Exploring inter- and intra- heterogeneity in childhood neuroblastoma and pheochromocytoma

Abstract: Neuroblastoma is the most common extra-cranial solid tumor of the sympathoadrenal cell lineage, which is a unique pediatric malignancy with remarkable inter- and intra-tumoral heterogeneity. In infants, neuroblastoma can regress spontaneously without treatment, while in older patients, neuroblastoma can develop with lethal progression with less than a 50% survival rate. Age at diagnosis is a clinically relevant prognosis factor. Chemoresistance in neuroblastoma patients has been associated with tumor cell plasticity and intratumoral heterogeneity. However, the causes of tumor cell plasticity and intratumoral heterogeneity are not well understood. In Paper I, we aim to investigate the in vivo function of KIF1Bβ in the sympathoadrenal system. KIF1Bβ locates on chromosome 1p36. Loss of heterozygosity at 1p36 strongly correlates with a poor prognosis of neuroblastoma. KIF1Bβ has been suggested to be a 1p36 tumor suppressor gene and demonstrated to be required for neuroblast developmental apoptosis. We showed that the loss of KIF1Bβ impairs neuroblast differentiation during development and causes misexpression of genes required for sympathoadrenal differentiation. We demonstrated that KIF1Bβ mediates neuroblast differentiation by transporting the NGF receptor TRKA. Transcriptomic analysis revealed that KIF1Bβ deficient sympathetic neuroblasts is similar to the profile in non-MYCN amplified high-risk neuroblastoma, independent of the loss of KIF1Bβ neighboring genes on 1p36. In Paper II, we found that loss of NF1 alone or in combination NF1 with KIF1Bβ in mouse sympathoadrenal lineage leads to neuroblastoma, pheochromocytoma, and composite tumors. In addition, mice with NF1 and KIF1Bβ loss have earlier tumor death onset and lower survival probability compared to NF1 loss alone. The single-cell sequencing of tumors and embryonic(E17) adrenal medulla showed a remarkable heterogeneity of chromaffin cells and neuroblasts. Importantly, we observed abundant neuroblasts born in the adrenal medulla in the E17 embryo. Furthermore, computational analysis reveals a cell state transition from chromaffin cells to neuroblasts in embryonic and pre-malignant stages. The chromaffin neuroblasts transition has also been observed in a three-segment structure in which chromaffin cells break through the cortex, suggesting that chromaffin cells acquire neuroblast signature and continue to form neuroblastoma, pheochromocytoma and composite tumors. In Paper III, we explored the inter- and intra-tumoral heterogeneity in human neuroblastoma and why age at diagnosis is one of the important prognostic factors. We analyzed the singlenuclei transcriptomes of human healthy fetal and postnatal adrenal glands and primary neuroblastomas from different stages and risk groups. We found two disease entities with varying signatures of cells in low and high-risk neuroblastoma. Notably, the transcriptome of high-risk neuroblastoma resembles a TRKB+ cholinergic progenitor cell population characterized specifically in the human postnatal adrenal glands. Thus, our study reveals two cellular identities reflecting the clinical heterogeneity of neuroblastoma tumors.