## 3 Results
### RNAi Screening in Mutant DNMT3A-Driven Primary AML Cells
Continuous expression of Cas9 and a gRNA against _MDM4_ led to the depletion of targeted cells over time (Figure S5b), although the dynamics of the cell depletion here was slower than upon shRNA-mediated knockdown  
Figure 3: Validation of the screen hit MDM4. (**a**) Volcano plot visualizing the results of the combined RNAi screens in 9 primary patient samples. FC—fold change; LUC—luciferase. The \(p\)-values were calculated with the Wilcoxon rank-sum test and corrected for multiple testing using [PERSON]’s method to obtain the Q-value. (**b**) Expression data for the candidate gene MDM4 and the housekeeping genes PSMD6, EIF4H, PSMA1 obtained from RNA-Seq of _37 DNMT3A_ R882X AML samples, including 18 samples in which the functional screens were performed. CPM—counts per million. (**c**) Growth dynamic of two primary _DNM73A_ R882X AML primary samples (SAL1788 and SYT59) expressing an shRNA MSM4 (shRNA1 and shRNA2). The positive control PSMA1 (shPSMA1) and the non-targeting control (shScramble) are indicated. \(n=1\). (shRNA vs. gRNA: depletion to 50% within 9 days vs. 14 days). To test the efficiency of the employed gRNA, we transduced HEK293T cells with a GFP-reporter construct expressing Cas9 and MDM4-gRNA and analyzed the targeted genomic locus by Sanger sequencing 5 days after infection. The sequencing revealed an in-frame:frameshift ratio of 1.86 (Figure S4c). Thus, the slow dynamics of cell depletion can be explained by the low efficiency of the gRNA-mediated disruptive indel generation. A recent report from [PERSON] et al. demonstrated that MDM4 is particularly highly expressed in AML cell lines and patient-derived AML cells [28]. This study did not, however, investigate the potential differences between the cytogenetic subgroups of AML. Using a publicly available RNA-Seq dataset of AML samples (GSE67040), we compared the expression of MDM4 in _DNMT3 AWT/R882X_ and _DNMT3 AWT/WT_ normal karyotype (NK) AML. This analysis revealed a clear, albeit not statistically significant, trend towards higher MDM4 expression in _DNMT3 AWT/R882X_ samples (Figure 4). This trend could not, however, be confirmed in the normal karyotype AML samples from the TCGA cohort (data not shown). [PERSON] and colleagues further reported that the dual inhibition of MDM2/MDM4 with a novel inhibitor ALRN-6924 induced apoptosis and cell cycle arrest in leukemic cells accompanied by a survival advantage in xenotransplantation mouse models [28]. Of note, this study also provided evidence for the sensitivity of enriched leukemic progenitor populations to MDM2/4 inhibition and nominated MDM2/4 as therapeutically actionable targets for p53 wild type tumors [28]. Strikingly, [PERSON] and colleagues observed that the leukemic cell line OCI-AML3 which, similar to our tested cohort, bears the mutations _DNMT3 AWT/F.R882C_ and _NPM1 WT/F.W288X/F12_, had a high resistance to ALRN-6924 treatment compared to other leukemic cell lines, despite all having the p53 signaling pathway intact [28]. Although the study also reports the response of several primary AML samples to ALRN-6924 treatment, it does not indicate the mutational profiles of these cells. Thus, how distinct genetic subgroups of AML respond to ALRN-6924 treatment remains unknown. As our screen data suggested that primary _DNMT3 AWT/R882H_/ _NPM1 WT/F.W288X/F12_ AML cells are sensitive to MDM4 depletion, we decided to investigate whether MDM2/4 inhibition is effective in these cells. We applied the dual chemical inhibitor ALRN-6924 described in [28] on two primary samples--SAL1788 (_DNMT3 AWT/F.R882C_, _NPM1 WT/WT_, _FLT3 WT/WT_) and SYT50 (_DNMT3 AWT/F.R882H_, _NPM1 WT/F.W288X/F12_, _FLT3-ITD_). Figure 4: Comparison of MDM4 transcript expression in NK-AML samples with and without the _DNMT3A_ R882H mutation. The expression values across samples are normalized to the housekeeping gene _EIF4H_. PSMA1 and PSMD6 are housekeeping controls. n is the number of samples in each group. The data was retrieved from the publicly available dataset GSE67040.