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Background: Most liver tumors arise on the basis of chronic liver diseases that trigger inflammatory responses. Besides inflammation, subsequent defects in the p53-signaling pathway frequently occurs in liver cancer. In this study, we analyzed the consequences of inflammation and p53 loss in liver carcinogenesis. Methods: We used inducible liver-specific transgenic mouse strains to analyze the consequences of NF-&kappa, B/p65 activation mimicking chronic inflammation and subsequent p53 loss. Results: Ikk2ca driven NF-&kappa, B/p65 activation in mice results in liver fibrosis, the formation of ectopic lymphoid structures and carcinogenesis independent of p53 expression. Subsequent deletion of Trp53 led to an increased tumor formation, metastasis and a shift in tumor differentiation towards intrahepatic cholangiocarcinoma. In addition, loss of Trp53 in an inflammatory liver resulted in elevated chromosomal instability and indicated a distinct aberration pattern. Conclusions: In conclusion, activation of NF-&kappa, B/p65 mimicking chronic inflammation provokes the formation of liver carcinoma. Collateral disruption of Trp53 supports tumor progression and influences tumor differentiation and heterogeneity.

Liver cholestasis is a chronic liver disease and a major health problem worldwide. Cholestasis is characterised by a decrease in bile flow due to impaired secretion by hepatocytes or by obstruction of bile flow through intra- or extrahepatic bile ducts. Thereby cholestasis can induce ductal proliferation, hepatocyte injury and liver fibrosis. Notch signalling promotes the formation and maturation of bile duct structures. Here we investigated the liver regeneration process in the context of cholestasis induced by disruption of the Notch signalling pathway. Liver-specific deletion of recombination signal binding protein for immunoglobulin kappa j region (Rbpj), which represents a key regulator of Notch signalling, induces severe cholestasis through impaired intra-hepatic bile duct (IHBD) maturation, severe necrosis and increased lethality. Deregulation of the biliary compartment and cholestasis are associated with the change of several signalling pathways including a Kyoto Encyclopedia of Genes and Genomes (KEGG) gene set representing the Hippo pathway, further yes-associated protein (YAP) activation and upregulation of SRY (sex determining region Y)-box 9 (SOX9), which is associated with transdifferentiation of hepatocytes. SOX9 upregulation in cholestatic liver injury in vitro is independent of Notch signalling. We could comprehensively address that in vivo Rbpj depletion is followed by YAP activation, which influences the transdifferentiation of hepatocytes and thereby contributing to liver regeneration.

Aneuploidy can instigate tumorigenesis. However, mutations in genes that control chromosome segregation are rare in human tumors as these mutations reduce cell fitness. Screening experiments indicate that the knockdown of multiple classes of genes that are not directly involved in chromosome segregation can lead to aneuploidy induction. The possible contribution of these genes to cancer formation remains yet to be defined. Here we identified gene knockdowns that lead to an increase in aneuploidy in checkpoint-deficient human cancer cells. Computational analysis revealed that the identified genes overlap with recurrent mutations in human cancers. The knockdown of the three strongest selected candidate genes (ORP3, GJB3, and RXFP1) enhances the malignant transformation of human fibroblasts in culture. Furthermore, the knockout of Orp3 results in an aberrant expansion of lymphoid progenitor cells and a high penetrance formation of chromosomal instable, pauci-clonal B-cell lymphoma in aging mice. At pre-tumorous stages, lymphoid cells from the animals exhibit deregulated phospholipid metabolism and an aberrant induction of proliferation regulating pathways associating with increased aneuploidy in hematopoietic progenitor cells. Together, these results support the concept that aneuploidy-inducing gene deficiencies contribute to cellular transformation and carcinogenesis involving the deregulation of various molecular processes such as lipid metabolism, proliferation, and cell survival.

SummaryThe p53 tumor suppressor coordinates a series of antiproliferative responses that restrict the expansion of malignant cells, and as a consequence, p53 is lost or mutated in the majority of human cancers. Here, we show that p53 restricts expression of the stem and progenitor-cell-associated protein nestin in an Sp1/3 transcription-factor-dependent manner and that Nestin is required for tumor initiation in vivo. Moreover, loss of p53 facilitates dedifferentiation of mature hepatocytes into nestin-positive progenitor-like cells, which are poised to differentiate into hepatocellular carcinomas (HCCs) or cholangiocarcinomas (CCs) in response to lineage-specific mutations that target Wnt and Notch signaling, respectively. Many human HCCs and CCs show elevated nestin expression, which correlates with p53 loss of function and is associated with decreased patient survival. Therefore, transcriptional repression of Nestin by p53 restricts cellular plasticity and tumorigenesis in liver cancer.

