Sequential expression of lineage specific SOX factors drives differentiation in High-Risk Neuroblastoma

Authors

• D Banerjee
• BE Gryder
• Z Liu
• S Bagchi
• HC Chou
• S Sindiri
• J Khan
• CJ Thiele

Abstract

Neuroblastoma is a pediatric tumor of the peripheral sympathetic nervous system, where less than 50% high-risk cases survive. It arises from an inability of sympathoadrenal progenitors to exit a self-renewal program and terminally differentiate. However, retinoids (RA) can arrest NB growth and induce neural differentiation. A network of super enhancer(SE) driven transcription factors(TFs) underlies lineage identity and their dysregulation leads to cancer. We analyzed SE dynamics using H3K27ac ChIP-Seq in matched control (self-renewal) and RA treated NB cells[2,4,8 days(D)] to define core regulatory circuitries controlling self-renewal and differentiation. K-means clustering identified 4 temporal patterns of dynamically RA regulated SEs(n=940/1199). One cluster(n=254) decreasing with RA included TFs like MYCN, GATA3, ID2, SOX11, known to be involved in stem cell development and differentiation. The 3 other SE clusters showed sequential waves of activation at 2D(n=174), 4D(n=355) or 8D(n=157) and included TFs like GATA2, SOX4, and TWIST1 which are involved in regulation of neuron-projection. Under self-renewal conditions, SEs were maximally enriched for MYCN and SOX11 motifs, whereas with differentiation SEs were enriched for SOX4 motifs. The MYCN and SOX11 SEs disappeared with RA, corresponding to decreased expression. CRISPR-Cas9 screen of NB cell lines showed preferential dependencies on MYCN and SOX11. Inhibition of SOX11 led to 30% decreased cell growth without evidence of differentiation indicating SOX11 may be a key regulator of self-renewal. In RA activated clusters, increase in SE activity driving SOX4 was observed with increased SOX4 expression. The cis-interactions between promoter and SEs driving SOX11 and SOX4 were confirmed using 4C-seq. In a cohort of 498 NB patients, SOX11 is overexpressed in HR cases and associated with poor prognosis(p=6.582e-09), whereas SOX4 is overexpressed in more differentiated low-risk cases and is associated with good prognosis(p=6.188e-17), implicating SOX4 as a regulator of NB differentiation. Patients with low SOX11 and high SOX4 levels showed increased survival(p

Scientific Focus Area: Cancer Biology

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