Triad1 regulates myelopoiesis and functions as a leukemia suppressor

Triad1 is an E3 ubiquitin ligase that impairs proliferation of bone marrow progenitor cells and increases in expression during granulopoiesis. We found that Triad1 enhances ubiquitin (Ub) mediated degradation of Fgf- R1, Flt3 and αv integrin in myeloid cells. Fgf-R1 and Flt3 activate phospho-inositol-3-kinase; resulting in stabilization of βcatenin and expression of βcatenin-target-genes involved in proliferation/survival. Syk is activated by αvβ3 integrin, resulting in Pak1-dependent proliferation and PLCγ-dependent survival. Triad1 also inhibits Ub of p53 by Mdm2. We hypothesize that increasing Triad1 expression during granulopoiesis favors degradation of Fgf-R1, Flt3 and αvβ3 integrin, but stabilizes p53; decreasing proliferation and enhancing sensitivity to apoptosis. This identifies a possible leukemia suppressor function for Triad1, since impaired activity would sustain Fgf-R1, Flt3 and αvβ3 signaling and destabilize p53. Consistent with this, Triad1 is specifically decreased in subsets of acute myeloid leukemia (AML) with MLL-translocations (i.e. 11q23-AML) or activating FLT3 mutation. Our studies suggest a mechanism for this. 11q23-AML is characterized by increased expression of a set of HOX genes, including HoxA9 and A10. We found that HoxA9 and A10 regulate transcription of ARIH2 (encoding Triad1) in a manner that requires cytokine-induced tyrosine phosphorylation of HoxA9 and A10. We found that constitutive activation of Shp2-PTP blocks ARIH2 transcription by preventing Hox phosphorylation. Interestingly, FLT3 mutations are frequent in Hox-over- expressing and activate Shp2. A myeloproliferative neoplasm (MPN) develops in mice transplanted with bone marrow expressing MLL fusion proteins or overexpressing HoxA9 or A10. This MPN evolves to AML over time, suggesting that Hox-overexpression is inadequate for AML in the absence of cooperating mutations. We find constitutive Shp2-activation performs this function. We hypothesize Triad1 is a leukemia suppressor that decreases proliferation/survival of cytokine-stimulated progenitor cells, and impaired Triad1-activity facilitates disease progression/drug resistance in Hox-overexpressing AML. We will pursue this via 3 aims: Aim 1: Identify functionally significant, Triad1-regulated events during myelopoiesis. The functional impact of Triad1-related Ub/degradation of Fgf-R1, αvβ3, Flt3 and p53 will be investigated in vitro and in vivo. Aim 2: Determine if Triad1 is a leukemia suppressor in AML with Hox-overexpression and Shp2 activation. The contributions of Triad1-expression and Shp2-activation to drug resistance/disease progression will be explored in studies with murine AML models and human AML bone marrow samples. Aim 3: Define mechanisms of leukemia suppression by Triad1. The contributions of Fgf-R1, Flt3, αv integrin and p53 to the leukemia suppressor effect of Triad1 will be explored in vivo in murine AML models. Hox-overexpressing AML has poor prognosis and is treatment refractory. Clarifying cooperating lesions and down-stream events may suggest therapeutic targets for this subset of individuals.