Daniel Dauch

Senescence surveillance of pre-malignant hepatocytes limits liver cancer development.

Upon the aberrant activation of oncogenes, normal cells can enter the cellular senescence program, a state of stable cell-cycle arrest, which represents an important barrier against tumour development in vivo. Senescent cells communicate with their environment by secreting various cytokines and growth factors, and it was reported that this ‘secretory phenotype’ can have pro- as well as anti-tumorigenic effects. Here we show that oncogene-induced senescence occurs in otherwise normal murine hepatocytes in vivo. Pre-malignant senescent hepatocytes secrete chemo- and cytokines and are subject to immune-mediated clearance (designated as ‘senescence surveillance’), which depends on an intact CD4+ T-cell-mediated adaptive immune response. Impaired immune surveillance of pre-malignant senescent hepatocytes results in the development of murine hepatocellular carcinomas (HCCs), thus showing that senescence surveillance is important for tumour suppression in vivo. In accordance with these observations, ras-specific Th1 lymphocytes could be detected in mice, in which oncogene-induced senescence had been triggered by hepatic expression of NrasG12V. We also found that CD4+ T cells require monocytes/macrophages to execute the clearance of senescent hepatocytes. Our study indicates that senescence surveillance represents an important extrinsic component of the senescence anti-tumour barrier, and illustrates how the cellular senescence program is involved in tumour immune surveillance by mounting specific immune responses against antigens expressed in pre-malignant senescent cells.

A Differentiation Checkpoint Limits Hematopoietic Stem Cell Self-Renewal in Response to DNA Damage

Checkpoints that limit stem cell self-renewal in response to DNA damage can contribute to cancer protection but may also promote tissue aging. Molecular components that control stem cell responses to DNA damage remain to be delineated. Using in vivo RNAi screens, we identified basic leucine zipper transcription factor, ATF-like (BATF) as a major component limiting self-renewal of hematopoietic stem cells (HSCs) in response to telomere dysfunction and γ-irradiation. DNA damage induces BATF in a G-CSF/STAT3-dependent manner resulting in lymphoid differentiation of HSCs. BATF deletion improves HSC self-renewal and function in response to γ-irradiation or telomere shortening but results in accumulation of DNA damage in HSCs. Analysis of bone marrow from patients with myelodysplastic syndrome supports the conclusion that DNA damage-dependent induction of BATF is conserved in human HSCs. Together, these results provide experimental evidence that a BATF-dependent differentiation checkpoint limits self-renewal of HSCs in response to DNA damage.

In vivo RNAi screening identifies a mechanism of sorafenib resistance in liver cancer

In solid tumors, resistance to therapy inevitably develops upon treatment with cytotoxic drugs or molecularly targeted therapies. Here, we describe a system that enables pooled shRNA screening directly in mouse hepatocellular carcinomas (HCC) in vivo to identify genes likely to be involved in therapy resistance. Using a focused shRNA library targeting genes located within focal genomic amplifications of human HCC, we screened for genes whose inhibition increased the therapeutic efficacy of the multikinase inhibitor sorafenib. Both shRNA-mediated and pharmacological silencing of Mapk14 (p38α) were found to sensitize mouse HCC to sorafenib therapy and prolong survival by abrogating Mapk14-dependent activation of Mek-Erk and Atf2 signaling. Elevated Mapk14-Atf2 signaling predicted poor response to sorafenib therapy in human HCC, and sorafenib resistance of p-Mapk14-expressing HCC cells could be reverted by silencing Mapk14. Our results suggest that a combination of sorafenib and Mapk14 blockade is a promising approach to overcoming therapy resistance of human HCC.

A Direct In Vivo RNAi Screen Identifies MKK4 as a Key Regulator of Liver Regeneration

The liver harbors a distinct capacity for endogenous regeneration; however, liver regeneration is often impaired in disease and therefore insufficient to compensate for the loss of hepatocytes and organ function. Here we describe a functional genetic approach for the identification of gene targets that can be exploited to increase the regenerative capacity of hepatocytes. Pools of small hairpin RNAs (shRNAs) were directly and stably delivered into mouse livers to screen for genes modulating liver regeneration. Our studies identify the dual-specific kinase MKK4 as a master regulator of liver regeneration. MKK4 silencing robustly increased the regenerative capacity of hepatocytes in mouse models of liver regeneration and acute and chronic liver failure. Mechanistically, induction of MKK7 and a JNK1-dependent activation of the AP1 transcription factor ATF2 and the Ets factor ELK1 are crucial for increased regeneration of hepatocytes with MKK4 silencing.

