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SummaryWe created a mouse model wherein conditional expression of an Mll-AF4 fusion oncogene induces B precursor acute lymphoblastic (ALL) or acute myeloid leukemias (AML). Gene expression profile analysis of the ALL cells demonstrated significant overlap with human MLL-rearranged ALL. ChIP-chip analysis demonstrated histone H3 lysine 79 (H3K79) methylation profiles that correlated with Mll-AF4-associated gene expression profiles in murine ALLs and in human MLL-rearranged leukemias. Human MLL-rearranged ALLs could be distinguished from other ALLs by their H3K79 profiles, and suppression of the H3K79 methyltransferase DOT1L inhibited expression of critical MLL-AF4 target genes. We thus demonstrate that ectopic H3K79 methylation is a distinguishing feature of murine and human MLL-AF4 ALLs and is important for maintenance of MLL-AF4-driven gene expression.

In this chapter, we will review how signal transduction pathways have been assembled in the past, bringing us to our present understanding of this area of research. The methods employed have relied heavily upon the genetics of yeast, worms, flies, mice, and humans. The use of second site suppressors and epistasis has permitted the detection of interacting elements and the sequence of genetic activities. Biochemistry has been employed to elucidate metabolic pathways, demonstrate protein complexes, and identify functions of gene products. The tools of molecular biology-knocking concentration of protein products down or up-have been helpful to trace the function of pathways in vivo. The study of disease states has led to the identification of a set of altered genes and helped define a network that is altered and gives rise to the disease. We will also discuss some serious limitations in these approaches. After reviewing how signal transduction pathways are constructed and investigated, we will turn our attention to an example that demonstrates the inter-relationships between pathways and the regulation of a specific set of pathways. We will examine how the p53 pathway in responding to stress shuts down the AKT-1 and mTOR pathways so as to limit the error frequency of cell growth and division during a stressful time where homeostatic mechanisms are required to respond and increase the fidelity of these processes.

The transcription factor p53 has been studied extensively as a tumour suppressor, but little is known about its normal physiological role. A study in mice now links normal p53 function to reproduction and fertility. A p53 deficiency results in poor implantation of embryos in female mice, low pregnancy rates and small litter sizes. In this role p53 acts by regulating LIF (leukaemia inhibitory factor), a cytokine involved in blastocyst implantation. This work raises the possibility that the normal function of p53 is important for the success of implantation particularly in women undergoing in vitro fertilization or embryo transfer, and suggests a potential strategy to improve implantation efficiency in women with recurrent implantation failure. The p53 gene has been extensively studied for its role in tumour prevention but little is known about its normal physiological function. A crucial role of this factor in fecundity and reproduction is now reported. Extensive studies have shown that p53 is important in tumour prevention1. However, little is known about its normal physiological function. Here we show that p53 is important in reproduction, in a gender-specific manner. Significant decreases in embryonic implantation, pregnancy rate and litter size were observed in matings with p53-/- female mice but not with p53-/- male mice. The gene encoding leukaemia inhibitory factor (LIF), a cytokine critical for implantation2, was identified as a p53-regulated gene that functions as the downstream mediator of this effect. p53 can regulate both basal and inducible transcription of LIF. Loss of p53 decreased both the level and function of LIF in uteri. Lower LIF levels were observed in the uteri of p53-/- mice than in those of p53+/+ mice, particularly at day 4 of pregnancy, when transiently induced high levels of LIF were crucial for embryonic implantation. This observation probably accounts for the impaired implantation of embryos in p53-/- female mice. Administration of LIF to pregnant p53-/- mice restored maternal reproduction by improving implantation. These results demonstrate a function for p53 in maternal reproduction through the regulation of LIF. Evidence is accumulating that p53 may have a similar function in humans.

The insulin-like growth factor 1 (IGF-1)-AKT-mTOR pathways sense the availability of nutrients and mitogens and respond by signaling for cell growth and division. The p53 pathway senses a variety of stress signals which will reduce the fidelity of cell growth and division, and responds by initiating cell cycle arrest, senescence, or apoptosis. This study explores four p53-regulated gene products, the β1 and β2 subunits of the AMPK, which are shown for the first time to be regulated by the p53 protein, TSC2, PTEN, and IGF-BP3, each of which negatively regulates the IGF-1-AKT-mTOR pathways after stress. These gene products are shown to be expressed under p53 control in a cell type and tissue-specific fashion with the TSC2 and PTEN proteins being coordinately regulated in those tissues that use insulin-dependent energy metabolism (skeletal muscle, heart, white fat, liver, and kidney). In addition, these genes are regulated by p53 in a stress signal–specific fashion. The mTOR pathway also communicates with the p53 pathway. After glucose starvation of mouse embryo fibroblasts, AMPK phosphorylates the p53 protein but does not activate any of the p53 responses. Upon glucose starvation of E1A-transformed mouse embryo fibroblasts, a p53-mediated apoptosis ensues. Thus, there is a great deal of communication between the p53 pathway and the IGF-1-AKT and mTOR pathways. [Cancer Res 2007;67(7):3043–53]

Oscillations of both p53 and MDM2 proteins have been observed in cells after exposure to stress. A mathematical model describing these oscillations predicted that oscillations occur only at selected levels of p53 and MDM2 proteins. This model prediction suggests that oscillations will disappear in cells containing high levels of MDM2 as observed with a single nucleotide polymorphism in the MDM2 gene (SNP309). The effect of SNP309 upon the p53-MDM2 oscillation was examined in various human cell lines and the oscillations were observed in the cells with at least one wild-type allele for SNP309 (T/T or T/G) but not in cells homozygous for SNP309 (G/G). Furthermore, estrogen preferentially stimulated the transcription of MDM2 from SNP309 G allele and increased the levels of MDM2 protein in estrogen-responsive cells homozygous for SNP309 (G/G). These results suggest the possibility that SNP309 G allele may contribute to gender-specific tumorigenesis through further elevating the MDM2 levels and disrupting the p53-MDM2 oscillation. Furthermore, using the H1299-HW24 cells expressing wild-type p53 under a tetracycline-regulated promoter, the p53-MDM2 oscillation was observed only when p53 levels were in a specific range, and DNA damage was found to be necessary for triggering the p53-MDM2 oscillation. This study shows that higher levels of MDM2 in cells homozygous for SNP309 (G/G) do not permit coordinated p53-MDM2 oscillation after stress, which might contribute to decreased efficiency of the p53 pathway and correlates with a clinical phenotype (i.e., the development of cancers at earlier age of onset in female). [Cancer Res 2007;67(6):2757–65]

