It has become apparent that microRNAs (miRs), a class of small non-coding RNAs, are important regulators of cellular systems. We have found that certain miRs are profoundly important to the development and function of the immune system, coupling with transcription factors to provide precise control. The focus of this proposal is on understanding the roles of two particular miRs, miR-146a and miR-125b, in orchestrating immunity, with an emphasis on understanding why either deletion or overproduction of these miRs causes leukemia. The broad goal of this proposed research is to determine the cellular and molecular mechanisms by which miR-146a and miR-125b orchestrate blood cell formation, immune function, and cancer occurrence. Previously, we generated miR-146a-/- mice and found that they become depleted of hematopoietic stem cells and develop chronic inflammation with an overproduction of myeloid cells leading to frank myeloid leukemia. We found that another cell type, lymphocytes, contribute importantly to the development of myeloid proliferative disease and hematopoietic stem cell abnormalities in miR-146a-/- mice. We will now discover what type of lymphocyte is important to this process. We will also identify the pro-inflammatory cytokines produced by the lymphocytes to cause these disorders and the molecular pathways involved in leukemia induction. Additionally, we will test the hypothesis that multiple miRNAs function cooperatively to regulate immune cell development and function. In this regard, our focus will be on the cooperativity between miR-146a and miR-155, as well as miR-146a and miR-125b. In addition to miR-146a, we previously found that miR-125b is an effector of immunity by augmenting macrophage function, inhibiting B cell development, and impairing T cell function. In contrast to miR-146a, constitutive over-expression of miR-125b rapidly induces aggressive myeloid leukemia through a cell intrinsic process not requiring lymphocytes. Later, B cell cancers also develop in miR-125b over-expressing mice, likely through the occurrence of secondary genetic mutations within these cells. In our proposed research, we will determine the cellular and molecular mechanisms by which miR-125b inhibits B cell development and impairs T cell function. We will also identify genetic cancer 'driver' mutations and dysregulated signal pathways in miR-125b-induced leukemic cells and determine which of these abnormalities induces tumors. Over the next five years, we hope to generate some principles of the regulation of immune cell development and functions by focusing on miR-146a and miR-125b. A better understanding of the control exerted by miRNAs on the immune system will potentially open up new therapeutic avenues for treating immunologic diseases and cancer. The expression of miR-146a and miR-125b is often dysregulated in human leukemia, linking our research directly to public health needs.