A MYC-aurora kinase A protein complex represents an actionable drug target in p53-altered liver cancer

MYC oncoproteins are involved in the genesis and maintenance of the majority of human tumors but are considered undruggable. By using a direct in vivo shRNA screen, we show that liver cancer cells that have mutations in the gene encoding the tumor suppressor protein p53 (Trp53 in mice and TP53 in humans) and that are driven by the oncoprotein NRAS become addicted to MYC stabilization via a mechanism mediated by aurora kinase A (AURKA). This MYC stabilization enables the tumor cells to overcome a latent G2/M cell cycle arrest that is mediated by AURKA and the tumor suppressor protein p19ARF. MYC directly binds to AURKA, and inhibition of this protein–protein interaction by conformation-changing AURKA inhibitors results in subsequent MYC degradation and cell death. These conformation-changing AURKA inhibitors, with one of them currently being tested in early clinical trials, suppressed tumor growth and prolonged survival in mice bearing Trp53-deficient, NRAS-driven MYC-expressing hepatocellular carcinomas (HCCs). TP53-mutated human HCCs revealed increased AURKA expression and a positive correlation between AURKA and MYC expression. In xenograft models, mice bearing TP53-mutated or TP53-deleted human HCCs were hypersensitive to treatment with conformation-changing AURKA inhibitors, thus suggesting a therapeutic strategy for this subgroup of human HCCs.

Optimized Target Residence Time: Type I1/2 Inhibitors for p38α MAP Kinase with Improved Binding Kinetics through Direct Interaction with the R-Spine

Skepinone-L was recently reported to be a p38α MAP kinase inhibitor with high potency and excellent selectivity in vitro and in vivo. However, this class of compounds still act as fully ATP-competitive Type I binders which, furthermore, suffer from short residence times at the enzyme. We herein describe a further development with the first Type I1/2 binders for p38α MAP kinase. Type I1/2 inhibitors interfere with the R-spine, inducing a glycine flip and occupying both hydrophobic regions I and II. This design approach leads to prolonged target residence time, binding to both the active and inactive states of the kinase, excellent selectivity, excellent potency on the enzyme level, and low nanomolar activity in a human whole blood assay. This promising binding mode is proven by X-ray crystallography.

Assessment of mutation probabilities of KRAS G12 missense mutants and their long-timescale dynamics by atomistic molecular simulations and Markov state modeling

A mutated KRAS protein is frequently observed in human cancers. Traditionally, the oncogenic properties of KRAS missense mutants at position 12 (G12X) have been considered as equal. Here, by assessing the probabilities of occurrence of all KRAS G12X mutations and KRAS dynamics we show that this assumption does not hold true. Instead, our findings revealed an outstanding mutational bias. We conducted a thorough mutational analysis of KRAS G12X mutations and assessed to what extent the observed mutation frequencies follow a random distribution. Unique tissue-specific frequencies are displayed with specific mutations, especially with G12R, which cannot be explained by random probabilities. To clarify the underlying causes for the nonrandom probabilities, we conducted extensive atomistic molecular dynamics simulations (170 μs) to study the differences of G12X mutations on a molecular level. The simulations revealed an allosteric hydrophobic signaling network in KRAS, and that protein dynamics is altered among the G12X mutants and as such differs from the wild-type and is mutation-specific. The shift in long-timescale conformational dynamics was confirmed with Markov state modeling. A G12X mutation was found to modify KRAS dynamics in an allosteric way, which is especially manifested in the switch regions that are responsible for the effector protein binding. The findings provide a basis to understand better the oncogenic properties of KRAS G12X mutants and the consequences of the observed nonrandom frequencies of specific G12X mutations.

Abstract 3820: Epigenetic re-expression of fetal IGF2 as therapeutic target in hepatocellular carcinoma

