Therapeutic Targeting of HDAC3 in FOXO1-Mutant DLBCL

Histone deacetylase inhibitors have tremendous potential as cancer therapeutics, yet as single agents they have had limited success in the clinic outside of cutaneous T cell lymphoma and multiple myeloma. One of the major issues is toxicity, as the current compounds are broad-spectrum inhibitors that target all of the class 1 HDACs (HDAC1-3 and 8). Thus, using more selective inhibitors could have a major impact in the right genetic landscape. HDAC3-selective inhibitors are being developed and this deacetylase targets both histone and non-histone substrates in conjunction with its co-factors, NCOR or SMRT. NCOR or SMRT bind to and activate HDAC3, and this association is mediated by an inositol phosphate-4 (IP₄) molecule, suggesting that HDAC3 is a sensor of cellular metabolism in the same way that Sirtuins (Class 3 HDACs) sense NAD/NADH levels.

Diffuse large B cell lymphoma (DLBCL) is a particularly aggressive form of non-Hodgkin lymphoma (NHL), accounting for approximately 30% of new NHL diagnoses. Recurrent activating mutations in the transcription factor, FOXO1, are present in nearly 10% of DLBCLs at diagnosis. Importantly, these mutations are associated with a poor response to standard therapy, indicating a pressing need for the development of novel treatments for FOXO1 mutant disease. While direct therapeutic targeting of transcription factors has proven largely unsuccessful, indirect disruption of transcription factor function through the inhibition of associated chromatin-modifying enzymes has shown promise. Therefore, a mechanistic understanding of FOXO1 transcriptional complexes relevant for B cell function and DLBCL initiation and/or maintenance will provide insights for the development of new strategies for targeting FOXO1 mutant lymphoma.

The germinal center (GC) reaction is critical for the development of high affinity, class-switched antibodies and provides the cell of origin for the majority of diffuse large B cell lymphomas (DLBCLs). We used conditional Hdac3 deletion to assess the requirement of Hdac3 for germinal center formation in mice. In contrast to Bcl6, which is required for germinal center formation and recruits Hdac3, we found that Hdac3 -deficient cells gave rise to germinal centers, albeit in fewer numbers. RNA-seq analysis of Hdac3^-/- GC B cells revealed an upregulation of only a subset of Bcl6-regulated genes associated with terminal plasma cell differentiation (e.g. Prdm1, Xbp1) without any changes in other canonical Bcl6 target genes associated with DNA damage checkpoints (e.g. Trp53, Atr). In addition, deletion of Hdac3 resulted in a dramatic reduction in the numbers of GC dark zone B cells and an accumulation of cells as light zone centrocytes. This phenotype, as well as the associated gene expression changes, bore striking similarity to that found upon deletion of Foxo1 from germinal centers. Therefore, we performed further transcriptional analyses in germinal center-derived DLBCL to examine the coordination of FOXO1 and HDAC3 function at shared gene targets.

We demonstrate that HDAC3 and FOXO1 associate and disruption of HDAC3 function in DLBLC cells by a selective small molecule led to the upregulation of a FOXO1-regulated gene signature. ChIP-exo revealed localization of FOXO1 at critical BCL6:SMRT-bound enhancers. Furthermore, precision nuclear run-on transcription sequencing (PRO-seq) showed that inhibition of HDAC3 caused an increase in eRNA synthesis that was associated with increased H3K27ac at these enhancers and accompanied an increase in gene body transcription rates. Combined, studies in mouse germinal centers and DLBCL cell lines suggest that HDAC3 is critical for FOXO1-mediated transcriptional control. Therefore, we treated a panel of human DLBCL cell lines with an HDAC3-selective inhibitor, RGFP966, and examined its effect on cell growth and viability. While the response of DLBCL cells to HDAC3 inhibition was highly varied, those cell lines with FOXO1 mutations were the most sensitive to RGFP966 treatment. Thus, HDAC3 regulates FOXO1 gene targets within germinal centers and diffuse large B cell lymphoma, and HDAC3 is a promising therapeutic target for FOXO1-mutant disease.