Abstract
<title>Abstract</title> <p> FLT3-ITD mutations in acute myeloid leukemias (AMLs) cause ligand-independent signaling. One way signaling pathways potently and immediately influence cell fates is by phosphorylating key fate-determining proteins to trigger their proteolysis. We investigated the master transcription factor (MTF) driver of granulo-monocytic lineage-fates, CEBPA, for regulation by this mechanism because we found high CEBPA mRNA but little CEBPA protein in <italic>FLT3-ITD</italic> versus <italic>FLT3</italic> -wildtype AML cells, and inhibiting FLT3-ITD signaling with tyrosine kinase inhibitors (TKI) rapidly rescued CEBPA protein. Mass spectrometry revealed that CEBPA interacts with major ubiquitin-proteasome pathway (UPP) components: the ubiquitin-ligase UHRF1 and the deubiquitinase USP7. TKI treatment decreased the phosphorylation of CEBPA (Ser21) and USP7 (Ser18) alongside shifts in CEBPA interactions from degradative UHRF1 to protective USP7, stabilizing CEBPA and activating differentiation. Similarly, TKIs and UPP inhibitors stabilized the USP7 client p53, triggering apoptosis specifically in FLT3-ITD cells. Notably, UPP inhibitors (such as bortezomib) successfully stabilized CEBPA and p53 even in TKI-resistant <italic>FLT3-ITD</italic> cells. Because FLT3-ITD signaling functionally suppresses CEBPA and p53, genetic mutations in <italic>CEBPA</italic> or <italic>TP53</italic> were mutually exclusive with FLT3-ITD in clinical series. In summary, FLT3-ITD drives the UPP-mediated destruction of CEBPA and p53, positioning UPP inhibitors as promising therapeutic candidates acting downstream of TKIs. </p>