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Cellular senescence acts as a potent barrier to tumorigenesis and contributes to the anti-tumor activity of certain chemotherapeutic agents. Senescent cells undergo a stable cell cycle arrest controlled by RB and p53 and, in addition, display a senescence-associated secretory phenotype (SASP) involving the production of factors that reinforce the senescence arrest, alter the microenvironment, and trigger immune surveillance of the senescent cells. Through a proteomics analysis of senescent chromatin, we identified the nuclear factor-κB (NF-κB) subunit p65 as a major transcription factor that accumulates on chromatin of senescent cells. We found that NF-κB acts as a master regulator of the SASP, influencing the expression of more genes than RB and p53 combined. In cultured fibroblasts, NF-κB suppression causes escape from immune recognition by natural killer (NK) cells and cooperates with p53 inactivation to bypass senescence. In a mouse lymphoma model, NF-κB inhibition bypasses treatment-induced senescence, producing drug resistance, early relapse, and reduced survival. Our results demonstrate that NF-κB controls both cell-autonomous and non-cell-autonomous aspects of the senescence program and identify a tumor-suppressive function of NF-κB that contributes to the outcome of cancer therapy.

SummaryThe monomeric RalGTPases, RalA and RalB are recognized as components of a regulatory framework supporting tumorigenic transformation. Specifically, RalB is required to suppress apoptotic checkpoint activation, the mechanistic basis of which is unknown. Reported effector proteins of RalB include the Sec5 component of the exocyst, an octameric protein complex implicated in tethering of vesicles to membranes. Surprisingly, we find that the RalB/Sec5 effector complex directly recruits and activates the atypical IκB kinase family member TBK1. In cancer cells, constitutive engagement of this pathway, via chronic RalB activation, restricts initiation of apoptotic programs typically engaged in the context of oncogenic stress. Although dispensable for survival in a nontumorigenic context, this pathway helps mount an innate immune response to virus exposure. These observations define the mechanistic contribution of RalGTPases to cancer cell survival and reveal the RalB/Sec5 effector complex as a component of TBK1-dependent innate immune signaling.

The monomeric RAL (RAS-like) GTPases have been indirectly implicated in mitogenic regulation and cell transformation. Here, we show that RALA and RALB collaborate to maintain tumorigenicity through regulation of both proliferation and survival. Remarkably, this task is divided between these highly homologous isoforms. RALB is specifically required for survival of tumour cells but not normal cells. RALA is dispensable for survival, but is required for anchorage-independent proliferation. Reducing the 'oncogenic burden' in human tumour cells relieves the sensitivity to loss of RALB. These observations establish RAL GTPases as crucial components of the cellular machinery that are exploited by factors that drive oncogenic transformation.

Expression of an oncogene in a primary cell can, paradoxically, block proliferation by inducing senescence or apoptosis through pathways that remain to be elucidated. Here we perform genome-wide RNA-interference screening to identify 17 genes required for an activated BRAF oncogene (BRAFV600E) to block proliferation of human primary fibroblasts and melanocytes. Surprisingly, we find a secreted protein, IGFBP7, has a central role in BRAFV600E-mediated senescence and apoptosis. Expression of BRAFV600E in primary cells leads to synthesis and secretion of IGFBP7, which acts through autocrine/paracrine pathways to inhibit BRAF-MEK-ERK signaling and induce senescence and apoptosis. Apoptosis results from IGFBP7-mediated upregulation of BNIP3L, a pro-apoptotic BCL2 family protein. Recombinant IGFBP7 (rIGFBP7) induces apoptosis in BRAFV600E-positive human melanoma cell lines, and systemically administered rIGFBP7 markedly suppresses growth of BRAFV600E-positive tumors in xenografted mice. Immunohistochemical analysis of human skin, nevi and melanoma samples implicates loss of IGFBP7 expression as a critical step in melanoma genesis.

The Ras-like GTPases, RalA and RalB, are key components of the oncogenic Ras signaling network. Recent evidence suggests that RalA and RalB collaborate to support tumorigenic transformation through distinct cell regulatory events. While RalA is apparently required to bypass normal restraints on cell proliferation, RalB is required to bypass normal restraints on cell survival. A direct Ral effector protein, Sec5, is a subunit of the exocyst complex, and is required to mediate RalB-dependent survival signals in transformed cells. Further analysis identified TBK1, a key mediator of the host defense response to viral challenge, as a novel Sec5 interacting protein essential for the capacity of RalB and Sec5 to deflect cell death in transformed cells. RalB activation promotes a direct interaction between Sec5 and TBK1 that results in TBK1 kinase activation via an unknown mechanism. Accordingly, both RalB and Sec5 are required for initiating host defense pathway activation upon virus infection. These observations revealed a novel relationship between molecular components of cell-autonomous innate immune signaling pathways and oncogenic transformation, and identified TBK1 as a potential target for therapeutic intervention in cancer. Here we describe details of methods, including protein complex analysis, protein kinase assays, host defense-response pathway activation, and cell transformation analysis, that can be used to investigate the contribution of the RalB-Sec5-TBK1 signaling cascade to both innate immune signaling and cell transformation.

