Victoria Hwang, a senior at the Arkansas School for Mathematics, Sciences, & the Arts, has been named a top 300 Scholar in the 79^th Regeneron Science Talent Search. Victoria was selected for her work entitled “POLK^KO GBM-derived Cells Exhibit Increased Replication Catastrophe” in Dr. Robert Eoff’s laboratory.  Congratulations Victoria!

Mechanistic Insights into Chemoresistance Mediated by Oncogenic Viruses in Lymphomas.

Duah Alkam, a Ph.D. student in Mark Smeltzer‘s and Dave Ussery‘s laboratories was invited to present her work at the Nanopore Community Meeting in New York. A description of her presentation from Oxford Nanopore Technologies is below.

Duah Alkam – PCR-free transposon sequencing (TnSeq): Cas9/dCas9-mediated transposon enrichment

Duah Alkam (University of Arkansas for Medical Sciences) opened the Targeted Sequencing breakout session by introducing the goal of her team: to find therapeutic targets for the prominent bacterial pathogen Staphylococcus aureus. Staphylococcus bacteria are a leading cause of healthcare-associated infections, with Methicillin-resistant S. aureus (MRSA) causing over 80,000 severe infections and over 11,000 deaths per year.

Duah and the team take a whole-genome screening approach to the search for novel therapeutics via transposon sequencing (TnSeq), enabling analysis of the genes that affect the fitness of microorganisms in a particular condition. She described how, in a transposon library, each cell is mutagenized by a transposon – a DNA segment that inserts into a gene, resulting in dysfunction. The transposon library used by Duah and her colleagues is in a background of a clinically-relevant strain of S. aureus; Duah gave an overview of a typical workflow, in which the libraries are grown in different conditions, then enriched for the region of interest. The current protocol for TnSeq requires PCR amplification of the transposon-library junctions prior to sequencing; Duah noted that this method has been used in many papers. However, Duah explained, there’s a problem with this method: inherent bias is introduced through the PCR steps. Looking to overcome this, Duah asked: “can we enrich for the transposon via a PCR-free method?”. Here, Duah explained, the team turned to Oxford Nanopore and decided to explore PCR-free dCas9/Cas9-mediated transposon enrichment.

Duah outlined the four methods tested to investigate Cas9-mediated enrichment and nanopore sequencing of S. aureus TnSeq libraries; in each case, samples were prepared for sequencing without PCR using the Ligation Sequencing Kit and sequenced on the MinION device.

In the first condition, the TnSeq library was sequenced without any enrichment. This no-enrichment condition resulted in just 8,566, or 0.2% of the 5.1m reads representing transposons, as expected – transposons are “pretty rare in the library”. This confirmed the need for enrichment of the transposons.

In the second condition, CRISPR/Cas9 was used to induce a double-stranded cut targeting transposon insertion sites, then the library was sequenced. Duah outlined the CRISPR/Cas9-mediated enrichment method: firstly, the 5′ ends of all strands in the sample are dephosphorylated. Next, the Cas9 enzyme is directed to and cleaves DNA at transposon sites via RNAs including custom transposon-specific probes. This reveals phosphorylated ends, to which sequencing adapters can be ligated so that the enriched DNA can be sequenced. In this dataset, transposons were successfully enriched, representing 45% of reads in sequencing – “we were very excited when we saw this” – but sequencing yield was depleted, so that this was comprised of 3.7k reads.

To investigate whether yield could be improved, in the third condition, dCas9 – “dead” Cas9, an inactive form of the enzyme which does not cut the DNA – and biotinylated RNA probes were used, so that the biotinylated targets could be captured using Streptavidin beads then prepared for sequencing. Whilst this protocol resulted in only 0.8% transposon reads, sequencing output was higher, resulting in 94.6k reads.

Aiming to make the most of the enrichment from Cas9 and yield from dCas9, in the last condition, a combination of these methods were used. dCas9 and biotinylated probes were first used to capture the DNA, then active Cas9 was used to cleave the transposons at a single site prior to sequencing. Here, enrichment was lower than when using Cas9 alone, with 31% of reads representing transposons, but as sequencing output was higher, 19.4k reads were on-target.

Summarising, Duah concluded that she and her team had successfully devised and utilized a PCR-free method of transposon enrichment from a TnSeq S. aureus library, representing the first time this has been achieved without PCR. Furthermore, the use of dCas9 in pulldown prior to cleavage by Cas9 improved sequencing yield, enabling a greater number of transposon reads. They are now optimizing their protocol to further improve sequencing yield and recover greater numbers of insertions.

Congratulations to Tresor O. Mukiza, who successfully defended his dissertation entitled “Multiple DNA Sequence-Specific Protein-DNA Complexes Activate Meiotic Recombination Hotspots Via Chromatin Remodeling”. Tresor is a student in Wayne Wahls‘ laboratory.

