## 2 Results
### MYC Targeting by CRISPR-Cas9 Gene Editing
In addition, successfully MYC-repaired subclones displayed a similar CD19 and CD38 expression as parental OCI-LY18 WT cells (Figures S3D and 3E,G). Figure 3: MYC downregulation increases lysis by conventional and T cell-redirecting therapeutic antibodies independent of target antigen upregulation. **(A)** OCI-LY18 wild type (WT, black) and MYC  
targeted clones (colored) were incubated with 100 ng/mL daratumumab (x-axis) in the presence of PBMCs obtained from healthy donors as effector cells (E:T ratio 40:1) for 16 h. Percentage of tumor cell lysis is depicted on the y-axis. Tumor cell lysis was assessed using a flow cytometry-based cytotoxicity assay. Cell viability was calculated based on untreated cells (0 ng/mL), and the percentage of viable cells of the treated is normalized compared to OCI-LY18 WT. Data are presented as mean tumor cell lysis \(\pm\) SEM of 3-4 independent experiments performed in duplicate. * indicates \(p<0.05\); **\(p<0.01\), ns not significant. (**B**) OCI-LY18 WT (black) and a representative selection of MYC-targeted clones (colored) were incubated with 1 ng/mL blinatumomab in the presence of PBMCs obtained from healthy donors as effector cells (E:T ratio of 10:1) for 24 h. Details as in (**A**). (**C**) LY18 WT (black) and MYC-targeted clone #1 (red) and MYC-repaired subclones (grey) were incubated with 100 ng/mL daratumumab or blinatumomab in the presence of PBMCs obtained from healthy donors as effector cells (E:T ratio 40:1 (daratumumab) or 10:1 (blinatumomab)) for 16 h (daratumumab) or 24 h (blinatumomab). Details as in (**A**). (**D**) Representative flow-cytometry histograms depicting CD38 and CD19 expression in OCI-LY18 WT (black) and MYC-targeted clones (colored). (**E-G**) Representative flow-cytometry histograms depicting CD38 expression and CD19 expression (left) and corresponding mean fluorescent values (MFI) (right) in OCI-LY18 WT (black) and MYC-targeted clone #1 (red) and MYC-repaired subclones (grey). Expression data are representative histograms of at least 2 technical replicates. Single-nucleotide polymorphism (SNP)-based genomic array profiling revealed that all MYC downregulated clones had more or less the same chromosomal gains and losses as were present in OCI-LY18 WT cells and OCI-LY18-Cas9 cells. Clones #3, #5 and #9 showed additional chromosomal abnormalities, but these seemed to be nonspecific and not related to the CD20 locus on chromosome 11q12.2 or the other target loci for CD38 (4p15.32) or CD19 (16p11.2) (Supplemental Table S1). Therefore, the limited degree of genomic heterogeneity within the clones did not seem to affect the target antigen expression of CD20, CD38 and CD19. These results indicate that modulating target antigen expression was not the only mechanism for the evasion of immunotherapeutic approaches in MYC overexpressing tumor cells. We next aimed to further delineate these additional mechanisms. Increased Cytotoxicity in MYC Downregulated Cells Is Associated with Downregulation of Anti-Apoptotic Proteins  
In MM cells, the upregulation of anti-apoptotic pathways can mitigate NK cell- and T cell-mediated cytotoxicity [27; 28; 29]. To evaluate whether MYC could blunt the efficacy of immunotherapeutic approaches by modulation of apoptosis, we measured the expression of the most important apoptosis regulatory proteins BCL-2, MCL1, survivin and XIAP, and pro-apoptotic proteins PUMA and BIM in the representative clone #1 and its MYC-repaired subclones. The MYC downregulated clone #1 displayed lower expression levels of anti-apoptotic proteins XIAP, survivin, MCL-1 and BCL-2, and a higher expression level of the pro-apoptotic PUMA (Figure 4A) compared to parental OCI-LY18 WT cells. The direct involvement of MYC in these events was confirmed by the restored expression of XIAP, PUMA and, to a lesser extent, survivin and MCL-1 in both MYC-repaired subclones (Figure 4A).