Abstract PR005: Mutant FOXO1 licenses MYC transformation of human germinal center B cells into dark-zone MYC-BCL2 high-grade B cell lymphomas

Abstract

High-grade B-cell lymphomas with MYC and BCL2 rearrangements (HGBCL-DH-BCL2) represent a clinically aggressive entity with poor prognosis and limited therapeutic options. Progress in this disease has been hindered by the lack of robust preclinical systems that faithfully recapitulate its molecular, cellular, and microenvironmental complexity. We recently identified recurrent silencing of the B cell receptor (BCR) in primary HGBCL-DH-BCL2, associated with transcriptional programs and immune landscapes reminiscent of germinal center (GC) dark-zone (DZ) B cells. HGBCL-DH-BCL2 are characterized by recurrent gain-of-function mutations in FOXO1 (most commonly FOXOM1L), frequent disruption of the TP53 pathway, and expression of stabilized CCND3 variants, together with deregulated BCL2 expression driven by t(14;18) and MYC activation through chromosomal rearrangements, often reinforced by stabilizing mutations such as MYCT58A. Here, we aimed to develop a stepwise genetic engineering platform in primary human GC B cells to model transformation toward HGBCL-DH-BCL2. Adapting a previously described retroviral transduction system (Caeser R. et al., 2021), GC B cells were engineered to constitutively express BCL2, BCL6, FOXO1M1L, dominant-negative TP53, and CCND3T283A, each coupled to a reporter gene to enable tracking of combinatorial genetic programs. Engineered GC B cells exhibited robust proliferation under GC light-zone mimicking conditions conferred by contact with YK6 feeder cells expressing membrane-bound CD40L and IL-21. However, transduced cells remained strictly dependent on CD40L/IL-21 signals for in vitro survival and expansion. Notably, the subsequent introduction of MYCT58A relieves the requirement for exogenous mitogenic support, allowing for sustained feeder-free in vitro expansion and rapid lymphoma formation upon transplantation into NSG mice. Tumors arising from these cells closely recapitulated the histological architecture, immunophenotype, and transcriptional features of HGBCL-DH-BCL2, including reduced surface BCR expression and enrichment of DZ-associated markers. Mechanistically, omission of FOXO1M1L from the initial lymphoma-reproducing genetic module (BCL2/BCL6/TP53DN/ CCND3T283A) abrogated later MYC-driven transformation. This result indicates that mutant FOXO1 acts as a critical prerequisite that licenses GC B cells to undergo MYC-driven, microenvironment-independent transformation, likely by enforcing a DZ-like cellular state permissive for oncogenic reprogramming. Collectively, we define a hierarchical model of lymphomagenesis in which FOXO1 activation precedes and enables MYC-driven transformation. This human GC B cell-based transduction system provides a powerful platform for dissecting the mechanisms underlying BCR silencing in HGBCL-DH-BCL2 and identifying novel therapeutic vulnerabilities. AI has been used to improve the clarity of the text and for the production of new text.