Science Spotlight

Oncogenic role of a developmentally regulated NTRK2 splice variant

From the Holland Lab, Human Biology Division

Alternative splicing allows one gene to encode multiple variant proteins that have similar, distinct, or even opposite functions. Alternative splicing plays an important role in embryonic development and organogenesis; however, it can be harmful if it occurs at the wrong time or in the wrong type of cell. According to a previous report from the Holland lab in the Human Biology Division at Fred Hutch, the NTRK2 gene, which encodes the tropomyosin receptor kinase (TrkB.FL- a protein well known to play a role in neurodevelopment), produces several splice variants, including TrkB.T1, a truncated isoform that lacks the kinase domain and contains a unique 11 amino-acid sequence at the C terminus. Across species, this unique domain has been conserved, suggesting an important biological function. Interestingly, the TrkB.T1 variant is commonly expressed in gliomas, one of the most common tumors of the central nervous system (CNS), suggesting it may also play a role in oncogenesis. A recent study aimed to characterize the role of TrkB.T1 during development and oncogenesis was published in Science Advances and led by Dr. Siobhan Pattwell, a former postdoctoral fellow in the Holland lab and now a principal investigator at Seattle Children’s Research Institute.

 

To investigate the expression of TrkB.T1 transcript variant during mouse development, Dr. Pattwell, Sonali Arora, and the Holland team performed a transcript analysis of single cell RNA sequencing datasets comprising the public resource from Dr. Jay Shendure’s lab at UW Genome Sciences, a MOCA (Mouse Organogenesis Cell Atlas)- or single cell transcriptional atlas of mouse organogenesis. The researchers assessed the expression levels of TrkB.FL and TrkB.T1 in single cells at discrete stages of early mouse embryonic development. Although TrkB.FL and TrkB.T1 were highly expressed during embryonic development, their cellular localization differed. TrkB.FL was mainly found in cells in the developing central nervous system, whereas TrkB.T1 was expressed in a variety of embryonic cell types, including radial glia, immature oligodendrocytes, and isthmic organizer cells, among others. To validate these results, the researchers compared protein expression of TrkB.FL and TrkB.T1 in histological sections of mouse embryos throughout development. Their results confirmed high protein expression of TrkB.FL, only within the central nervous system, and found TrkB.T1 widespread across the different embryonic cell types. According to the authors “…widespread embryonic expression pattern of TrkB.T1 suggested that TrkB.T1 plays a role in both development and cancer and may not be restricted to the CNS, highlighting the possibility that it may also influence additional tumor types beyond what has been shown for gliomas”.

Gene expression of TrkB.FL and TrkB.T1 during early mouse embryonic development. Dr. Pattwell and colleagues showed that the expression of TrkB.FL (shown in orange-left) was restricted to neurons and the expression of TrkB.T1 (shown in green-right) was expressed broadly across different cell types.
Gene expression of TrkB.FL and TrkB.T1 during early mouse embryonic development. Dr. Pattwell and colleagues showed that the expression of TrkB.FL (shown in orange-left) was restricted to neurons and the expression of TrkB.T1 (shown in green-right) was expressed broadly across different cell types. Image taken from original article.

The authors then explored the oncogenic potential of TrkB.T1 in vivo. This was accomplished by using a RCAS/TVA gene delivery system in a sensitized background mouse model. Their goal was to overexpress TrkB.T1 and knock down PTEN in nestin-positive progenitor cells by injecting the constructs into these mice. PTEN, a tumor suppressor that negatively regulates the PI3K/AKT pathway, was knocked down because TrkB.T1 has been reported to play a role in the PI3K/AKT pathway. Nestin cells were targeted as they are widely expressed both within and outside of the central nervous system. The delivery of TrkB.T1, as well as PTEN loss, caused various types of tumors to develop from nestin-positive cells outside of the central nervous system, including soft tissue sarcomas, renal tumors, lymphoid leukemias and lymphomas. In vitro studies using primary neural stem cells overexpressing platelet-derived growth factor beta (PDGFB- an oncogene known to drive glioma formation) with TrkB.T1 knockdown showed a decrease in neurosphere formation. Their results show the oncogenic potential of TrkB.T1 within and outside of the central nervous system.

 

To further dissect the role of TrkB.T1 in development and oncogenesis, the authors aimed to identify TrkB.T1 interactors. They performed pulldown assays on cells expressing TrkB.T1-HA and TrkB.FL-HA and found that TrkB.T1 has both unique binding partners as well as some binding partners that are shared with TrkB.FL. Gene ontology (GO) and Reactome pathway enrichment analysis revealed that those binding partners are associated with known developmental and oncogenic signaling pathways, such as Gli, Hedgehog, Wnt, RAS, mitogen-activated protein kinase (MAPK) among others,  that have not been previously reported to interact with TrkB.T1. The unique set of interactors of TrkB.T1 suggests that it functions through a different signaling pathway from TrkB.FL.

 

Finally, to understand the potential importance of TrkB.T1 in human cancers, the authors compared the expression of NTRK2 transcripts across adult and pediatric cancer data sets from the TCGA (The Cancer Genome Atlas) and TARGET (Therapeutical Applicable Research to Generate Effective Treatments) databases. The authors found that TrkB.T1 is expressed across a wide range of pediatric and adult tumors, including rhabdoid tumor, neuroblastoma, or clear-cell sarcoma of the kidney. In contrast, TrkB.FL is detected at much lower levels. Additionally, they examined the protein expression of TrkB.T1 in different types of tumors using tissue microarrays which confirmed high expression of TrkB.T1 across different tumor types. Together, the findings of this study demonstrate the significance of the TrkB.T1 splice variant in early embryonic development and oncogenesis.

This study was supported by grants from the National Institutes of Health, a Jacobs Foundation Research Fellowship, an NSF Graduate Research Fellowship, a grant from the Department of Defense, a grant from Safeway Foundation, an AACR-QuadW Foundation Fellowship for Clinical/Translational Sarcoma Research, a grant from the Kuni Foundation, and a grant from the Paul G. Allen Frontiers Group Allen Discovery Center.

Fred Hutch/UW Cancer Consortium members Eric Holland, Siobhan Pattwell, Jay Shendure, Michael Haffner, Keith Loeb and Michael Wagner contributed to this study.

Pattwell SS, Arora S, Nuechterlein N, Zager M, Loeb KR, Cimino PJ, Holland NC, Reche-Ley N, Bolouri H, Almiron Bonnin DA, Szulzewsky F, Phadnis VV, Ozawa T, Wagner MJ, Haffner MC, Cao J, Shendure J, Holland EC. Oncogenic role of a developmentally regulated NTRK2 splice variant. Sci Adv. 2022 Oct 7;8(40):eabo6789. doi: 10.1126/sciadv.abo6789. Epub 2022 Oct 7. PMID: 36206341