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Cancer cells show increases in protein degradation pathways, including autophagy, during progression to meet the increased protein degradation demand and support cell survival. On the other hand, reduced autophagy activity during aging is associated with a reduced DNA damage response and increased genomic instability. Therefore, it is a puzzling how DNA repair can be increased in cancer cells that are resistant to chemotherapies or during progression when autophagy activity is intact or increased. We discovered that tripartite motif containing 44 (TRIM44) is a pivotal element regulating the DNA damage response in cancer cells with intact autophagy. TRIM44 deubiquitinates p62, an autophagy substrate, which leads to its oligomerization. This prevents p62 localization to the nucleus upon irradiation. Increased cytoplasmic retention of p62 by TRIM44 prevents the degradation of FLNA and 53BP1, which increases DNA damage repair. Together, our data support TRIM44 a potential therapeutic target for therapy-resistant tumor cells with intact autophagy.

A defective homologous recombination (HR) repair program increases tumor incidence as well as providing a survival advantage in patients with breast and ovarian cancers. Here, we hypothesize that the tumor-promoting side of genome maintenance programs may be contributed by a self-renewal protein, nucleostemin (NS). To address this issue, we established its functional importance in mammary tumor progression in mice and showed that mammary tumor cells become highly susceptible to replicative DNA damage following NS depletion and are protected from hydroxyurea-induced damage by NS overexpression. Breast cancer cells with basal-like characters display more reliance on NS for genome maintenance than those with luminal characters. Mechanistically, NS-deficient cells demonstrate a significantly reduced HR repair activity. TCGA analyses of human breast cancers revealed that NS is co-enriched positively with HR repair proteins and that high NS expression correlates with low HR defects and predicts poor progression-free survival and resistance to knockdown of cell cycle checkpoint genes in triple-negative/basal-like breast cancers. This work indicates that NS constitutes a tumor-promoting genome maintenance program required for mammary tumor progression.

Despite progress in the treatment of MM, including the use of high-dose chemotherapy and autologous stem cell transplantation, a considerable proportion of patients are refractory to all therapies. This resistance is related to the molecular genetic heterogeneity in MM cells as well as to the contributions from the BM, which is one of the key determinants of treatment outcome. Our previous studies using fluorescent tracers revealed that MM heterogeneity is correlated with the presence of quiescent stem-like cancer cells, which prefer to reside within the osteoblastic niche of the BM. In this report, we identified a novel protein, tripartite motif containing 44 (TRIM44), which is overexpressed in the osteoblastic niche of the BM, enabling MM cells to compete with HSCs for niche support. TRIM44 expression in MM cells promoted cell quiescence but increased bone destruction in xenograft mice, similar to what is observed in MM patients. TRIM44 functions as a deubiquitinase for hypoxia inducible factor-1α (HIF-1α), which stabilizes HIF-1α expression during hypoxia and normoxia. Stabilized HIF-1α stimulates MM cell growth and survival during hypoxia. Our work is the first report to reveal signaling in quiescent MM cells and the functions of TRIM44.

Let-7a-3 transcribes the miRNA let-7a, of which the expression is dysregulated in cancer. We evaluated the significance of let-7a-3 gene methylation in patients with de novo acute myeloid leukemia (AML). Let-7a-3 was methylated in 81.1% (73/90), partially methylated in 12.2% (11/90), or unmethylated in 6.7% (6/90) of patients. Let-7a-3 methylation correlated with AML karyotyping and CCAAT/enhancer binding protein α ( CEBPA ) methylation. Kaplan–Meier survival analysis predicted that let-7a-3 hypermethylation correlated with better survival in AML with hypomethylated CEBPA or with hypomethylated CEBPA without the favorable karyotype. We conclude that let-7a-3 methylation is a positive prognosticator for AML patients with hypomethylated CEBPA .