Tumor suppressor p53 is frequently mutated in human cancers. Many tumor-associated mutant p53 (mutp53) proteins gain new functions in promoting tumorigenesis, defined as gain of function (GOF). The mechanisms for mutp53 GOF are not well-understood. Mutp53 has been reported to interact with some proteins and it has been suggested that some interactions of mutp53 with its binding partners are important for mutp53 GOF through regulating the levels or functions of mutp53 and/or its binding proteins. To understand the molecular mechanisms for mutp53 GOF, we searched for mutp53 binding proteins in thymic lymphomas of R172H mutp53 knock-in (p53R172H/R172H) mice with significant mutp53 protein accumulation by employing an unbiased immunoprecipitation (IP) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) screening approach. Normal tissues of p53R172H/R172H mice with low mutp53 levels and tumors of p53-/- mice were used as controls. Using this approach, we identified Pontin, a highly conserved AAA+ ATPase important for various cellular functions, as a new mutp53 binding protein. This Pontin-mutp53 is conserved in both humans and mice. Importantly, this Pontin-mutp53 interaction promotes mutp53 GOF in invasion, migration, and anchorage-independent growth of tumor cells. Furthermore, we found that the C-terminus of Pontin is required for Pontin-mutp53 interaction and mutp53 GOF. The ATPase domain in the C-terminus of Pontin is crucial for its promoting effect on mutp53 GOF; blocking the ATPase activity of Pontin by a Pontin specific ATPase inhibitor or an ATPase deficient dominant-negative Pontin expression vector greatly diminished mutp53 GOF. We found that Pontin was largely co-localized with mutp53 in the nucleus and promotes mutp53 GOF through regulation of mutp53 transcriptional activity; knock-down of Pontin abolished the transcriptional regulation of mutp53 towards a group of genes involved in many important signaling pathways, including gap junction signaling, IGF-1 signaling, EGF signaling etc. Furthermore, overexpression of Pontin is associated with poor survival in cancer patients, especially those containing mutp53 in tumors. This work demonstrates that Pontin promotes mutp53 GOF in tumorigenesis in an ATPase-dependent manner, and raises interesting and intriguing possibilities to block mutp53 GOF in tumors through targeting Pontin or its ATPase activity. In summary, our results highlight an important role and mechanism for Pontin, a new p53 partner, in promoting mutp53 GOF in tumorigenesis. Citation Format: Yuhan Zhao, Cen Zhang, Xuetian Yue, Xiaoyan Li, Juan Liu, Haiyang Yu, Qifeng Yang, Zhaohui Feng, Wenwei Hu. Pontin, a new mutant p53 binding protein, promotes gain-of-function of mutant p53. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1221. doi:10.1158/1538-7445.AM2015-1221

Tumor suppressor p53 plays a key role in maintaining genomic stability and preventing tumorigenesis through its regulation of cellular stress responses, including apoptosis, cell cycle arrest and senescence. To ensure its proper levels and functions in cells, p53 is tightly regulated mainly through post-translational modifications. Ubiquitination is the most important post-translational modification for p53. TRIM32 is a protein of the tripartite motif (TRIM) family. The proteins of TRIM family have been reported to be involved in different biological processes, including cell growth, differentiation, development, muscular physiology, innate immune response and cancer. However, like the majority of TRIM family proteins, the biological function of TRIM32 is not well-understood. Here, we identified TRIM32 as a novel p53 target gene and E3 ubiquitin ligase for p53. In response to stress, including ionizing radiation (IR) and chemotherapeutic agents (e.g. etoposide and 5-fluorouracil), p53 protein binds to the p53 responsive element in the promoter of the TRIM32 gene and transcriptionally induces the expression of TRIM32 in cells. In turn, TRIM32 protein interacts with p53 protein and promotes p53 degradation through ubiquitination to negatively regulate p53-mediated apoptosis, cell cycle arrest and senescence in response to stress. Thus, p53 and TRIM32 form a novel negative feedback loop in cells to regulate p53 protein levels and function. TRIM32 is frequently overexpressed in different types of human tumors, including lung, colorectal and liver cancers. TRIM32 overexpression promotes the oncogenic transformation of primary mouse embryonic fibroblasts in a largely p53-dependent manner. Furthermore, TRIM32 overexpression promotes tumorigenesis in mice through the down-regulation of p53 function. Taken together, our results demonstrated that as a novel p53 target and a novel negative regulator for p53, TRIM32 plays an important role in regulation of p53 and p53-mediated cellular stress responses. Furthermore, our results revealed that impairing p53 function is a novel mechanism for TRIM32 in tumorigenesis. Citation Format: Cen Zhang, Juan Liu, Xiaolong Wang, Ken H. Young, Wenwei Hu, Zhaohui Feng. E3 ubiquitin ligase TRIM32 negatively regulates tumor suppressor p53 to promote tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1229. doi:10.1158/1538-7445.AM2015-1229

