AR-V7 utilizes a noncanonical nuclear localization signal to sustain androgen-independent nuclear import and signaling

Prostate cancer is the second leading cause of cancer death and the most common non-cutaneous malignancy in men in the United States. Its initiation and progression are regulated by the oncogenic transcriptional program of the androgen receptor (AR). Standard-of-care therapies inhibit AR signaling by blocking androgen synthesis or competing with androgen binding at the ligand-binding domain, thereby preventing AR nuclear import and stalling tumor growth. However, inevitable therapeutic resistance enables progression to lethal metastatic castration-resistant prostate cancer (mCRPC). This is largely driven by the expression of truncated, alternatively spliced variants of AR that localize to the nucleus independently of androgen binding. Among these, AR splice variant 7 (AR-V7) is the most prevalent, detectable in approximately 75% of mCRPC patients. AR-V7 arises through alternative splicing and excludes the ligand-binding domain while incorporating a unique 16-amino acid cryptic exon (CE3) at its C-terminus. Although nuclear localization is essential for AR-V7 to drive transcriptional programs that support tumor progression, the mechanism underlying its androgen-independent nuclear import has remained undefined. Here, we report newly generated mechanistic data defining the molecular basis of AR-V7 nuclear trafficking. Using systematic truncation and alanine-scanning mutagenesis, we mapped a previously unrecognized non-canonical, composite NLS spanning basic residues R607 to P643 in the DNA-binding and CE3 domains. To identify the nuclear transport receptors and trafficking machinery that mediate AR-V7 nuclear import, we employed μMap, a novel antibody-based photocatalytic proximity labeling technology coupled with mass spectrometry (MS), which enables highly specific, noninvasive analysis of dynamic protein-protein interactions under near-physiological conditions. Using this approach, we performed comparative proteomic and transcriptomic analyses of full-length and nuclear-localized AR-V7 relative to a newly engineered NLS-deficient AR-V7 mutant that is retained in the cytoplasm. This direct nuclear versus cytoplasmic comparison of AR-V7 enabled the selective identification of compartment-specific AR-V7 interactors. Together, these findings define a noncanonical NLS that is required for androgen-independent AR-V7 nuclear import and establish an experimentally tractable cytoplasmic AR-V7 platform that reveals distinct nuclear and cytosolic interaction networks. These data uncover previously unrecognized non-transcriptional functions of AR-V7 and nominate nuclear import machinery as a therapeutic vulnerability in lethal prostate cancer. By shifting therapeutic focus from the undruggable receptor itself to its nuclear import machinery and associated trafficking dependencies, these findings reveal new, actionable vulnerabilities for targeting AR-V7-driven lethal prostate cancer.