Abstract B015: Exploiting polyamine driven translational reprogramming as a vulnerability in neuroblastoma

Abstract

MYC genes drive neuroblastoma (NB) oncogenesis and redirect metabolism to support the synthesis of cellular building blocks, including proteins and polyamines (PAs). Our prior work in complementary murine models demonstrated that PA depletion extends survival, with the benefit correlating with the extent of PA depletion. Combined PA substrate restriction (dietary or pharmacologic arginine depletion) with high-dose DFMO (α-difluoromethylornithine), a covalent inhibitor of ODC1, the rate-limiting enzyme in PA synthesis, markedly enhanced anti-tumor activity. In PA-depleted tumors, multiomic analyses revealed a profound translation defect characterized by ribosome stalling at codons with adenosine in the third position (A-end codons). This novel PA-dependent translational reprogramming leads to loss of cell-cycle proteins, permissiveness for neuronal differentiation, and tumor regression. However, the mechanisms by which PA availability is coupled to translational control and tumor cell death remain incompletely defined. To mechanistically interrogate this response, we developed an in vitro model of PA depletion that recapitulates key features of the in vivo system. During DFMO exposure, we find that NB cells are particularly sensitive to arginine (PA substrate) deprivation. Growth suppression is rescued by exogenous polyamines and by pathway substrate repletion, supporting on-target effects rather than nonspecific nutrient toxicity. PA depletion in vivo and in vitro disrupts eIF5A hypusination and promotes the accumulation of inactive K47-acetylated eIF5A. While not directly linked to A-end stalling, this biomarker of PA depletion, coupled with real-time cell analysis, provides a kinetic framework to define the onset of growth arrest and select time points for biochemical and translational profiling before secondary cell death pathways initiate. This system reveals that NB cell lines with high versus low MYC activity display distinct sensitivity and growth kinetics during PA depletion, suggesting that MYC-driven translational demand influences vulnerability to disrupted PA homeostasis. Biochemically, PA depletion activates stress pathways associated with impaired elongation, including p38 and eIF2α-associated translational stress. These changes occur downstream of ZAKα phosphorylation, consistent with a model in which PA deprivation impairs decoding or elongation at A-end codons, leading to collided ribosomes and activation of ribotoxic and integrated stress signaling. This work develops a physiologically relevant in vitro platform for dissecting PA-dependent translational control in neuroblastoma and provides a framework for understanding how PA depletion reprograms translation in MYC-driven tumors to identify translational stress responses that may be exploited therapeutically.