The tumor suppressor gene p53 is frequently mutated in cigarette smoke (CS)-related lung cancer. The p53 binding pattern of carcinogenic polycyclic aromatic hydrocarbons (PAHs) found in CS coincides with the p53 mutational pattern found in lung cancer, and PAHs have thus been considered to be major culprits for lung cancer. However, compared with other carcinogenic compounds, such as aldehydes, the amount of PAHs in CS is minute. Acrolein (Acr) is abundant in CS, and it can directly adduct DNA. Acr–DNA adducts, similar to PAH–DNA adducts, induce predominantly G-to-T transversions in human cells. These findings raise the question of whether Acr–DNA adducts are responsible for p53 mutations in CS-related lung cancer. To determine the role of Acr–DNA adducts in p53 mutagenesis in CS-related lung cancer we mapped the distribution of Acr–DNA adducts at the sequence level in the p53 gene of lung cells using the UvrABC incision method in combination with ligation-mediated PCR. We found that the Acr–DNA binding pattern is similar to the p53 mutational pattern in human lung cancer. Acr preferentially binds at CpG sites, and this enhancement of binding is due to cytosine methylation at these sequences. Furthermore, we found that Acr can greatly reduce the DNA repair capacity for damage induced by benzo[ a ]pyrene diol epoxide. Together these results suggest that Acr is a major etiological agent for CS-related lung cancer and that it contributes to lung carcinogenesis through two detrimental effects: DNA damage and inhibition of DNA repair.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental carcinogens. PAHs are classified into bay and fjord region compounds according to structural differences in the molecule region where enzymatic epoxidation occurs. Dibenzo[a,l]pyrene (DB[a,l]P), one of the fjord region compounds, has been demonstrated to be the most carcinogenic PAH known to date. DB[a,l]P is activated to fjord region (+)-syn and (-)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DB[a,l]PDE) metabolites. In this study, we analyzed mutagenesis induced by (+)-syn- and (-)-anti-DB[a,l]PDE at the cII transgene in Big-Blue mouse cells. The mutant frequency of untreated cells (background level) was 6.53 x 10(-5). This level increased 3.7-fold for 20 nmol/L, 5.3-fold for 50 nmol/L, and 7.9-fold for 100 nmol/L (+)-syn-DB[a,l]PDE, respectively. In the case of (-)-anti-DB[a,l]PDE it increased 4.5-fold for 20 nmol/L, 6.7-fold for 50 nmol/L, and 10.6-fold for 100 nmol/L, respectively, indicating that (-)-anti-DB[a,l]PDE is slightly more mutagenic than (+)-syn-DB[a,l]PDE. The mutational spectra of (+)-syn- and (-)-anti-DB[a,l]PDE were quite similar except for several hotspots, specific for either (+)-syn-DB[a,l]PDE or (-)-anti-DB[a,l]PDE. The most frequently induced mutations were A to T transversions, which were 43.9% for (+)-syn- and 38.8% for (-)-anti-DB[a,l]PDE. In addition, G to T transversions were induced significantly, at frequencies of 18.5% by (+)-syn- and 18.1% by (-)-anti-DB[a,l]PDE. Using UvrABC cleavage and ligation-mediated PCR or the terminal transferase-dependent PCR method, we have determined DB[a,l]PDE-DNA adduct formation sites and repair rates in carcinogen-exposed cells. The mutation hotspots coincided with sites of strong adduct formation, but not all of the adduct hotspots were mutational hotspots. Slow adduct removal occurred for both (+)-syn- and (-)-anti-DB[a,l]PDE adducts over a time period of up to 72 hours. The data suggest that, although the (-)-anti-isomer is slightly more mutagenic, DNA adducts of both DB[a,l]PDE stereoisomers may have similar biological properties. We discuss the implications of these findings for human cancer mutagenesis.

Polycyclic aromatic hydrocarbons (PAHs) and their derivatives, such as benzo[a]pyrene (B[a]P), (+/-)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE), and 5-methylchrysene-1,2-diol-3,4-epoxide (5-MCDE), are complete carcinogens. However, the tumor promotion effects of PAHs remain unclear. We therefore investigated the possible activation of activator protein-1 (AP-1) and nuclear factor-kappaB (NFkappaB) in mouse epidermal Cl41 cells after different PAHs treatments, including B[a]P, B[a]PDE, chrysene-1,2-diol-3,4-epoxid (CDE), and 5-MCDE. The results showed that B[a]PDE and 5-MCDE were able to activate AP-1 and NF-kappaB, whereas B[a]P showed only marginal effect on AP-1 activation, and B[a]P and CDE had no effect on NF-kappaB activation. Treatment with either B[a]PDE or 5-MCDE also resulted in mitogen-activated protein kinases (MAPKs) activation as well as inhibitory subunit kappa-B (IkappaBalpha) phosphorylation and degradation, whereas B[a]P and CDE had no effect. Pretreatment with PD98059, a specific inhibitor for extracellular signal-regulated protein kinases (ERKs) upstream kinase MEK1/2, or SB202190, a p38 kinase inhibitor, resulted in a dramatic inhibition of B[a]PDE-induced AP-1 transactivation. In addition, B[a]PDE-induced AP-1 activation was also inhibited by overexpressing a dominant negative mutant of JNK1 in the cells. All these suggest ERKs, c-jun N-terminal kinases (JNKs), and p38 kinase signal transduction pathways are required for AP-1 induction by B[a]PDE. Taken together, B[a]PDE and 5-MCDE are the active compounds of PAHs to initiate signaling pathways. Considering the important roles of AP-1 and NF-kappaB in tumor promotion, we speculated the activation of AP-1 and NF-kappaB by B[a]PDE and 5-MCDE may involve in their or their parent compounds' tumor promotion effects. This study may help in better understanding the tumor promotion effects of PAHs.

