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Abstract Background Asparagine depletion with asparaginase exemplifies the successful therapeutic exploitation of metabolic dependencies in acute lymphoblastic leukaemia (ALL). However, its clinical utility is frequently constrained by toxicity, resistance mechanisms, and heterogeneous responses; underscoring a need for more precise, stratified approaches to targeting metabolic vulnerabilities in ALL. Arginine auxotrophy is a metabolic limitation of cancers deficient in the enzyme argininosuccinate synthase (ASS1), that renders a dependence on extracellular arginine for survival. This trait is therapeutically exploitable with arginine depleting enzymes, most notably pegylated arginine deiminase (pegargiminase), which has proven clinical efficacy in arginine auxotrophic cancers. Here, we investigate arginine auxotrophy in adult B-ALL as a target for personalised metabolic therapy. Methods Molecular predictors of arginine auxotrophy were derived from publicly available transcriptomic and genomic data from over 800 adult patients (Gu et al. Nat. Genet. 2019, Kim et al. Nat. Genet. 2023). Pre-clinical drug evaluation was performed in Philadelphia-chromosome positive (Ph+) ALL cell line and primary tumour models in vitro, and with patient derived xenografts (PDXs) and the Arf-/-p185+ tyrosine kinase inhibitor (TKI) resistant murine Ph+ ALL cell line (Boulos et al. Blood 2011) in vivo. Results Ranked ASS1 expression varied significantly across B-ALL subtypes (p< 2.2×10⁻¹⁶), with lowest ranks in Ph+ and Ph-like cases, suggestive of arginine auxotrophy as a class-specific metabolic state. In ASS1-low Ph+ cases, we found a coordinated downregulation of other genes involved in amino acid biosynthesis (GPT2, PHGDH, PSAT1, PYCR1 and SHMT2), which was validated in other ASS1-low, non-Ph+ cases, consistent with a putative arginine auxotrophic gene signature and thus bona fide metabolic target within B-ALL. We further evaluated ASS1 expression as a function of copy number, relapse, cell of origin and molecular subtypes in Ph+ ALL. Low ASS1 expression correlated with both early-progenitor phenotype (p= 0.015) and poor-risk HBS1L gene deletions (p< 0.001), highlighting potential biomarkers for objective identification of arginine auxotrophy in the clinical setting. Pegargiminase robustly induced apoptosis in ASS1-low Ph+ cell lines (n=2, range 68.6-87.4% apoptotic cells), while ectopic ASS1 expression reduced inhibition by 41.4% (p< 0.001) and ASS1-high cell lines were intrinsically resistant; confirming ASS1-low as a functionally arginine auxotrophic state in Ph+ ALL. Susceptibility was recapitulated in 4/5 unselected primary Ph+ tumours ex vivo (mean cellular inhibition 45.5%, range 24.1–69.4%), with no-response only seen in a single case with markedly elevated ASS1 expression. In two independent Ph+ PDX models, pegargiminase monotherapy elicited marked in vivo anti-tumour responses in both bone marrow (proportional cytoreduction 84.7%, p= 0.029) and spleen (93.7%, p= 0.029), as well as significantly extending host survival (p= 0.048); consistent with a robust single-agent effect. To evaluate therapeutic potential in combination with TKI, we first used the Arf-/-p185+ cell line that has Il-7 inducible in vitro TKI resistance. The addition of pegargiminase to dasatinib overcame Il-7 driven TKI resistance, eliminating dasatinib-resistant cultures (91.2% relative cytoreduction, p< 0.001), and thus supporting a TKI-independent therapeutic effect. To reinforce this concept, we tested pegargiminase-TKI therapy in 5 primary samples and showed a consistent additive effect ex vivo when combined with dasatinib (8.5% inhibition increase, p= 0.034) and ponatinib (22.4% increase, p= 0.011). Finally, we tested combined pegargiminase-dasatinib in vivo using the Arf-/-p185+ cell line that rapidly evolves ABL1-mutation mediated resistance under TKI therapy. Compared to dasatinib alone, combined therapy induced a deeper response (p< 0.001), prevented on-treatment relapse, and prolonged mouse survival (p= 0.018); demonstrating activity against canonical TKI resistance. Conclusions Our study uncovers a novel, targetable metabolic vulnerability in Ph+ ALL. Pegargiminase demonstrates robust pre-clinical activity in arginine auxotrophic Ph+ ALL, including in the context of TKI resistance. Together, these findings provide a strong rationale for clinical evaluation of arginine depletion as a novel chemotherapy-light treatment paradigm for Ph+ ALL.