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Characterization of MYC function in KPC cells.

Deregulated expression of the MYC oncoprotein enables tumor cells to evade immune surveillance, but the mechanisms underlying this surveillance are poorly understood. We show here that endogenous MYC protects pancreatic ductal adenocarcinoma (PDAC) driven by KRASG12D and TP53R172H from eradication by the immune system. Deletion of TANK-binding kinase 1 (TBK1) bypassed the requirement for high MYC expression. TBK1 was active due to the accumulation of double-stranded RNA (dsRNA), which was derived from inverted repetitive elements localized in introns of nuclear genes. Nuclear-derived dsRNA is packaged into extracellular vesicles and subsequently recognized by toll-like receptor 3 (TLR3) to activate TBK1 and downstream MHC class I expression in an autocrine or paracrine manner before being degraded in lysosomes. MYC suppressed loading of dsRNA onto TLR3 and its subsequent degradation via association with MIZ1. Collectively, these findings suggest that MYC and MIZ1 suppress a surveillance pathway that signals perturbances in mRNA processing to the immune system, which facilitates immune evasion in PDAC.Significance:This study identifies a TBK1-dependent pathway that links dsRNA metabolism to antitumor immunity and shows that suppression of TBK1 is a critical function of MYC in pancreatic ductal adenocarcinoma.

Histological analyses of transplanted tumors.

Characterization of dsRNA and ssDNA in KPC cells.

Characterization of MYC depletion.

Role of MIZ1 in gene regulation.

Further characterization of dsRNA species.

Characterization of MYC depletion.

TBK1-dependent regulation of NF-kB-dependent gene expression.

Deregulated expression of the MYC oncoprotein enables tumor cells to evade immune surveillance, but the mechanisms underlying this surveillance are poorly understood. We show here that endogenous MYC protects pancreatic ductal adenocarcinoma (PDAC) driven by KRASG12D and TP53R172H from eradication by the immune system. Deletion of TANK-binding kinase 1 (TBK1) bypassed the requirement for high MYC expression. TBK1 was active due to the accumulation of double-stranded RNA (dsRNA), which was derived from inverted repetitive elements localized in introns of nuclear genes. Nuclear-derived dsRNA is packaged into extracellular vesicles and subsequently recognized by toll-like receptor 3 (TLR3) to activate TBK1 and downstream MHC class I expression in an autocrine or paracrine manner before being degraded in lysosomes. MYC suppressed loading of dsRNA onto TLR3 and its subsequent degradation via association with MIZ1. Collectively, these findings suggest that MYC and MIZ1 suppress a surveillance pathway that signals perturbances in mRNA processing to the immune system, which facilitates immune evasion in PDAC. Significance: This study identifies a TBK1-dependent pathway that links dsRNA metabolism to antitumor immunity and shows that suppression of TBK1 is a critical function of MYC in pancreatic ductal adenocarcinoma.

The success of molecular therapies targeting specific metabolic pathways in cancer is often limited by the plasticity and adaptability of metabolic networks. Here we show that pharmacologically induced lipotoxicity represents a promising therapeutic strategy for the treatment of hepatocellular carcinoma (HCC). LXRα-induced liponeogenesis and Raf-1 inhibition are synthetic lethal in HCC owing to a toxic accumulation of saturated fatty acids. Raf-1 was found to bind and activate SCD1, and conformation-changing DFG-out Raf inhibitors could disrupt this interaction, thereby blocking fatty acid desaturation and inducing lethal lipotoxicity. Studies in genetically engineered and nonalcoholic steatohepatitis-induced HCC mouse models and xenograft models of human HCC revealed that therapies comprising LXR agonists and Raf inhibitors were well tolerated and capable of overcoming therapy resistance in HCC. Conceptually, our study suggests pharmacologically induced lipotoxicity as a new mode for metabolic targeting of liver cancer. Zender, Dauch and colleagues demonstrate that pharmacologically induced lipotoxicity by activating LXRα and Raf-1 inhibition provides a metabolic therapeutic strategy for hepatocellular carcinoma.