Nickel (II), a ubiquitous environmental and industrial contaminant, is a well-known human carcinogen, particularly in human lung cancer. Although by itself it is a weak mutagen, nickel (II) is able to significantly enhance the genotoxicity of other mutagens and carcinogens, such as polycyclic aromatic hydrocarbons (PAHs) and ultraviolet light. Certain human populations, especially cigarette smokers, are frequently exposed to both nickel (II) and PAHs. To understand the interplay of nickel (II) and PAHs in mutagenesis and human carcinogenesis, we used a shuttle vector mutagenicity assay to examine the effect of nickel (II) on (+/-) anti-7beta, 8alpha-dihydroxy-9alpha, 10alpha-epoxy-7,8,9,10-tetrahydroxybenzo[a]pyrene (BPDE)-induced mutagenesis in human cells. BPDE is an activated metabolite of benzo[a]pyrene (BP), a major carcinogen in cigarette smoke. The shuttle vector pSP189 modified with BPDE was transfected into human cells with and without nickel (II) exposure. We found that nickel (II) exposure significantly enhanced BPDE-induced mutation frequency, but did not change BPDE-induced mutational spectrum in the supF gene of pSP189 plasmids replicated in nucleotide excision repair (NER)-proficient human cells. However, the enhancing effect of nickel (II) on BPDE-induced mutation frequency was not observed in NER-deficient human XPA cells. We also found that nickel (II) exposure of human cells did not change the spontaneous mutation frequency of the supF gene in NER-proficient or NER-deficient human cells, indicating that nickel (II) did not affect the replication fidelity in human cells. Using a plasmid containing a luciferase reporter gene and a host cell reactivation assay, we have found that nickel (II) exposure greatly inhibited the repair of BPDE-DNA adducts in NER-proficient but not in NER-deficient cells. Together these results strongly suggest that nickel (II) can greatly enhance the mutagenicity and genotoxicity of PAHs by inhibiting the NER pathway in human cells, and this may constitute an important mechanism for nickel (II)-induced human carcinogenesis.

In the ras gene superfamily, codon 12 (-TGGTG-) of the K-ras gene is the most frequently mutated codon in human cancers. Recently, we have found that bulky chemical carcinogens preferentially form DNA adducts at codons 12 and 14 (-CGTAG-) in the K-ras gene in normal human bronchial epithelial (NHBE) cells. Furthermore, DNA adducts formed at codon 12 of the K-ras gene are poorly repaired compared with those at other codons including codon 14. These results suggest that targeted carcinogen-DNA adduct formation is a major reason for the observed high mutation frequency at codon 12 of the K-ras gene in human cancers. This preferential carcinogen-DNA adduct formation at codons 12 and 14 could result from effects of (1) primary sequences of these codons and their surrounding codons in the K-ras gene, (2) the chromatin structure, and/or (3) epigenetic factors such as C5 cytosine methylation or other DNA modifications at these codons and their surrounding codons. To distinguish these possibilities, we have introduced modifications with benzo[a]pyrene diol epoxide, N-hydroxy-2-aminofluorene, and aflatoxin B1 8,9-epoxide in (1) naked intact genomic DNA isolated from NHBE cells, (2) fragmented genomic DNA digested by restriction enzymes, and (3) in vitro synthesized DNA fragments containing the K-ras gene exon 1 sequence with or without methylation of the cytosines at CpG sites and the cytosines pairing with the guanines of codons 12 and 14. The distribution of carcinogen-DNA adducts in the K-ras gene was mapped at the nucleotide sequence level using the UvrABC nuclease incision method with or without the ligation-mediated polymerase chain reaction technique. We have found that carcinogens preferentially form adducts at codons 12 and 14 in the K-ras gene exon 1 in intact as well as in fragmented genomic DNA. In contrast, this preferential DNA adduct formation at codons 12 and 14 was not observed in PCR-amplified DNA fragments containing the K-ras gene exon 1 sequence. Methylation of the cytosine at the CpG site of codon 14, or the cytosine pairing with guanine of codon 14, greatly enhanced carcinogen-DNA adduct formation at codon 14 but did not affect carcinogen-DNA adduct formation at codon 12. Methylation of the cytosine pairing with the guanine of codon 12 also did not enhance carcinogen-DNA adduct formation at codon 12. Furthermore, we found that the cytosine at the CpG site of codon 14 is highly methylated in NHBE cells. These results suggest that cytosine methylation at the CpG site is the major reason for the preferential DNA damage at codon 14 and that epigenetic modification(s) other than cytosine methylation may contribute to the preferential DNA damage at codon 12 of the K-ras gene.

Striking regional differences in the prevalence of coal workers' pneumoconiosis (CWP) have been observed but not fully understood. This study investigated the early biological responses of primary lung cells to treatment with coal dusts from various seams. High-density oligoarray technology (GeneChip, Affymetrix, Santa Clara, CA) was used to compile gene expression profiles of primary human bronchial epithelial cells to low concentrations (2 microg/cm(2)) of coals for 6 h or 24 h of treatment. Data showed that a total of 1050 out of 12,000 genes on the chip were altered by 2 coal dusts. The coal from the Pennsylvania (PA) coal-mine region with a high prevalence of CWP altered 908 genes, many more than the coal from Utah (UT) with a low prevalence of CWP, which affected 356 genes. Many genes decreased their expression levels in response to the PA coal at 6 h and/or 24 h of treatment. For example, transferrin receptor, a gene known to control cellular iron uptake, was downregulated in the cells treated with the iron-containing PA coal in order to protect cells from iron overload. The UT coal without bioavailable iron had no such effect. The downregulation patterns of genes were confirmed by reverse-transcription polymerase chain reaction (RT-PCR). This study is one of the first in profiling gene expressions of primary bronchial epithelial cells treated with coals from various seams, which may set stages for future studies on specific genes.

Trans-4-hydroxy-2-nonenal (4-HNE), a major electrophilic by-product of lipid peroxidation, is able to interact with DNA to form exocyclic guanine adducts. 4-HNE is a mutagen and a significant amount of 4-HNE-guanine adduct has been detected in normal cells. Recently, it has been reported that exposure of the wild-type p53 human lymphoblastoid cell line to 4-HNE causes a high frequency of G to T transversion mutations at the third base of codon 249 (-AGG*-) in the p53 gene, a mutational hotspot in human cancers, particularly hepatocellular carcinoma. These findings raise a possibility that 4-HNE could be an important etiological agent for human cancers that have a mutation at codon 249 of the p53 gene. However, to date, the sequence specificity of 4-HNE-DNA binding remains unclear due to the lack of methodology. To address this question, we have developed a method, using UvrABC nuclease, a nucleotide excision repair enzyme complex isolated from Escherichia coli, to map the distribution of 4-HNE-DNA adducts in human p53 gene at the nucleotide sequence level. We found that 4-HNE-DNA adducts are preferentially formed at the third base of codon 249 in the p53 gene. The preferential binding of 4-HNE was also observed at codon 174, which has the same sequence and the same nearest neighbor sequences (-GAGG*C-) as codon 249. These results suggest that 4-HNE may be an important etiological agent for human cancers that have a mutation at codon 249 of the p53 gene.

