Exploration of novel therapeutic targets in neuroblastoma

Neuroblastoma is a malignancy of the sympathetic nervous system occurring in early childhood. It accounts for approximately 6% of all childhood cancers and is a highly heterogeneous disease. While patients with low- and intermediate-risk disease have a good prognosis, high-risk disease remains a therapeutic challenge. High-risk neuroblastoma often presents with metastatic spread and therapy resistance already at diagnosis. Consequently, there is a great need for improved treatment options to improve patient survival but also to reduce side effects and chronic health conditions. Human neuroblastomas bear recurrent genetic alternations in neuritogenesis-associated genes such as teneurins (TENM1- TENM4) and components of Rho/Rho-associated Coiled-Coil Kinase (ROCK1 and ROCK2) signaling, pathways associated with differentiation and proliferation. This thesis aims to identify and characterize novel targets and therapeutic approaches for neuroblastoma and to investigate the microenvironmental factors.

In Paper I, the efficacy of the ROCK1/2 inhibitor RKI-1447 was assessed in vitro and in vivo using preclinical neuroblastoma models. Treatment with RKI-1447 repressed tumor growth by inhibiting N-MYC in vitro and in the transgenic TH- MYCN mouse model. A combinational drug screen revealed RKI-1447 to be a beneficial combinational partner for bromodomain and extra terminal domain (BET) inhibitors. Synergistic effects between RKI-1447 and the BET inhibitor ABBV- 075 (Mivebresib) were confirmed in different neuroblastoma models, including a zebrafish-embryo xenograft model. Furthermore, our studies demonstrated that BET inhibitor treatment increased the phosphorylation of ROCK targets, which could be prevented by combinational treatment, proposing a possible mechanism of action.

With the aim of gaining insight into neuroblastoma bone marrow metastases, the bone marrow microenvironment of neuroblastoma patients with and without metastases was compared, using single-cell RNA-sequencing, in Paper II. The results indicated a niche-dependent remodeling of immune cell populations and shifted transcriptomic profiles. Several immune cell populations were enriched in the metastatic bone marrow, including tumor-associated neutrophils, macrophages, and exhausted T cells. Additionally, an increased number of regulatory T cells as well as a decreased number of B cells was observed. Furthermore, this work identified potential therapeutic interventions, including markers of poor clinical prognosis occurring in malignant cells, and communications pathways between tumor and immune cells.

Paper III explores the function of teneurins (TENMs) in neuroblastoma tumorigenesis. Transient knockdown of TENM4 significantly decreased proliferation in all investigated neuroblastoma cell lines, in contrast to knockdown of TENM1, TENM2 and TENM3. To verify these results, we generated TENM4-/- clones from the neuroblastoma cell line SK-N-BE(2) using CRISPR-Cas9; the clones demonstrated a neuronal differentiation-like morphology with reduced clonogenic capacity and proliferation compared to wild-type cells. Gene set enrichment analysis of RNA-sequencing data from neuroblastoma cells with TENM4-knockdown identified key components including induced differentiation, inhibited mTOR signaling, and epithelial to mesenchymal transition, as TENM4 targets. TENM4-/- cells did not lead to tumor formation when grafted into nude mice as opposed to wild-type cells. Furthermore, using a CRISPR-Cas13d-model with a TENM4 knockdown under a doxycycline-inducible promoter, we observed a delay until tumors were established in mice receiving doxycycline, compared to the control counterparts. Finally, we detected a significantly higher number of TENM4-positive tumor cells in high-risk vs. non-high-risk and MYCN-amplified vs. non-MYCN-amplified human neuroblastomas.

This thesis covers various topics, from an explorative study searching for novel therapeutic targets in a common site of neuroblastoma metastasis, the bone marrow microenvironment, to interventional studies on novel molecular targets, using genetic inhibition or pharmacological drugs. I hope this research has increased our understanding of neuroblastoma and can support further studies on novel therapies in neuroblastoma.

List of scientific papers

I. Pepich A, Tümmler C, Ajamieh SA, Treis D, Boje AS, Vellema Q, Tsea I, Åkerlund E, Seashore-Ludlow B, Fard SS, Kogner P, Johnsen JI, Wickström M. The ROCK-1/2 inhibitor RKI-1447 blocks N-MYC, promotes cell death, and emerges as a synergistic partner for BET inhibitors in neuroblastoma. Cancer Letters. 2024 Nov 28;605: 217261. https://doi.org/10.1016/j.canlet.2024.217261

II. Mei S, Alchahin AM, Embaie BT, Gavriliuc IM, Verhoeven BM, Zhao T, Li X, Jeffries NE, Pepich A, Sarkar H, Olsen TK, Wickström M, Stenman J, Reina-Bedoya O, Kharchenko PV, Saylor PJ, Johnsen JI, Sykes DB, Kogner P, and Baryawno N. Single-cell analyses of metastatic bone marrow in human neuroblastoma reveals microenvironmental remodeling and metastatic signature. JCI Insight. 2024 Feb 15;9(6): e173337. https://doi.org/10.1172/jci.insight.173337

III. Andonova* T, Abu Ajamieh* S, Pepich A, Olsen TK, Tümmler C, Liu M, Thankaswamy-Kosalai S, Kanduri C, Djos A, Fransson S, Martinsson T, Baryawno N, Wilhem M, Näsman A, Kogner P, Johnsen JI, Wickström M. Inhibition of Teneurin 4 expression suppresses growth and induces differentiation in neuroblastoma. [Manuscript]
*These authors contributed equally to the manuscript and share primary authorship.