In chronic lymphocytic leukemia (CLL) short telomeres are associated with other adverse prognostic factors and poor survival. We and others have described association of telomere length with genomic complexity and clonal evolution in CLL. To understand if telomere shortening rather than being only a marker of cell proliferation, could also functionally contribute to disease progression, we generated Terc knock out in the Eµ-TCL1 murine CLL model (Eµ-TCL1 mTerc-/- mice). Comparison of the Eµ-TCL1 mTerc-/- mice from the generations G1, G2 and G3 with that of Eµ-TCL1 (TCL1+) did not show a difference in disease initiation, progression as well as survival even though a significant decrease in telomere length of tumor cells was observed with increasing generations. Of interest, the Eµ-TCL1 mTerc-/- G3 tumors (n=8; G3) more frequently showed defective DNA damage response compared to TCL1+ tumors (n=8), as analysed by changes in phosphorylation of gamma-H2AX, ATM and p53 measured by FACS at 1, 3, 6, 16 and 24 hours after gamma irradiation. Despite this predisposition to undergo genomic complexity, the G3 mice showed no difference in disease development compared to TCL1+ mice. Therefore we investigated if cell extrinsic factors in Terc-/- mice could affect tumor development. The Terc-/- microenvironment is known to be restrictive to B and T-lymphopoiesis and could hence inhibit CLL development. To study the impact of Terc-/- microenvironment, splenic tumors from the Eµ-TCL1 mTerc-/- G3 (n=11; G3) mice were transferred into syngeneic Terc+/+ C57Bl6 mice and compared with that of TCL1+ (n=11). No significant difference in disease burden and survival was observed between these 2 cohorts, indicating that there is no adverse influence of the Terc-/- microenvironment on the tumor growth in the Eµ-TCL1 mTerc-/- mice. Further, we hypothesized that the telomere length in the G3 mice may not be short enough to induce genomic instability and enhance disease aggressiveness. The mice were thus crossed to obtain the Eµ-TCL1 mTerc-/- generation G4 (G4). Though the survival of these mice were similar to that of Eµ-TCL1 controls (median survival :49 vs. 51 weeks; P=0.301), the G4 mice showed significantly decreased disease burden as measured by spleen weight, liver weights and tumor cell fraction. The G4 mice showed decreased fertility and hence further crosses to generate G5 were not performed. However, further shortening of telomeres was achieved by serially transplanting the tumors from G3 into syngeneic recipient mice for 2 generations. Telomere length of the tumor cells analysed by Q-PCR showed a significant decrease with increasing transfer rounds, compared to primary TCL1+ tumors. Interestingly, the 2nd round transfer of the G3 tumors led to a less severe disease and significantly longer survival of the recipient mice compared to 2nd round TCL1+ tumors transplants (median survival from date of transplantation: 15 vs. 7 weeks; P=0.030), indicating that cell intrinsic factors in the G3 tumors hamper proliferation of these cells. We finally analyzed if absence of telomerase could mask disease aggressiveness of the G3 tumors by crossing the G3 mice with wildtype Terc+/+ mice. The resulting TCL1+ mTerc+/- G4 mice showed an increased telomerase activity but had significantly shorter telomere length compared to TCL1+ mice. Strikingly, the TCL1+ mTerc+/- G4 mice showed faster disease development and significantly shorter survival (median survival: 42 vs. 51 weeks; p In summary, the TCL1+ mTerc-/- mice crossed through generations G1 to G4 did not show a difference in disease initiation, progression or survival despite significant shortening of telomeres. However, the tumors from G3 had defective DDR, indicating a potential for accumulating genetic aberrations and clonal evolution. But the absence of functional telomerase decreased the growth potential of these genomic instable tumors. Reconstitution of telomerase in G3 mice resulted in aggressive tumors with short telomere length and could therefore be a valuable murine model for genomic instability and aggressive CLL. Disclosures Tausch: Roche: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Other: travel support, Speakers Bureau. Schneider:Celgene: Other: travel grant. Döhner:AbbVie, Agios, Amgen, Astellas, Astex, Celator, Janssen, Jazz, Seattle Genetics: Consultancy, Honoraria; AROG, Bristol Myers Squibb, Pfizer: Research Funding; Celgene, Novartis, Sunesis: Honoraria, Research Funding. Stilgenbauer:AbbVie, AstraZeneca, Celgene, Gilead Sciences, Inc., GSK, Hoffmann La-Roche, Janssen, Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Background and aims The cyclin-dependent kinase inhibitor p21 has been implicated as a tumour suppressor. Moreover, recent genetic studies suggest that p21 might be a potential therapeutic target to improve regeneration in chronic diseases. The aim of this study was to delineate the role of p21 in chronic liver injury and to specify its role in hepatocarcinogenesis in a mouse model of chronic cholestatic liver injury. Methods The degree of liver injury, regeneration and tumour formation was assessed in Mdr2 −/− mice and compared with Mdr2/ p21 −/− mice. Moreover, the role of p21 was evaluated in hepatoma cells in vitro and in human hepatocellular carcinoma (HCC). Results Mdr2 −/− mice developed HCCs as a consequence of chronic inflammatory liver injury. In contrast, tumour development was profoundly delayed in Mdr2/ p21 −/− mice. Delayed tumour development was accompanied by markedly impaired liver regeneration in Mdr2/ p21 −/− mice. Moreover, the regenerative capacity of the Mdr2/ p21 −/− livers in response to partial hepatectomy declined with age in these mice. Hepatocyte transplantation experiments revealed that impaired liver regeneration was due to intrinsic factors within the cells and changes in the Mdr2/ p21 −/− microenvironment. In human HCCs, a subset of tumours expressed p21, which was associated with a significant shorter patient survival. Conclusions We provide experimental evidence that p21 is required for sustained liver regeneration and tumour development in chronic liver injury indicating that p21 needs to be tightly regulated in order to balance liver regeneration and cancer risk. Moreover, we identify p21 as a negative prognostic marker in human HCC.