4-aminobiphenyl (4-ABP) is a major etiological agent of human bladder cancer, and its metabolites are able to form DNA adducts that may induce mutation and initiate bladder carcinogenesis. Thirty to sixty percent of human bladder cancer has a mutation in the p53 gene, and the mutational spectrum bears two characteristics: compared with other cancers, the pattern of mutations is more evenly distributed along the p53 gene, and the mutational hotspots occur at both CpG sites, such as codons 175, 248 and 273, and non-CpG sites, such as codons 280 and 285, the latter two being unique mutational hotspots for bladder and other urinary tract cancers. These findings raise the possibility that the special p53 mutational features in human bladder cancer are due to the unique binding spectrum of metabolically activated 4-ABP in bladder cells. To address this question, here we have mapped the 4-ABP-DNA adduct distribution in the p53 gene at the nucleotide sequence level in human bladder cells. We found that, unlike benzo[a]pyrene trans-7,8-dihydrodiol-9,10-epoxide-DNA adduction, which preferentially occurs at CpG sites, 4-ABP-DNA adduction is not biased for CpG sites, and the adducts are more evenly distributed along the p53 gene; nonetheless, the p53 mutational hotspots in bladder cancer at codons 175, 248, 280 and 285 are also the preferential sites for 4-ABP adduct formation. These results strongly suggest that the unique binding spectrum of 4-ABP contributes greatly to the unique mutational spectrum in the p53 gene of human bladder cancer, and provide further molecular evidence to directly link 4-ABP to bladder cancer.

Obesity is the result of an imbalance between energy intake and energy expenditure. Using high-density DNA microarrays and Northern analyses, we demonstrated that the activation of a nutrient-sensing pathway, the hexosamine biosynthesis pathway (HBP), rapidly decreased the expression of a cluster of nuclear-encoded mitochondrial genes involved in skeletal muscle oxidative phosphorylation. Conversely, the expression of uncoupling protein-1 and of the same mitochondrial genes was increased in brown adipose tissue. Most important, these transcriptional changes were accompanied by a marked decrease in whole-body energy expenditure. Short-term overfeeding replicated this transcriptional pattern, suggesting that this adaptation to nutrient abundance occurs under physiological conditions. Thus, the activation of the HBP by nutrients represents a biochemical link between nutrient availability, mitochondrial proteins, and energy expenditure, and it is likely to play an important role in the regulation of energy balance.

Genomic injury induced by environmental carcinogens, such as polycyclic aromatic hydrocarbons and aromatic amines, is the initial step that can trigger mutagenesis and carcinogenesis. In addition to the physico-chemical property of DNA damaging agents, several important factors such as primary sequence, chromatin structure, methylation, protein association, and transcriptional activity can affect not only the initial level and distribution of DNA damage but also the efficiency of repair. Therefore, mapping the DNA damage induced by environmental agents in cancer-related genes such as p53 and ras at the sequence level provides essential information for assessing their carcinogenic potential. Recently, using the E. coli nucleotide excision enzyme complex, UvrABC nucleases in combination with ligation-mediated polymerase chain reaction, we developed a method to map DNA damage in the p53 and ras genes. These studies led us to conclude that targeted DNA damage, in combination with growth selection, contributes greatly in shaping the mutation spectrum in these genes in human cancer. Here we present the rationale and details of this approach, typical experimental results and necessary precautions.

DNA damage is preferentially repaired in the transcribed strand of many active genes. Although the concept of DNA repair coupled with transcription has been widely accepted, its mechanisms remain elusive. We recently reported that in Chinese hamster ovary cells while ultraviolet light-induced cyclobutane pyrimidine dimers (CPDs) are preferentially repaired in the transcribed strand of dihydrofolate reductase gene, CPDs are efficiently repaired in both strands of adenine phosphoribosyltransferase (APRT) locus, in either a transcribed or nontranscribed APRT gene (1). These results suggested that the transcription dependence of repair may depend on genomic context. To test this hypothesis, we constructed transfectant cell lines containing a single, actively transcribed APRT gene, integrated at different genomic sites. Mapping of CPD repair in the integrated APRT genes in three transfectant cell lines revealed two distinct repair patterns, either preferential repair of CPDs in the transcribed strand or very poor repair in both strands. Similar kinetics of micrococcal nuclease digestion were seen for all three transfectant APRT gene domains and endogenous APRT locus. Our results suggest that both the efficiency and strand-specificity of repair of an actively transcribed gene are profoundly affected by genomic context but do not reflect changes in first order nucleosomal structure.

To ensure proper function, the tumor suppressor p53 is tightly regulated through different post-translational modifications, particularly ubiquitination. Recently, TRIM32 was identified as a p53-regulated gene and an E3 ubiquitin ligase of p53. Thus, TRIM32 and p53 form a novel auto-regulatory negative feedback loop for p53 regulation in cells.

