## 3 Results
### RNAi Screening in Mutant DNMT3A-Driven Primary AML Cells
To emphasize common trends and increase the statistical power of the analysis, we pooled the count data of the nine successful screens and compared the shRNA counts between Day 20 and day 0, using the Wilcoxon rank sum test. After this step, all positive control shRNAs were among the top-scoring hits, demonstrating that the obtained results appear valid (Figure 3a). Additionally, known common essential genes appeared as hits, such as the cohesin complex genes _SMC1a/2/3/4_. Based on this analysis, we selected nine candidate genes for validation experiments (Figure S3). To further shortlist candidates to study in primary cells, we used the leukemic cell line OCI-AML3, which genetically closely resembles the primary patient cohort (\(DNMT3A^{WT\!P\!-\!R882C}\), \(NPM1^{WT\!P\!-\!W288X\!S\! *\!12}\)), but is easier to maintain and transduce. We tested the on-target activity of the two highest-scoring shRNAs from the screening library per gene of interest in a qPCR and a GFP reporter assay. An shRNAs was considered validated, when the reduction of mRNA levels upon the knock down was >50% and the depletion of the GFP reporter signal was >50% by day 9 post transduction. The qPCR revealed that the knock down was on target in 15 out of 18 tested shRNAs (Figure S3a). In the GFP reporter assay, a consistent depletion phenotype was achieved with four hits--DBF4 zinc finger (DBF4), Glucocorticoid Modulatory Element Binding protein 1 (GMEB1), Thioredoxin Domain Containing Protein 9 (TXNDC9) and Mouse Double Minute 4 (MDM4) (Figure S3b). We decided to focus on MDM4, because it is a well characterized protein with a prominent role in cell cycle regulation and has potential for chemical inhibition. In summary, our results show that cell viability is the bottleneck of in vitro functional screens in primary AML cells. Nevertheless, depletion of positive controls can be used to nominate screening samples that were successful. Additionally, we could demonstrate that, in the absence of biological replicates, pooling of the count data obtained in different patient lines emphasized the common trends and allowed for nominating potential common dependencies of the analyzed sample group. Loss of MDM2/4 Activity Stops the Proliferation of DNMT3 AWT/B-R882C/NPMI1 WT/B-V288X5%12 Primary AML Cells  
One of the strongest hits that emerged from the screen was Mouse Double Minute 4 (MDM4) (Figure 3a). MDM4 is a known regulator of the activity of the major tumor suppressor p53. Like its paralog Mouse Double Minute 2 (MDM2), it has an inhibitory effect on p53, preventing it from initiating a stress response program (reviewed in [31]). To confirm that _MDM4_ was expressed in our primary samples, we analyzed RNA-Seq data available for our extended _DNM73 AWT/B882X_ patient cohort. All the samples expressed _MDM4_, to a level comparable to that of the housekeeping genes _PSMD6_, _EIF4H_ and _PSMA132_ (Figure 3b). Next, we validated the phenotype of _MDM4_ knockdown in two primary patient samples from the screened cohort, SAL1788 (_DNM73 AWT/B-R882C_, _NPM1 WT/WT_, _FIT3 WT/WT_) and SYT59 (_DNM73 AWT/B-R882H_, _NPM1 WT/B-V288X5%12_, _FLT3 WT/WT_), using a lentiviral GFP reporter system. The expression of either the two shRNAs against _MDM4_ led to a rapid depletion of primary AML cells in vitro. Of note, primary cells from different patients displayed unequal sensitivity to _MDM4_ knock down, possibly reflecting the variable dependency of the original tumors, underlined by their genetic and epigenetic profiles (Figure 3c). We confirmed the on-target activity of the employed shRNAs on mRNA (Figure S4b) and protein (Figure S4c) levels. Furthermore, we utilized the CRISPR/Cas9 system to independently validate the dependency of _DNM73 AWT/B-R882X_-driven leukemic cells on MDM4, using OCI-AML3 cells as a model (Figure S5a).