The overexpression of HOXA9 in acute myeloid lymphoma (AML) is linked to poor outcomes for patients. Researchers at St. Jude Children’s Research Hospital sought ways to indirectly extinguish HOXA9, as it is a hard protein to target. Researchers identified RBM5 using CRISPR/Cas9, demonstrating that RBM5 expression is linked to leukemia cell proliferative. This link is driven primarily by RBM5’s dual function as a DNA and an RNA handler for gene expression. This research was published in Genome Biology.
The overexpression of the protein HOXA9, which is present in more than 70% of AML cases and often associated with a poor prognosis, is a hallmark. Despite its role as a transcriptional factor, the protein is “undruggable,” as a drug that interferes would have many other side effects. Researchers were inspired to take a new approach to the problem by examining the proteins that HOXA9 relies on and works with. Chunliang Li, Ph.D., St. Jude Department of Tumor Cell Biology, is a researcher who has done this. In his recent work, he devised an unbiased CRISPR screen strategy to identify targets for HOXA9. This led him to uncover a vast network of opportunities.
Since my lab’s establishment in 2017, this has been an ongoing effort. In early 2019, we developed this unique reporter system, which is the only reporter that authentically represents HOXA9 in these leukemias systems.”
Chunliang Li, PhD, St. Jude Department of Tumor Cell Biology
CRISPR/Cas9 is incredibly simple to use but extremely effective. The HOXA9 is tagged with a fluorescent tag and inserted into leukemia cells. Researchers can track the differences in expression by examining fluorescence within cells.
We wanted to find a novel or more targetable regulator. I explained that we performed a whole genome CRISPR screen to target all genes expressed by cells. The researchers were able to investigate different pathways in which HOXA9 had left its fluorescent footprint.
The researchers were surprised to find that splicing was the most common pathway.
This was quite surprising because the splicing factor usually regulates different combinations of transcripts, but not the level. I said that our data indicated these proteins control the HOXA9 level of expression. We hypothesized that the splicing factor may have a second function.
The Immunology eBook is a compilation of the most important interviews, articles, and news from the past year. Download the latest version. RBM5 was the protein that really stood out. Researchers found that RBM5 was highly expressed in leukemia compared to other cell types and that its oncogenic function is dependent on both DNA and RNA binding sites. The RBM family includes RNA splicing factors, but their role in DNA transcription is unknown. Researchers created a system that allowed the rapid degradation of RBM5 to address the direct transcriptional control of RBM5/HOXA9.
Li said that “HOXA9 mRNA was significantly reduced as soon as RBM5 protein had been removed from the cells,” Li explained. “This reduction occurred as early as 2 hours later, but it did not affect splicing of HOXA9.” Overexpression of HOXA9 was able to rescue leukemia cells that had lost their ability to produce RBM5. This further demonstrated the link between these two proteins.
Li is now exploring the protein as a potential drug target for AML.
He said: “We believe RBM5 is an excellent dependency gene and should be a great target based upon our functional assays.” If we target the DNA-binding affinity of these proteins synergistically, we could target HOXA9-driven leukemia.