New P-TEFb–BHLHE40–Tim8/Tim13 Axis Reveals HIF-Independent Hypoxic Gene Regulation in Treatment-Resistant Tumors
核心洞察
Northwestern Medicine scientists identified a previously unrecognized regulatory axis—P-TEFb (搜索), BHLHE40 (搜索), and Tim8-Tim13 complexes—that controls rapid gene expression changes during hypoxia, functioning partially independently of HIF.
The mechanism may explain why some treatment-resistant tumors respond poorly to HIF-targeted therapies, with particular relevance for clear cell renal cell carcinoma (搜索) where hypoxia signaling is chronically activated.
The study, published in Science Advances, used proteomics, CRISPR gene editing, RNA interference, and ChIP-seq to characterize how cancer cells activate hypoxia-responsive genes beyond the well-known HIF pathway.
Northwestern Medicine scientists have, for the first time, described the underlying mechanisms that regulate how cells rapidly change gene expression in response to hypoxia, a key feature of many treatment-resistant tumors, according to a study published in Science Advances. The work, led by Ali Shilatifard, PhD, chair and Robert Francis Furchgott Professor of Biochemistry and Molecular Genetics, identifies a novel regulatory pathway that could inform future treatment opportunities for patients who respond poorly to HIF-targeted treatments.
Hypoxia—a period of low oxygen within solid tumor microenvironments—reduces the effectiveness of chemotherapy, radiation therapy, and immunotherapy. Yet how cancer cells rapidly shift gene expression in response to hypoxia has remained unclear. The P-TEFb (搜索) transcriptional kinase complex regulates the pause-release checkpoint step in transcription by RNA polymerase II, a multiprotein complex that transcribes DNA into precursors of mRNA as well as most small nuclear RNA (snRNA) and microRNA. P-TEFb is also essential for gene expression during hypoxia.
"We wanted to understand how cells activate hypoxia-responsive genes beyond the well-known HIF pathway. Since P-TEFb (搜索) is essential for hypoxic gene expression, we used an unbiased proteomics approach to identify proteins that interact with P-TEFb specifically during hypoxia," said Shimaa Soliman, PhD, a former postdoctoral research fellow in the Shilatifard laboratory and lead author of the study.
A Multi-Pronged Approach to Map the Hypoxic Transcriptional Response
The scientists used a combination of proteomics, genetics, and genomics approaches to better characterize the P-TEFb (搜索) pathway. First, they compared proteins associated with the transcriptional regulator P-TEFb under normal and low-oxygen conditions using immunoprecipitation and mass spectrometry. Next, they used CRISPR gene editing and RNA interference to remove or reduce these proteins and determine their functional importance. Finally, they used genome-wide chromatin profiling (ChIP-seq) to measure how these factors affect the recruitment of RNA polymerase II and the activation of hypoxia-responsive genes.
Using these approaches, the team found that Tim8-Tim13 proteins are present in the nucleus and help regulate hypoxic gene expression. They also discovered that the DNA binding transcription factor BHLHE40 (搜索) acts as a bridge between Tim8-Tim13 complexes and P-TEFb (搜索), creating a novel regulatory pathway that supports the hypoxic transcriptional response.
A HIF-Independent Mechanism with Clinical Relevance
Together, the findings identify a new regulatory axis, in which Tim8-Tim13 complexes and BHLHE40 (搜索) modulate P-TEFb (搜索) activity in the transcriptional response to hypoxia. This mechanism functions partially independently of HIF, a distinction with important therapeutic implications.
"Our work expands that framework by identifying a previously unrecognized regulatory axis involving P-TEFb (搜索), BHLHE40 (搜索) and Tim8-Tim13 complexes. Importantly, this mechanism functions partially independently of HIF. From a therapeutic perspective, this is particularly relevant for cancers such as clear cell renal cell carcinoma (搜索), where hypoxia signaling is chronically activated," Soliman said.
The discovery is especially significant given the broader context of hypoxia as a driver of treatment resistance. Hypoxia-inducible factors (HIFs), particularly HIF-1α and HIF-2α (搜索), stabilize under low-oxygen conditions and activate transcriptional programs supporting tumor-cell survival, including angiogenesis, glycolysis, metabolic adaptation, and immune escape. The selective HIF-2α inhibitor belzutifan has validated HIF-2α as a clinically actionable target in von Hippel–Lindau disease (搜索)-associated tumors and renal cell carcinoma. However, the newly identified P-TEFb (搜索)–BHLHE40 (搜索)–Tim8/Tim13 axis operates partially outside this HIF framework, suggesting it may account for tumors that remain resistant even when HIF signaling is targeted.
An Unexpected Link Between Mitochondria and the Nucleus
"This work uncovers an unexpected link between mitochondria and gene regulation in the nucleus, highlighting new layers of complexity in how cells sense and respond to low oxygen," Soliman said.
Building on these findings, Soliman said that her team seeks to better understand how Tim8-Tim13 complexes function in the nucleus, define how they regulate transcription, and determine whether disrupting the BHLHE40 (搜索)-Tim8/Tim13-P-TEFb (搜索) interaction could be therapeutically beneficial in hypoxia-driven cancers.
Co-authors of the study include Marta Iwanaszko, PhD, research associate professor of Biochemistry and Molecular Genetics; Grant Andersen, a student in the Driskill Graduate Program in Life Sciences (DGP); and Navdeep Chandel, PhD, the David W. Cugell, MD, Professor of Medicine in the Division of Pulmonary and Critical Care and of Biochemistry and Molecular Genetics. This work was supported by the National Cancer Institute of the National Institutes of Health under award numbers 5T32CA281953-02, R35CA197569 and R50CA265372.