## 2 Results  
MYC Downregulation by Synthetic MYC Inhibitor 10058-F4 Sensitizes Lymphoma Cells Towards Rituximab-Mediated Antibody-Dependent Cellular Cytotoxicity Through CD20 Upregulation  
To study the impact of _MYC_ on conventional and T cell-redirecting therapeutic antibodies, we first inhibited MYC in an extensive panel of DLBCL, BL and MM cell lines using the small molecule 10058-F4. This MYC inhibitor specifically inactivates MYC, but not its down- or upstream products, by inhibiting the dimerization of MYC with MYC-associated factor X (MAX) [20]. First, we confirmed decreased MYC protein expression at concentrations of 25-150 \(\upmu\)M in DLBCL cell lines OCI-LY18, WSU-DLCL2, OCI-LY7 and SU-DHL-6; in BL cell lines Daudi and Raji; and in MM cell lines MM1S and RPMI8226 (Figures 1A and 5A). 10058-F4 inhibited cell proliferation at concentrations of 100-150 \(\upmu\)M (Figure 1B). Rituximab as a single agent induced moderate lysis in the majority of the cell lines (Figure 1C). In cytotoxicity assays, we pre-incubated tumor cells with 10058-F4 and washed it away to avoid toxicity on immune effector cells. The pre-incubation of DLBCL cell lines OCI-LY18, WSU-DLCL2, OCI-LY7 and BL cell lines Daudi and 10058-F4 synergistically improved rituximab-mediated tumor cytotoxicity (Figure 1B,C). Additive effects were observed in SU-DHL6 and Raji. While the MYC protein expression level was not associated with CD20 expression in a selection of DLBCL cell lines (Figure 1D), the increased rituximab-mediated antibody-dependent cellular cytotoxicity (ADCC) was associated with increased CD20 surface expression (Figure 1D), pointing to a MYC-dependent regulation of CD20 expression, as previously suggested [21]. MYC Downregulation by Synthetic MYC Inhibitor 10058-F4 Sensitizes Lymphoma Cells Towards Dartumumab-Mediated and T Cell-Redirected Cytotoxicity Independent of Target Antigen Expression  
After showing enhanced rituximab-mediated ADCC, we tested whether the efficacy of the monoclonal anti-CD38 antibody dartumumab and the CD3 xCD19 bispecific T-cell engager blinatumombab could be improved by MYC downregulation. MYC downregulation by 10058-F4 also synergistically enhanced dartumumab-mediated ADCC in both MM cell lines (Figure 1E). In DLBCL cell lines, 10058-F4 did not affect CD38 expression (Figure S1E). Figure 1: Combination of MYC inhibitor 10058-F4 with conventional and T cell-redirecting therapeutic antibodies increases tumor cell kill in various B-cell malignancies. (**A**) Immunoblot analysis of MYC protein and \(\beta\)-actin levels after treatment with indicated concentrations of 10058-F4 for 96 h (WSU-DLCL2, OCI-LY7 and SU-DHL-6), 48 h (OCI-LY18, Daudi, Raji and MM1.S) or 24 h RPMI826. (**B,C**) Diffuse large B-cell lymphoma (DLBCL) cell lines (**B**) and Burkitt lymphoma (BL) cell lines (**C**) were pre-incubated with indicated concentrations of 10058-F4 (12.5-150 uM) for 96 h (WSU-DLCL2, OCI-LY7 and SU-DHL-6) or 48 h (Daudi and Raji) and thereafter incubated with predetermined concentrations of rituximab (rituximab, 1–1000 ng/mL) in the presence of PBMCs obtained from healthy donors as effector cells (E:T ratio of 25:1) for 16 h. Tumor cell lysis was assessed using a bioluminescence-based cytotoxicity assay. Cell viability was calculated based on untreated cells. Observed [O] lysis upon combined treatment was compared to the expected [E] lysis, which was calculated according to the BLISS model (% expected lysis = ((% lysis 10058-F4/100) + (% lysis antibody/100)) – ((% lysis 10058-F4/100) \(\times\) (% lysis antibody/100)) \(\times\) 100). The null hypothesis of  
\"additive effects\" was rejected if the observed values were significantly different than the expected values. (**D**) Representative flow-cytometry histograms depict CD20 expression in DLBCL cell lines after 24 h of treatment with indicated concentrations (0-50 uM) of 10058-F4. Dotted line represents isotype staining control.