagy via facititating autophagosome-lysosome fusion. Furthermore, we delineate that Kras and p53 mutations cooperate to induce autophagic flux. B) To establish Plac8’s role in PDA growth and autophagy we use murine cell lines and human PDA cell lines to determine 1) the lysosomal localization of Plac8, 2) its role in regulating autophagy using both loss of function and gain of function approaches, 3) the impact on lysosomal biology, and 4) the relationship between Plac8 and other genetic pathways governing autophagy. Using genetically engineered models of PDA we determine the timing of autophagy activation in PDA progression and the impact of Plac8 mutation. C) We identify Plac8 as a novel regulator of autophagosome-lysosome fusion required for PDA growth, thus providing a mechanistic link between oncogenic mutations and the activation of autophagy in cancer. Plac8 expression is required for growth of human PDA cells as xenografts in mice, as well as activation of autophagy. We find that concurrent mutation of KRAS and p53 is critical for maximal induction of autophagy in vitro. Correspondingly, using genetically engineered mouse models of PDA (Pdx1-Cre; LSL-KrasG12D; p53L/+), in which loss of p53 function occurs in a step-wise manner relying on the spontaneous loss of a heterozygous WT p53 allele, we see a step-wise incremental increase in LC3 puncta in vivo with each histological stage through the course of PDA progression. Thus, we find that the cooperative effects of KRAS and p53 drive activation of autophagy rather than either mutation alone. The overall survival of a Pdx1-Cre; LSL-KrasG12D; p53L/+; Plac8null murine cohort (OS 27.9 wks) was significantly longer than a Pdx1-Cre; LSL-KrasG12D; p53L/+; Plac8wt cohort (OS 17.0 wks, p=0.0006) demonstrating in vivo that genetic inactivation of Plac8 impedes cancer progression and resulting death. Our data suggest that the role of Plac8 in facilitating autophagy is critical to cancer, as the requirement of Plac8 for both tumorigenicity and autophagy can be compensated by over-expression of Atg12, a gene critical for autophagosome formation or by constitutively activated Rab7, a gene encoding a GTP-binding protein stimulating autophagosome-lysosome fusion. D) We conclude that Plac8 may offer a potential therapeutic window and point of intervention, as Plac8 mutation in the engineered PDA model inhibits cancer progression and significantly improves survival while having a minimal impact on the overall fitness of the animals. In fact, Plac8, and regulation of autophagosome-lysosome fusion, has specific relevance to regulation of autophagy during malignant cell transformation as Plac8 and the processes it regulates, appear to be largely dispensable to many normal physiologic processes. This abstract is also presented as Poster B36. Citation Format: Vijaya Balakrishnan, Kinsey Conan, Michael O'Dell, Jing Li Huang, Laurel Newman, Christa Whitney-Miller, Hartmut Land, Aram Hezel. Plac8 links oncogenic mutations to regulation of autophagy and is critical to pancreatic cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr PR09.