The FOXO1^TAD-p300-Mediated Lineage Survival Transcription Program Is a Targetable Dependency of Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is a non-Hodgkin's lymphoma that remains incurable due to the development of resistance to immuno-chemotherapy and targeted agents. There is a significant unmet need to develop new therapeutic strategies to bring forward with curative potential for MCL patients.

Targeting lineage vulnerabilities driven by specific ligand-activated transcription factors has proven an effective intervention in human cancers. However, targeting non-liganded transcription factors that were traditionally viewed as undruggable has been a challenge. Through conducting an unbiased domain-focused CRISPR-Cas9 screen against 1,434 transcription factors, we identified the transcription program dependency on Forkhead Box O1 (FOXO1), EBF1, PAX5, and IRF4 for MCL cell survival and growth. Integrated chromatin immunoprecipitation sequencing (ChIP-seq) and transcriptional network reconstruction analysis further revealed that the four transcription factors act together to orchestrate B cell lineage transcriptional program and MCL cell survival. Our hierarchical interaction analysis showed that FOXO1 acts upstream in the regulatory hierarchy of B cell lineage survival networks that facilitates the access of other B cell lineage transcription factors to chromatin and stabilizes the complexes that support MCL proliferation. Along this line, we showed that enforced expression of FOXO1 in myeloid leukemia cells induces B-cell specific gene expression and lymphoid transdifferentiation. Mechanistically, we demonstrated through tiling CRISPR-Cas9 scanning screens combined with FOXO paralog domain swapping assays that the c-terminal transactivation domain of FOXO1 (FOXO1^TAD) is specifically required for MCL cell viability.

To determine the mechanism of FOXO1^TAD action, we performed TurboID-tagging and proximity labeling-based identification of transcriptional coregulators of FOXO1 in MCL cells. Through conducting a CRISPR-Cas9 screen against 455 epigenetic regulators, including those discovered through proximity labeling of FOXO1, we identified the histone acetyltransferase p300 (EP300), but not its close paralog, CBP (CREBBP), as a selective dependency in MCL cell lines. Mechanistically, p300 interacts with FOXO1^TAD and activates FOXO1-dependent transcription by acetylating and stabilizing MCL lineage survival transcription factors.

Given our finding of FOXO1 as a lineage-selective survival factor of MCL, we next explored the possibility of disrupting FOXO1^TAD-p300 interaction by small molecule inhibitors. We screened a library of 144 FOXO1 inhibitor candidates and identified cpd10 as a potent and selective FOXO1 inhibitor (IC₅₀=76 ×/÷ 1.7. nM) which works through interfering with FOXO1^TAD-p300 interaction. We demonstrated that cpd10 disrupts FOXO1 interaction with p300 to suppress H3K27 acetylation on FOXO1-occupied promoters and enhancers. Our mechanistic studies revealed that cpd10 treatment suppresses pro-survival B cell receptor (BCR)-PI3K-AKT signaling cascades in MCL cells. Cpd10 treatment induced a robust cytotoxic response in cultured primary MCL cells and cell lines. Using in vivo xenograft and preclinical models of MCL, we found that cpd10 was well tolerated with an on-target effect on MCL cells. Most notably, cpd10 treatment suppressed MCL progression in vivo and prolonged the survival of the treated group. Our findings establish FOXO1^TAD-p300-mediated transcription dependency as a MCL lineage-survival mechanism that can be exploited as a therapeutic target.

Baiocchi:Atara Biotherapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; eLife (Journal): Other: Editorial board; Viracta Therapeutics: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; CODIAK Biosciences: Research Funding. Elemento:AstraZeneca: Research Funding; Eli Lilly: Research Funding; One Three Biotech: Consultancy, Current equity holder in publicly-traded company; Owkin: Consultancy, Current equity holder in publicly-traded company; Freenome: Consultancy, Other: Current equity holder in a privately-held company; Johnson and Johnson: Research Funding; Volastra Therapeutics: Consultancy, Current equity holder in publicly-traded company, Research Funding; Champions Oncology: Consultancy; Janssen: Research Funding.