The Ras-like GTPases, RalA and RalB, are key components of the oncogenic Ras signaling network. Recent evidence suggests that RalA and RalB collaborate to support tumorigenic transformation through distinct cell regulatory events. While RalA is apparently required to bypass normal restraints on cell proliferation, RalB is required to bypass normal restraints on cell survival. A direct Ral effector protein, Sec5, is a subunit of the exocyst complex, and is required to mediate RalB-dependent survival signals in transformed cells. Further analysis identified TBK1, a key mediator of the host defense response to viral challenge, as a novel Sec5 interacting protein essential for the capacity of RalB and Sec5 to deflect cell death in transformed cells. RalB activation promotes a direct interaction between Sec5 and TBK1 that results in TBK1 kinase activation via an unknown mechanism. Accordingly, both RalB and Sec5 are required for initiating host defense pathway activation upon virus infection. These observations revealed a novel relationship between molecular components of cell-autonomous innate immune signaling pathways and oncogenic transformation, and identified TBK1 as a potential target for therapeutic intervention in cancer. Here we describe details of methods, including protein complex analysis, protein kinase assays, host defense-response pathway activation, and cell transformation analysis, that can be used to investigate the contribution of the RalB-Sec5-TBK1 signaling cascade to both innate immune signaling and cell transformation.

We have previously identified the Ras-like GTpases, RalA and RalB as linchpin modulators of human tumor cell proliferation and survival. Using RNAi-mediated loss-of-function analysis, we find that RalA is critical for anchorage-independent growth whereas RalB is required for tumor cell survival. The collaboration of these isoform-specific contributions generates a minimal oncogenic platform, which results in aberrant proliferation coupled with suppression of apoptosis. We have begun to characterize the molecular basis of the collaborative contributions of RalA and RalB to cell regulation. These studies have surprisingly revealed that RalB and Sec5 are mediating tumor cell survival whereas RalA and STAT3 contribute to anchorage-independent proliferation. Understanding how the regulation occurs will be the focus of my future work.

MDM2, the most prominent natural inhibitor of the p53 tumor suppressor pathway, and BCL2, an anti-apoptotic regulator and important player in preventing induction of apoptosis in tumor cells, both play a pivotal role in leukemia cell survival and response to therapy in the clinic. The principal objectives of this study were to assess a synergistic antitumor effect by combining the clinical stage compounds RG7388, a MDM2 antagonist reactivating p53, and the BCL2 selective inhibitor GDC-0199 and to elucidate the underlying mechanism of action. Further insight into the biological rationale for this potential therapeutic strategy may pave the path for clinical combination studies in AML patients. We first investigated the effect of co-treatment with RG7388 and GDC-0199 on the viability and induction of apoptosis in AML tumor cell lines followed by high-resolution cell cycle kinetic analysis using BrdU-Hoechst quenching technology in-vitro. Cell viability assays demonstrated synergistic potency of combined RG7388 and GDC-0199 treatment with low nM IC50 values. Subsequent annexin binding assays confirmed the efficacy and revealed different kinetics of apoptosis induction. To further explain the mode of action, high-resolution cell cycle compartment analysis of consecutive cell cycles was performed. Mechanistically, RG7388 induces G1 arrest and preferentially causes nuclear fragmentation in G1 phase of the second cycle. The BCL2 inhibitor GDC-0199 mainly causes apoptosis in G1 compartments and hence cells transiently protected from apoptosis by RG7388 induced G1 arrest are hit by GDC-0199. In addition we analyzed changes in expression of pro- and anti-apoptotic genes and proteins upon combined treatment using qRT-PCR, Western Blot and Luminex technology. We identified down-modulation of MCL-1 levels, a known GDC-0199 resistance factor, as one potential mechanism for the synergistic efficacy and confirmed this finding by shRNA experiments in-vitro. Furthermore, the efficacy of the compounds was assessed in subcutaneous and orthotopic AML tumor cell line based xenograft models in-vivo, confirming the synergy observed in-vitro. Taken together, our study demonstrates that combined treatment with RG7388 and GDC-0199 shows a synergistic anticancer effect on AML cells. This indicates that activating the p53 tumor suppressor pathway and inhibiting a key anti-apoptosis target simultaneously induces a more than additive inhibitory effect by modulating the levels of key pro-survival proteins, such as Mcl-1, thereby further lowering the apoptotic threshold level for tumor cell death. Based on the observations from our study, the combined administration of RG7388 and GDC-0199 might offer a promising new therapeutic regimen for treating AML patients. Citation Format: Markus Dangl, Yuchen Chien, Christian Lehmann, Thomas Friess. Synergistic anticancer activity of clinical stage, non-genotoxic apoptosis inducing agents RG7388 (MDM2 antagonist) and ABT-199 (GDC-0199, BCL2 inhibitor) in p53 wild-type AML tumor models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5505. doi:10.1158/1538-7445.AM2014-5505