Native homing endonucleases can target conserved genes in humans and in animal models

In recent years, both homing endonucleases (HEases) and zinc-finger nucleases (ZFNs) have been engineered and selected for the targeting of desired human loci for gene therapy. However, enzyme engineering is lengthy and expensive and the off-target effect of the manufactured endonucleases is difficult to predict. Moreover, enzymes selected to cleave a human DNA locus may not cleave the homologous locus in the genome of animal models because of sequence divergence, thus hampering attempts to assess the in vivo efficacy and safety of any engineered enzyme prior to its application in human trials. Here, we show that naturally occurring HEases can be found, that cleave desirable human targets. Some of these enzymes are also shown to cleave the homologous sequence in the genome of animal models. In addition, the distribution of off-target effects may be more predictable for native HEases. Based on our experimental observations, we present the HomeBase algorithm, database and web server that allow a high-throughput computational search and assignment of HEases for the targeting of specific loci in the human and other genomes. We validate experimentally the predicted target specificity of candidate fungal, bacterial and archaeal HEases using cell free, yeast and archaeal assay

Native homing endonucleases can target conserved genes in humans and in animal models

In recent years, both homing endonucleases (HEases) and zinc-finger nucleases (ZFNs) have been engineered and selected for the targeting of desired human loci for gene therapy. However, enzyme engineering is lengthy and expensive and the off-target effect of the manufactured endonucleases is difficult to predict. Moreover, enzymes selected to cleave a human DNA locus may not cleave the homologous locus in the genome of animal models because of sequence divergence, thus hampering attempts to assess the in vivo efficacy and safety of any engineered enzyme prior to its application in human trials. Here, we show that naturally occurring HEases can be found, that cleave desirable human targets. Some of these enzymes are also shown to cleave the homologous sequence in the genome of animal models. In addition, the distribution of off-target effects may be more predictable for native HEases. Based on our experimental observations, we present the HomeBase algorithm, database and web server that allow a high-throughput computational search and assignment of HEases for the targeting of specific loci in the human and other genomes. We validate experimentally the predicted target specificity of candidate fungal, bacterial and archaeal HEases using cell free, yeast and archaeal assays

Inflammatory mediators in breast cancer: Coordinated expression of TNFα & IL-1β with CCL2 & CCL5 and effects on epithelial-to-mesenchymal transition

<p>Abstract</p> <p>Background</p> <p>The inflammatory chemokines CCL2 (MCP-1) & CCL5 (RANTES) and the inflammatory cytokines TNFα & IL-1β were shown to contribute to breast cancer development and metastasis. In this study, we wished to determine whether there are associations between these factors along stages of breast cancer progression, and to identify the possible implications of these factors to disease course.</p> <p>Methods</p> <p>The expression of CCL2, CCL5, TNFα and IL-1β was determined by immunohistochemistry in patients diagnosed with: (1) Benign breast disorders (=healthy individuals); (2) Ductal Carcinoma <it>In Situ </it>(DCIS); (3) Invasive Ducal Carcinoma without relapse (IDC-no-relapse); (4) IDC-with-relapse. Based on the results obtained, breast tumor cells were stimulated by the inflammatory cytokines, and epithelial-to-mesenchymal transition (EMT) was determined by flow cytometry, confocal analyses and adhesion, migration and invasion experiments.</p> <p>Results</p> <p>CCL2, CCL5, TNFα and IL-1β were expressed at very low incidence in normal breast epithelial cells, but their incidence was significantly elevated in tumor cells of the three groups of cancer patients. Significant associations were found between CCL2 & CCL5 and TNFα & IL-1β in the tumor cells in DCIS and IDC-no-relapse patients. In the IDC-with-relapse group, the expression of CCL2 & CCL5 was accompanied by further elevated incidence of TNFα & IL-1β expression. These results suggest progression-related roles for TNFα and IL-1β in breast cancer, as indeed indicated by the following: (1) Tumors of the IDC-with-relapse group had significantly higher persistence of TNFα and IL-1β compared to tumors of DCIS or IDC-no-relapse; (2) Continuous stimulation of the tumor cells by TNFα (and to some extent IL-1β) has led to EMT in the tumor cells; (3) Combined analyses with relevant clinical parameters suggested that IL-1β acts jointly with other pro-malignancy factors to promote disease relapse.</p> <p>Conclusions</p> <p>Our findings suggest that the coordinated expression of CCL2 & CCL5 and TNFα & IL-1β may be important for disease course, and that TNFα & IL-1β may promote disease relapse. Further <it>in vitro </it>and <it>in vivo </it>studies are needed for determination of the joint powers of the four factors in breast cancer, as well as analyses of their combined targeting in breast cancer.</p

Inflammatory Factors of the Tumor Microenvironment Induce Plasticity in Nontransformed Breast Epithelial Cells: EMT, Invasion, and Collapse of Normally Organized Breast Textures