Jacy Wagnon, who earned her Ph.D. in Dr. Wayne Wahls’ laboratory, has now obtained a tenure-track faculty position at The Ohio State University.  Doctor Wagnon is an Assistant Professor in the Department of Neuroscience, with research interests in epilepsy, neurodegenerative disorders, and genetics of neurological diseases.

Nov. 27, 2019 | Scientists conducting cancer research in the lab, clinic and community gathered Nov. 20 at the 26^th UAMS Showcase of Medical Discoveries to share their projects and discuss ways to work together.

A quarterly event, the showcase is designed to highlight research areas across UAMS and encourage collaboration among scientists. Cancer research served as the focus of the event held at the UAMS Winthrop P. Rockefeller Cancer Institute. It attracted about 170 attendees from research and clinical areas to learn about the university’s wide scope of cancer research projects.

The showcase was co-sponsored by the UAMS Office of Research in conjunction the Cancer Institute, College of Medicine and Office of Institutional Advancement.

“This is an excellent time to highlight our cancer research programs, as we focus our efforts on attaining National Cancer Institute Designation and transition to new Cancer Institute leadership under director Dr. Michael Birrer. As we take stock of our strengths, we look forward to advancing our research programs on a strong new trajectory in the upcoming decade,” said Shuk-Mei Ho, Ph.D., vice chancellor of research.

Birrer, a medical oncologist who specializes in gynecologic cancers, joined the Cancer Institute as vice chancellor and director in late 2019.

In early 2019, the UAMS Cancer Institute received unanimous support in both the Arkansas Senate and House for its quest to achieve designation by the National Cancer Institute. An annual state allocation of $10 million will support this effort.

Ho stressed that the allocation designated by the Legislature is essential and illustrates the overarching support for designation among leaders and citizens in Arkansas.

“We have established a good foundation at UAMS toward achieving NCI Designation. The state’s support and enthusiasm for this important goal is a vital part of that,” Ho said.

To achieve designation, cancer centers undergo a highly competitive assessment process that demonstrates an outstanding depth and breadth of basic laboratory, patient/clinical and population-based research. The designation brings with it many benefits, including expanded access to federal funding for researchers and improved access to clinical trials for patients.

At the showcase, researchers displayed 40 scientific posters representing Cancer Institute programs and numerous UAMS colleges and academic departments. Together with their colleagues, the scientists joined in lively discussions about the implications and goals of their research. Of the 26 showcases the UAMS Office of Research and Innovation has presented, this one was by far the largest, Ho said.

Genetic counselor Mindy Simonson and Kristin Zorn, M.D., chair of the Division of Gynecologic Oncology, shared a poster they recently presented at the National Society of Genetic Counselors Annual Meeting in Salt Lake City.

Their project examines how better to identify and streamline the referral process for patients with hereditary cancers.

“We are working to build an adaptive questionnaire using electronic medical records to automatically generate a referral for patients who meet certain qualifications related to hereditary cancers,” Simonson said.

Although the process has presented technological challenges, the idea generated much excitement among participants at the Utah conference.

“A lot of people are interested in this concept and have talked to us about how it could work in various settings,” Simonson said.

Other scientists shared information related to their laboratory research, including Samantha Kendrick, Ph.D., assistant professor in the UAMS College of Medicine Department of Biochemistry and Molecular Biology.

Kendrick’s research focuses on treatment resistance in aggressive diffuse large B-cell lymphoma.

“We are working to find a way to shut off B-cell receptor signaling pathways at the DNA level. If we can develop a drug to target these genes, the disease could become more sensitive to chemotherapy and lead to better outcomes for patients,” Kendrick said.

In addition to viewing the wide range of cancer research programs and gathering information about potential collaborations, attendees also learned about the Cancer Institute’s infrastructure and research core facilities available to support their work.

In a brief presentation, Ho recognized Laura Hutchins, M.D., for her more than 30 years of service to UAMS and its cancer programs. Hutchins served in multiple roles at UAMS, including division director of the UAMS Division of Hematology/Oncology for 15 years and program director of the UAMS Hematology/Oncology Fellowship Program for 10 years.

She held the prestigious Virginia Clinton Kelley Endowed Chair for Clinical Research from March 2007-December 2018 and was associate director for clinical research in the Cancer Institute for 20 years. For 18 months, she served as Cancer Institute interim director while the search for the new director was underway.

“Dr. Hutchins is one the strongest friends of research we have at UAMS. She champions this place in many ways and we are so thankful for her contributions,” Ho said. Hutchins plans to retire in early 2020.

Congratulations to graduate student Tresor Mukiza and his co-authors, Reine Protacio, Mari Davidson, Walter Steiner and Wayne Wahls.  Cover art for their new article on chromosome dynamics is featured on the home page of Genetics for November, 2019.  The editors also selected this paper to be featured in a highlight in Genetics.