In the era of precision medicine, cancer researchers and oncologists are eagerly searching for more realistic, cost effective, and timely tumor models to aid drug development and precision oncology. Tumor models that can faithfully recapitulate the histological and molecular characteristics of various human tumors will be extremely valuable in increasing the successful rate of oncology drug development and discovering the most efficacious treatment regimen for cancer patients. Two-dimensional (2D) cultured cancer cell lines, genetically engineered mouse tumor (GEMT) models, and patient-derived tumor xenograft (PDTX) models have been widely used to investigate the biology of various types of cancers and test the efficacy of oncology drug candidates. However, due to either the failure to faithfully recapitulate the complexity of patient tumors in the case of 2D cultured cancer cells, or high cost and untimely for drug screening and testing in the case of GEMT and PDTX, new tumor models are urgently needed. The recently developed patient-derived tumor organoids (PDTO) offer great potentials in uncovering novel biology of cancer development, accelerating the discovery of oncology drugs, and individualizing the treatment of cancers.In this review, we will summarize the recent progress in utilizing PDTO for oncology drug discovery. In addition, we will discuss the potentials and limitations of the current PDTO tumor models. K E Y W O R D S drug testing, patient derived tumor organoids, precision oncology, tumor models | 151 GRANAT eT Al. not be present in cells when grown in vivo. 4 Third, the growth medium used for culturing cancer cell lines is not able to completely mirror the conditions and environment that tumor cells naturally reside in. In vivo, tumor cells are surrounded by fibroblasts, blood vessels, and immune cells, and their collective interactions are important; this aspect is unfortunately missing in the cultured cancer cell lines. 5 Therefore, the in vitro cultured 2D cancer cell lines are the least faithful tumor model to be able to recapitulate patient tumors. By growing the established cancer cell lines in a three-dimensional (3D) environment, which mimics the in vivo extracellular matrix, the so called 3D cell culture moves a step closer to the in vivo tumors. 6However, the 3D cell culture still lacks the complex tissue hierarchy comparing to the primary tumors. 6 Therefore, the 3D cell culture is not ideal for investigating the tumor biology and testing oncology drugs. | Genetically engineered mouse tumor modelsDue to the aforementioned limitations, another commonly utilized model in cancer research is the genetically engineered mouse tumor (GEMT) model. In contrast to transplanting cancer cell lines into mice, which requires an immunocompromised status of the host mice to prevent rejection, GEMT is immunocompetent. 7 Therefore, GEMT can be potentially used for the investigation of immunotherapy. However, mouse tumor models often do not faithfully recapitulate the human cancers. Furthermore, generat...

In Escherichia coli, DNA cytosine methyltransferase (Dcm) methylates the second cytosine in the sequence 5’CCWGG3’ generating 5-methylcytosine. Dcm is not associated with a cognate restriction enzyme, suggesting Dcm impacts facets of bacterial physiology outside of restriction-modification systems. Other than gene expression changes, there are few phenotypes that have been identified in strains with natural or engineered Dcm loss, and thus Dcm function has remained an enigma. Herein, we demonstrate that Dcm does not impact bacterial growth under optimal and selected stress conditions. However, Dcm does impact viability in long-term stationary phase competition experiments. Dcm+ cells outcompete cells lacking dcm under different conditions. Dcm knockout cells have more RpoS-dependent HPII catalase activity than wild-type cells. Thus, the impact of Dcm on stationary phase may involve changes in RpoS activity. Overall, our data reveal a new role for Dcm during long-term stationary phase.

All cancer cells need to maintain their telomeres in order to continue proliferating. In 85–90% of all human cancers, maintenance and extension of these telomeres is accomplished by telomerase, which adds a short repetitive sequence to the ends of chromosomes. 10–15% of cancers adopt another method, called the Alternative Lengthening of Telomeres (ALT) pathway. ALT pathway uses homologous recombination to maintain the telomere length of ALT cancers and is vital for the continued growth of ALT cancers. Previously, our lab reported that depletion of FANCM, a key member of the Fanconi Anemia family of genes, induced replication stress response primarily at the ALT telomeres (Pan, PNAS 2017). The objectives of our study were: (1) Elucidate the biological function of human Timeless (Tim1) protein, a very important DNA repair protein, in DNA replication stress response at ALT telomeres. (2) Determine whether depletion of Tim1 affects the viability of ALT cells. To accomplish these goals, we first used siRNA transfection to deplete either Tim1, or FANCM, or both in ALT cells. These cells were then co‐stained with antibodies recognizing TRF2 (a telomere marker) and either BLM (a DNA repair marker) or pRPA (a DNA damage checkpoint marker). The percentage of cells with TRF2 & BLM as well as TRF2 & pRPA foci were quantified. In addition, we also monitored the amount of micronuclei formed in those cells. Formation of micronuclei often represent the unrepaired DNA damages. Finally, cell viability assays of the siRNA transfected cells were done. Cells were allowed to grow for 10 days, fixed and stained with crystal violet. Our results show that: siRNA depletion of FANCM and Tim1 in ALT cells had synergistic/additive foci formation for TRF2 & BLM, and TRF2 & pRPA. siRNA depletion of FANCM and Tim1 in ALT cells had no significant effect on micronuclei formation alone, but increased formation when combined. siRNA depletion of FANCM and Tim1 in ALT cells appears to decrease cell viability to the greatest extent when co‐depleted, as opposed to either one depleted alone. Our findings suggest: (1) ALT cancer cells utilize both FANCM and Tim1 to resolve the replication stress at ALT telomeres; (2) co‐depletion of FANCM and Tim1 is synthetically lethal in ALT cancers. (3) Targeting both FANCM and Tim1 is a potential efficacious strategy in treating ALT cancers. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

