## 3 Results
### RNAi Screening in Mutant DNMT3A-Driven Primary AML Cells  
To investigate other potential dependencies of mutant _DNMT3A_-driven leukemic cells, we decided to perform RNAi-based functional screens on primary AML cells. The primary bone marrow samples selected for screening all harbored a _DNMT3A_ p.R882 mutation. All the samples were additionally carrying a frameshift mutation in _NPM1_ (p.W288 Xfs*12; also called NPM1c). The mutational information of the 30 samples selected for the screening is summarized in the Table S4. The screen procedure is illustrated in Figure 2a. Figure 1: Testing the dependency of SET2 cells on mutant DNMT3A. (**a**) Chromatograms visualizing the Sanger sequencing reads of the PCR-amplified _DNMT3A_ R882H genomic locus in SET2-ABE8e-GFP-Puro transgenic cell line expressing a tdTomato reporter alone (no gRNA) or in combination with a gRNA targeting the R882H mutation (R882H gRNA). (**b**) tdTomato reporter assay tracking the percentage of gRNA-expressing SET2-ABE8e-GFP-Puro cells over time (\(n=2\)). Because the availability of the patient samples was limited and did not allow for the isolation of a sufficient number of _DNMT3 AWT/R88 ZX_ progenitor cells, the screens were performed on the bulk mononuclear cells. Based on the available cell number and the screen coverage requirements, we opted for a focused library. The rationale for the library composition was the known role of epigenetic regulators during early steps of leukemogenesis [30]. Thus, we aimed to probe the dependency of _DNMT3 AWT/R88 ZX_ AML cells on epigenetic and transcriptional factors. The library consisted of ~6500 shRNAs targeting 540 genes (average 12 shRNA per gene), including one negative and five positive controls [21]. Figure 2: RNAi screen in primary AML samples. (**a**) Overview of the experimental procedure for RNAi-mediated in vitro screening of primary AML cells. Created with BioRender.com (**b**) Cell viability measured via trypan blue exclusion assay immediately after thawing of the samples (0 h) and after overnight cytokine induction (24 h). Each data point represents one primary AML sample. (**c**) Dot plot visualizing the shRNA representation, calculated based on the number of cells collected for gDNA isolation, in every primary sample at the beginning (Day 0) and the end (Day 20) of the screen. Solid lines mark the arbitrary threshold of 60\(\times\) representation. Unlike cancer cell lines, in vitro cultured primary cells are sensitive to manipulation procedures and have a limited proliferation capacity. Thus, functional screens in primary AML cells are a challenging task. Overall, 30 unique \(DNMT3A^{WT,R882X}\) patient samples were screened. The total number of screened samples amounted to 39, considering replicate screens for some of the patient lines. From these, 34 samples, or 90%, survived the in vitro propagation, despite the initial drop of viability (Figure 2b, 42% median viability directly after thawing vs. 14% median viability 24 h after thawing; range at 24 h after thawing: 1-55%). In turn, 59% of these samples were proliferative in culture, i.e., the number of live cells increased between Day 0 and Day 20 of the screen (see Table S5 and Figure S2a). As expected for primary material, the achieved library coverage was highly variable across samples (Figure 2c; 56\(\times\) median representation at T\({}_{0}\), range 10\(\times\) to 300\(\times\)). Compared to cell viability and the proliferation rate in culture, library coverage appeared to have the biggest impact on the behavior of positive controls (Figure S2b), suggesting that the absolute number of shRNA-carrying viable cells is a major determinant of the screen success. Based on the performance of the shRNAs targeting the positive control genes _PSMA1_, _PSMA3_, _RPS13_, _RPL6_ and _RPL30_ compared to the non-targeting control shRNAs against luciferase (_LUC_), nine screens were considered successful. (Figure 2c and Figure S1).