To date, the precise role of ion channels in developmental processes has remained elusive, although it is well accepted that cell differentiation and maturation affect the expression patterns of ion channels [1]. Calcium-activated potassium channels (SKCas) exhibit small (SK1-3, Kcnn1-3) or intermediate (SK4, IK, Kcnn4) unitary conductance for Stem Cell Rev and Rep (2012) 8:720–740 DOI 10.1007/s12015-011-9324-9

Telomere shortening impairs liver regeneration in mice and is associated with cirrhosis formation in humans with chronic liver disease. In humans, telomerase mutations have been associated with familial diseases leading to bone marrow failure or lung fibrosis. It is currently unknown whether telomerase mutations associate with cirrhosis induced by chronic liver disease. The telomerase RNA component (TERC) and the telomerase reverse transcriptase (TERT) were sequenced in 1,121 individuals (521 patients with cirrhosis induced by chronic liver disease and 600 noncirrhosis controls). Telomere length was analyzed in patients carrying telomerase gene mutations. Functional defects of telomerase gene mutations were investigated in primary human fibroblasts and patient-derived lymphocytes. An increased incidence of telomerase mutations was detected in cirrhosis patients (allele frequency 0.017) compared to noncirrhosis controls (0.003, P value 0.0007; relative risk [RR] 1.859; 95% confidence interval [CI] 1.552-2.227). Cirrhosis patients with TERT mutations showed shortened telomeres in white blood cells compared to control patients. Cirrhosis-associated telomerase mutations led to reduced telomerase activity and defects in maintaining telomere length and the replicative potential of primary cells in culture. Conclusion: This study provides the first experimental evidence that telomerase gene mutations are present in patients developing cirrhosis as a consequence of chronic liver disease. These data support the concept that telomere shortening can represent a causal factor impairing liver regeneration and accelerating cirrhosis formation in response to chronic liver disease. (HEPATOLOGY 2011;)