The c-myc proto-oncogene is activated by translocation in Burkitt's lymphoma and substitutions in codon 58 stabilize the Myc protein or augment its oncogenic potential. In wild-type Myc, phosphorylation of Ser 62 and Thr 58 provides a landing pad for the peptidyl prolyl-isomerase Pin1, which in turn promotes Ser 62 dephosphorylation and Myc degradation. However, the role of Pin1 in Myc-induced lymphomagenesis remains unknown. We show here that genetic ablation of Pin1 reduces lymphomagenesis in Eμ-myc transgenic mice. In both Pin1-deficient B-cells and MEFs, the proliferative response to oncogenic Myc was selectively impaired, with no alterations in Myc-induced apoptosis or mitogen-induced cell cycle entry. This proliferative defect wasn't attributable to alterations in either Ser 62 phosphorylation or Myc-regulated transcription, but instead relied on the activity of the ARF-p53 pathway. Pin1 silencing in lymphomas retarded disease progression in mice, making Pin1 an attractive therapeutic target in Myc-driven tumors.

Although it is well established that TNFα contributes to hepatitis, liver failure and associated hepatocarcinogenesis via the regulation of inflammation, its pro-apoptotic role in the liver has remained enigmatic. On its own, TNFα is unable to trigger apoptosis. However, when combined with the transcriptional inhibitor GaLN, it can cause hepatocyte apoptosis and liver failure in mice. Moreover, along with others, we have shown that TNFα is capable of sensitizing cells to FasL- or drug-induced cell death via c-Jun N-terminal kinase (JNK) activation and phosphorylation/activation of the BH3-only protein Bim. In this context, TNFα could exacerbate hepatocyte cell death during simultaneous inflammatory and T-cell-mediated immune responses in the liver. Here we show that TNFα sensitizes primary hepatocytes, established hepatocyte cell lines and mouse embryo fibroblasts to FasL-induced apoptosis by the transcriptional induction and higher surface expression of Fas via the NFκB pathway. Genetic deletion, diminished expression or dominant-negative inhibition of the NFκB subunit p65 resulted in lower Fas expression and inhibited TNFα-induced Fas upregulation and sensitization to FasL-induced cell death. By hydrodynamic injection of p65 shRNA into the tail vein of mice, we confirm that Fas upregulation by TNFα is also NFκB-mediated in the liver. In conclusion, TNFα sensitization of FasL-induced apoptosis in the liver proceeds via two parallel signaling pathways, activation of JNK and Bim phosphorylation and NFκB-mediated Fas upregulation.

Comment in :- Neighbourhood deaths cause a switch in cancer subtype. [Nature. 2018]- Neighbourly deaths dictate fate. [Nat Rev Cancer. 2018]- Bad neighborhoods: apoptotic and necroptotic microenvironments determine liver cancer subtypes. [Hepatobiliary Surg Nutr. 2019]- Viewpoint: necroptosis influences the type of liver cancer via changes of hepatic microenvironment. [Hepatobiliary Surg Nutr. 2019]; International audience; Primary liver cancer represents a major health problem. It comprises hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC), which differ markedly with regards to their morphology, metastatic potential and responses to therapy. However, the regulatory molecules and tissue context that commit transformed hepatic cells towards HCC or ICC are largely unknown. Here we show that the hepatic microenvironment epigenetically shapes lineage commitment in mosaic mouse models of liver tumorigenesis. Whereas a necroptosis-associated hepatic cytokine microenvironment determines ICC outgrowth from oncogenically transformed hepatocytes, hepatocytes containing identical oncogenic drivers give rise to HCC if they are surrounded by apoptotic hepatocytes. Epigenome and transcriptome profiling of mouse HCC and ICC singled out Tbx3 and Prdm5 as major microenvironment-dependent and epigenetically regulated lineage-commitment factors, a function that is conserved in humans. Together, our results provide insight into lineage commitment in liver tumorigenesis, and explain molecularly why common liver-damaging risk factors can lead to either HCC or ICC.