Autophagy is a catabolic process that functions in recycling and degrading cellular proteins, and is also induced as an adaptive response to the increased metabolic demand upon nutrient starvation. However, the prosurvival role of autophagy in response to metabolic stress due to deprivation of glutamine, the most abundant nutrient for mammalian cells, is not well understood. Here, we demonstrated that when extracellular glutamine was withdrawn, autophagy provided cells with sub-mM concentrations of glutamine, which played a critical role in fostering cell metabolism. Moreover, we uncovered a previously unknown connection between metabolic responses to ATG5 deficiency and glutamine deprivation, and revealed that WT and atg5 (-/-) MEFs utilized both common and distinct metabolic pathways over time during glutamine deprivation. Although the early response of WT MEFs to glutamine deficiency was similar in many respects to the baseline metabolism of atg5 (-/-) MEFs, there was a concomitant decrease in the levels of essential amino acids and branched chain amino acid catabolites in WT MEFs after 6 h of glutamine withdrawal that distinguished them from the atg5 (-/-) MEFs. Metabolomic profiling, oxygen consumption and pathway focused quantitative RT-PCR analyses revealed that autophagy and glutamine utilization were reciprocally regulated to couple metabolic and transcriptional reprogramming. These findings provide key insights into the critical prosurvival role of autophagy in maintaining mitochondrial oxidative phosphorylation and cell growth during metabolic stress caused by glutamine deprivation.

Epigenetic inactivation of tumor-suppressor genes, often in association with aberrant DNA methylation of CpG islands in the promoter region of these genes, is a key factor in tumorigenesis. CCAAT/enhancer binding protein alpha (CEBPA) methylation is a favorable prognostic biomarker for acute myeloid leukemia; however, rather than the complete methylation observed in inherited disorders, CEBPA methylation is heterogeneous. In this study, we established an algorithm called the “methylation index,” deduced from high-resolution melting profiles, which includes Tm shifting (ΔTm) and Tm width ratio (fold of width), to evaluate the heterogeneous methylation status. The methylation index was highly correlated with the exact methylation levels detected by using the MassARRAY method (R2 = 0.80; P < 0.001). Within-run reproducibility for the methylation index was 0.9% as the coefficient of variation, and between-run reproducibility was 2.6%. It was determined that with a cutoff methylation index of 1.412, the best measures of sensitivity and specificity could be obtained (97.14% and 95.89%, respectively) to discern low or high CEBPA methylation status. This novel algorithm for calculation of the methylation index from high-resolution melting profiles for CEBPA methylation is compatible with measurement of the methylation level as assayed using MassARRAY and could be a simple and efficient screening method for determination of CEBPA methylation status in acute myeloid leukemia.

Hypermethylation of the distal CEBPA promoter region has been reported to result in the downregulation of CEBPA expression in several malignancies. However, the clinical implication of CEBPA hypermethylation in acute myeloid leukemia (AML) remains unclear. To investigate the correlation between CEBPA hypermethylation and clinical features in AML, quantitative MassARRAY analyses for CEBPA methylation status were performed on a cohort of 193 patients. High CEBPA methylation group (CEBPA(high-meth), n=28) and low methylation group (CEBPA(low-meth), n=165) were defined by using two-way hierarchical clustering. With a median follow-up of 48 months, among the 125 patients receiving standard induction therapy, CEBPA(high-meth) was associated with better treatment response (complete remission rate 93.3% versus 73.6%, P=0.116). In patients with normal karyotype and without CEBPA and NPM1 mutations, the CEBPA(high-meth) had longer overall survival (OS) than the CEBPA(low-meth) (P=0.028). Multivariate analysis further supported that the CEBPA methylation was an independent prognostic factor for disease free-survival (hazard ratio=0.416; 95% confidence interval, 0.223-0.777, P=0.006) and OS (hazard ratio=0.406; 95% confidence interval, 0.166-0.996, P=0.050). We conclude that CEBPA methylation status is a useful prognostic biomarker for AML patients.

Correction to: Leukemia (2007) 20, 1899–1903. doi:10.1038/sj.leu.2404331 Since the publication of the above paper, the authors have noticed an error in the sequence of primers used for TAD1, TAD2 and bZIP amplification in Table 1. The correct Table 1 is shown below: The authors apologize for any inconvenience caused.

