β-Catenin Determines Developmental Stage Specific Transformation by Hox Genes.
Authors: Wang Yingzi , Krivtsov Andrei V. , Sinha Amit U. , North Trista , Goessling Wolfram , Zon Leonard I. , Armstrong Scott 
Published: October 2019

Abstract
Abstract 385
Leukemia stem cells (LSC) possess extensive proliferative and self-renewal potential similar to normal hematopoietic stem cells (HSC). Therefore understanding the similarities and differences between HSC and LSC is critical if LSC specific therapies are to be developed. Hox genes represent a group of genes that can influence both normal HSC and LSC self-renewal, and are critical targets of leukemogenic MLL fusion proteins. Previous reports have described the ability of Hoxa9 and Meis1a (HoxA9/M) to induce leukemia when expressed in mouse bone marrow (BM). However, whether HoxA9/M can fully recapitulate the leukemogenic activity of MLL fusion proteins remains unclear. In this study, we show that HoxA9/M, unlike MLL-AF9, fails to induce leukemia from granulocyte-macrophage progenitors (GMP) but does so from HSC. Immunophenotypic analysis and in vivo limiting dilution transplantation of HSC-derived leukemias demonstrate heterogeneity with only a subset of cells possessing leukemia-propagating activity. The LSC in this model have an immunophenotype consistent with differentiating myeloid cells. Gene expression analysis of LSC induced by MLL-AF9 expression in GMP and HoxA9/M expression in HSC demonstrate an approximately 10-fold increase in prostaglandin-endoperoxide synthase 1 (PTGS1) (also known as Cycloxygenase-1 or Cox-1) and prostaglandin E receptor 1 (PTGER1) expression. As recent studies have highlighted a critical connection between prostaglandin synthesis and Wnt/ β-catenin signaling pathway, we hypothesized that β-catenin is aberrantly activated in LSC derived from either GMP expressing MLL-AF9 or HSC expressing HoxA9/M. Western blots and immunofluorescence using an antibody specific for dephosphorylated (activated) β-catenin identified active β-catenin in MLL-AF9-driven and HoxA9/M-driven LSC but not normal GMP. These data suggested that insufficient β-catenin activity might be a contributing factor to the inability of HoxA9/M to transform GMP and thus we sought to determine if activated β-catenin cooperated to induce leukemia from GMP. We found that co-expression of HoxA9/M and activated β-catenin efficiently induced leukemia from GMP whereas neither expressed alone had leukemogenic activity. Next, we assessed if β-catenin is required for HoxA9/M-mediated leukemogenesis initiated from HSC. Conditional β-catenin loss-of-function experiments demonstrated impaired in vivo expansion of cells derived from HoxA9/M transduced HSC, and β-cat-/- cells did not induce leukemia. This defect could be rescued by expression of a constitutively active form of β-catenin. Finally, we demonstrate that continued β-catenin activity is required for LSC maintenance by chemical suppression of the β-catenin pathway with indomethacin (a cox-1/cox-2 inhibitor), which shows remarkable selective elimination of the LSC fraction in mice transplanted with HoxA9/M transduced HSC. Our gain and loss-of-function studies demonstrate that β-catenin activity is required for leukemia initiation from HSC, and that constitutively active β-catenin can cooperate with HoxA9/M to efficiently transform GMP. Thus, Wnt/β-catenin activity makes cells permissive to transformation, which suggests that its restricted activation to stem cell populations in normal hematopoietic development limits the permissiveness of developmental cell types to transformation by specific oncogenes. These data have important implications for tumor development in other tissues/organs and for the development of β-catenin pathway antagonists in AML.

Disclosures:
No relevant conflicts of interest to declare.