Protein hydroxylation affects protein stability, activity, and interactome, therefore contributing to various diseases including cancers. However, the transiency of the hydroxylation reaction hinders the identification of hydroxylase substrates. By developing an enzyme-substrate trapping strategy coupled with TAP-TAG or orthogonal GST- purification followed by mass spectrometry, we identify adenylosuccinate lyase (ADSL) as an EglN2 hydroxylase substrate in triple negative breast cancer (TNBC). ADSL expression is higher in TNBC than other breast cancer subtypes or normal breast tissues. ADSL knockout impairs TNBC cell proliferation and invasiveness in vitro and in vivo. An integrated transcriptomics and metabolomics analysis reveals that ADSL activates the oncogenic cMYC pathway by regulating cMYC protein level via a mechanism requiring ADSL proline 24 hydroxylation. Hydroxylation-proficient ADSL, by affecting adenosine levels, represses the expression of the long non-coding RNA MIR22HG, thus upregulating cMYC protein level. Our findings highlight the role of ADSL hydroxylation in controlling cMYC and TNBC tumorigenesis.

Cancer Research UK Edinburgh Centre IGMM University of Edinburgh Edinburgh EH4 2XR UK

Department of Biochemistry and Biophysics University of North Carolina Chapel Hill NC 27599 USA

Department of Clinical Genetics Maastricht University Medical Centre Maastricht The Netherlands

Department of Genetics Neuroscience Center University of North Carolina Chapel Hill NC 27599 USA

Department of Pathology and Laboratory Medicine University of North Carolina Chapel Hill NC 27599 USA

Department of Pathology UT Southwestern Medical Center Dallas TX 75390 USA

Department of Pediatrics School of Medicine Emory University Atlanta GA 30322 USA

Department of Pharmacology and Cancer Biology Duke University School of Medicine Durham NC 27710 USA

Department of Pharmacology University of North Carolina Chapel Hill NC 27599 USA

Lineberger Comprehensive Cancer Center University of North Carolina School of Medicine Chapel Hill NC 27599 USA

Research Unit for Rare Diseases Department of Pediatrics and Adolescent Medicine 1st Faculty of Medicine Charles University and General University Hospital Prague Prague Czechia

UNC Neuroscience Center Carolina Institute for Developmental Disabilities University of North Carolina Chapel Hill NC 27599 USA

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Pathway-specific effects of ADSL deficiency on neurodevelopment