Tumor suppressor p53 plays a crucial role in preventing tumor formation. p53 responds to stress signals and transcriptionally regulates its target genes to start various cellular responses, thus preventing tumor initiation and/or progression. p53 is the most frequently-mutated gene in tumors; approximately 50% of human tumors harbor inactivating mutations in p53. The p53 protein is under tight regulation in cells. Loss or the attenuation of p53 function affects cancer risk and tumor progression. Epidemiological studies strongly suggest that chronic psychological stress has negative influences on the onset, progression and mortality of cancers. Studies employing chronic restraint or social isolation in mice, two well-established chronic stress models, have shown that chronic stress enhances tumor growth and metastasis in xenograft tumor assays. However, the direct evidence that chronic stress promotes tumorigenesis in vivo is still lacking, and furthermore, the molecular mechanisms by which chronic stress promotes tumorigenesis remain unclear. To investigate the impact of chronic stress upon tumorigenesis in vivo, we established a mouse model that combines chronic restraint and ionizing radiation (IR)-induced tumorigenesis. IR induces tumorigenesis in both mice and humans. p53+/- mice are susceptible to IR-induced tumorigenesis; a single dose of 4 Gy IR results in the development of tumors, mainly lymphomas, in p53+/- mice. By employing this mouse model system, we found that in p53+/- C57BL6/J mice, chronic restraint stress greatly promoted IR-induced tumorigenesis; chronic restraint reduced IR-induced tumor latency from ∼49 weeks to ∼38 weeks of median survival age (p=0.004). This result provides direct evidence that chronic stress enhances tumorigenesis in vivo. Furthermore, chronic restraint decreased p53 protein levels and function in mice, and promoted the growth of human xenograft tumors in a largely p53-dependent manner. Chronic restraint greatly promotes the growth rate of HCT116 p53+/+ tumors (increased by 3.77-fold) in mice; and has significantly less pronounced promoting effect on the growth of HCT116 p53-/- tumors (increased by 1.52-fold) (p Citation Format: Cen Zhang, Meihua Lin, Yuhan Zhao, Tongsen Zheng, Jiabei Wang, Arnold Levine, Zhaohui Feng, Wenwei Hu. Chronic stress promotes tumorigenesis through attenuation of p53 function . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 784. doi:10.1158/1538-7445.AM2013-784

Tumor suppressor p53 is frequently mutated in tumors. Mutant p53 (Mutp53) proteins often gain new activities in promoting tumorigenesis, defined as gain-of-function (GOF). Mutp53 often accumulates at high levels in tumors, which promotes mutp53 GOF in tumorigenesis. The mechanism of mutp53 accumulation is poorly understood. Here we find that MDM2 isoforms promote mutp53 accumulation in tumors. MDM2 isoform B (MDM2-B), the MDM2 isoform most frequently over-expressed in human tumors, interacts with full-length MDM2 to inhibit MDM2-mediated mutp53 degradation, promoting mutp53 accumulation and GOF in tumorigenesis. Furthermore, MDM2-B over-expression correlates with mutp53 accumulation in human tumors. In mutp53 knock-in mice, a MDM2 isoform similar to human MDM2-B is over-expressed in the majority of tumors, which promotes mutp53 accumulation and tumorigenesis. Thus, over-expression of MDM2 isoforms promotes mutp53 accumulation in tumors, contributing to mutp53 GOF in tumorigenesis. Furthermore, promoting mutp53 accumulation and GOF is an important mechanism by which MDM2 isoforms promote tumorigenesis.

Abstract 93 CML is driven by the oncogenic fusion kinase BCR-ABL which is capable of corrupting and transforming hematopoietic stem cells (HSC) and thus inititating myeloproliferative disease (MPD). While imatinib (IM) has become an effective treatment for CML, the leukemia stem cells (LSC) which reside in the bone marrow (BM) remain largely unaffected by imatinib treatment. As LSC rely on their ability to self-renew, several genes/pathways responsible for self-renewal properties have been investigated as potential targets of LSCs in myeloid neoplasia. Recent studies clearly indicate that canonical Wnt/β-catenin-signaling plays a pivotal role in initiation and development of BCR-ABL induced MPD (Zhao, Cancer Cell 2009; Hu, Leukemia 2009). However, the impact of β-catenin (Ctnnb1) on the maintenance of LSC in established disease, which more accurately models the clinical scenario, has not been extensively studied. To address the impact of targeting Ctnnb1 in CML-LSC, we used a conditional Ctnnb1 knockout mouse with an estrogen-receptor driven Cre-recombinase as BM-donors. CML was induced by retroviral transduction of either Ctnnb1flox/flox ER-Cre+, Ctnnb1flox/wt ER-Cre+, or Ctnnb1wt/wt ER-Cre+ murine bone marrow with a virus encoding BCR-ABL (MSCV-BCR-ABLp210-GFP), and injection of transduced cells into wildtype syngeneic recipient mice. Administration of tamoxifen to recipient mice with CML led to excision of Ctnnb1 in the Ctnnb1flox/flox homozygous or Ctnnb1flox/wt heterozygous cells. Interestingly genetic inactivation of Ctnnb1 after onset of CML in primary recipient mice resulted only in minor prolongation of survival. The animals died due to organ infiltration (primarily lung) by differentiated progenitor cells that were presumably not affected by Ctnnb1 deletion. However, in the BM and peripheral blood, a clear reduction of GFP+ cells was evident in heterozygous - and even more pronounced - in homozygous animals, indicating a potential effect on more immature cells and perhaps LSC. To determine if LSC were compromised and to investigate the efficacy of a combination treatment with IM, we used a serial BM-transplantation assay. Consistent with previously published data, we found that leukemia cells in the Sca-1/GFP+ fraction are able to re-establish CML in secondary recipient mice and thus contain LSC. IM treatment alone led to a relative increase of Sca-1/GFP+ LSC over time. However, we found a significant reduction of Sca-1/GFP+ bone marrow cells after inactivation of Ctnnb1 during IM-treatment. Mice transplanted with Ctnnb1flox/flox ER-Cre+ CML cells had on average 0.025% Sca-1/GFP+ (total of 7,375 cells/mouse) cells in the bone marrow after treatment of the recipient mice with IM and tamoxifen, mice that received Ctnnb1flox/wt ER-Cre+ CML had 0.036% Sca-1/GFP+ cells (total of 10,584 cells/mouse) and mice that received Ctnnb1wt/wt ER-Cre+ CML had 0.075% Sca-1/GFP+ cells (total of 24,375 cells/mouse). As indomethacin has recently been shown to inhibit Ctnnb1 in hematopoietic and AML stem cells (Goessling, Cell 2009; Wang, Science 2010), we aimed to investigate the efficacy of indomethacin combined with IM. We were able to demonstrate a decrease in Ctnnb1 protein after indomethacin treatment of human cell lines and transformed murine BM cells by either immunoblot, flow-cytometry or immunofluorescence. Moreover, we were able to show synergy between indomethacin and IM co-treatment which produced almost complete abrogation of Ctnnb1 protein. IM/Indomethacin co-treatment of mice with CML decreased the Kit+Sca1+ fraction of the Lin-GFP+ bone marrow population to 0.92% (1799 cells/mouse) whereas treatment with IM alone was similar to untreated mice with 2.8% of the Lin-GFP+ population being Kit+Sca1+ (22,601 cells/mouse). Importantly, transplantation of the bone marrow from treated mice into tertiary recipients showed significantly prolonged survival of tertiary recipient mice that received bone marrow from donors treated with the combination therapy (IM/INDO) compared to IM/DMSO controls (p=0.0038*). In summary, we demonstrate that inhibiting Ctnnb1 by genetic inactivation or drug treatment is an effective combination therapy with imatinib mesylate and targets CML leukemia stem cells. This provides strong evidence for the development and clinical use of β-catenin-targeted therapies in CML. Disclosures: No relevant conflicts of interest to declare.

