No abstract

Activated interleukin-7 receptor signaling drives B-cell acute lymphoblastic leukemia in mice.

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.

Chimeric antigen receptor (CAR) T cell immunotherapies targeting CD19 or CD22 induce remissions in the majority of patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL), although relapse due to target antigen loss or downregulation has emerged as a major clinical dilemma. Accordingly, great interest exists in developing CAR T cells directed against alternative leukemia cell surface antigens that may help to overcome immunotherapeutic resistance. The fms-like tyrosine kinase 3 receptor (FLT3) is constitutively activated via FLT3 mutation in acute myeloid leukemia (AML) or wild-type FLT3 overexpression in KMT2A (lysine-specific methyltransferase 2A)-rearranged B-acute lymphoblastic leukemia (ALL), which are associated with poor clinical outcomes in children and adults. We developed monovalent FLT3-targeted CAR T cells (FLT3CART) and bispecific CD19xFLT3CART and assessed their anti-leukemia activity in preclinical models of FLT3-mutant AML and KMT2Arearranged infant ALL. We report robust in vitro FLT3CART-induced cytokine production and cytotoxicity against AML and ALL cell lines with minimal cross-reactivity against normal hematopoietic and non-hematopoietic tissues. We also observed potent in vivo inhibition of leukemia proliferation in xenograft models of both FLT3-mutant AML and KMT2A-rearranged ALL, including a post-tisagenlecleucel ALL-to-AML lineage switch patient-derived xenograft model pairing. We further demonstrate significant in vitro and in vivo activity of bispecific CD19xFLT3CART against KMT2A-rearranged ALL and posit that this additional approach might also diminish potential antigen escape in these high-risk leukemias. Our preclinical data credential FLT3CART as a highly effective immunotherapeutic strategy for both FLT3-mutant AML and KMT2A-R ALL that is poised for further investigation and clinical translation.