![]() ![]() Whenever possible, primary AML cells were maintained at high density (5−10 × 10 6 cells/mL) and the medium was changed every second day. Primary AML cells were cultured in StemSpan Serum-Free Expansion Medium II (STEMCELL Technologies, Vancouver, BC, Canada) containing L-Glutamine (2 mM), Penicillin/Streptomycin (50 U/mL), the cytokines Flt3-Ligand (50 ng/mL), TPO (50 ng/mL), SCF (100 ng/mL) and IL-3 (20 ng/mL) (all from PeproTech Inc., Cranbury, NJ, USA) and the compounds StemRegenin1 (500 nM) and UM729 (50 nM) (both from STEMCELL Technologies, Vancouver, BC, Canada). All cell lines were kept at 37 ☌ and 5% CO 2 and were split twice a week. HEK293T and HeLa cells were cultured in DMEM (Gibco TM Thermo Fisher Scientific, Waltham, MA, USA) supplemented with Penicillin/Streptomycin (50 U/mL) and 10% FBS. ![]() The leukemic cell line SET-2 was maintained in RPMI (Gibco TM Thermo Fisher Scientific, Waltham, MA, USA) supplemented with Penicillin/Streptomycin (50 U/mL) and 20% heat-inactivated FBS. The leukemic cell line OCI-AM元 was maintained in Alpha-MEM (Biochrom AG, Berlin, Germany) supplemented with L-Glutamine (2 mM Invitrogen TM Thermo Fisher Scientific, Waltham, MA, USA), Penicillin/Streptomycin (50 U/mL Invitrogen TM) and 20% heat-inactivated FBS (Gibco TM Thermo Fisher Scientific, Waltham, MA, USA). Here, we describe a high-throughput RNAi-based screening approach to functionally profile primary DNMT3A WT/R882X cells from AML patients. Previous work from our lab has shown that high-throughput genetic screens in primary AML cells are feasible and a powerful tool to nominate patient-specific targets. While AML cell lines largely reflect the cytogenetics of the original malignancy, the transcriptional and epigenetic profiles of cancer cell lines may differ dramatically from those of primary cancer cells. Nevertheless, confirming these dependencies in patient-derived material remains a challenging task, exposing the drawbacks of cell lines as AML models. Genetic screens in cancer cell lines have been used to identify cancer vulnerabilities and in recent years very comprehensive databases of such vulnerabilities have been generated. High-throughput genetic screens are a well-established technique for revealing genotype–phenotype connections. Thus, there is a clinical need for discovering vulnerabilities of this cell population that can be exploited as a therapeutic approach. The mutant DNMT3A R882X protein induces epigenetic changes in HSCs that distort the balance between self-renewal and differentiation, however detailed knowledge about the downstream targets of these changes remains scarce. Remarkably, AML patients with DNMT3A mutations have shorter survival than DNMT3A WT patients, and this effect seems to be independent of the characteristic late co-mutations in FLT3 or NPM1. ![]() Recent studies in primary cells from AML patients have shown that the DNMT3A R882 mutation marks immature cells that are resistant to chemotherapy and represent a reservoir for AML relapse. A prominent example of this is normal karyotype AML (NK-AML) with mutations in DNMT3A. Unlike leukemic blasts that harbor several driver mutations, which can potentially be exploited as therapeutic targets (e.g., FLT3-ITD ), leukemic progenitors often lack therapeutically actionable mutated targets. Our results suggest that MDM4 inhibition is a potential target in NK-AML patients bearing DNMT3A R882X mutations. Moreover, we found that the MDM2/4 inhibitor ALRN-6924 impairs growth of DNMT3A WT/R882X primary cells in vitro by inducing cell cycle arrest through upregulation of p53 target genes. We analyzed a publicly available RNA-Seq dataset of primary normal karyotype (NK) AML samples and found a trend towards MDM4 transcript overexpression particularly in DNMT3A-mutant samples. As one of the strongest hits, we identified MDM4 as a gene essential for proliferation of primary DNMT3A WT/R882X AML cells. To unravel therapeutically actionable targets in mutant DNMT3A-driven AML cells, we have performed a focused RNAi screen in a panel of 30 primary AML samples, all carrying a DNMT3A R882 mutation. Currently, there are no therapeutic approaches directed specifically against this cell population. ![]() Recent evidence suggests that these mutations arise early in leukemogenesis, marking leukemic progenitors and stem cells, and persist through consolidation chemotherapy, providing a pool for AML relapse. DNA-methyltransferase 3A ( DNMT3A) mutations belong to the most frequent genetic aberrations found in adult acute myeloid leukemia (AML). ![]()
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