Distinct transcriptomic programs and proximal AKT activation associated with AKT1 E17K and PIK3CA hotspot mutations in HR-positive/HER2-negative breast cancer Running title: Molecular differences between AKT1- and PIK3CA-mutant breast cancer

Background PIK3CA and AKT1 alterations are commonly grouped within the therapeutically relevant PI3K-AKT pathway, but whether they represent equivalent breast-cancer states remains uncertain. We compared molecular features associated with AKT1 E17K and recurrent PIK3CA hotspots. Methods METABRIC was the discovery cohort and TCGA-BRCA the independent validation cohort. Prespecified hotspots were AKT1 E17K and PIK3CA E542K, E545K, H1047R, or H1047L. Primary transcriptomic analyses were restricted to HR-positive/HER2-negative tumors and adjusted for age and PAM50 subtype. Differential expression, Hallmark/Reactome enrichment, genomic context, and TCGA reverse-phase protein array data were analyzed. Results AKT1 hotspots occurred in 79 of 2,433 METABRIC tumors (3.2%) and 19 of 816 TCGA tumors (2.3%); PIK3CA hotspots occurred in 733 (30.1%) and 210 (25.7%), respectively. The alterations were largely mutually exclusive. In adjusted HR-positive/HER2-negative analyses, 341 genes differed between AKT1-mutant and PIK3CA-mutant-only METABRIC tumors; 280 showed concordant TCGA directions and 53 achieved same-direction nominal replication. Nine pathways achieved FDR-controlled TCGA replication, including higher G2M, glycolysis, chromosome-segregation, and ER-Golgi transport and lower translation-elongation and nonsense-mediated-decay programs in AKT1-mutant tumors. TCGA RPPA showed a higher AKT activation score (0.98 SD; FDR = 0.0025), AKT pS473 (1.00 SD; FDR = 0.0107), and pT308 (0.90 SD; FDR = 0.0166), without differences in pan-AKT, aggregate AKT-substrate, or mTOR-effector scores. Conclusions AKT1 E17K and PIK3CA hotspots are associated with overlapping but non-equivalent molecular states. AKT1-mutant tumors show distinct transcriptional programs and preferential proximal AKT phosphorylation without uniform downstream mTOR activation. Confirmation in treatment-annotated and experimental cohorts is required.