Liver cholestasis is a chronic liver disease and a major health problem worldwide. Cholestasis is characterised by a decrease in bile flow due to impaired secretion by hepatocytes or by obstruction of bile flow through intra- or extrahepatic bile ducts. Thereby cholestasis can induce ductal proliferation, hepatocyte injury and liver fibrosis. Notch signalling promotes the formation and maturation of bile duct structures. Here we investigated the liver regeneration process in the context of cholestasis induced by disruption of the Notch signalling pathway. Liver-specific deletion of recombination signal binding protein for immunoglobulin kappa j region (Rbpj), which represents a key regulator of Notch signalling, induces severe cholestasis through impaired intra-hepatic bile duct (IHBD) maturation, severe necrosis and increased lethality. Deregulation of the biliary compartment and cholestasis are associated with the change of several signalling pathways including a Kyoto Encyclopedia of Genes and Genomes (KEGG) gene set representing the Hippo pathway, further yes-associated protein (YAP) activation and upregulation of SRY (sex determining region Y)-box 9 (SOX9), which is associated with transdifferentiation of hepatocytes. SOX9 upregulation in cholestatic liver injury in vitro is independent of Notch signalling. We could comprehensively address that in vivo Rbpj depletion is followed by YAP activation, which influences the transdifferentiation of hepatocytes and thereby contributing to liver regeneration.

Overexpression of survivin, a crucial regulator of the mitotic spindle checkpoint with additional antiapoptotic effects, is associated with poor prognosis in neuroblastoma (NB) and other cancers. Thus, survivin is a promising therapeutic target in NB. Transcriptional inhibitors of survivin show preclinical efficacy and have been tested in clinical trials. The results of these trials, however, have been disappointing, due to modest efficacy and unexpected off-target effects. We thus investigated the consequences of marked, yet incomplete decrease of survivin on NB in a novel transgenic mouse model. To this end, we crossed mice haploinsufficient for survivin with TH-MYCN mice to generate BIRC5+/- MYCN tg/+ mice. These mice developed NB with markedly decreased mRNA and protein expression of survivin. Surprisingly, incidence and latency of the NB that developed in the BIRC5+/- MYCN tg/+ mice were not different from those in the TH-MYCN mice. Growth of tumors was not decreased in BIRC5+/- MYCN tg/+ mice, as assessed by serial MRI imaging, and survival of the mice was not increased. Immunohistochemistry of the NB did not show decreased proliferation (Ki67), increased apoptosis (activated caspase 3) or a change in vascularity (CD31). No difference in copy number variations between the two NB mouse models was discernible using CGH arrays. To determine whether dysfunctional T cells described in mice haploinsufficient for survivin could have masked any loss of aggressiveness of NB cells haploinsufficient for survivin, NB of BIRC5+/- MYCN tg/+ mice were retransplanted into survivin sufficient mice. No difference in tumor incidence, latency and growth was detected. Taken together, despite a marked decrease in the expression of BIRC5/survivin by haploinsufficiency development and growth of NB were not inhibited. While a further decrease of survivin beyond that seen in haploinsufficiency will inhibit NB, it most likely will also elicit marked side effects, given that survivin is essential for normal cells. Our results cast doubt on the efficacy of inhibitors of survivin expression in NB and call for targeting survivin by alternative approaches, such as inhibiting protein-protein interactions of survivin. Citation Format: Carmen Dorneburg, Volker Rasche, Andre Lechel, Sarah-Fee Katz, Klaus-Michael Debatin, Christian Beltinger. Marked decrease of BIRC5/Survivin by haploinsufficiency does not inhibit neuroblastoma in transgenic mice: Implications for survivin as a therapeutic target in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4136.

Exploratory behavior and string agility. Exploratory behavior of the mice has been analysed using the Open Field System. Several parameters, e.g. the activity, duration and the totally travelled distance were measured in three different zones of the open field: Border, intermediate and center of the open field. 7 to 10 mice of the indicated genotypes were recorded each for 10 min. (A) Activity in the open field and (B) duration of stay in the three indicated zones. αSYNtg/tg G3Terc-/- mice did not show a difference in comparison to αSYNtg/tg mice and the two control groups. Although a trend can be seen in duration, αSYNtg/tg and αSYNtg/tg G3Terc-/- stay even shorter in the intermediate and the center zone and prefer the border zone in comparison to Terc+/+ or G3Terc-/- mice. (C) String agility test shows no difference between the different groups. (D) Activated astrocytes were measured using GFAP immunohistochemistry. The histogram depicts GFAP positive astrocytes of the brainstem (n = 4–6 mice). αSYNtg/tg mice show a significant increase in astrocyte activation (62.35 ± 5.656) in comparison to αSYNtg/tg G3Terc-/- mice (39 ± 2.383, P = 0.0159). Furthermore, αSYNtg/tg mice with phenotype (85 weeks) show a significant activation (62.35 ± 5.65) if compared with non-phenotypic mice (75 weeks, 33.37 ± 4.086, P = 0.0095). Terc+/+ (14.3 ± 7.6) and G3Terc-/- mice (10.07 ± 7.10) show low activation levels. (PDF 33 kb)