The mammalian nucleolar proteins nucleostemin and GNL3-like (GNL3L) are encoded by paralogous genes that arose from an ancestral invertebrate gene, GNL3. Invertebrate GNL3 has been implicated in ribosome biosynthesis, as has its mammalian descendent, GNL3L. The paralogous mammalian nucleostemin protein has, instead, been implicated in cell renewal. Here, we found that depletion of nucleostemin in a human breast carcinoma cell line triggers prompt and significant DNA damage in S-phase cells without perturbing the initial step of ribosomal (r)RNA synthesis and only mildly affects the total ribosome production. By contrast, GNL3L depletion markedly impairs ribosome production without inducing appreciable DNA damage. These results indicate that, during vertebrate evolution, GNL3L retained the role of the ancestral gene in ribosome biosynthesis, whereas the paralogous nucleostemin acquired a novel genome-protective function. Our results provide a coherent explanation for what had seemed to be contradictory findings about the functions of the invertebrate versus vertebrate genes and are suggestive of how the nucleolus was fine-tuned for a role in genome protection and cell-cycle control as the vertebrates evolved.

Generally speaking, traffic headway comprises distance headway and time headway, which varies according to velocity and acceleration. Distance headway is a part of the traffic following model as an expression of the stimulus-response factor. A more responsive driver accelerates and decelerates more frequently in response to the vehicle in front, thereby reducing the capacity of the traffic lane. This study attempts to calculate the distance headway in both the Macro Traffic Model and the Micro Traffic Model, and thus determine a steady state for the distance headway. The research results show that the Macro Traffic Model reaches Steady State at 20-23 seconds and the Micro Traffic Model reaches Steady State at 19-23 seconds. This research can be applied to traffic planning, calculation of crossroad traffic saturation rates, or in traffic simulation applications.

A backward safety analysis model that can deal with dangerous status by virtue of fuzzy theory is proposed in this paper. Fuzzy Petri Nets (FPN) is developed and applied to the safety analysis for three types of level crossing surveillance systems of Taiwan Railway Administration. The numerical results of proposed FPN models are fairly plausible.

The transcription factor Twist protein has been found to be correlated with metastasis in various carcinomas, including hepatocellular, breast and prostate carcinomas. However, the role of Twist in head and neck squamous cell carcinomas (HNSCC) remains unknown. Head and neck cancer tissue microarrays (TMAs) of tumors from 50 patients with HNSCC were examined. Immunohistochemical (IHC) stain analysis showed that, out of the 50 patients, twenty (40%) showed Twist-positive staining in the tumor cells, and Twist expression was positively associated with differentiation status (p=0.027), lymph node metastasis (p=0.032) and disease progression (p=0.029). Further analysis revealed that the expression of Twist was positively correlated with CXCR4 (Spearman, r=0.408, p=0.003) and CCR7 (r=0.417, p=0.003). FindPatterns analysis suggested that the transcription factor Twist, as a basic helix-loop-helix (bHLH) protein, might regulate CXCR4 and CCR7 expression in squamous cell carcinomas, which in turn might be associated with lymph node metastasis.

