## 4 Discussion  
Since the _DNMT3A_ R882 mutation was found to mark early leukemic progenitors [5], efforts have been directed towards finding and exploiting vulnerabilities of this cell population for therapeutic applications. In this work, we have shown that mutant DNMT3A is not required for in vitro survival and proliferation of leukemic blasts by correcting the R882H mutation in SET2 cells with an adenine base editor. More subtle molecular changes  
Figure 5: Effects of MDM2/4 inhibition in primary _DNMT3 AWT,R882_ AML cells. (**a**) Normalized counts of live primary AML cells (SAL1788) after the treatment with indicated concentrations of ALRN-6924 (\(n=2\)). The samples were compared to the DMSO treated sample with a parametric two-sided Student’s t-test (**:**\(p\leq 0.05\); **:**\(p\leq 0.01\); ns—non-significant). The error bars represent the standard deviation of the mean. (**b**) Relative mRNA expression levels of p53 target genes in SAL1788 patient cells treated with DMSO or with 5 uM ALRN-6924 for 24 h (\(n=2\), a representative plot from one experiment is shown). (**c**) Flow cytometric analysis of the cell cycle distribution in primary AML cells from two patients (SAL1788 and SYT50) treated with DMSO or with 5 uM ALRN-6924 for 24 h (\(n=2\) for SAL1788, a representative plot is shown, \(n=1\) for SYT50). resulting from the inactivation of this mutation need to be investigated in follow-up studies. Single-cell RNA-Seq/CHIP-Seq experiments could help delineate the role of DNMT3A in distinct cell populations. The response of early _DNMT3A_-mutant leukemic progenitors to the restoration of the wild type DNMT3A activity also remains to be investigated. Because the acquisition of _DNMT3A_ mutations in HSCs leads to clonal hematopoiesis and often represents the first step of leukemogenesis [35; 36; 37; 38; 39], we speculate that reinstatement of the normal DNMT3A functions would be sufficient for the affected cell to return to its native state, i.e., revert the methylation state and the proliferation rate back to the wild type levels. Isolation of leukemic progenitor cells followed by the correction of the _DNMT3A_ R882 mutation and a comparative analysis of methylation patterns and proliferative behavior in the corrected vs. non-corrected cells would be required to test this hypothesis. _DNMT3A_-mutant clonal cell populations have been implicated in leukemogenesis initiation [40; 5], chemotherapy resistance [5; 30] and relapse [5; 30; 41]. Nevertheless, a targetable vulnerability of _DNMT3A_-mutant leukemic cells has not yet been found. We have functionally profiled primary _DNMT3 AWT3 AWT3 AWT3A_ AML cells, aiming to identify cellular dependencies that can be exploited as therapeutic targets. To the best of our knowledge, such a large panel of primary human AML cells has not been subjected to functional screening before. Apart from the limited sample availability, such experiments are constrained by the viability and in vitro growth capacity of primary cells. Our data show that large cell numbers are required to account for the viability drop as the primary cells are adapting to in vitro culture conditions. Furthermore, some primary AML samples tend to rapidly differentiate in culture (data not shown), calling for improvements of the culture conditions to allow long term expansion. Methods such as co-culture with feeder cells [42; 43; 44] or in vivo propagation [45; 46; 47] have been shown to be superior to the in vitro culture with regard to the viability and proliferation capacity of primary cells. However, technical requirements, such as isolating of the population of interest in sufficient numbers/purity and ensuring high library coverage with large-scale screens, respectively, may be more challenging to meet with these methods. Using our screen setting (~6500 shRNAs), we have experimentally demonstrated that with the current culturing techniques a library representation of ~60\(\times\) can be achieved and is sufficient to detect phenotypic effects (positive controls).