Classification and scoring of phospho-α-synuclein and PK-PET Blot. (A) Classification of phospho-α-synuclein staining into four different scores. Representative pictures for scoring. Score 0: no p-α-synuclein staining, score 1: little staining in brainstem and DpMe, score 2: strong staining in brainstem and DpMe, score 3: strong p-α-synuclein staining in brainstem, DpMe, and cerebellum indicating severe disease progression. (B) Scoring to classify PK-PET Blot. Score 0: no PK resistant aggregates, score 1: light aggregates in brainstem and Deep Mesencepahlic nucleus (DpMe), score 2: clear PK resistant aggregates in brainstem and DpMe, score 3: dominant aggregates in brainstem and DpMe. Score 4: Prominent aggregates in brainstem, DpMe and cerebellum. (PDF 125 kb)

Mating scheme for the generation of mouse cohorts. (A) Breeding scheme for generating 3rd generation telomerase knockout and α-synuclein transgenic mice (αSYNtg/tg G3Terc-/-) and corresponding control cohorts. Heterozygous α-synuclein (αSYN) mice were crossed with heterozygous telomerase knockout mice (Terc) to generate double transgenic αSYNtg/tg G1Terc-/- and single transgenic αSYNtg/tg, G1Terc-/- and Terc+/+ mice (G1 = first generation of telomerase knockout). These double transgenic αSYNtg/tg G1Terc-/- mice were crossed with each other to produce αSYNtg/tg G2Terc-/- mice and finally αSYNtg/tg G3Terc-/- mice. (B) Telomere length was measured in neurons of the brainstem using qFISH telomere staining double-stained with Cy5-Neuron N dye. The graph represents telomere length in brainstem in 75 weeks old mice (n = 5 mice per group). Analyzed were αSYNtg/tg G3Terc-/- in comparison to αSYNtg/tg mice (P = 0.0012) and G3Terc-/- in comparison to Terc+/+ mice (P = 0.0079) as well as αSYNtg/tg G3Terc-/- compared with G3Terc-/- (P = 0.03). (PDF 29 kb)

Primer sequences which were used for RT-PCR. (DOC 40 kb)

Schematic representation of the parameter analyzed in 3D-reconstructed microglia. Automated analysis of each structural characteristic was performed using Imaris Bitplane software. (PDF 50 kb)

Dynamic regulation of cell-cell adhesion by the coordinated formation and dissolution of E-cadherin-based adherens junctions is crucial for tissue homeostasis. The actin-binding protein cortactin interacts with E-cadherin and enables F-actin accumulation at adherens junctions. Here, we were interested to study the broader functional interactions of cortactin in adhesion complexes. In line with literature, we demonstrate that cortactin binds to E-cadherin, and that a posttranslational modification of cortactin, RhoA-induced phosphorylation by protein kinase D1 (PKD1; also known as PRKD1) at S298, impairs adherens junction assembly and supports their dissolution. Two new S298-phosphorylation-dependent interactions were also identified, namely, that phosphorylation of cortactin decreases its interaction with β-catenin and the actin-binding protein vinculin. In addition, binding of vinculin to β-catenin, as well as linkage of vinculin to F-actin, are also significantly compromised upon phosphorylation of cortactin. Accordingly, we found that regulation of cell-cell adhesion by phosphorylation of cortactin downstream of RhoA and PKD1 is vitally dependent on vinculin-mediated protein interactions. Thus, cortactin, unexpectedly, is an important integration node for the dynamic regulation of protein complexes during breakdown and formation of adherens junctions. ispartof: Journal of Cell Science vol:129 issue:12 pages:2416-29 ispartof: location:England status: published

