Variety is the spice of life, and one of the drivers of genetic variation is gene splicing.
After a gene is transcribed, there are alternatively spliced transcripts that add even more variety to that gene’s expression and its menu of phenotypes.
It appears that there are types of disorders that take advantage of these varieties. Top amongst them are myeloid disorders, where somatic mutations in splicing factors lead to cell proliferation in myelodysplastic syndromes (MDS) and blood cancers.
Christopher R. Cogle, a physician-scientist at the University of Florida, would like to understand why, in hopes that such knowledge could be used to develop new therapeutic strategies to target acute myeloid leukemia (AML).
With the help of the Icahn Institute for Data Science and Genomic Technology at Mount Sinai, the 2019 RNA Sequencing SMRT Grant recipient will be able to interrogate the differential isoforms within AML cell lines and test the effects of a novel splicing factor depletion agent his lab has created.
AML is the result of a multistep transforming process of hematopoietic stem and progenitor cells (HSPCs) which enables them to proceed through limitless numbers of cell cycles and to become resistant to cell death. Interference with DNA replication using a combination of chemotherapy drugs has been the mainstay in AML therapy for more than fifty years, but the relapse rate is still very high.
Cogle’s lab has found that some of these leukemia cells embed within blood vessels to protect themselves from these drugs, so he developed a vascular disrupting agent to disrupt such sanctuary. He treated around 40 patients with the therapy, with success, but also some side effects.
Seeking a similar, but better tolerated alternative, he returned to the lab, growing AML cells on endothelial cells and then testing the leukemia killing activity of 31 million compounds. He identified several promising compounds that selectively killed AML cells within the vascular niche, while sparing endothelial cells and normal lymphocytes.
Extensive proteomic studies on one of the hit compounds showed that it binds and inhibits a splicing repressor. To understand the role of this splicing repressor in AML, Cogle’s team generated cell lines of human AML with and without knock-down of the splicing repressor and found that depletion of the splicing repressor leads to AML cell death and failure to engraft in mice. But downregulation of the splicing repressor in normal hematopoietic cells don’t affect cell viability or proliferation.
Indispensible – or not
Why is the splicing repressor seemingly indispensable in AML, yet dispensable in normal hematopoietic cells? This is the question Cogle is hoping the Iso-Seq method will answer.
He plans to use the PacBio RNA Sequencing technique to compare and contrast the gene expression and transcript isoform expressions in AML versus normal HSPC with and without knock-down of the splicing repressor.
“We’ve used conventional RNA sequencing to examine the splicing repressor depleted AML cell lines, and wished we had longer and more reads to detect the full variety of isoforms under the control of the splicing repressor,” Cogle said.
The initial RNA sequencing data was able to illuminate some biology — and several gaps that Cogle hopes to fill with more robust Iso-Seq data. He will be working with his UF colleague Ana Conesa, who has experience in functional RNA splicing as well as Big Data, including the development of a newly released computational tool, tappAS.
“In order to get to the resolution needed, you need to move beyond conventional RNA sequencing. Iso-Seq will be an important tool in dissecting these splicing mechanisms and matching them to cancer phenotypes.”
Cogle’s ultimate goal is to get these new therapies into the clinic, and full-length transcript sequencing and isoform analysis will help this endeavor in many ways. It will help explain the oncogenic mechanisms of blood cancer and how his pharmacological agents work — information that can be used in validation studies, toxicology studies, designing bioactivity assays for early phase clinical trials, and expansion campaigns to identify additional compounds.
It will also help answer some fundamental biological questions.
“This is where PacBio will have one of its greatest impacts in science,” Cogle said. “It allows people to look at alternative splicing to a depth and breadth that conventional RNA sequencing cannot.”
We’re excited to support this research and look forward to seeing the results. Check out our website for more information on upcoming SMRT Grant Programs for a chance to win free sequencing. Thank you to our co-sponsor, the Icahn Institute for Data Science and Genomic Technology at Mount Sinai, for supporting the 2019 RNA Sequencing SMRT Grant Program.