Carl E. Freter

Non-Homologous End Joining and Homology Directed DNA Repair Frequency of Double-Stranded Breaks Introduced by Genome Editing Reagents

Genome editing using transcription-activator like effector nucleases or RNA guided nucleases allows one to precisely engineer desired changes within a given target sequence. The genome editing reagents introduce double stranded breaks (DSBs) at the target site which can then undergo DNA repair by non-homologous end joining (NHEJ) or homology directed recombination (HDR) when a template DNA molecule is available. NHEJ repair results in indel mutations at the target site. As PCR amplified products from mutant target regions are likely to exhibit different melting profiles than PCR products amplified from wild type target region, we designed a high resolution melting analysis (HRMA) for rapid identification of efficient genome editing reagents. We also designed TaqMan assays using probes situated across the cut site to discriminate wild type from mutant sequences present after genome editing. The experiments revealed that the sensitivity of the assays to detect NHEJ-mediated DNA repair could be enhanced by selection of transfected cells to reduce the contribution of unmodified genomic DNA from untransfected cells to the DNA melting profile. The presence of donor template DNA lacking the target sequence at the time of genome editing further enhanced the sensitivity of the assays for detection of mutant DNA molecules by excluding the wild-type sequences modified by HDR. A second TaqMan probe that bound to an adjacent site, outside of the primary target cut site, was used to directly determine the contribution of HDR to DNA repair in the presence of the donor template sequence. The TaqMan qPCR assay, designed to measure the contribution of NHEJ and HDR in DNA repair, corroborated the results from HRMA. The data indicated that genome editing reagents can produce DSBs at high efficiency in HEK293T cells but a significant proportion of these are likely masked by reversion to wild type as a result of HDR. Supplying a donor plasmid to provide a template for HDR (that eliminates a PCR amplifiable target) revealed these cryptic DSBs and facilitated the determination of the true efficacy of genome editing reagents. The results indicated that in HEK293T cells, approximately 40% of the DSBs introduced by genome editing, were available for participation in HDR.

Acute myeloid leukemia induction with cladribine: Outcomes by age and leukemia risk

Acute myeloid leukemia (AML) induction traditionally includes seven days of cytarabine and three days of an anthracycline (7 + 3). Because of evidence of increased efficacy of cladribine combined with this regimen, we conducted a retrospective analysis of 107 AML patients treated with idarubicin, cytarabine and cladribine (IAC) at our institution. Complete remission (CR) occurred in 71%, with overall response of 79%. One-year survival overall was 59%, with 47% (27/57) among patients ≥60 years old and 72% (36/50) in those <60 (Relative Risk [RR] 1.9, 95% CI 1.2-3.2). Median overall survival was 17.3 months in all patients and Cox proportional hazard ratio (HR) for death was 2.2 (95% CI 1.3-3.6) for age ≥60 years compared to <60 years. One year survival was 100% among favorable NCCN risk patients versus 64% in intermediate-risk and 35% in poor-risk patients (p < 0.001). HR for death in intermediate- risk (4.2, 95% CI 1.5-12) and poor-risk (8.4, 95% CI 3.0-24) compared to favorable risk AML was higher than that associated with age ≥60 years (HR 2.2). We conclude that IAC is an effective AML induction regimen, NCCN leukemia risk predicts survival better than age in our population, and high intensity regimens can be justified in selected older patients.

Gaussian decomposition of high-resolution melt curve derivatives for measuring genome-editing efficiency

We describe a method for measuring genome editing efficiency from in silico analysis of high-resolution melt curve data. The melt curve data derived from amplicons of genome-edited or unmodified target sites were processed to remove the background fluorescent signal emanating from free fluorophore and then corrected for temperature-dependent quenching of fluorescence of double-stranded DNA-bound fluorophore. Corrected data were normalized and numerically differentiated to obtain the first derivatives of the melt curves. These were then mathematically modeled as a sum or superposition of minimal number of Gaussian components. Using Gaussian parameters determined by modeling of melt curve derivatives of unedited samples, we were able to model melt curve derivatives from genetically altered target sites where the mutant population could be accommodated using an additional Gaussian component. From this, the proportion contributed by the mutant component in the target region amplicon could be accurately determined. Mutant component computations compared well with the mutant frequency determination from next generation sequencing data. The results were also consistent with our earlier studies that used difference curve areas from high-resolution melt curves for determining the efficiency of genome-editing reagents. The advantage of the described method is that it does not require calibration curves to estimate proportion of mutants in amplicons of genome-edited target sites.

Fludarabine-resistance associates with ceramide metabolism and leukemia stem cell development in chronic lymphocytic leukemia

Fludarabine (flu) -containing regimens such as flu, cyclophosphamide and rituximab have been established as one of the standard first line therapy in medically-fit chronic lymphocytic leukemia (CLL) patients. Therefore, flu-refractory (primary flu-insensitivity or flu-caused relapse) remains a major problem causing treatment failure for CLL patients. We isolated the peripheral blood mononuclear cells (PBMCs) from CLL patients and treated with flu to find flu-refractory cases, and established flu-resistant clonal cells to study molecular mechanism of flu-resistance. By comparing parental MEC-2 cells, a human CLL cell line, we found that flu-resistant clonal cells were significantly increased lethal dose 50 of flu concentration, and up-regulated expression of P-glycoprotein, a drug-resistant marker, glucosylceramide synthase (GCS), an enzyme that can convert ceramide to glucosylceramide, and CD34, a leukemia stem cell marker. Overexpression of GCS leads to promptly elimination of cellular ceramide levels and accumulation of glucosylceramide, which reduces apoptosis and promotes survival and proliferation of flu-resistant clonal cells. Furthermore, we demonstrated that the accumulation of glucosylceramide can be blocked by PDMP to restore flu-sensitivity in flu-resistant clonal cells. We also found that elevating glucosylceramide levels in flu-resistant clonal cells was associated with up-regulation of GCS and CD34 expression. Importantly, overexpression of GCS or CD34 was also determined in flu-refractory PBMCs. Our results show that flu-resistance is associated with the alteration of ceramide metabolism and the development of leukemia stem cell-like cells. The flu-resistance can be reversed by GCS inhibition as a novel strategy for overcoming drug resistance.