Background— Ion channels are key determinants for the function of excitable cells, but little is known about their role and involvement during cardiac development. Earlier work identified Ca 2+ -activated potassium channels of small and intermediate conductance (SKCas) as important regulators of neural stem cell fate. Here we have investigated their impact on the differentiation of pluripotent cells toward the cardiac lineage. Methods and Results— We have applied the SKCa activator 1-ethyl-2-benzimidazolinone on embryonic stem cells and identified this particular ion channel family as a new critical target involved in the generation of cardiac pacemaker-like cells: SKCa activation led to rapid remodeling of the actin cytoskeleton, inhibition of proliferation, induction of differentiation, and diminished teratoma formation. Time-restricted SKCa activation induced cardiac mesoderm and commitment to the cardiac lineage as shown by gene regulation, protein, and functional electrophysiological studies. In addition, the differentiation into cardiomyocytes was modulated in a qualitative fashion, resulting in a strong enrichment of pacemaker-like cells. This was accompanied by induction of the sino-atrial gene program and in parallel by a loss of the chamber-specific myocardium. In addition, SKCa activity induced activation of the Ras-Mek-Erk signaling cascade, a signaling pathway involved in the 1-ethyl-2-benzimidazolinone–induced effects. Conclusions— SKCa activation drives the fate of pluripotent cells toward mesoderm commitment and cardiomyocyte specification, preferentially into nodal-like cardiomyocytes. This provides a novel strategy for the enrichment of cardiomyocytes and in particular, the generation of a specific subtype of cardiomyocytes, pacemaker-like cells, without genetic modification.

Telomeres are nucleo-protein complexes at the ends of the chromosomes that play a key role in protection of the ends from being recognized as DNA damage and to prevent fusion of the chromosomes. The telomeric DNA shortens with each cell division in the absence of telomerase, due to end replication problem. In chronic lymphocytic leukemia (CLL), short telomeres were found to be associated with poor prognostic factors and poor survival in various univariable and multivariable analyses. Short telomeres in CLL are known to be frequently associated with increased DNA damage response and to undergo fusion events, conferring genomic instability. But the contribution of telomere dysfunction to CLL pathogenesis and disease progression has never been studied in vivo using mouse models. Here, we hypothesized that genomic instability resulting from telomere dysfunction could drive acquisition of genetic lesions, contributing to CLL pathogenesis, progression and disease evolution. Thus, the CLL mouse model with telomere dysfunction was generated by crossing the Eµ-TCL1 (TCL1+) mouse with mTerc-/- mouse. The first generation TCL1+ mTerc-/- (G1) mice were inter-crossed to obtain generations G2 and G3, as telomeres are known to shorten with subsequent generations. The TCL1+ mTerc-/- mice from the generations G1 (N=14), G2 (N=33) and G3 (N=26), including TCL1+ (N=34), wildtype (WT, N=18) and mTerc-/- G1 (N=4), G2 (N=5) and G3 (N=13) as controls were initially analyzed for disease burden in peripheral blood (PB) by bleeding at an interval of 4 weeks, starting from 12 weeks and the percentage of CD19+ CD5+ cells was estimated by FACS. No difference in disease onset or progression was observed between the TCL1+ mTerc-/- G1, G2 and G3 in comparison toTCL1+ mice (Fig. 1a). Similarly, analysis of survival showed no significant difference between the TCL1+ mTerc-/- G1 (N=14), G2 (N=33) and G3 (N=26) mice, compared to TCL1+ (N=34) (median: 53, 55, 52 weeks vs. 50.5 weeks, Fig. 1b). Spleen and liver weights in the TCL1+ mTerc-/- G1 (N=12), G2 (N=33) and G3 (N=26) mice were highly variable (spleen: 0.1g to 3.5g, liver: 0.1g to 8.0g) as in the TCL1+ (N=27, spleen: 0.3g to 5.0g, liver: 1.7g to 7.4g) mice but no significant difference in spleen (Fig. 1d) and liver weights was observed between the subgroups. Interestingly, spleen weights were associated with survival only in the TCL1+ mice, with larger spleens associated with worse survival (48.5 vs. 57.5 weeks, P=0.091). Since no difference in disease characteristics was observed, it was verified using Q-PCR, if telomere lengths vary in the tumors from the different subgroups. Telomere lengths of CLL cells from the spleen were significantly shorter (Fig. 1c) in the G1 (median: 20.5kb, P=0.0002), G2 (median: 18.5kb, P=0.0016) and G3 (median: 13.2kb, P Additionally, the G3 mTerc-/- microenvironment is known to restrict B and T lymphopoiesis and thus might influence CLL cell proliferation, masking disease aggressiveness in the TCL1+ mTerc-/- G3 mice. To overcome the influence of mTerc-/- microenvironment, CLL cells obtained from spleens of TCL1+ and TCL1+ mTerc-/- G3 mice were transferred into syngeneic C57Bl6 mice. Briefly, 20 million cells were intravenously injected into the tail vein and disease was monitored by analysis of CD19+ CD5+ cells in PB, once every 4 weeks. Early follow up of 8 weeks clearly show a trend towards increase in CLL cells in PB of mice transferred with TCL1+ mTerc-/- G3 tumors compared to those with TCL1+ tumors (median tumor load: 15.75% vs. 6.1%, P=0.0553). Longer follow up of the experiment is ongoing. In summary, the TCL1+ mTerc-/- mice across the generations G1, G2 and G3 showed no difference in disease onset, progression, disease burden and survival in comparison to TCL1+ mice. The absence of increased disease manifestation in the TCL1+ mTerc-/- may be attributed to the microenvironmental influence on lymphopoiesis, as syngeneic transfer of CLL from TCL1+ mTerc-/- G3 mice showed an increase in tumor load compared to that of TCL1+ tumors, indicating a contribution of telomere shortening to disease aggressiveness in CLL. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Background & Aims p53 limits the self-renewal of stem cells from various tissues. Loss of p53, in combination with other oncogenic events, results in aberrant self-renewal and transformation of progenitor cells. It is not known whether loss of p53 is sufficient to induce tumor formation in liver. Methods We used AlfpCre mice to create mice with liver-specific disruption of Trp53 ( AlfpCre + Trp53 Δ2-10/Δ2-10 mice). We analyzed colony formation and genomic features and gene expression patterns in liver cells during hepatocarcinogenesis in mice with homozygous, heterozygous, and no disruption of Trp53. Results Liver-specific disruption of Trp53 consistently induced formation of liver carcinomas that had bilineal differentiation. In nontransformed liver cells and cultured primary liver cells, loss of p53 (but not p21) resulted in chromosomal imbalances and increased clonogenic capacity of liver progenitor cells (LPCs) and hepatocytes. Primary cultures of hepatocytes and LPCs from AlfpCre + Trp53 Δ2-10/Δ2-10 mice, but not Cdkn1a −/− mice, formed tumors with bilineal differentiation when transplanted into immunocompromised mice. Spontaneous liver tumors that developed in AlfpCre + Trp53 Δ2-10/Δ2-10 mice had significant but complex alterations in expression of Rb checkpoint genes compared with chemically induced liver tumors that developed mice with wild-type Trp53. Conclusions Deletion of p53 from livers of mice is sufficient to induce tumor formation. The tumors have bilineal differentiation and dysregulation of Rb checkpoint genes.

