Aberrant activation of the innate immune response is hypothesized to contribute to leukemogenesis in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Patients with MDS were described with mutations that constitutively activated such pathways. Our studies suggested that mutations which impair termination of emergency (stress) granulopoiesis (EG) are also a possible mechanism. EG is the process for rapid, episodic granulocyte (PMN) production during infectious challenge and a key component of the innate immune response. We previously determined that Triad1, an E3 ubiquitin (Ub) ligase, was essential for EG- termination. Consistent with a role for a sustained EG response in leukemogenesis, we found Triad1 functioned as a leukemia suppressor for AML with increased expression of homeodomain transcription factors. An adverse prognosis subset of clonal myeloid malignancies, including MDS/AML with MLL1/KMT2A rearrangements, is characterized by overexpression of a group of these proteins (e.g HoxB3, B4, A9-11, Cdx1 and 2, Meis1). Mice transplanted with bone marrow expressing leukemia-associated Mll1-fusion proteins develop AML after a lag time of months; suggesting leukemogenesis requires accumulation of mutations in addition to those involving MLL1/KMT2A-rearrangement. We found Triad1 expression decreased during leukemogenesis in a mice with expression of an Mll1-fusion protein in the bone marrow. We also found that either Triad1 knockdown or EG episodes accelerated AML development in such mice. We demonstrated HoxA10 enhanced, but HoxA9 repressed, Triad1 gene transcription. And, Triad1 re-expression rescued EG termination in Hoxa10-/- mice. We performed a screen to identify proteins with Triad1-dependent Ub. In addition to inflammatory mediators and RTKs, we identified proteins involved in the integrated stress response (ISR; Gcn1, eIF2B4 and eIF4G1). The ISR prevents metabolic exhaustion and cell death during sustained inflammation by modulating translation to correct metabolic defects and enhance proliferation once defects are corrected. Gcn1 functions as a primary regulator of this process by activating Gcn2/eIF2B4. We found Triad1-knockdown in myeloid cells altered the profile of mRNAs undergoing translation. However, combined knockdown of Triad1 and Gcn1 in these cells reversed abnormalities in translation of mRNAs involved in cellular response to stress, cellular response to DNA damage, cell cycle progression, translation, protein metabolism and ISR termination with Triad1 knockdown alone. We found Gcn1 knockdown delayed AML development in mice transplanted with bone marrow expressing an Mll1-fusion oncoprotein, and reversed the effect of Triad1-knockdown on accelerating leukemogenesis. We hypothesize that inhibition of the ISR by Triad1 facilitates emergency granulopoiesis (EG)-termination and suppresses leukemogenesis in disorders with increased Hox expression. This will be pursued by 3 Aims. Aim 1: Define the role of ISR in...