The t(4:11) is the most common chromosomal rearrangement associated with acute lymphoblastic leukemia (ALL) in infants and predicts a poor outcome. We created a conditional knock-in mouse model that expresses the Mll-AF4 fusion protein generated by the t(4;11). The mice developed acute leukemia consisting of bone marrow replacement, splenomegaly and variable lymphadenopathy with a latency from 3–6 months depending upon the method of cre recombinase expression. Immunophenotypic analysis demonstrated the development of acute lymphoblastic leukemia (mALL), acute myeloid leukemia (mAML) or occasionally mixed lineage (biphenotypic) leukemia (mMLL). Further immunophenotypic analysis of mALL demonstrated the majority of the cells to be B220(+) CD43(low/−) IgM(−) IgD(−), pre-B cells. Unsupervised gene expression analysis confirmed that mALL cells are more similar to pre-B cells than to pre-pro-B, immature B or mature B-cells. Supervised gene expression analysis demonstrated that mALL cells had increased expression of genes including HoxA3/4/5/7/9/10, Meis1, and Myc, similar to previously described human ALL profiles. Furthermore gene set enrichment analysis demonstrated significant overlap between the murine and human MLL-AF4 leukemia gene expression profiles. As it has been previously shown that MLL-AF10 recruits the K79 methyltransferase DOT1L to HOXA cluster genes, we assessed whether Mll-AF4 mediates gene expression through similar chromatin modifications. We assayed H3K4; H3K27; and H3K79 methylation levels on HoxA cluster genes in mALL and pre-B cells. While H3K4 and H3K27 levels were not significantly different between pre-B and mALL cells, H3K79 was increased on HoxA3 through HoxA10 but not HoxA1/2/11/13. Assessment of genome wide distribution of H3K79 mark demonstrated a dramatic increase in H3K79 methylation on HoxA loci, and hundreds of other loci throughout the mouse genome. To determine whether findings in our mouse model have relevance for human ALL we assessed genome wide distribution of the H3K79 modification in MLL-AF4 rearranged ALL, non MLL-rearranged ALL, and human bone marrow CD34(+) CD19(+) pre-B cells. We found approximately 5000 genes in MLL-AF4 ALL cells possessed enhanced levels of the H3K79 modification as compared to normal human CD34(+) CD19(+) cells whereas MLL-germline ALLs did not possess a global enhancement. Moreover, MLL-AF4 ALL samples possessed the H3K79 modification on the promoters of over-expressed genes that distinguish MLL-rearranged ALL from other ALL. Finally, shRNA mediated knockdown of the K79 methyltransferase DOT1 suppressed growth of MLL-rearranged but not non MLL-rearranged ALL cell lines. These data demonstrate that MLL-AF4 induces genome wide abnormalities in H3K79 chromatin modification, which is likely a critical component of its leukemogenic mechanism. Targeting DOT1 with specific inhibitors may be therapeutically useful for the most common MLL-rearranged leukemias.

Recent experiments have demonstrated that MLL-translocation associated fusion proteins can transform either hematopoietic stem cells (HSC) or granulocyte macrophage progenitors (GMP) into leukemia stem cells. However, it may be that leukemogenic process differs when HSC are the cell of origin as compared to myeloid progenitors. We transduced either HSC or GMP with a retrovirus expressing MLL-AF9 and GFP followed by single cell sorting of transduced cells. Approximately 80% of singly sorted MLL-AF9 transduced GMP (MLL-AF9-GMP) and about 25% of MLL-AF9 transduced HSC (MLL-AF9-HSC) could be serially re-plated over 9 passages. Upon transplantation into syngeneic mice, 83% (n=12) of MLL-AF9-HSC single cell derived clones induced AML with a median latency 70 days. Approximately 30% (n=20) of MLL-AF9-GMP single cell derived clones induced AML, with median latency 112 days. When MLL-AF9-GMP single cell derived clones were co-infected with an empty retrovirus (to provide additional oncogenic events as a result of retroviral integration) before transplantation into recipient mice, 93% of the transplanted mice (n=15) developed AML with mean latency 65 days, similar to leukemia initiated from HSC. This suggests that either single GMP or HSC can be transformed into leukemia initiating cells. However, extra mutations appear to be required to induce leukemia from committed progenitors. Consistent with this hypothesis, southern blot analysis performed on leukemias initiated from 5,000 and 15,000 MLL-AF9 transduced HSC or GMP demonstrated polyclonal AML arising from HSC compared to oligoclonal AML arising from GMP. Next, we used bioluminescent imaging to follow disease kinetics. When 15,000 MLL-AF9 transduced HSC were injected into recipient mice, the disease accumulated in a linear fashion over 42 days. However, when 15,000 MLL-AF9 transduced GMP were injected the disease developed more slowly over 75 days. Immunophenotypic analysis of the resultant leukemias demonstrated that the HSC-derived and GMP-derived leukemias were quite similar, with a GMP-like population containing LSC in both cases. Globally, the two cell types were also very similar with their gene expression profile being more similar to GMP than any other progenitor or stem cell population. However, we found that in addition to the previously reported 363-gene “self-renewal associated signature” LSC derived from HSC also possessed high-level expression of genes such as Flt3, Mcl1, and Notch-1. Preliminary analysis also suggests that gene expression differences between HSC and GMP-derived leukemia stem cells may have prognostic significance in human AML. These data suggest that AML derived from different cells of origin, while globally quite similar, require a different number of genetic events, and have gene expression differences that may influence drug response.

