Signaling pathways regulated by mutant Fms-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD), which mediate resistance to acute myeloid leukemia (AML) cell death, are poorly understood. Here, we reveal that pro-cell death lipid ceramide generation is suppressed by FLT3-ITD signaling. Molecular or pharmacologic inhibition of FLT3-ITD reactivated ceramide synthesis, selectively inducing mitophagy and AML cell death. Mechanistically, FLT3-ITD targeting induced ceramide accumulation on the outer mitochondrial membrane, which then directly bound autophagy-inducing light chain 3 (LC3), involving its I35 and F52 residues, to recruit autophagosomes for execution of lethal mitophagy. Short hairpin RNA (shRNA)-mediated knockdown of LC3 prevented AML cell death in response to FLT3-ITD inhibition by crenolanib, which was restored by wild-type (WT)-LC3, but not mutants of LC3 with altered ceramide binding (I35A-LC3 or F52A-LC3). Mitochondrial ceramide accumulation and lethal mitophagy induction in response to FLT3-ITD targeting was mediated by dynamin-related protein 1 (Drp1) activation via inhibition of protein kinase A-regulated S637 phosphorylation, resulting in mitochondrial fission. Inhibition of Drp1 prevented ceramide-dependent lethal mitophagy, and reconstitution of WT-Drp1 or phospho-null S637A-Drp1 but not its inactive phospho-mimic mutant (S637D-Drp1), restored mitochondrial fission and mitophagy in response to crenolanib in FLT3-ITD AML cells expressing stable shRNA against endogenous Drp1. Moreover, activating FLT3-ITD signaling in crenolanib-resistant AML cells suppressed ceramide-dependent mitophagy and prevented cell death. FLT3-ITD AML drug resistance is attenuated by LCL-461, a mitochondria-targeted ceramide analog drug, in vivo, which also induced lethal mitophagy in human AML blasts with clinically relevant FLT3 mutations. Thus, these data reveal a novel mechanism which regulates AML cell death by ceramide-dependent mitophagy in response to FLT3-ITD targeting.

To my entire family: for your loving support and tireless encouragement, I dedicate this work to you.iii Acknowledgements: Abstract:The precise mechanism regulating focal adhesion disassembly has yet to be elucidated. Recently

These authors contributed equally to this work.Keywords: Ste20-like kinase, cancer and metastasis, development, cell migration, cell cycle, apoptosis, focal adhesion turnover, cytoskeletal dynamics, HER2/Neu/ErbB2 signaling, FAK/src signaling Abbreviations: AT 2 R, angiotensin II type 2 receptor; ASK1, apoptosis signal-regulating kinase-1; ATH, AT1-46 homology; CHK2, checkpoint kinase-2; DAPK3, death-associated protein kinase-3; ER, endoplasmic reticulum; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; EMT, epithelial-to-mesenchymal transition; FA, focal adhesion; FAK, focal adhesion kinase; GCKs, germinal center kinases; JNK1, c-Jun N-terminal kinase-1; Ldb, LIM domain binding protein; LOK, lymphocyte oriented kinase; MST1, mammalian sterile twenty 1; MKK, MAPK kinase; MEK1, MAPK kinase 1; MAPK, mitogen-activated protein kinase; NLS, nuclear localization signal; PAK, p21-activated kinase; PH-PAK, Pleckstrin-homology domain-containing PAK; Plk1, polo-like kinase homolog 1; RTK, receptor tyrosine kinase; SLK, Ste20-like kinase; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling; TNFa, tumor necrosis factor a; xPlkk1, Xenopus polo-like kinase kinase-1Reorganization of the cytoskeleton is necessary for apoptosis, proliferation, migration, development and tissue repair. However, it is well established that mutations or overexpression of key regulators contribute to the phenotype and progression of several pathologies such as cancer. For instance, c-src mutations and the overexpression of FAK have been implicated in the invasive and metastatic process, suggesting that components of the motility system may represent a new class of therapeutic targets. Over the last several years, we and others have established distinct roles for the Ste20-like kinase SLK, encompassing apoptosis, growth, motility and development. Here, we review the SLK field from its initial cloning to the most recent findings from our laboratory. We summarize the various roles of SLK and the biochemical mechanisms that regulate its activity. These various findings reveal very complex functions and pattern of regulation for SLK in development and cancer, making it a potential therapeutic target.