Alternative splicing of its 5' untranslated region controls CD20 mRNA translation and enables resistance to CD20-directed immunotherapies

CD20-directed immunotherapies, such as rituximab, are mainstays of clinical management of mature B-cell neoplasms, previously known as non-Hodgkin lymphomas (NHL). More recently, the bispecific CD20/CD3 antibody mosunetuzumab was approved in the EU and US for treatment of relapsed or refractory follicular lymphoma (FL). The clinical efficacy of these CD20-directed therapies is limited by instances of intrinsic or acquired resistance, frequently mediated by complete or partial loss of CD20 expression. For example, in most chronic lymphocytic leukemia (CLL) at diagnosis and in a subset of post-mosunetuzumab FL relapses, CD20 protein levels are inexplicably low, despite near normal levels of CD20 mRNA and no detectable CD20 genetic variants. Here, we show that in both normal and malignant B-cells, CD20 down-regulation commonly occurs via alternative splicing in its 5’ UTR. This event generates an extended 5’ UTR isoform (V1) with a stem-loop structure and upstream open reading frames (uORFs), which cooperatively inhibit translation. This translation-deficient V1 was abundant in most CLL, in a CD20-negative post-mosunetuzumab FL relapse, and in a subset of diffuse large B-cell lymphoma (DLBCL) where it correlated with low CD20 protein levels. In a panel of B-cell lines, knockout of the Sam68/KHDRBS1 splicing factor with multiple putative binding sites in V1 shifted CD20 splicing toward translation-competent variants and increased CD20 levels. Even more profound effects on CD20 expression were observed by modulating CD20 splicing with Morpholino oligomers, which led to enhanced rituximab-mediated cytotoxicity. Thus, this widespread splicing-mediated mechanism of CD20 antigen loss potentially could be targeted to enhance CD20-directed immunotherapies.