While the role of DNA methylation in eukaryotes is well known, little is known about the role of DNA methylation in prokaryotes. Dcm is a DNA cytosine methyltransferase in E. coli that methylates the second cytosine in the sequence 5′CCWGG3′. Although the methylation recognition sites are known, the function of methylation catalyzed by Dcm and the mechanism by which methylation impacts bacterial physiology is not yet known. Our laboratory's microarray data indicates numerous gene expression changes at stationary phase upon addition of a methylation inhibitor, 5‐azacytidine, suggesting a role for Dcm in stationary phase. Stationary phase competition experiments were conducted with a wild‐type strain and a kanamycin resistant dcm knockout strain and demonstrated that the dcm knockout strain was less fit during stationary phase. In subsequent experiments, a kanamycin resistant manA knockout strain was utilized in place of the wild‐type strain to control for possible effects of the addition of a kanR gene on stationary phase fitness. Competition experiments between the manA knockout strain and dcm knockout strain indicated that the lack of a dcm gene conferred a decrease in stationary phase fitness. As previous data have indicated that the dcm knockout strain is less fit than strains with a functional dcm gene at stationary phase, the laboratory is working to determine the mechanism by which cells lacking Dcm are disadvantaged. Dcm‐mediated methylation may regulate genes important to cell survival or play a role in growth advantage in stationary phase (GASP) mutation generation. Cells in stationary phase are characterized by an increase in rpoS expression, a change that can be determined indirectly by measuring the activity of the RpoS‐dependent catalase enzyme. Catalase activity was measured in a wild‐type strain, a dcm knockout strain, and a rpoS knockout strain; the experiments demonstrated that there was significantly more catalase activity in the dcm knockout strain compared to the wild‐type strain and that the rpoS knockout strain had minimal catalase activity. These data suggest that RpoS activity increases significantly in cells lacking the dcm gene. In order to test that the increase in catalase activity was due to the lack of the dcm gene, catalase activity was measured in a dcm knockout strain complemented with an empty plasmid and a knockout strain complemented with a dcm containing plasmid. The dcm knockout strain with the empty plasmid showed increased catalase activity compared to wild‐type strains, but when complemented with a dcm containing plasmid, levels of catalase activity were reduced to that seen in wild‐type strains. Additionally, colonies from the last day of each stationary phase competition experiment are currently being assayed for catalase activity to determine if the colonies that won the competition experiments contain GASP mutations. Published data indicate that rpoS is mutated during GASP, but retains a small amount of activity. In summary, we have demonstrated that lacking functional Dcm results in reduced fitness at stationary phase. Experiments that measure catalase activity as a proxy for RpoS function may implicate rpoS in the process through which loss of the dcm gene confers its effects.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

There has been a recent increase in the incidence of urinary tract infections (UTIs) caused by extended spectrum beta-lactamase (ESBL) producing Enterobacteriaceae, which are resistant to third-generation cephalosporins. Our goal was to compare the clinical responses of patients with ESBL UTI and non-ESBL UTI who received empiric third-generation cephalosporins. A retrospective analysis was performed on data collected between June 1, 2013, and June 30, 2017, from children aged 0 days to 19 years old who presented to NYU Langone Long Island Hospital’s pediatric ED and/or were admitted with a UTI caused by Enterobacteriaceae. There was no significant difference in median length of fever duration. However, ESBL patients had significantly longer hospital stays, higher 30-day readmission rate, and higher 7-day revisit rate. It is reasonable to maintain an empiric UTI antibiotic choice rather than selecting a broad-spectrum antibiotic, such as carbapenem for children at high risk of ESBL UTI.