There has been a missing puzzle piece in phase separation and its role in cancer. Now, researchers at the University of North Carolina (UNC) at Chapel Hill and the UNC Lineberger Comprehensive Cancer Center report phase separation as a driver in cancer formation.
Their findings are published in the journal Nature in a paper titled, “Phase separation drives aberrant chromatin looping and cancer development.”
“The development of cancer is intimately associated with genetic abnormalities that target proteins with intrinsically disordered regions (IDRs),” wrote the researchers. “In human hematological malignancies, recurrent chromosomal translocation of nucleoporin (NUP98 or NUP214) generates an aberrant chimera that invariably retains the nucleoporin IDR—tandemly dispersed repeats of phenylalanine and glycine residues. However, how unstructured IDRs contribute to oncogenesis remains unclear.”
The researchers demonstrate that a mutation that fuses two unrelated genes can promote a process called liquid-liquid phase separation. The process occurs inside a cell’s nucleus and enables the formation of compartments with various physical properties that can promote cancers such as acute leukemias.
“Phase separation and its role in cancer has been a missing puzzle piece in understanding this disease,” explained UNC Lineberger’s Greg Wang, PhD, associate professor of biochemistry and biophysics and pharmacology at the UNC School of Medicine and co-lead author of this article. “This finding is among the first to link phase separation to cancer formation.”
The researchers performed lab experiments in cancer cells that carried a common gene fusion called NUP98-HOXA9. The fusion is found solely in blood cells of patients that develop leukemia.
“Here we show that IDRs contained within NUP98–HOXA9, a homeodomain-containing transcription factor chimera recurrently detected in leukemias, are essential for establishing liquid–liquid phase separation (LLPS) puncta of chimera and for inducing leukemia transformation,” wrote the researchers. “Notably, LLPS of NUP98–HOXA9 not only promotes chromatin occupancy of chimera transcription factors, but also is required for the formation of a broad ‘super-enhancer’-like binding pattern typically seen at leukemogenic genes, which potentiates transcriptional activation.”
“Because similar gene fusions have been observed in other malignancies, the mechanism we elucidated could explain other types of cancer as well,” said UNC Lineberger’s Douglas H. Phanstiel, PhD, assistant professor of cell biology and physiology at the UNC School of Medicine and co-lead author of this article. “We believe that our research could open up new and innovative avenues to attack cancer cells.”
Within the proteins produced by NUP98-HOXA9 are unstructured stretches, known as intrinsically disordered regions, or IDRs. The role of IDRs has been a mystery, but the researchers showed that IDRs promote liquid-liquid phase separation of NUP98-HOXA9 proteins when they reach critical concentrations in the nucleus, causing NUP98-HOXA9 to become phased, or compartmentalized.
“The way that liquid-liquid phase separation alters the behavior of NUP98-HOXA9 proteins is that it makes them bind to target genes much more strongly,” said Jeong Hyun Ahn, PhD, a postdoctoral fellow at UNC and first author of this article. “DNA binding of NUP98-HOXA9 proteins, when they are phase-separated, generates a unique pattern called a ‘super-enhancer.’ Stronger, super enhancer-like binding by NUP98-HOXA9 proteins to DNA leads to more potent activity of this factor, which underlies the formation of aggressive blood cancers.”
“Theoretically, a drug that specifically disrupts or dissolves the phase-separated liquid droplets formed by NUP98-HOXA9 could be a therapeutic agent,” Wang said. The researchers hope they can look into potential therapeutics that target phase separation, as this process also impacts neurodegenerative diseases such as Alzheimer’s disease.
The researchers also found that phase separation can influence the three-dimensional structure of the genome by creating chromatin loops, which organize the genome and help control which regions are active and inactive.
“Our discovery is the first clear evidence of chromatin loops formed by phase separation,” Phanstiel said. “This new class of loops appears to be driving cancer development by connecting regulatory regions of chromatin to cancer genes, thereby increasing cancer gene expression and lethality.”