Prognosis of combined hepatocellular carcinoma-intrahepatic cholangiocarcinoma, a type of primary liver cancer comprising areas with HCC and ICC histopathology, is dismal, and it is unclear if such tumors develop clonally and how they should be treated. In this issue of Cancer Cell, Xue et al. (2019) provide answers to these questions.

A precise balance between quiescence and proliferation is crucial for the lifelong function of hematopoietic stem cells (HSCs). Cyclins E1 and E2 regulate exit from quiescence in fibroblasts, but their role in HSCs remains unknown. Here, we report a non-redundant role for cyclin E1 in mouse HSCs. A long-term culture-initiating cell (LTC-IC) assay indicated that the loss of cyclin E1, but not E2, compromised the colony-forming activity of primitive hematopoietic progenitors. Ccne1(-/-) mice showed normal hematopoiesis in vivo under homeostatic conditions but a severe impairment following myeloablative stress induced by 5-fluorouracil (5-FU). Under these conditions, Ccne1(-/-) HSCs were less efficient in entering the cell cycle, resulting in decreased hematopoiesis and reduced survival of mutant mice upon weekly 5-FU treatment. The role of cyclin E1 in homeostatic conditions became apparent in aged mice, where HSC quiescence was increased in Ccne1(-/-) animals. On the other hand, loss of cyclin E1 provided HSCs with a competitive advantage in bone marrow serial transplantation assays, suggesting that a partial impairment of cell cycle entry may exert a protective role by preventing premature depletion of the HSC compartment. Our data support a role for cyclin E1 in controlling the exit from quiescence in HSCs. This activity, depending on the physiological context, can either jeopardize or protect the maintenance of hematopoiesis.

MYC genes have both essential roles during normal development and exert oncogenic functions during tumorigenesis. Expression of a dominant-negative allele of MYC, termed OmoMYC, can induce rapid tumor regression in mouse models with little toxicity for normal tissues. How OmoMYC discriminates between physiological and oncogenic functions of MYC is unclear. We have solved the crystal structure of OmoMYC and show that it forms a stable homodimer and as such recognizes DNA in the same manner as the MYC/MAX heterodimer. OmoMYC attenuates both MYC-dependent activation and repression by competing with MYC/MAX for binding to chromatin, effectively lowering MYC/MAX occupancy at its cognate binding sites. OmoMYC causes the largest decreases in promoter occupancy and changes in expression on genes that are invaded by oncogenic MYC levels. A signature of OmoMYC-regulated genes defines subgroups with high MYC levels in multiple tumor entities and identifies novel targets for the eradication of MYC-driven tumors.

Oncogene-induced replicative stress activates an Atr- and Chk1-dependent response, which has been proposed to be widespread in tumors. We explored whether the presence of replicative stress could be exploited for the selective elimination of cancer cells. To this end, we evaluated the impact of targeting the replicative stress-response on cancer development. In mice (Mus musculus), the reduced levels of Atr found on a mouse model of the Atr-Seckel syndrome completely prevented the development of Myc-induced lymphomas or pancreatic tumors, both of which showed abundant levels of replicative stress. Moreover, Chk1 inhibitors were highly effective in killing Myc-driven lymphomas. By contrast, pancreatic adenocarcinomas initiated by K-Ras(G12V) showed no detectable evidence of replicative stress and were nonresponsive to this therapy. Besides its impact on cancer, Myc overexpression aggravated the phenotypes of Atr-Seckel mice, revealing that oncogenes can modulate the severity of replicative stress-associated diseases.

In this Article, the pCaMIN construct consisted of ‘mouse MYC and mouse NrasG12V’ instead of ‘mouse Myc and human NRASG12V; and the pCAMIA construct consisted of ‘mouse Myc and human AKT1’ instead of ‘mouse Myc and Akt1’ this has been corrected online.