Breast Cancer and Lipid Metabolism

Alteration of lipid metabolism plays a critical role in the development of many types of cancer including breast cancer. Lipid metabolism can be regulated by a variety of signaling pathways with proliferative stimuli, apoptotic stimuli or environment changes under physiological, pathophysiological or therapeutic conditions. On the other hand, many lipids and lipid intermediate metabolites are also important signaling molecules involved in cell signaling that regulate cell proliferation, differentiation, apoptosis and migration as well as responses to drug treatment. In physiological condition, lipid metabolism (anabolism and catabolism) is tightly regulated by signaling network in the cell under designed path to growth, proliferation, differentiation and migration. Dysregulation of lipid metabolism under pathological condition leads to alteration of lipid profiles and accumulation of some “bad” lipids which can cause cell overgrowth and hyper-proliferation such as cancer and cell death such as tissue injury (Fig. 8.3). This chapter focuses on the emerging understanding of the role of lipid metabolic pathway in breast carcinogenesis and tumor progression. The update information indicates that the modulation of lipid metabolism can potentially be exploited to prevent breast cancer and enhance efficacy of breast cancer therapy.

Signaling regulation and role of filamin A cleavage in Ca2+-stimulated migration of androgen receptor-deficient prostate cancer cells.

Ca2+, a ubiquitous cellular signal, and filamin A, an actin-binding protein, play an important role in the regulation of cell adhesion, shape and motility. Using transwell filters to analyze cell migration, we found that extracellular Ca2+ (Cao2+) promotes the migration of androgen receptor (AR)-deficient and highly metastatic prostate cancer cell lines (DU145 and PC-3) compared to AR-positive and relatively less metastatic prostate cancer cells (LNCaP). Furthermore, we found that expression of filamin A is up-regulated in DU145 and PC-3 cells, and that Cao2+ significantly induces the cleavage of filamin A. Silencing expression of Ca2+-sensing receptor (CaR) and p115RhoGEF, and treating with leupeptin, a protease inhibitor, and ALLM, a calpain specific inhibitor, we further demonstrate that Cao2+-induced filamin A cleavage occurs via a CaR- p115RhoGEF-calpain dependent pathway. Our data show that Cao2+ via CaR- mediated signaling induces filamin A cleavage and promotes the migration in AR-deficient and highly metastatic prostate cancer cells.

Abstract B78: The interplay between smoking at diagnosis, marital status, and head and neck cancer survivorship

BACKGROUND There are currently almost 300,000 head and neck cancer survivors in the United States, and that number is increasing due to a nationwide decrease in smoking incidence. Reports on whether smoking at time of diagnosis is associated with adverse head and neck cancer outcomes are inconsistent. There is also a paucity of information on whether marital status of head and neck cancer patients affects tobacco use. In the era of personalized cancer care, it is important to examine whether smoking status at diagnosis negatively affects survival of head and neck cancer patients to develop interventions that improve survivorship. It is also important to examine how marital status is associated with smoking, since spousal support is known to improve survival outcomes among head and neck cancer patients. PURPOSE Examine the association between smoking status at diagnosis and survival among head and neck cancer patients, as well as the association between marital status and smoking in these patients. METHODS This retrospective cohort study included 463 patients aged 20 to 87 (59.31 ± 11.42) with a diagnosis of head and neck squamous cell carcinoma (HNSCC) who attended an academic medical center between 1997 and 2012. For the first aim, the outcome was overall mortality. Main predictor variable was smoking at diagnosis (dichotomized as yes/no) and covariates included age, gender, race, marital status, insurance, alcohol use, stage of cancer, and treatment type received. Descriptive statistics and Cox proportional hazards regression analysis were used to evaluate predictors of survival based on smoking status at diagnosis and covariates. For the second aim, the outcome was smoking status at diagnosis, and the main predictor variable was marital status using the same covariates in the first aim. Multivariate logistic regression models were used to assess whether marital status was a predictor of smoking at diagnosis. RESULTS Approximately 56% of patients were smokers at diagnosis, and 50% were married. We found a statistically significant difference in median survival time between smokers vs. nonsmokers (50 months vs. 80 months, p CONCLUSIONS Smoking status at diagnosis independently predicts survival of head and neck cancer patients, and those who were smokers were almost twice as likely to die earlier than those who did not smoke. We also found that those who were married were less likely to be smokers at diagnosis. Our study suggests that individualized cancer care should incorporate social support in managing cancer risk behaviors. Both smoking and marital status predicted survival outcomes; thus, our findings highlight the need for providers to discuss and encourage spousal involvement in tobacco cessation efforts to mitigate risky behaviors that negatively affect head and neck cancer survival. Citation Format: Nosayaba Osazuwa-Peters, Eric Adjei Boakye, Betelihem B. Tobo, Kara M. Christopher, Betty Y. Chen, Carl E. Freter, Mark A. Varvares. The interplay between smoking at diagnosis, marital status, and head and neck cancer survivorship. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B78.