The RNA Splicing Factor RBM25 in Preleukemic Clonal Expansion

molecular changes: molecular changesThe process of clonal expansion, where cells undergo rapid replication, can pave the way for cancer development by allowing molecular changes to accumulate. Certain genetic mutations, like TET2, are known to trigger clonal expansion and malignancy. However, such mutations are frequently observed in healthy individuals as well. So why do some individuals with a genetic mutation associated with leukemia remain healthy, while others with that mutation develop blood cancer? A team of scientists from the USC Stem Cell laboratory conducted a study that sheds light on this phenomenon. Their findings, published in the journal Blood, revealed a mechanism that links the leukemic mutation to varying potentials for disease development (2023; https://doi.org/10.1182/blood.2023019620). The researchers believe this finding could help identify patients with the mutation at the highest risk of developing leukemia. To delve deeper into this mystery, researchers used genetic barcoding to track individual blood stem cells in mice with a TET2 mutation, which is prevalent in myeloid leukemia patients. The study involved an inducible TET2 knockout mouse model. Study Details In their study, researchers discovered that certain blood stem cells and their offspring, referred to as clones, had a disproportionately large impact on the overall count of blood and immune cells. These clones tended to produce a large number of “myeloid” cells, including granulocytes, which could potentially increase the risk of developing myeloid leukemia. Additionally, the scientists observed noteworthy distinctions in the gene activity between the over-contributing clones and the rest of the clones. The over-contributing clones displayed lower activity in various genes recognized for inhibiting the progression of leukemia and other cancers. They also demonstrated decreased activity in genes associated with RNA splicing, which is the process of removing non-coding segments from RNA that carries instructions from DNA to the cell's protein synthesis machinery. The team found that only a small subset of hematopoietic stem cells (HSCs) overexpanded upon knockout of the TET2 gene. Among these overexpanded cells, there was a notable reduction in the activity of a specific RNA splicing gene called RBM25. The repression of RBM25 triggers clonal expansion of TET2 knockout hematopoietic cells both in vitro and in vivo. Interestingly, these overexpanded HSCs had lower levels of genes associated with leukemia and RNA splicing than non-overexpanded HSCs. To understand the effect of RBM25, the researchers used CRISPR/Cas9 gene editing to manipulate the activity of RBM25 in cells with TET2 mutations. They discovered that increasing RBM25 activity slowed cell proliferation, while reducing RBM25 activity made cells multiply more quickly. This reduction in RBM25 activity also caused changes in the RNA splicing of the gene BCL2L1, which regulates apoptosis or programmed cell death. To gain additional insights about the research, HemOnc Times spoke with the senior author, Rong Lu, PhD, Associate Professor of Stem Cell Biology and Regenerative Medicine, Biomedical Engineering, Medicine, and Gerontology at the University of Southern California, and a Leukemia & Lymphoma Society Scholar. Her area of expertise involves the study of stem cells and their coordination, regulation, and malfunction at the single-cell level. She primarily investigates the dissimilarities among individual stem cells and how they work together to maintain the blood supply using mouse hematopoietic stem cells as a model system. HemOnc Times: What is the role of RNA splicing in the regulation of programmed cell death and how does this relate to the development of leukemia? Lu: “RNA splicing plays crucial roles in many biological processes, including programmed cell death, also known as apoptosis. Many apoptotic genes, including BCL2L1, are regulated by RNA splicing. Taking BCL2L1 as an example, a short splicing isoform of this gene promotes apoptosis, and a long splicing isoform inhibits apoptosis. RNA splicing modulates the ratio of these isoforms and, therefore, influences apoptosis resistance. Apoptosis is a critical process that eliminates damaged cells in the body. Its resistance can lead to the accumulation of damaged cells and increased cell proliferation, ultimately leading to the development of cancer, including leukemia.” HemOnc Times: How does RBM25 influence the proliferation of cells with TET2 mutations and what is the mechanism behind this effect? Lu: “Our study suggests that cells with TET2 mutations exhibit increased variability in the expression level of RBM25, which regulates RNA splicing of many genes, including BCL2L1 that regulates apoptosis. Our data indicate that TET2 mutant cells with low RBM25 expression have less short splicing isoform and more long splicing isoform of BCL2L1. Since the former is known to promote apoptosis and the latter is known to inhibit apoptosis, the overall effect of reduced RBM25 expression would lead to apoptosis resistance that ultimately increases cell proliferation. Other splicing targets of RBM25 may also contribute to this process.” HemOnc Times: What are the potential clinical implications of this study for the diagnosis and treatment of myeloid leukemia? Lu: “Our findings suggest that RNA splicing regulators such as RBM25 could serve as diagnostic markers for identifying patients with varying leukemia risks. We found that RBM25 expression is significantly lower in human acute myeloid leukemia (AML) cells that carry mutations in TET2 and other AML-associated genes encoding epigenetic regulators such as DNMT3A, ASXL1, IDH2, and NPM1. Moreover, reduced RBM25 expression is associated with poor survival among AML patients. RBM25 and its splicing target genes such as BCL2L1 may also be used as therapeutic targets for treating myeloid leukemia. Our data suggest that overexpression of RBM25 inhibits cell proliferation.” HemOnc Times: How can future research build on the findings of this study to further investigate the relationship between TET2 mutations, RNA splicing factors, and the development of myeloid leukemia? Lu: “There are many questions remaining to be addressed regarding how RNA splicing factors mediate leukemia development initiated by TET2 mutations. For example, there may be other RNA splicing factors involved in this process, in addition to RBM25. Our data revealed significant downregulation of many RNA splicing factors in TET2 mutant cells that exhibited excessive proliferation. “Furthermore, dysregulation of RNA splicing following dysregulation of epigenetics could be a general mechanism for leukemia development. Mutations of other leukemia-associated epigenetic regulators such as DNMT3A, ASXL1, IDH2, and NPM1 may also induce abnormal expression of RNA splicing factors, which could be a crucial step in the development of myeloid leukemia. Moreover, much remains to be done to leverage the interplay between RNA splicing factors and epigenetic regulators in the diagnosis and treatment of leukemia.” Dibash Kumar Das is a contributing writer.