Open Access

Susanna Irene Hock

• Aktivierende Mutationen der Fms-like tyrosine kinase (FLT3) treten bei 25 % der Patienten mit akuter myeloischer Leukämie (AML) auf und begünstigen die unkontrollierte Proliferation maligner Blasten. Autophagie ist ein intrazellulärer Prozess, durch den zytoplasmatische Bestandteile lysosomal abgebaut werden und fungiert als intrazellulärer Homöostase-Mechanismus unter Stress-Bedingungen. Ziel dieser Arbeit war es, herauszufinden, ob FLT3-ITD+-AML-Zellen vulnerabel gegenüber Autophagie-Hemmung sind. Hierzu wurde zunächst untersucht, wie sich FLT3-ITD-Signaling und Autophagie unter basalen Wachstumsbedingungen gegenseitig beeinflussen. In einem genetischen Modell zeigte sich, dass FLT3-ITD-transformierte wachstumsfaktorunabhängige Zellen während ihrer fortgesetzten Proliferation vermehrt Autophagie betreiben. Lysosomale Autophagie-Inhibitoren zeigten jedoch unter diesen Bedingungen keine erhöhte Wirksamkeit gegenüber FLT3-ITD-positiven Zellen. Humane FLT3-ITD-positive AML-Zellen zeigten nach genetischer Deletion von ULK1 ebenfalls nur transiente und milde Proliferationsdefizite. Unter basalen Wachstumsbedingungen zeigte sich also keine erhöhte Vulnerabilität FLT3-ITD-exprimierender Zellen gegenüber Autophagie-Inhibition. Daraufhin wurde die Bedeutung von Autophagie während pharmakologischer Hemmung von FLT3 untersucht. FLT3-Inhibition mittels AC220, einem FLT3-spezifischer Tyrosinkinase-Inhibitor, induzierte bzw. steigerte die autophagische Aktivität ähnlich stark wie eine direkte mTOR-Inhibition. Dies ließ sich im Zellmodell therapeutisch ausnutzen: eine Kombinationsbehandlung mit AC220 und einem lysosomalen Autophagie-Inhibitor zeigte eine synergistische antiproliferative Wirkung. Dies stellt möglicherweise einen neuen rationalen Kombinationsbehandlungsansatz für die Therapie FLT3-ITD-positiver AML-Patienten dar.
• Mutations of Fms-like tyrosine kinase (FLT3) account for 25 % of cases of acute myeloid leukemia (AML) and lead to uncontrolled proliferation of malignant blasts. AML patients are often unfit to receive intensive chemotherapy needed to convalesce. When diagnosing AML testing for mutations such as the most frequent FLT3-internal tandem duplication (FLT3-ITD) is routine. It disrupts the autoinhibitory domain resulting in constitutive activation of the receptor itself, upregulation of MAPK and PI3K/AKT pathway and aberrant activation of STAT5. Besides a poor prognosis FLT3-ITD-AML provides pharmacologic opportunities like FLT3 inhibitors and/or inhibition of autophagy which describes a degradative process that recycles dispensable bulk to useable substrates in situations of stress such as nutrient deprivation or exposition to toxic agents as chemotherapeutics to maintain viability. This thesis aims at elucidating whether FLT3-ITD+ AML provides a vulnerability to the inhibition of autophagy. Using a genetic model, the impact of FLT3-ITD on the autophagic flux and the response to lysosomal inhibitors was characterized. Furthermore, the relevance of the protein kinase ULK1 was investigated by generating a genetic knockout as well as testing ULK1/2-inhibitors on AML-cells. FLT3-ITD-signaling turned out to be influenced by the autophagic dynamics and vice versa. Suppression of autophagy by lysosomal inhibitors, Chloroquine and Bafilomycin A1, reduced the proportion of phosphorylated, thus activated, FLT3 as well as AKT, downstream of FLT3-ITD. In turn, specific inhibition of FLT3 by AC220 increased the autophagic flux, determined on the decreased GFP/mCherry ratio measured by flow cytometry. As autophagy presents a dynamic process that is rapidly adapting to changes within the cellular metabolism these interactions seem to be crosstalk between main pathways to maintain homeostasis. Transformation with FLT3-ITD confers growth factor independent proliferation to the murine myeloblasts 32D. Upon deprivation of the cytokine mIL-3 32DFLT3-ITD displayed increased autophagy, providing a rationale for inhibiting autophagy to impair FLT3-ITD-mediated proliferation. However, although 32DFLT3-ITD were more effectively restrained from degrading autophagosomes following treatment with lysosomal inhibitors, Chloroquine and its derivates Lys05 and ROC-325 diminished the proliferation of 32DFLT3-ITD no more than 32Dctrl. Neither the additional deprivation of mIL-3 augmented the potency. Nevertheless, malignancies often reveal their dependency to autophagy once they are exposed to severe stress conditions like cytotoxic substances and to date not a single compound was found to be effective alone against AML. Given the fact of ULK1/2 being de- or activated through phosphorylation by mTORC1 during abundance of nutrients or by AMPK because of starvation, this protein kinase takes on a pivotal role in the regulation of autophagy. Activating phosphorylation of ATG13, FIP200 and Beclin-1 by ULK1/2 is necessary for the initiation of building an autophagosome in the canonical way. Thus, ULK1/2 offers a promising target for small molecules aiming at the inhibition of autophagy. Several highly selective ULK1/2-inhibitors turned out to potently impair proliferation of AML-cells harboring FLT3-ITD while those with WT-FLT3 were less vulnerable. In order to relate this observation to the specific inhibition of ULK1/2 the Aurora kinase A-inhibitor Alisertib was implemented to rule out this major off target. Indeed, this inhibitor without affinity for ULK1/2 displayed no greater impact on FLT3-ITD+ cells. However, a CRISPR/Cas-mediated genetic knockout of ULK1 neither impaired the proliferation nor the ability of autophagy, underlining the widely observed redundancy of ULK1 and ULK2 in many different mammalian cells. Lastly the FLT3-inhbitor AC220 was proven to be highly selective for FLT3 and to induce autophagy in FLT3-expressing cells. Finally, AC220 synergized with chloroquine to impair preferably FLT3-ITD+ cells’ viability. Therefore, simultaneous induction and inhibition of autophagy provides a novel strategy for treating AML. The compounds still have to be optimized to specifically target malignant blasts while sparing normal hematopoietic cells.