ABSTRACT
RUNX1 is frequently mutated in T-cell acute lymphoblastic leukemia (T-ALL). The spectrum of RUNX1 mutations has led to the notion that it acts as a tumor suppressor in this context; however, other studies have placed RUNX1, along with transcription factors TAL1 and NOTCH1, as core drivers of an oncogenic transcriptional program. To reconcile these divergent roles, we knocked down RUNX1 in human T-ALL cell lines and deleted Runx1 or Cbfb in primary mouse T-cell leukemias. RUNX1 depletion consistently resulted in reduced cell proliferation and increased apoptosis. RUNX1 upregulated variable sets of target genes in each cell line, but consistently included a core set of oncogenic effectors including insulin-like growth factor 1 receptor (IGF1R) and NRAS. Our results support the conclusion that RUNX1 has a net positive effect on cell growth in the context of established T-ALL.
Subject(s)
Core Binding Factor Alpha 2 Subunit/physiology , Gene Expression Regulation, Leukemic/genetics , Neoplasm Proteins/physiology , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Animals , Cell Division , Cell Line, Tumor , Cell Size , Core Binding Factor Alpha 2 Subunit/deficiency , Core Binding Factor Alpha 2 Subunit/genetics , Core Binding Factor beta Subunit/deficiency , Core Binding Factor beta Subunit/genetics , Gene Deletion , Gene Knockdown Techniques , Heterografts , Humans , Leukemia, Experimental/genetics , Leukemia, Experimental/pathology , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, SCID , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , RNA Interference , RNA, Small Interfering/genetics , Transcription, Genetic , Transcriptome , Tumor BurdenABSTRACT
Reactive oxygen species (ROS), a byproduct of cellular metabolism, damage intracellular macromolecules and, when present in excess, can promote normal hematopoietic stem cell differentiation and exhaustion. However, mechanisms that regulate the amount of ROS in leukemia-initiating cells (LICs) and the biological role of ROS in these cells are largely unknown. We show here that the ROS(low) subset of CD44(+) cells in T cell acute lymphoblastic leukemia (T-ALL), a malignancy of immature T cell progenitors, is highly enriched in the most aggressive LICs and that ROS accumulation is restrained by downregulation of protein kinase C θ (PKC-θ). Notably, primary mouse T-ALLs lacking PKC-θ show improved LIC activity, whereas enforced PKC-θ expression in both mouse and human primary T-ALLs compromised LIC activity. We also show that PKC-θ is regulated by a new pathway in which NOTCH1 induces runt-related transcription factor 3 (RUNX3), RUNX3 represses RUNX1 and RUNX1 induces PKC-θ. NOTCH1, which is frequently activated by mutation in T-ALL and required for LIC activity in both mouse and human models, thus acts to repress PKC-θ. These results reveal key functional roles for PKC-θ and ROS in T-ALL and suggest that aggressive biological behavior in vivo could be limited by therapeutic strategies that promote PKC-θ expression or activity, or the accumulation of ROS.
Subject(s)
Core Binding Factor Alpha 2 Subunit/metabolism , Core Binding Factor Alpha 3 Subunit/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma , Protein Kinase C , Reactive Oxygen Species/metabolism , Receptor, Notch1 , Animals , Cells, Cultured , Gene Expression Regulation, Leukemic , Humans , Jurkat Cells , Leukocytes, Mononuclear/metabolism , Mice , Mice, Knockout , Molecular Targeted Therapy , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/genetics , Protein Kinase C/metabolism , Receptor, Notch1/genetics , Receptor, Notch1/metabolism , Signal TransductionABSTRACT
Malignant transformation of normal hematopoietic progenitors is a multistep process that likely requires interaction between collaborating oncogenic signals at critical junctures. For instance, the MLL-AF9 fusion oncogene is thought to contribute to myeloid leukemogenesis by driving a hematopoietic stem cell-like "self-renewal" gene expression signature in committed myeloid progenitors. In addition, insulin-like growth factor (IGF) signaling has been implicated in self-renewal/pluripotency in hematopoietic and embryonic stem cell contexts and supports cell growth/survival by activation of downstream pathways, including phosphatidylinositol 3-kinase/Akt and Ras/Raf/extracellular signal-regulated kinase. We hypothesized that IGF signaling could be an important contributor in the process of cellular transformation and/or clonal propagation. Utilizing an MLL-AF9 mouse bone marrow transplantation model of acute myelogenous leukemia, we discovered that committed myeloid progenitor cells with genetically reduced levels of IGF1R were less susceptible to leukemogenic transformation due, at least in part, to a cell-autonomous defect in clonogenic activity. Rather unexpectedly, genetic deletion of IGF1R by inducible Cre recombinase had no effect on growth/survival of established leukemia cells. These findings suggest that IGF1R signaling contributes to transformation of normal myeloid progenitor cells, but is not required for propagation of the leukemic clone once it has become established. We also show that treatment of mouse MLL-AF9 acute myelogenous leukemia cells with BMS-536924, an IGF1R/insulin receptor-selective tyrosine kinase inhibitor, blocked cell growth, suggesting its efficacy in this model may be due to inhibition of insulin receptor and/or related tyrosine kinases, and raising the possibility that similar IGF1R inhibitors in clinical development may be acting through alternate/related pathways.