## 2 Results
### MYC Targeting by CRISPR-Cas9 Gene Editing  
To study the effects of MYC targeting and to investigate the mechanisms underlying the increased susceptibility to conventional and T cell-redirecting therapeutic antibodies, we next targeted the _MYC_ gene in OCI-LY18 using CRISPR-Cas9 gene-editing technology. OCI-LY18 overexpressed MYC due to a pathogenic translocation of the _MYC_ gene with the immunoglobulin heavy chain enhancer gene (_IGH_, t(8;14)(q24;q32)). We observed that all viable _MYC-targeted_ daughter clones retained at least a _MYC_ open reading frame, confirming that a complete loss of function of _MYC_ is detrimental to cell proliferation (Table 1). We selected 10 clones which retained a single MYC ORF for further evaluation to overcome any possible bias introduced by clonal heterogeneity. Immunoblot showed MYC protein downregulation in all clones (Figure 2A). We, therefore, refer to these clones as \"MYC downregulated\". As expected, the proliferation rate of these MYC downregulated clones was lower compared to the parental OCI-LY18 wild type (WT) cells after 2-3 days (Figure S2A). Given the association between MYC and decreased expression of MHC molecules, we explored the phenotype of our clones. In agreement with other reports [22;23], MYC downregulated clones showed upregulation of HLA-DR (Figure S2B). Data on the association between the expression of intracellular MYC and cell surface immune checkpoint molecules are conflicting in lymphoma [24]. In accordance with a previous study [25], we observed no changes in PD-L1 in MYC downregulated clones compared to parental OCI-LY18 WT cells. We observed no changes in CD47 expression (Figure S2B). Increased Rituximab-Mediated ADCC and CD20 Surface Expression Are a Direct Consequence of MYC Downregulation  
After the initial characterization of MYC downregulated cells, we tested their susceptibility against rituximab-mediated antibody-dependent cellular cytotoxicity (ADCC). While the introduction of Cas9 did not have any effect on rituximab-mediated ADCC or target antigen expression (Figure S2C), rituximab-mediated ADCC was increased in nine out of ten MYC downregulated clones (Figure 2B). Again, the increased rituximab-mediated ADCC was associated with increased CD20 surface expression (Figure 2C,D), pointing to a MYC-dependent regulation of CD20 expression. In light of current antibody development for the treatment of DLBCL, we evaluated the expression of other pan-B-cell targetable antigens CD37, CD79b and CD22. Interestingly, CD37, which co-localizes with CD20 [26], was also upregulated in MYC downregulated clones in a similar manner as CD20 (Figure S2D). The expression of CD79b and CD22 was not affected (Figure S2E). To confirm the direct involvement of MYC in the increased rituximab-mediated ADCC and CD20 expression, we repaired the mutated _MYC_ coding sequence in the representative MYC downregulated clone #1 by homologous recombination (Table 2). Several successfully \"MYC-repaired\" subclones displayed a similar proliferation rate, a similar sensitivity towards rituximab-mediated cytotoxicity and similar CD20 expression levels as OCI-LY18 WT (Figure S3A,B). We selected two representative repaired subclones (\"repair 1\" and \"repair 2\") for further experiments. These subclones showed similar MYC protein expression compared to parental OCI-LY18 WT cells (Figure 2E). In both repaired subclones, rituximab-mediated cytotoxicity and CD20 expression were reversed towards the parental OCI-LY18 WT phenotype (Figure 2F,G), confirming that the increase in rituximab-mediated ADCC and CD20 surface expression were a direct consequence of MYC downregulation. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Clone Nr** & \multicolumn{2}{c}{**5’ MYC-1 Target Site 3’**} & **Basepair Nr** \\ \hline MYC1 & CTCCCTTCGGGGAGACAACGACGGCGGTGCCGGGAGCTTCTCACGGG & \\ \hline Clone \#1 & \begin{tabular}{c} CTCCCTTCGGGGAGACAACG.CGGCGGGGGGAGCTTCTCCACAGC \\ CTCCCTTTGGGAGACAACG....GTGGCGGAGCTTCTCCACAGGC \\ \end{tabular}  & \(-1\) \\ \hline Clone \#3 & \