Prolyl-tRNA synthetase inhibition disrupts oncogenic translation in T-cell acute lymphoblastic leukemia

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive blood cancer marked by the abnormal growth of immature thymocytes. 5-year survival rate in T-ALL is about 85% in children and less than 50% in adults. Relapsed/refractory T-ALL remains a major clinical challenge, with long-term remission rates falling below 50% in children and <10% in adults. Activating mutations in NOTCH1 are present in ~70% of T-ALL cases. However, attempts to therapeutically target NOTCH1 signaling, including with γ-secretase inhibitors, have been limited by dose-limiting gastrointestinal toxicity, underscoring the need for alternative downstream targets. We previously demonstrated an extensive oncogenic NOTCH1-dependent rewiring of global tRNA biogenesis and demonstrated the importance of aberrant tRNA deregulation in the disease pathogenesis of T-ALL. In this study, we evaluated specific tRNA pathways regulated by NOTCH1 signaling, which play a significant role in T-ALL pathogenesis. We identified several tRNA-Pro genes and the associated bifunctional glutamyl-prolyl-tRNA synthetase (EPRS1) to be transcriptionally sensitive to NOTCH1 modulation. These genes exhibit reduced expression upon NOTCH1 inhibition and are rapidly restored upon inhibitor withdrawal. NOTCH1 inhibition using a small molecule inhibitor CB-103 significantly reduced expression of EPRS1 mRNA and protein levelsin T-ALL cell lines harboring activating NOTCH1 mutations, further confirming EPRS1 as a downstream target of NOTCH1 signaling. Moreover, expression of EPRS1 and tRNA-Pro was significantly upregulated in primary T-ALL samples relative to thymocytes and mature T-cell subsets from healthy human donors. Functional interrogation using CRISPR-Cas9 mediated knock out EPRS1, and CRISPR interference (CRISPRi) mediated depletion of tRNA-Pro reduced cell fitness and viability, underscoring the critical role of this pathway in T-ALL proliferation and survival. The pyrazinamide analog NCP26, which targets the ProRS aminoacylation activity of EPRS1, has demonstrated potent anti-tumor activity in preclinical models of multiple myeloma. In this study, NCP26 significantly inhibited proliferation and induced apoptosis in T-ALL cell lines and patient derived xenograft (PDX) samples. Notably, T cells from healthy donors showed minimal response, indicating low systemic toxicity. Global proteomic analysis using Tandem Mass Tag (TMT) labelling following NCP26 treatment revealed significant downregulation of several proline rich and/or polyproline motif-containing oncogenic proteins including c-MYC, HES1 and CCND3, which are known to play pivotal roles in T-ALL disease progression. No significant changes in the mRNA levels were detected for these genes from the RNA-seq data, suggesting a reduction in translation efficiency mediated by charged proline tRNA availability. To validate Eprs1 as a therapeutic target in T-ALL, we generated an inducible Eprs1 conditional knockout miceEprs1fl/+ ubc cre-ERT2to assess the effects of Eprs1 deletion on established, NOTCH1-induced leukemias in vivo. Hematopoietic stem and progenitor cells (HSPCs) from C57BL/6 mice were transduced with constitutively active NOTCH1 (ICN) and transplanted into lethally irradiated wild-type recipients to induce primary leukemia. Primary leukemia was then secondary transplanted in wildtype recipients, which were treated with either vehicle or tamoxifen to induce Cre-mediated deletion of Eprs1. Tamoxifen-induced heterozygous loss of Eprs1 delayed disease progression and significantly increased overall survival in this murine model. Eprs1+/-heterozygous mice were fertile and had normal T cell development. These studies highlight a potential dosage dependency and a therapeutic window to target Eprs1 in T-ALL blasts without observable systemic toxicity. Collectively, our study reveals that upregulated proline tRNA biogenesis directly contributes to the maintenance of key oncogenic gene expression programs. Importantly, pharmacologic inhibition of the ProRS activity of Eprs1 impaired leukemic growth in vitro and patient-derived xenograft (PDX) models. These findings were corroborated in vivo, where hemizygous Eprs1 deletion delayed leukemia progression and extended survival. Together, our findings identify enhanced proline tRNA biogenesis as a critical adaptation in T-ALL pathogenesis and provide a strong molecular and preclinical rationale for therapeutically targeting ProRS in T-ALL.