Abstract 1569 Poster Board I-593 Acute myeloid leukemia (AML) is a clonal disorder characterized by acquired genetic abnormalities in hematopoietic progenitors. CCAAT/enhancer binding protein α (C/EBPα) is a basic leucine zipper transcription factor that regulates differentiation-dependent genes during granulocyte differentiation. CEBPA mutations have been observed in several human malignancies. Epigenetic modification of the distal CEBPA promoter region (-1422 to -896) has been reported to result in down-regulation of CEBPA expression in lung cancer, pancreatic cancer, and head and neck squamous cell carcinoma. Apart from solid tumors, epigenetic modification of CEBPA in AML has also been reported. Chim et al. reported aberrant CEBPA core promoter methylation in a small proportion (2.85%) of patients with AML. Wouters et al. identified a subgroup of AML patients (1.4%) in which the CEBPA gene was silenced in association with CEBPA core promoter methylation. Hackanson et al. observed CEBPA methylation in the distal promoter region in 51% of selected AML patients. Due to the selection of differing patient populations and CEBPA promoter regions for analysis in past studies, the clinical significance of CEBPA methylation in AML remains unclear. To evaluate the CEBPA methylation status of patients with AML, 193 patients with de novo AML were evaluated for CEBPA methylation by bisulfite sequencing and MSP methods. A substantial proportion of patients were noted to have some degree of heterogeneous methylation in the distal region (-1423 to -1121), but not in the proximal region (-1121 to -896) or in the core region (-141 to +103), of the CEBPA promoter. Only one patient was noted to have broad methylation from the distal region to the core of the CEBPA promoter, and extending into exon1 (-25 to +103). This patient was a 61-year-old female with M1 subtype AML, a normal karyotype and an absence of CEBPA, NPM1 or FLT3 mutations. We further correlated the CEBPA methylation levels with CEBPA transcript levels in leukemic cells from 12 patients with AML and found a negative correlation (coefficient of correlation = -0.722, P = 0.017). To clarify the correlation between CEBPA methylation and clinical features in AML, quantitative MassARRAY analyses for CEBPA methylation were performed. Among the 193 patients studied, methylation levels ranged from 0.073 to 0.971 with a median value of 0.229, compared to normal bone marrow cells (NBM) with levels ranging from 0.207 to 0.269 (n=5, median: 0.218) and mature leukocytes (NPB) which ranged from 0.144 to 0.201 (n=5, median 0.161). Using complete linkage clustering, we divided the patients into two groups: one with high CEBPA methylation levels (≥ 0.55, n = 28), and the other with low methylation levels (< 0.55, n = 165). Clinical and laboratory features of these two groups are explored. Gene alteration analysis revealed NPM1 mutation was mutually exclusive with CEBPA methylation (P = 0.004), so was similar trend in CEBPA mutation. Of the 28 patients with high CEBPA methylation, only one patient had a CEBPA mutation. immunophenotyping work revealed that leukemic blasts from patients with high CEBPA methylation were predominately negative for HLA-DR (P = 0.005) and CD56 (P = 0.015) expression. Of the 140 patients receiving standard induction therapy, 110 (79.3%) patients achieved CR. With a median follow-up of 17.5 months, patients with high CEBPA methylation demonstrated a greater probability of survival (median >60 months vs. 20 months, P = 0.020) than those with low CEBPA methylation. In addition, in the subgroup of AML patients without favorable karyotypes as well as NPM1 and CEBPA mutations, patients with high CEBPA methylation (n = 14) still had better five-year survival (median 34 months vs. 11 months, P = 0.048) than those with low CEBPA methylation. Intriguingly, among the cytogenetically normal AML patients, except those with CEBPA and NPM1 mutations, the five-year survival of patients with high CEBPA methylation (n=8) was remarkably better than those with low CEBPA methylation (n=19; median >60 months vs. 11 months, P = 0.002). These results suggest that, in addition to current cytogenetic and molecular markers, CEBPA methylation status could emerge as a prognostic factor for survival advantage in AML patients. Disclosures No relevant conflicts of interest to declare.

An increasing number of epigenetic deregulation was reported to be associated with acute myeloid leukemia (AML). Hypermethylation of several genes is associated with hematologic malignancies, such as the calcitonin gene, p15INK4B, CEBPA, SOCS1, DAPK, p21Cip/Waf1, the ER gene, SDC4, MDR. MicroRNAs (miRNAs) are endogenous nonconding small RNAs that inhibit the translation of genes whose 3′-UTR are targeted. Many miRNA genes, including let-7a-3, are located in CpG islands, suggesting possible epigenetic regulation of their expression. The let-7a-3 gene has been reported to be heavily methylated in normal human placenta, brain, bone marrow, colon, skin, and lung tissue but unmethylated in some lung cancer samples. In this communication, we used bisulfite sequencing and real-time reverse transcription-PCR to analyze methylation status in 33 CpG sites of the let-7a-3 gene and miRNA expression of let-7a in four myeloid cell lines, HEL, HL60, K562 and U937. All of them showed hypermethylation (93%, 80%, 71% and 90%, respectively) in let-7a-3 promoter region (Figure 1) and displayed little let-7a miRNA expression. To evaluate the significance of let-7a-3 methylation status in patients with AML, 90 patients with de novo AML were evaluated for let-7a-3 methylation status by using combined bisulfite restriction analysis (COBRA) and confirmation with bisulfite sequencing. Most AML patients were noted to have hypermethylation in the promoter region of let-7a-3 gene. We divided these patients into three groups: demethylated (n=6), hypomethylated (n=11) and hypermethylated (n=73). Correlation of let-7a-3 methylation with biological and clinical features was evaluated. We found that the methylation status was not associated with patient age at diagnosis, sex, FAB classification and cytogenetic change. Further sequential study revealed that no association was found between let-7a-3 methylation and disease status at either complete remission or relapse stage. We previously demonstrated that 25% AML patients showed hypermethylation in distant region of CEBPA promoter and the methylation status correlated with disease status. In this study, we found that up to 81% AML patients showed let-7a-3 methylation. In the meanwhile, further analysis revealed inverse association between CEBPA and let-7a-3 methylation (p=0.007). It has be widely known that CpG islands hypermethylation of tumor-suppressor genes is a common alteration in cancer cells, leading to the transcriptional inactivation of these genes and normal cellular malfunctions. At the same time, it is also known that the genome of the cancer cell undergoes global hypomethylation at repetitive sequences, leading to genome fragility and tumorigenesis. CEBPA is widely known to be tumor suppressor gene, and let-7a-3 was previously identified as with oncogenic function in lung adenocarcinomas. Consistent to the results shown in lung adenocarcinomas, our results indicate that the miRNA let-7a might play an oncogenic function in AML. Figure 1 let-7a-3 methylation status in 33 CpG sites of promoter region in four myeloid cell lines, HEL, HL60, K562 and U937. The nucleotide numbering are according to the DNA sequence from NT_011523. Figure 1. let-7a-3 methylation status in 33 CpG sites of promoter region in four myeloid cell lines, HEL, HL60, K562 and U937. The nucleotide numbering are according to the DNA sequence from NT_011523.

