The MYCN/Aurora-A/CDK12 axis as a critical target in MYCN amplified neuroblastoma

Neuroblastoma is the most common extracranial solid tumor of early childhood. Tumors driven by amplification of the oncogene MYCN exhibit aggressive clinical behavior and poor prognosis despite multimodal therapy. Previous in vitro studies have shown that complex formation between MYCN and the Aurora-A kinase stabilizes MYCN while also activating Aurora-A’s kinase activity. This allows high MYCN expression, uncoupled from intrinsic control mechanisms such as the cell cycle or growth factor signaling. The interaction of MYCN and Aurora-A occurs primarily in S phase, consistent with the critical role of MYCN-activated Aurora-A in S phase progression and protection from transcription-replication conflicts (TRCs) in MYCN amplified neuroblastoma cells. However, the exact mechanism by which the MYCN/Aurora-A complex coordinates transcription and replication and the critical substrates involved in S phase progression remained unknown. In the first part of this thesis, I demonstrate that MYCN-dependent Aurora-A functions as a cyclindependent kinase (CDK) activating kinase (CAK) for CDK12 and that the activation of CDK12 by Aurora-A regulates the phosphorylation of threonine 4 (T4) in the C-terminal domain (CTD) of RNA polymerase II (RNAPII). T4 phosphorylation is required for the recruitment of transcription termination complexes to RNAPII, which prevents TRCs. Enhanced crosslinking and immunoprecipitation demonstrated that Aurora-A is an RNA-binding protein (RBP), localizing predominantly to splice sites on nascent RNA. Complex formation between MYCN and Aurora-A displaces Aurora-A from RNA, enhancing CDK12 phosphorylation by Aurora-A. Further, combining Aurora-A inhibition with CDK12 degradation potently suppressed the growth of MYCN amplified neuroblastoma cells but not MYCN non-amplified cells and led to tumor control in first in vivo studies in MYCN amplified patient-derived xenografts (PDX). Collectively, this first part presents a novel transcription termination pathway that enables the MYCN/Aurora-A complex to cope with transcription and replication stress, thereby ensuring genomic stability in neuroblastoma cells. These findings emphasize the role of gene expression-independent functions of MYC proteins in tumorigenesis. In the second part of this thesis, I proposed a novel treatment strategy for targeting MYCN amplified neuroblastomas. The combination of Aurora-A and ATR inhibitors resulted in infiltration of CD45+ cells into the tumor tissue and tumor regression in TH-MYCN mice. However, I confirmed that T cell function is impacted by Aurora-A inhibition. Therefore, I investigated a novel strategy to combine immunotherapy with inhibitor treatment by using inhibitor-resistant T cells. This approach resulted in T cells capable of proliferating upon inhibitor treatment, enabling their use to maximize on-target tumor effects.