Background and aims Hepatocellular carcinoma (HCC) is a major health problem. Most patients are diagnosed at advanced stages when the only approved therapy is the multi-kinase inhibitor sorafenib. Consequently, there is a great need for the development of new effective treatments. IGF signaling pathway is aberrantly activated in HCC; however, its contribution to HCC pathogenesis is still unclear. Since IGF2 is overexpressed in HCC, we aimed to elucidate the oncogenic potential and mechanism of dis-regulation of this protein and determine the antitumoral efficacy of molecular abrogation of this ligand by targeted therapies. Methods Transcriptomic profiling, miRNAs expression, RNA- and whole exome- sequencing and methylation were analyzed in 228 HCCs with a focus on IGF-pathway. Gene Set Enrichment Analysis and Ingenuity Pathway Analysis were carried out in IGF2-overexpressing tumors. Stable HCC cell lines with knock-down and ectopic overexpression of IGF2 were generated. A chemically-induced mouse model of HCC, and two genetically-engineered mosaic mouse models (GEMM) overexpressing IGF2 specifically in the liver were generated to assess IGF2 oncogenicity in hepatocarcinogenesis. The therapeutic potential of a monoclonal-antibody against IGF-ligands (IGF1/2-mAb) alone or in combination with sorafenib was tested in a xenograft model of HCC. Results Here, IGF2-overexpression occurred in 15% of HCC patients as a result of the epigenetic reactivation of IGF2-fetal promoters, mainly through loss of promoters methylation (53% of cases) and deregulation of miR-216b, miR-483-5p and miR-let7-d (35% of cases). Re-expression of IGF2 was associated with a progenitor cell-like, poorly differentiated and aggressive subtype of HCC, and poor prognosis (p Conclusions IGF2 is the first validated epidriver in HCC and has a key role in the hepatocarcinogenic process. These results provide the rationale for testing IGF1/2-mAb in a selected subset of HCC patients. Citation Format: Iris Martinez-Quetglas, Roser Pinyol, Daniel Dauch, Sara Torrecilla, Victoria Tovar, Agrin Moeini, Clara Alsinet, Anna Portela, Augusto Villanueva, Manel Esteller, Lars Zender, Josep M Llovet. Epigenetic re-expression of fetal IGF2 as therapeutic target in hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3820.

Abstract 4876: Multiplex in vivo RNAi screening instructs combination therapies to increase the therapeutic efficacy of the multikinase inhibitor sorafenib

Hepatocellular carcinoma (HCC), the third leading cause of cancer death worldwide, represents a highly chemoresistant tumor and only recently the multikinase inhibitor sorafenib was approved as the first active systemic treatment against HCC. This illustrates that targeted therapies can be effective against HCC and therefore heralds a new era in HCC treatment. However, sorafenib monotherapy increases survival of HCC patients by less than 3 month, thus emphasizing the strong need to understand the molecular mechanisms of sorafenib sensitivity and resistance in order to inform new combination therapies with higher therapeutic efficacy. Activation of the Ras/MAPK signaling pathway was reported to play a major role in liver tumor development and progression. Taking advantage of genetically defined mosaic liver cancer mouse models, we found that sorafenib treatment of NrasG12V driven murine liver carcinomas results in moderate but distinct treatment responses resembling the response rates of sorafenib treated human HCCs. To identify genes mediating resistance or sensitivity towards sorafenib, we conducted an in vivo RNAi screen. Pools of shRNAs targeting genes found amplified in human hepatocellular carcinomas were applied and mice harboring NrasG12V expressing liver carcinomas with stable expression of shRNA library pools were either treated with sorafenib or carrier control. After 5 weeks of treatment, shRNA distribution was quantified in tumors from both cohorts using deep sequencing, whereas enriched shRNAs pinpoint potential resistance genes and depleted shRNAs pinpoint targets that may be exploited as sorafenib sensitizers. Functional validation experiments were performed with multiple independent single shRNAs against each “sensitizing” target and confirmed a significant survival benefit over sorafenib treated tumors that express non-targeting control shRNAs. Importantly, our top-scoring candidate represents a kinase and, taking advantage of readily available pharmacological inhibitors, we show that combination therapy with sorafenib is well tolerated by the mice and results in a significant survival advantage. We also expanded our new combination treatment to murine HCCs of additional genetic backgrounds, overexpressing c-myc or AKT. As therapeutic efficacy was also shown in these models, it is likely that our new combination therapy will be active against a broad range of human HCCs. Furthermore, we found that the newly identified combination therapy significantly decreased proliferation and induced cell death in a panel of well established human hepatoma cell lines. In summary, our study establishes a new sorafenib based combination therapy for the treatment of hepatocellular carcinoma and highlights the potential of in vivo RNAi screens to identify genes that modulate the treatment response of targeted therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4876. doi:1538-7445.AM2012-4876

204 IN VIVO RNAI SCREENING IDENTIFIES NEW MEDIATORS OF P53 INDEPENDENT TUMOR SUPPRESSIVE FUNCTIONS OF P19ARF IN THE LIVER

putative oncogenes byWestern blot. Protein expressions of NUCKS1, located on 1q32.1 were analyzed in additional 70 pairs of HCC and surrounding non-tumor tissue. Up-regulation of NUCKS1 was observed in the 52.8% (37/70) of HCC samples. Functional analysis in Hep3B cell line demonstrated that inhibition of NUCKS1 expression by siRNA decreased cell viability. Conclusion: We identified probable chromosomal regions associated with the development of tumor and patient survival in HCC subjects. NUCKS1 was potential target within the chromosomal gain at 1q32.1 and may be involved in development of HCC.