Tumor suppressor-like mutations mark a dosage-constrained dependency in leukemia

Abstract Cancer genes are classically categorized as oncogenes or tumor suppressors based on whether gain or loss of function promotes malignancy. Here we show that oncogene-imposed dosage constraints can produce mutations that mimic tumor suppressor loss yet mark essential genes. In acute promyelocytic leukemia (APL), the transcription factor EBF3 gene harbors recurrent heterozygous loss-of-function mutations, a tumor suppressor signature, and is essential for APL survival. We resolve this paradox by showing that while PML::RARA, the fusion oncoprotein defining APL, drives EBF3 expression, excess EBF3 suppresses proliferation. Patient mutations reduce EBF3 activity through heterozygous inactivation, optimizing its expression within a corridor bounded by fitness constraints at both extremes. PML::RARA activates EBF3 via a p300-dependent intronic enhancer, and p300 inhibition exploits this constraint by suppressing EBF3 below the viability threshold. Our findings reveal that oncogenes can impose dosage constraints that drive adaptive mutations conventionally misclassified as tumor suppressor loss, with implications for interpreting cancer genomes.