Role of RNA methylation in chemoresistant cancer cells Goal Based on our data, we propose that transiently quiescent populations in acute myeloid leukemia (AML) are maintained in part by RNA methylation, permitting synthesis of survival and tumor promoting regulators necessary for chemoresistance and subsequent AML persistence. The primary objective is to characterize the role of RNA methylation in regulating gene expression in resistant AML, which contributes to chemosurvival. Significance AML is a serious malignancy that displays clinical resistance due to heterogeneity. Resistant AML cells include quiescent (G0) cells that are transiently arrested states, and thus resist clinical therapy that targets only cycling cells; these cells re-enter proliferation to cause AML persistence. A primary issue is that resistance regulators remain to be uncovered. A second need is to identify markers to detect resistant cells. Such cells show distinct gene expression that permits chemosurvival (PNAS 2014, Mol. Cell 2016, Biorxiv/ 418715). Canonical translation is decreased; yet specific mRNAs are expressed by unknown mechanisms. Regulation of specific gene expression in resistant AML needs to be uncovered & targeted to curtail AML. Premise Profiling in resistant cells revealed expression of factors that affect cell survival. Expressed mRNAs in G0 resistant cells have extended 3? untranslated regions (UTRs) to include regulatory sites, such as for RNA methylation. Methylation at N6 position of adenosines (m6A) are unique marks on RNAs that regulate gene expression via RNA binding proteins called readers, to control distinct cell states. Role of m6A in resistant AML remains to be uncovered. We find the m6A methyltransferase increases due to therapy induced signals. Our data reveal that m6A is needed for specific gene expression & resistance. We developed inhibitors to block therapy induced signals & antisense to block unique m6A sites on extended 3?UTRs, which reduce resistance. These data suggest that m6A regulates specific gene expression to enable therapy survival. Characterization of mRNAs that are modified & expressed in resistant cells, in vivo & in patient samples by m6A, their readers, their role in resistance, & of inhibitors that block m6A, will provide markers & therapeutics to curb resistance. Method Aim I will characterize m6A readers that regulate known modified mRNAs, by in vivo crosslinking coupled RNA affinity purification in resistant AML cells & in vivo models. Chemical inhibitors to therapy induced signaling that promotes m6A and antisense to block mapped m6A sites will be tested to curb resistance.
Aim II will globally identify m6A targets that contribute to resistance in cell lines & in vivo models, using m6A antibody & methyltransferase immunoprecipitation. Role of readers & m6A mRNAs in resistant cells & in vivo is tested by depletions coupled with chemosurvival assays; their expression will be verified in resistant patient samples. Outcomes mRNAs expressed by m6A, their readers, their role in resistance in AML cells & in vivo, & inhibitors of m6A to curb resistance will be identified. Their expression & efficacy in patient samples will be uncovered.
Role of RNA methylation in chemoresistant cancer cells Resistant cells are sleeper cells within a tumor that are transiently arrested?and thus withstand chemotherapy that targets only active, dividing cells; these cells subsequently resurge and cause recurrence. Based on our findings, the factors expressed that maintain chemoresistant cells are distinct and are regulated by RNA methylation, a specific, chemical modification that controls critical factors for such cells to persist. The primary goal of this study is to characterize RNA methylation effectors and target genes that enable specialized expression of specific factors that cause persistence of chemoresistant cells in cancer, which will provide new therapeutic targets and markers against clinical resistance in cancer.
