Combination Epigenetic Therapy Using YF2, a First-in-Class Histone Acetyltransferase Activator, Restores Immunogenicity in Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma. Molecular studies have identified epigenetic dysregulation as a key driver in germinal center (GC)-derived DLBCL. Mutations in CREBBP/p300 and EZH2 often co-occur and lead to transcriptional repression and decreased immunogenicity as demonstrated by reduced major histocompatibility complex (MHC) expression. Our lab previously reported that YF2, a first-in-class histone acetyltransferase activator discovered at Columbia University, induces cytotoxicity in both cell and murine models of DLBCL. Other epigenetic therapies, such as tazemetostat and belinostat, are approved for use in follicular and T-cell lymphoma, respectively, but have limited single agent activity in DLBCL. We hypothesize that treatment with YF2 in combination with tazemetostat or belinostat increases transcriptional activity and restores tumor immunogenicity in CBP/p300- and EZH2-mutated DLBCL.

We utilized several DLBCL cell lines to assess YF2 combination therapy: OCI-Ly7 (CBP/p300 ^WT, EZH2 ^WT), SU-DHL-4 (CBP/p300 ^WT, EZH2 ^GOF), A20 (CBP/p300 ^-/-, EZH2 ^WT), and SU-DHL-6 (CBP/p300 ^-/-, EZH2 ^GOF). Cells were treated on day 0 with single agents or with YF2 in combination with either tazemetostat (YF2 + taz) or belinostat (YF2 + bel), retreated on day 3, and collected on day 6 for flow cytometry (n = 4) and western blot (n = 2) analyses. MHC class I/II and β₂ microglobulin (B2M) expression were measured as markers of immunogenicity. To preliminarily assess YF2 combination therapy in vivo, immunocompetent BALB/c mice were engrafted with syngeneic A20 cells, immunized, and segregated (n = 2-4) into treatment groups: control (saline), tazemetostat (300 mg/kg twice daily x 28 days), belinostat (40 mg/kg once daily x 7 days), YF2 (50 mg/kg once daily x 28 days), YF2 + taz, and YF2 + bel. Blood and spleen samples were collected on day 29 to measure T- and B-cell activity via flow cytometry. All fold changes are expressed relative to the corresponding control.

Combination therapy generally increased MHC-I, -II, and B2M expression across all cell lines as measured via flow cytometry. In double-mutated SU-DHL-6 cells, MHC-I and -II expression increased 2.0- ( P < 0.01) and 3.1-fold ( P = 0.04), respectively, following YF2 + taz treatment, and increased 1.3- ( P = 0.06) and 4.5-fold ( P < 0.01) after YF2 + bel ( Figure 1). Expression of B2M increased 2.3-fold ( P = 0.01) with YF2 + taz and 1.6-fold ( P = 0.07) with YF2 + bel. Interestingly, YF2 combination therapy was similarly effective in non-mutated OCI-Ly7 cells: MHC-I, -II, and B2M expression increased 1.9- ( P < 0.01), 3.0- ( P < 0.01), and 2.4-fold ( P < 0.01), respectively, following YF2 + taz treatment, and increased 1.5- ( P = 0.01), 3.3- ( P < 0.01), and 2.4-fold ( P < 0.01) with YF2 + bel. Similar expression trends were observed via western blots, but to a lesser degree; MHC-I, -II, and B2M protein increased 1.3-, 1.4-, 1.6-fold with YF2 + taz, and increased 1.1-, 1.6-, and 1.2-fold following YF2 + bel treatment in SU-DHL-6 cells.

The combination therapies were tolerated by the mice and decreased tumor size. Splenic MHC-I expression increased 1.2-fold with YF2 + taz and 1.4-fold with YF2 + bel, whereas MHC-II increased 1.3-fold for both combination therapies ( Figure 2). Similarly, circulating B cells displayed increased MHC-I and -II expression with combination therapy. Treatment with YF2 + taz increased the number of circulating CD4 ⁺ T cells 1.5-fold and the number of CD8 ⁺ T cells 3.5-fold, while YF2 + bel increased the number of central memory and effector memory T cells by 1.6- and 1.9-fold, respectively. To understand the effect of combination therapies on B cell development in the GC, splenic B cells were evaluated and exhibited a 1.9-fold increase of cells in the light zone following YF2 + taz treatment, and a 1.2-fold increase after YF2 + bel exposure. The number of splenic plasma cells increased 1.4- and 1.5-fold following YF2 + taz and YF2 + bel treatments, respectively ( Figure 2). These findings suggest that combination epigenetic therapy may restore homeostatic B cell function and promote B cell cycling through the GC.

In summary, these data propose YF2 combination epigenetic therapy as a means to increase immunogenicity in CBP/p300- and EZH2-mutated DLBCL. While our preliminary mouse experiment provided promising initial results, an expanded study is underway to statistically confirm the exciting trends that we observed.