Background: Defects in the p53-signaling checkpoint have been described in 30-60% of human hepatocellular carcinoma (HCC) and are associated with less differentiated tumors and poor prognosis. The contribution of p53 deletion by itself to hepatocarcinogenesis is not known. Furthermore it is not clear, if liver stem cells play a role in the onset of liver tumor formation. Methods: We generated mice with liver specific TRP53 deletion (TRP53−/−) by crossing conditional TRP53 KO mice with AlfpCRE transgenic mice (CRE expression under the albumin-alphafetoprotein-enhancer-promoter). In vitro cultures were established from the developing liver tumors and analyzed for potential tumor stem cells using xenotransplantation. Endogenous liver stem/progenitor cells were analysed using FACS purification and in vitro culturing of purified liver stem cells (CD13+CD133+CD49f+). Results: Mice with liver specific TRP53 deletion developed liver tumors with a mixed differentiation (hepatocellular and cholangiocellular carcinoma). Purification and xenotransplantation of subpopulations of tumor cells from primary tumor cell cultures (using following surface marker: CD13, CD90, CD133, or cKit) revealed no differences in tumor forming capacity in NMRI nu/nu mice. Moreover, tumor formation was seen after xenotransplantation of very small tumor cell numbers (2-cells) indicating that the tumors have an inherent high tumor forming capacity. An analysis of the colony formation capacity of endogenous liver stem/progenitor cells revealed a prolonged survival of bipotent liver stem cells in aged TRP53−/− mice compared to TRP53+/− or TRP53+/+ mice. The last two groups showed a complete loss of bipotent liver stem cells by 8 month of age, whereas bipotent liver stem cells were detected with a frequency of 0,4% in liver of 8-10 month old TRP53−/− mice. Conclusion: This study provides the first experimental evidence that TRP53 deletion leads to a prolonged survival of bipotent liver stem cells and the formation of bi-differentiated liver tumors. These data indicate that the p53-dependent tumor suppressor functions include the restriction of proliferation and survival of bipotent stem cells in the liver. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4242.