The c-myc proto-oncogene is activated by translocation in Burkitt's lymphoma, where it is also frequently subject to secondary mutations. Substitutions in codon 58, in particular, stabilize the Myc protein and augment its oncogenic potential. In wild-type Myc, phosphorylation of Ser 62 primes phosphorylation of Thr 58, providing a landing pad for the peptidyl prolyl-isomerase Pin1, which in turn promotes Ser 62 dephosphorylation and Myc degradation. However, genetic data are missing to address whether Pin1 influences Myc-induced lymphomagenesis. Here, we show that genetic ablation of Pin1 in Eµ-myc transgenic mice starkly reduced lymphoma onset and penetrance. In pre-malignant Pin1 deficient B cells, the proliferative response to Myc was selectively impaired, while Myc-induced apoptosis was intact. This phenotype was not the result of deregulated levels of Ser 62 phosphorylation in the Pin1-/- background. Similar results were observed in Pin1-/- mouse embryo fibroblasts (MEFs), where prolonged Myc activation inhibited proliferation. In either B cells or MEFs, loss of Pin1 did not affect mitogen-induced cell cycle entry, an effect that relies on endogenous Myc activity. Thus, our data unravel the existence of a synthetic-lethal interaction between deletion of Pin1 and oncogenic activation of Myc. Myc-induced oncogenic stress is linked to the induction of the Arf/p53 pathway, which is critical for tumor suppression in Eµ-myc mice. The selective pressure to inactivate Arf/p53 axis was preserved in Eµ-myc Pin1-/- lymphomas. In the absence of Arf or p53, the Pin1 deletion no longer reduced Myc-induced tumorigenesis or proliferation. Thus the proliferative arrest of pre-tumoral Eµ-myc Pin1-/- B cells required an active ARF-p53 dependent cytostatic response. Our data demonstrate that Pin1 is required to support the oncogenic activity of Myc in B cells and warrant further assessment of Pin1 as a therapeutic target in Myc-dependent malignancies. Citation Format: Luana D'Artista, Andrea Piontini, Theresia Kress, Marco Morelli, Alessandro Verrecchia, Mirko Doni, Stefano Campaner, Bruno Amati. Pin1 is required for sustained B cell proliferation upon oncogenic activation of Myc. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B03.

The c-myc proto-oncogene is frequently overexpressed in human hepatoblastoma due to gene amplification or constitutively active Wnt signaling. High Myc levels characterize an aggressive hepatoblastoma subtype, reproduced in mice with a liver-specific tetracyclin-regulated c-myc transgene (1, 2). Here, we used an analogous mouse model (tetO-MYC/Lap-tTA; ref. 3) to study the epigenomic and transcriptional mechanisms underlying tumor progression and maintenance. Tet-Myc induction in utero resulted in rapid and fully penetrant tumorigenesis by the age of six weeks. Pre-tumoral changes were detectable already at a late embryonic stage (E18.5) with increased liver size and hepatoblast fraction. As previously described (2), tumors remained dependent on the constitutive expression of the transgene. To study transcriptional changes during tumor progression and regression, we established RNA-seq profiles in control hepatoblasts, pre-tumoral Tet-Myc hepatoblasts and tumor nodules, in the latter before and after short-term inactivation of Tet-Myc. In parallel, we used ChIP-seq to profile the genome-wide distribution of Myc, RNAPII and several histone marks (H3K4me1, H3K4me3, H3K27ac), yielding comprehensive maps of Myc binding to regulatory elements (promoters and enhancers). This unique dataset is providing the basis for mechanistic studies addressing how Myc deregulates its target genes during liver tumorigenesis: comparison of differentially expressed mRNAs and Myc-binding profiles enables us to discriminate between directly and potentially indirectly regulated genes, for which we are currently investigating the underlying molecular basis. Furthermore, our genome-wide data are allowing us to unravel potentially druggable genes and pathways regulated by Myc during hepatoblastoma progression and maintenance. Our progress in characterizing this model will be presented at the meeting. References: 1. Cairo S et al. 2008, Cancer Cell, 14:471-84 2. Shachaf CM et al. 2004, Nature, 431:1112-7 3. van Riggelen J et al. 2010, Genes Dev, 24:1281-94 Citation Format: Theresia Kress, Valerio Bianchi, Luana D'Artista, Paola Nicoli, Mirko Doni, Enrico Radaelli, Bruno Amati. Epigenomic and transcriptional analyses in a Tet-Myc driven mouse model of liver cancer. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr A27.