CCAAT/enhancer binding protein a (C/EBPa) is a 42-kDa transcription factor that is selectively maintained during granulocyte differentiation. Loss of tumor suppressor C/EBPa expression is seen in several human malignancies, including acute myelogenous leukemia (AML). Conditional mouse models revealed that impaired C/EBPa function contributes directly to the development of AML. Previously, we identified CEBPA mutations in 15% patients with AML and sequential study showed the mutations would disappear at complete remission but the same mutations would reappear at relapse. When only considering the AML patients with normal karyotype, CEBPA mutations were identified in up to 35% patients in AML patients. The fusion proteins generated from common chromosome translocation t(8;21), t(15;17), or inv(16) have been reported to down regulate CEBPA expression or to inhibit C/EBPa function. These studies raised the importance of the C/EBPa function, alteration of which would tip the balance from differentiation toward cancer. Since an increasing number of epigenetic deregulation were reported to be associated with AML, we hypothesized that DNA methylation of CEBPA promoter may play roles in modulating expression in AML. In this communication, we showed that C/EBPa were well expressed in HL60 cells, few expressed in U937 cells and poor expressed in K562 cells; real-time RT PCR revealed that the relative RNA expression ratio of HL60, U937, and K562 cells was 673.6, 73.2 and 0.2, respectively. Subsequent DNA sequence analysis revealed that all three cell lines had no CEBPA mutation. Further methylation analysis revealed that the repressed expression in U937 and K562 cells were associated with hypermethylation in the upstream CEBPA promoter region (Figure 1), not the core promoter region (−141 to −15 and −32 to +103). The U937 cells demonstrated methylation pattern in region upstream to CEBPA promoter (−1422 to −1142); whereas K562 cells demonstrated methylation pattern in all three upstream region (−1423 to −1121, −1142 to −896, −918 to −725). These data highlight the significance of CEBPA hypermethylation in AML. In order to evaluate the significance of CEBPA hypermethylation in patients with AML, we studied the methylation status of the CEBPA gene in the bone marrow cells from 92 adult patients with AML at diagnosis using the methylation specific PCR method. Eighteen were M1, 33 M2, 2 M3, and 21 M4. Of these AML patients, 23 (25%) had methylation pattern in region upstream to CEBPA promoter (−1423 to −1121), one further showed methylation pattern in region upstream to CEBPA promoter (−1142 to −896). These results indicate that DNA hypemethylation of upstream CEBPA promoter region, especially from −1423 to −1121, might be involved in the regulation of CEBPA expression and play an important role in AML. Figure 1 Methylation pattern of upstream CEBPA promoter region was shown in U937, HL60 and K562 cells *T-C Lin and C-Y Lee contributed equally to this communication. Figure 1. Methylation pattern of upstream CEBPA promoter region was shown in U937, HL60 and K562 cells *T-C Lin and C-Y Lee contributed equally to this communication.