Leukemias are composed of a hierarchy of cells only a fraction of which have stem cell like properties, and are capable of self-renewal. MLL fusion proteins produced by translocations involving the Mixed Lineage Leukemia ( MLL ) gene on chromosome 11q23 confer stem cell-like properties on committed hematopoietic progenitors. This provides an opportunity to determine if global cellular reprogramming is necessary for leukemia stem cell (LSC) generation from committed progenitors or if induction of a more limited self-renewal signature in committed progenitors is sufficient. We transduced murine IL-7R − Lin − Sca-1 − c-Kit + CD34 + FcγRII/III hi granulocyte macrophage progenitors (GMPs) with retroviruses encoding the MLL-AF9 fusion protein, which led to the development of acute myelogenous leukemia. From the leukemias we isolated a population of IL-7R − Lin − Sca-1 − c-Kit + CD34 int. FcγRII/III int. LSCs which can transplant the disease when fewer than 20 cells are injected into secondary recipients. We used hierarchical clustering, K-means clustering and principal component analysis to compare gene expression profiles of the LSC population to the normal lin − sca-1 + c-kit + HSC-enriched population, IL-7R − Lin − Sca-1 − c-Kit + CD34 + FcγRII/III lo common myeloid progenitors (CMPs), IL-7R − Lin − Sca-1 − c-Kit + CD34 − FcγRII/III − megakaryocyte erythroid progenitors (MEPs) and GMPs and found that the global gene expression profile most resembles the normal GMP from which they arose. However, a leukemia self-renewal signature was identified that shows significant overlap with a group of genes normally highly expressed in HSCs whose expression decreases during the transition to normal committed progenitors. Supervised analysis and gene set enrichment analysis (GSEA) demonstrated approximately 300 genes in the leukemia self-renewal signature. This is only a subset of the approximately 1500 genes that are highly expressed in the normal HSC-enriched population that show decreased expression in CMPs, MEPs, and GMPs. Next, we determined if this 300-gene leukemia stem cell signature is directly regulated by MLL-AF9 or if there is a hierarchy of gene expression. Assessment of gene expression changes 48 hours after MLL-AF9 expression in isolated GMPs demonstrated increased expression of 23/300 genes in the leukemia self-renewal signature. Of interest, there is a high degree of similarity between the 23 MLL immediate response genes and human MLL -rearranged AMLs including HOXA 5, HOXA7 , HOXA9 , HOXA10 , MEIS1 and genes not previously known to have a role in MLL -mediated leukemogenesis such as myocyte enhancer factor 2C ( MEF2C ). Detailed loss-of-function studies using shRNA and dominant negative mutants show inhibition of MEF2C reduces LSC colony formation and serial replating in semi-solid culture to less than 20% of control. Furthermore shRNA mediated inhibition of MEF2C has a significant impact on proliferation of human MLL-AF9 dependent leukemia cell lines, but not cell lines from other subtypes of AML. These data demonstrate LSCs can be generated from committed progenitors without widespread reprogramming of gene expression, and a leukemia self-renewal signature is activated in the process. We have used this program to identify MEF2C as playing a role in MLL-AF9 induced AML. Identification of this program provides an opportunity to further assess its importance in normal tissue homeostasis and neoplastic self-renewal/proliferation, and defines the progression from normal hematopoietic progenitor to leukemia stem cell.

Human leukemias harboring chromosomal translocations involving the Mixed lineage leukemia (MLL, HRX, ALL-1) gene possess high-level expression, and frequent activating mutations of the receptor tyrosine kinase FLT3. We used a murine bone marrow transplant model to assess cooperation between MLL translocation and FLT3 activation, and demonstrate that MLL-AF9 expression induces leukemia in approximately 70 days whereas the combination of MLL-AF9 and FLT3-ITD does so in less than 30 days. In both cases, the resulting disease is a highly aggressive, clonal, acute myelogenous leukemia. Detailed immunophenotypic analysis demonstrates expansion of an IL-7Rα− Lin− Sca-1− c-Kit+ CD34int. granulocyte macrophage progenitor (GMP)-like population in both the MLL-AF9 and MLL-AF9/FLT3-ITD induced leukemias. Other progenitor populations such as IL-7Rα− Lin− Sca-1− c-Kit+ CD34+ FcγRII/IIIlo common myeloid progenitors (CMPs) and IL-7Rα− Lin− Sca-1− c-Kit+ CD34− FcγRII/III− megakaryocyte erythroid progenitors (MEPs) are absent in the bone marrow from leukemic mice. Secondary transplantation of splenic cells from diseased mice established that leukemic stem cells are present at a very high frequency of approximately 1:100 in both diseases. Cooperation between MLL-AF9 and FLT3-ITD was further verified by real-time assessment of leukemogenesis using non-invasive bioluminescence imaging. We developed a transgenic mouse model that expresses luciferase under control of the ubiquitin promoter to provide luminescent bone marrow cells that can be used as donor cells in bone marrow transplant assays. In these experiments, the development of leukemia was followed in secondary recipients via bioluminescent imaging. We used this model to confirm cooperation of MLL-AF9 and FLT3-ITD, and to demonstrate that MLL-AF9/FLT3-ITD induced leukemias are sensitive to FLT3 inhibition in a 2–3 week in vivo assay. These data (1) show that MLL-AF9 induces acute myelogenous leukemia with a high frequency of leukemic stem cells and expansion of a GMP-like progenitor population, (2) demonstrate that activated FLT3 cooperates with MLL-AF9 to accelerate leukemogenesis, and (3) provide a new genetically-defined model system that should prove useful for rapid assessment of potential therapeutics in vivo.