Project 3: Role of stromal cell-activated CNOT6L deadenylase in driving AML transformation

Role of stromal cell-activated CNOT6L deadenylase in driving AML transformation. AML has long been thought to be exclusively driven by genetic or epigenetic mutations within HSCs. Besides these classical mechanisms, the BM stromal microenvironment plays a crucial role in the pathogenesis, maintenance and resistance to treatment of malignant clones. Using osteoblast-specific β-catenin models of AML and MDS, and cells from MDS patients that progressed to AML, we have identified a transcriptional event that is highly associated with MDS to AML transformation. Compared to MDS, AML cells show increased expression of the deadenylase CNOT6L. CLOT6L expression is also upregulated in cells from AML versus MDS patients in several cohorts, and additional mouse models of AML transformation. Overexpression of Cnot6l in HSPCs of MDS mice induces AML evolution. Cnot6l-induced AML is passaged to secondary healthy recipients Conversely, CNOT6L inactivation reverses AML transformation. In contrast to AML cells, CNOT6L expression is downregulated in healthy myeloid progenitors as compared to HSCs of humans and mice and in megakaryocyte progenitors of subjects with DNMT3AR882 CH. Notably, Cnot6l expression in HSCs is increased upon chronic IL-1 treatment suggesting that it may also be part of the regenerative response that involves emergency myelopoiesis (EM) and promotes clonal expansion with inflammation. Thus, maintaining low CNOT6L mRNA levels may maintain myeloid lineage differentiation whereas increasing it may favor AML transformation of ARCH or MDS. Indeed, CNOT6L expression is highly associated with AML founder mutations in the TCGA database and with MDM4 expression. Our hypothesis is that stromal cell amplified CNOT6L- mediated deadenylation can drive AML development from pre-AML states of MDS and in the context of aging- related stress factors of ARCH and inflammatory stress in the marrow. We will determine the role of CNOT6L in AML initiation with aging and age-related loss factors of ARCH and inflammatory stress with EM engagement; determine how Cnot6l promotes clonal heterogeneity by defining and validating the mutational profile and transcriptional targets/mechanisms of CNOT6L-related AML evolution using mouse modeling along with single cell mutational and transcriptional sequencing to define clonal trajectory in mice and primary patient samples; and, characterize stromal cell mechanisms that induce Cnot6l-dependent MDS to AML transformation using primary patient samples and mouse models and scRNAseq and spatial mapping. These studies will identify transcriptional and clonal events driven by the stroma that are significant contributors to AML transformation. Early transformative events may be therapeutically targeted thus implementing a potentially more effective therapy that is preventive rather than curative.