Studies on the maintenance of differentiated neuronal identity and the functional role for HIF2α in neuroblastoma

The role of PRC2-mediated gene silencing for maintenance of differentiated dopaminergic and serotonergic neuronal identity and function in healthy aging and disease

The mechanisms controlling how differentiated cells can maintain their identity and function remain largely unknown. This is of particular relevance for neurons, cells that must remain functionally intact over an entire lifespan. Apart from instructive information provided by transcription factors controlling cell type-specific gene programs, there is also a need to maintain silencing of transcriptional programs governing other cellular fates a process which is poorly understood. Furthermore, the predominantly sporadic nature of several neurodegenerative and psychiatric disorders necessitates a better understanding of how epigenetic mechanisms contribute to the etiology of pathological conditions in the CNS as well as to normal aging.

We aim to understand fundamental molecular mechanisms underlying the role of gene repression for maintenance of neuronal identity and function. As model systems we use the well-defined midbrain dopaminergic (mDA)- and serotonergic (5HT)-neuronal subpopulations both in mouse and human. Both cell types are involved in several psychiatric disorders taking a huge toll on individuals and society. In addition, degeneration of mDA-neurons is a defining feature of Parkinson’s disease (PD) with different subtypes of mDA-neurons selectively sensitive to degeneration. In addition, revealing potential linked regulation of distinct heterochromatin modifications (e.g., H3K9me3 and H3K27me3) will provide a novel understanding of how mechanisms governing chromatin compaction dictates cell type specific gene expression. This is of particular significance in mDA- and 5HT-neurons, cells whose function is lost in several pathological conditions of sporadic nature, lacking obvoius genetic components in their aetiology.

We are focusing on the importance of Polycomb repressive complex 2 (PRC2)-mediated repression for maintaining chromatin structure gene expression, neuronal identity, neuronal population integrity and behavior. Our research integrates basal mechanistic research in transgenic mouse models with a focus on the role of PRC2-mediated gene repression in human mDA-neurons during normal aging and in PD-patients. In addition, unraveling these mechanisms may provide insights leading to novel ways to tackle mental and neurodegenerative disease.

The functional role of HIF2α in neuroblastoma

Neuroblastoma (NB) arises within the sympathetic nervous system and is the most frequent extracranial solid childhood cancer, exhibiting a high degree of clinical heterogeneity ranging from spontaneous regression to fatal progression. The quintessential NB oncogene is MYCN which is amplified in a large proportion of high-risk cases, of which less than 50% survive. The disease also presents itself with considerable intratumor heterogeneity of which sympathetic noradrenergic (NORAD) and mesenchymal (MES) cells are two identified entities. NORAD cells are characterized by expression of typical NORAD lineage determinants such as PHOX2B whereas MES cells express mesenchymal markers, e.g., VIM. The MES cells have been reported to have increased resistance to chemotherapy and recent studies have described how NB cells transit between these two states.

Retinoic acid (RA) is a strong driver of neuronal differentiation and used as adjuvant therapy for high-risk NB patients. However, efficacy is limited, and outcome often remains fatal. The heterogeneous response to RA is reflected in NB cell lines, some respond with cell cycle exit and differentiation whereas several other are non-responsive and continue to proliferate. We have previously showed that combined treatment with the DNA demethylating agent, 5-Aza-dC, and RA promoted differentiation and inhibited tumor growth of otherwise RA-resistant NB cells. This response was associated with an increase of the hypoxia inducible factor, HIF2α encoded by the EPAS1 gene. HIF2α has previously been proposed to be a NB oncogene, something that our and other studies question.

We are addressing the functional role of HIF2α in NB with a particular focus on if HIF2α is a negative regulator of the NB oncogene MYCN and MYCN target genes. In addition, we are exploring whether HIF2α is required for NB cells to respond to differentiation inducing therapy, e.g., RA, as well as whether EPAS1/HIF2α is a determinant of the NORAD vs. MES state.

Selected Publications

Toskas, K., Yaghmaeian-Salmani, B., J., Skiteva, O., Paslawski, W., Gilberg, L., Skara, V., Antoniou, I., Södersten, E., Svenningsson, P., Chergui, K., Ringnér, M., Perlmann, T, and Holmberg, J.^§ (2022). PRC2-mediated repression is essential to maintain identity and function of differentiated dopaminergic and serotonergic neurons Science Advances 2022 Aug 26;8(34):eabo1543 https://doi.org/10.1126/sciadv.abo1543.

Shi, Y^§., Yuan, J., Rraklli, V., Maxymovitz, E., Cipullo, M., Liu, M., Li, S., Westerlund, I., Bedoya Reina, O., Bullova, P., Rorbach, J., Juhlin, C.C., Stenman, A., Larsson, C., Kogner, P., O’Sullivan, M.J., Schlisio, S. and Holmberg, J.^§ (2021). Aberrant splicing in neuroblastoma generates RNA-Fusion transcripts and provides vulnerability to spliceosome inhibitors. Nucleic Acids Research. 2021 Mar 18;49(5):2509-2521.

Westerlund, I., Shi, Y. and Holmberg, J^§. (2019). EPAS1/HIF2α correlates with features of low-risk neuroblastoma and with adrenal chromaffin cell differentiation during sympathoadrenal development. Biochem Biophys Res Commun. 2019 Jan 22; 508(4):1233- 1239.

Södersten, E^§., Toskas, K., Rraklli, V., Tiklova, K., Björklund, Å., Ringnér, M., Perlmann, T. and Holmberg, J^§. (2018). A comprehensive map coupling histone modifications with gene regulation in adult dopaminergic and serotonergic neurons. Nat Commun. 2018 Mar 26;9(1):1226.

Westerlund, I., Shi, Y., Toskas, K., Fell, SM., Li, S., Södersten, E., Schlisio, S. and Holmberg, J^§. (2017). Reply to Mohlin et al.: High levels of EPAS1 are closely associated with key features of low-risk neuroblastoma. Proc Natl Acad Sci U S A. 2017 Dec 19;114(51):E10859-E10860.

Westerlund, I., Shi, Y., Toskas, K., Fell, SM., Li, S., Surova, O., Södersten, E., Kogner, P., Nyman, U., Schlisio, S. and Holmberg, J^§. (2017). Combined epigenetic and differentiation based treatment inhibits tumor growth and links HIF2α to tumor suppression. Proc Natl Acad Sci U S A. 2017 Jul 25;114(30):E6137-E6146.

Rraklli, V., Södersten, E., Nyman, U., Hagey, DW. and Holmberg, J. ^§ (2016). Elevated levels of ZAC1 disrupt neurogenesis and promote rapid in vivo reprogramming. Stem Cell Res. 2016 Jan;16(1):1-9.

Egan, CM., Nyman, U., Skotte, J., Streubel, G., Turner, S., O'Connell, DJ., Rraklli, V., Dolan, MJ., Chadderton, N., Hansen, K., Farrar, GJ., Helin, K., Holmberg, J.^§ and Bracken, AP^§ (2013). CHD5 is required for neurogenesis and has a dual role in facilitating gene expression and    Polycomb    gene    repression.    Developmental    Cell, 26(3), 223-226.

Holmberg, J^§. and Perlmann, T.^§ (2012). Maintaining differentiated cell identities. Nature Review Genetics, 13(6), 429-439.