Identification of b lineage commitment markers for gene expression-based risk stratification of infant kmt2a::aff1 acute lymphoblastic leukemia

Infant acute lymphoblastic leukemia is a devastating disease which arises mainly due to the occurrence of KMT2A rearrangements (KMT2A-r). In this context, risk stratification is an indispensable pre-treatment step enabling assignment of patients to appropriate intervention subgroups. KMT2A-r, young age, poor Glucocorticoid response rates, and high blast counts are the primarily applied risk-determining factors. Our group recently described 100-fold increased expression of the immediate early gene EGR3 at time of relapse compared to diagnosis. Here, we present the EGR3 regulome of infant KMT2A-r ALL which was assessed through integration of ChIP-seq and MACE-seq data of an EGR3-overexpressing KMT2A::AFF1 cell culture model. Binding and expression target analysis followed by gene set enrichment analysis, motif scanning, and flow cytometry identified EGR3 as a mediator of B lineage commitment. Moreover, EGR3 was found to collaborate with a set of B lineage transcription factors including PAX5. Gene expression analysis of patient cohorts from diagnosis (n=50) and relapse (n=18) uncovered strict bimodal clustering of patients determined by four EGR3-regulated B lineage marker genes. Although the whole diagnosis cohort was assigned to the high-risk group, event free survival analysis of patient clusters demonstrated absence of B lineage gene expression translating to clearly inferior long-term event-free survival. Possible confounders including age, blast percentage, and HOXA status did not affect dichotomous separation of patients. In conclusion, our study identified EGR3 as a mediator of B lineage specification and commitment in infant KMT2A::AFF1 ALL and characterized the EGR3 regulome in detail. Furthermore, we present four B lineage markers whose gene expression can be applied for substratification of high-risk patients potentially improving clinical decision making. Infant acute lymphoblastic leukemia is a devastating disease which arises mainly due to the occurrence of KMT2A rearrangements (KMT2A-r). In this context, risk stratification is an indispensable pre-treatment step enabling assignment of patients to appropriate intervention subgroups. KMT2A-r, young age, poor Glucocorticoid response rates, and high blast counts are the primarily applied risk-determining factors. Our group recently described 100-fold increased expression of the immediate early gene EGR3 at time of relapse compared to diagnosis. Here, we present the EGR3 regulome of infant KMT2A-r ALL which was assessed through integration of ChIP-seq and MACE-seq data of an EGR3-overexpressing KMT2A::AFF1 cell culture model. Binding and expression target analysis followed by gene set enrichment analysis, motif scanning, and flow cytometry identified EGR3 as a mediator of B lineage commitment. Moreover, EGR3 was found to collaborate with a set of B lineage transcription factors including PAX5. Gene expression analysis of patient cohorts from diagnosis (n=50) and relapse (n=18) uncovered strict bimodal clustering of patients determined by four EGR3-regulated B lineage marker genes. Although the whole diagnosis cohort was assigned to the high-risk group, event free survival analysis of patient clusters demonstrated absence of B lineage gene expression translating to clearly inferior long-term event-free survival. Possible confounders including age, blast percentage, and HOXA status did not affect dichotomous separation of patients. In conclusion, our study identified EGR3 as a mediator of B lineage specification and commitment in infant KMT2A::AFF1 ALL and characterized the EGR3 regulome in detail. Furthermore, we present four B lineage markers whose gene expression can be applied for substratification of high-risk patients potentially improving clinical decision making.