Acute myeloid leukemia (AML) is a clonal neoplastic disorder organized as a cellular hierarchy, with the self-renewing leukemia stem cell (LSC) at the apex. Recurrent mutations in transcription factors (TF) and epigenetic regulators suggest that AML is driven by aberrant transcriptional circuits, but these circuits have not been fully defined in an LSC model. To study transcriptional mechanisms relevant to leukemogenesis in vivo, we generated a murine serial transplantation model of MLL-AF9-driven, myelomonocytic leukemia with genetically- and phenotypically-defined LSCs. Using this model, we pursued two related lines of investigation. First, we performed an in vivo RNA interference (RNAi) screen to identify transcription factors required for LSC function. This screen highlighted the circadian rhythm TFs, Clock and Bmal1, as genes essential for the survival of murine leukemia cells, and we validated this finding with CRISPR/Cas-based genome editing and knockdown studies in AML cell lines. Utilizing luciferase reporter mice to track expression of the circadian target gene Per2, we demonstrated that both leukemic and normal hematopoietic cells have the capacity for oscillating, circadian-dependent gene expression. Importantly, using murine knockout models, we found that normal hematopoietic stem and progenitor cells (HSPC), in contrast to leukemia cells, do not depend on Bmal1. We further demonstrated that selective depletion of LSCs following circadian perturbation is mediated through enhanced myeloid differentiation. ChIP-Seq studies revealed that the circadian rhythm network is integrally connected to the LSC self-renewal circuitry and highlighted putative Clock/Bmal1 targets in leukemia, providing a mechanistic basis for our findings. Second, we performed a functional and genomic characterization of our MLL-AF9 serial transplantation model to explore mechanisms of disease evolution and clonal selection in AML. Limiting dilution studies demonstrated that serial transplantation results in a reduction in disease latency, dramatic enrichment of leukemia-initiating cells (LIC), and reconfiguration of the LSC hierarchy. While mutations in known AML-associated genes were not linked to disease progression, RNA-sequencing (RNA-Seq) demonstrated that the increase in LIC frequency in serially transplanted leukemias is driven by changes in cell cycle and differentiation. In aggregate, these studies offer insights into the biological mechanisms regulating LSC self-renewal and disease evolution in AML.

Indiana University-Purdue University Indianapolis (IUPUI) / The development and function of hematopoietic stem cells (HSCs) is regulated by numerous signaling pathways including Akt-mechanistic target of rapamycin complex1 (mTORC1) pathway. Dysregulation of this pathway results in impaired HSC function and contributes to the development of hematologic malignancies. Activated mTORC1 phosphorylates and subsequently activates ribosomal protein S6 kinase 1 (S6K1). To study the role of S6K1 in hematopoiesis as well as leukemogenesis, we used a genetic model of S6K1 deficient mice (S6K1-/-). We found that loss of S6K1 expression in HSCs results in reduction of absolute HSC number in bone marrow (BM). Following chemotherapy, cycling HSCs undergo apoptosis and quiescent HSCs are required to cycle to regenerate the hematopoietic system. S6K1 regulates the quiescence of HSCs and in the absence of S6K1, mice are more susceptible to repeated myeloablative stress. We also observed that loss of expression as well as gain of expression of S6K1 affects the self-renewal ability of HSCs. Interestingly, when we overexpressed S6K1, it also resulted in reduced self-renewal of HSCs. Next, we assessed the role of S6K1 in the propagation of acute myeloid leukemia (AML). The mixed-lineage leukemia (MLL) gene is required for the maintenance of adult HSCs. Translocations in MLL are detected in approximately 5-10% of adult acute leukemia patients and in approximately 70% of acute leukemias in infants. We expressed MLL-AF9 fusion oncoprotein in WT and S6K1-/- hematopoietic stem and progenitor cells (HSC/Ps) and performed serial transplantation. Upon secondary transplantation, recipients of S6K1 deficient AML cells survived significantly longer compared to controls. In vitro, pharmacological inhibition of S6K1 activity resulted in reduced growth of primary human cells expressing MLL-AF9. Both human and murine HSC/Ps expressing MLL-AF9 showed reduced mTORC1 activity upon inhibition of S6K1 suggesting that loss of S6K1 activity results in reduced Akt-mTORC1 activation both upstream and downstream of mTORC1. Overall, our studies establish a critical role of S6K1 activity in the maintenance of HSC function and in the propagation of leukemia.