FLT3-ITD Signaling Continuously Degrades p53 By the Ubiquitin-Proteosome Pathway Explaining Chemo-Resistance

Introduction. FLT3-ITD AMLs resist standard chemotherapy at high rates by unknown mechanisms. Chemotherapy aims to upregulate p53 (TP53) the master regulator of apoptosis (‘cytotoxicity’). In normal myelopoiesis, p53 is suppressed by continuous proteolysis, presumably to allow immediate-early stress-responses in rapidly dividing cells, avoiding transcription/translation delays. We therefore examined if FLT3-ITD signaling promotes p53 proteolysis, as is needed to expand myeloid progenitors even normally.

Methods and Results. (i) The deubiquitinase USP7 is a key component of the ubiquitin-proteosome pathway (UPP) and is known to regulate p53 proteolysis in cell contexts other than myeloid. To evaluate a potential role for USP7 in regulating p53 in FLT3-ITD AML cells (MOLM13), we immunoprecipitated endogenous USP7 and used liquid chromatography tandem mass spectrometry (IP-LCMS/MS) to analyze for post-translational modifications in presence/absence of FLT3-ITD kinase-inhibitors: USP7 was phosphorylated at serine-18 (S18-P), a highly conserved residue. Kinase-inhibition decreased S18-P by ~50%, increased USP7 and p53 protein (Western blot, WB), and activated apoptosis in FLT3-ITD (MOLM13, MV4-11) but not FLT3-wildtype (WT) (THP1, OCI-AML3) AML cells (annexin/PI staining).

(ii) The small molecule P22077 is a specific modifier of the catalytic site in USP7 but not other deubiquitinases (Pozhidaeva. Cell Chem Biol 2017;24(12):1501). P22077 2.5 µM increased p53 protein (WB), and activated apoptosis (annexin/PI staining), again in FLT3-ITD but not FLT3-WT AML cells.

(iii) We reasoned that if FLT3-ITD signaling continuously degrades p53, then inhibiting the proteosome should also increase p53, without need for kinase-inhibitors. Bortezomib to inhibit the 26S proteasome increased p53 and activated apoptosis specifically in FLT3-ITD but not FLT3-WT AML cells, results reproduced with MG-132 that inhibits the 20S proteosome.

(iv) Consistent with FLT3-ITD acting as an alternative to TP53 genetic alterations to suppress p53, FLT3-ITD and TP53 mutations or deletions were mutually exclusive, although all are highly recurrent (analyses of TCGA data).

(v) IP-LCMS/MS of endogenous USP7 revealed interactions also with CEBPA, the master transcription factor driver of granulocytic differentiation. USP7/CEBPA interactions were confirmed by reverse IP-LCMS/MS of endogenous CEBPA, and by bi-directional IP-WB of endogenous USP7 and CEBPA. Demonstrating functional significance, FLT3-ITD AML cells (MOLM13, MV4-11) contained high CEBPA mRNA but little CEBPA protein. Inhibiting FLT3-ITD signaling increased total CEBPA protein even as CEBPA S21-P decreased, and activated granulocytic differentiation (in addition to apoptosis). Further supporting that CEBPA S21-P is a degron, broad-spectrum proteosome inhibitors increased S21-P and total CEBPA protein. Extending these results beyond cell lines, FLT3-ITD and FLT3-wildtype (WT) primary AML cells expressed similar amounts of CEBPA mRNA, but CEBPA target-genes were ~2-fold suppressed in FLT3-ITD (n=27) vs FLT3-WT (n=90) cells, with a significant positive correlation between CEBPA and CEBPA target-gene activation in FLT3-WT but not FLT3-ITD cells. Consistent with FLT3 and CEBPA operating in the same pathway, FLT3-ITD was mutually exclusive with biallelic CEBPA mutations (analyses of TCGA data).

Results in Context. Normal FLT3 signaling expands granulo-monocytic progenitors. Thus, FLT3-ITD promotion of p53 and CEBPA proteolysis may represent continuous imposition of molecular actions that are normally ligand-activated and transient. These mechanisms support evaluation of proteosome inhibitors, approved for other indications, as candidate treatments for relapsed/refractory FLT3-ITD AMLs, supported also by other reports of specific sensitivity of FLT3-ITD AML cells to proteosome inhibitors (Larrue. Blood 2016;127(7):882. Long. Cell Rpts Med 2023;4(11):101286). Presently, midostaurin to inhibit FLT3-ITD signaling is ingested on days after infusional chemotherapy - the mechanisms shown here suggest ingestion simultaneous or immediately-after chemotherapy, so that p53 is more available to respond to chemotherapy stress and activate apoptosis – timings again supported by in vitro studies by others (Levis. Blood 2004;104(4):1145. Schittenhelm. Cell cycle 2009;8(16):2621).