Nontransformed breast epithelial cells that are adjacent to tumor cells are constantly exposed to tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β), two inflammatory cytokines identified as having pro-tumoral causative roles. We show that continuous stimulation of nontransformed breast epithelial cells by TNFα + IL-1β for 2 to 3 weeks induced their spreading and epithelial-to-mesenchymal transition (EMT). The mechanistic bases for this slow induction of EMT by TNFα + IL-1β are: 1) it took 2 to 3 weeks for the cytokines to induce the expression of the EMT activators Zeb1 and Snail; 2) although Twist has amplified the EMT-inducing activities of Zeb1 + Snail, its expression was reduced by TNFα + IL-1β; however, the lack of Twist was compensated by prolonged stimulation with TNFα + IL-1β that has potentiated the EMT-inducing activities of Zeb1 + Snail. Stimulation by TNFα + IL-1β has induced the following dissemination-related properties in the nontransformed cells: 1) up-regulation of functional matrix metalloproteinases; 2) induction of migratory and invasive capabilities; 3) disruption of the normal phenotype of organized three-dimensional acini structures typically formed only by nontransformed breast cells and spreading of nontransformed cells out of such acini. Our findings suggest that TNFα + IL-1β induce dissemination of nontransformed breast epithelial cells and their reseeding at the primary tumor site; if, then, such detached cells are exposed to transforming events, they may form secondary malignant focus and lead to disease recurrence. Thus, our study reveals novel pathways through which the inflammatory microenvironment may contribute to relapsed disease in breast cancer

Can you hear me now? Tools for cultivating a culture of respect, value, and appreciation within pediatric hematology, oncology, and cellular therapy

The American Society of Pediatric Hematology/Oncology (ASPHO) conducted a workshop Can you hear me now? Cultivating a culture of respect, value, and appreciation within pediatric hematology/oncology at their annual meeting in May 2022 in hopes of exploring how the members can enhance wellness in a climate of increasing diversity. Initiatives in the past have focused on personal care, but it has been widely shown that administrative and institutional driven initiatives are essential to create an environment of wellness. In this interactive workshop, we discovered that 22% of participants felt their institution does not instill a culture of respect. We offered tools to the audience on multiple levels: graceful self-promotion, diversity and inclusion, and leadership perspective on creating a culture of respect to address the individual, local community, and top-down leadership approaches. Here, we offer a summary on the content of the workshop, and expand upon many of the discussion points that were raised during the workshop. We bring forth novel information on each topic individually from diverse points of view, specific to the field of pediatric hematology/oncology (PHO). We aim to highlight the importance of creating a diverse and respectful work environment in PHO in hopes of ensuring motivated, satisfied, and fulfilled healthcare providers who feel appreciated and valued

Tumor-Promoting Circuits That Regulate a Cancer-Related Chemokine Cluster: Dominance of Inflammatory Mediators Over Oncogenic Alterations

Here, we investigated the relative contribution of genetic/signaling components versus microenvironmental factors to the malignancy phenotype. In this system, we took advantage of non-transformed fibroblasts that carried defined oncogenic modifications in Ras and/or p53. These cells were exposed to microenvironmental pressures, and the expression of a cancer-related chemokine cluster was used as readout for the malignancy potential (CCL2, CCL5, CXCL8, CXCL10). In cells kept in-culture, synergism between Ras hyper-activation and p53 dysfunction was required to up-regulate the expression of the chemokine cluster. The in vivo passage of RasHigh/p53Low-modified cells has led to tumor formation, accompanied by potentiation of chemokine release, implicating a powerful role for the tumor microenvironment in up-regulating the chemokine cluster. Indeed, we found that inflammatory mediators which are prevalent in tumor sites, such as TNFa and IL-1β, had a predominant impact on the release of the chemokines, which was substantially higher than that obtained by the oncogenic modifications alone, possibly acting through the transcription factors AP-1 and NF-kB. Together, our results propose that in the unbiased model system that we were using, inflammatory mediators of the tumor milieu have dominating roles over oncogenic modifications in dictating the expression of a pro-malignancy chemokine readout

V-SYMPHONY Career Development Series: A Collaboration to Enhance Professional Awareness for Pediatric Hematology Oncology Trainees

BACKGROUND: A recent survey of pediatric hematology oncology (PHO) physicians identified that a majority believe fellows are struggling to find jobs that align with their goals. Career development for trainees has historically been home institution-specific, limiting fellows\u27 exposures to career path possibilities. The virtual-Symposium of Pediatric Hematology/Oncology of New York (v-SYMPHONY) instituted a tristate Career Development Series for PHO trainees to better address their needs and increase awareness of the variety of PHO career opportunities. PROCEDURE: The v-SYMPHONY Career Development Series incorporated three sessions: (a) institutional perspective, (b) individual perspectives, and (c) nuts and bolts of job search. Pre- and post-series surveys were administered to participants to measure impact. RESULTS: Forty-one fellows registered for the series and completed a pre-survey. Over half (54%) were in their third or later year of fellowship. Careers with a clinical focus were the most commonly desired career path (59%). Most had received career development advice only from faculty within their institutions (90%). Post-surveys were completed by 11 PHO fellows. Overall, 100% of respondents reported benefiting from the career sessions and recommended the series should be repeated annually. Over 90% learned new information to prepare for the job search. CONCLUSIONS: The v-SYMPHONY Career Development Series for PHO fellows across multiple institutions was established and was extremely well received by its participants. PHO fellows agreed that these sessions were beneficial in helping prepare them for the job search process. An annual regional Career Development Series is feasible and is strongly suggested to support PHO fellows