Open Access

Srinivasa Rao Bandi

• Acute myeloid leukemia (AML) is a hematopoietic cell disorder characterized by a block in differentiation and increased proliferation and survival of malignant blasts. Expansion of the malignant cell clone effects the normal production of blood cells and – if left untreated – leads to death. Receptor tyrosine kinases (RTKs) play an important role in the pathogenesis of AML, as they are either often mutated or overexpressed. In normal hematopoiesis, RTK signal termination is tightly controlled, and involves ubiquitination, internalization, endocytosis and degradation. Cbl proteins are E3 ligases and have been shown to ubiquitinate several activated RTKs, including Flt3 and Kit, targeting them for degradation. Recently, several Cbl mutations have been identified: Cbl-R420Q was identified in an AML patient and Cbl-70Z was identified in a mouse lymphoma model. In this thesis work, the role of these Cbl mutants in Kit signaling and in a mouse transplantation model was studied. Cbl mutants (Cbl-R420Q, Cbl-70Z) have the ability to transform the myeloid 32D cell line in cooperation with Kit WT. Cbl mutants along with Kit promoted interleukin-3 (IL3)-independent proliferation and enhanced the cell survival of 32D cells. In contrast, expression of the Cbl mutants alone did not confer IL3-independent growth. Stem cell factor (SCF, the Kit ligand) dependent growth was enhanced in the presence of Cbl mutants and Cbl mutants promoted colonogenic growth in the presence of Kit. Furthermore, Cbl mutants inhibited the ubiquitination of the activated Kit receptor. In addition, Cbl mutants inhibited the endocytosis of the activated Kit receptor. Retroviral expression of Cbl mutants in transplanted bone marrow induced a generalized mastocytosis, a myeloproliferative disease and, in rare care cases, myeloid leukemia. Splenomegaly was observed in the presence of Cbl mutants. Furthermore, mast cells with variable range of infiltration were noticed in all the vital organs (spleen, liver, bone marrow, lung, kidney, heart) of Cbl (mutant) transplanted mice. Almost all recipients of bone marrow cells transduced with Cbl mutants developed a lethal hematologic disorder with a mean latency of 341 days in the Cbl-R420Q group and 395 days in the Cbl-70Z group. This is the first published report on a hematological disease with Cbl mutants in a mouse model. Co-immunoprecipitation studies indicated that Cbl-70Z binds to Kit, even in the absence of Kit ligand. Cbl-R420Q also bound to Kit in the absence of SCF, albeit to a lesser extent. Association of Cbl mutants to Kit was enhanced in the presence of SCF. Signaling studies demonstrated the constitutive activation of Akt and Erk in the presence of Cbl mutants and Kit. In addition, Cbl mutants enhanced the SCF-dependent Kit, Akt and Erk activation. Cbl-70Z, in association with kinase-dead Kit (Kit-KD) or kinase-dead Flt3 (Flt3-KD), conferred IL3-independent growth and survival to the myeloid 32D cell line. Cbl-R420Q provided only a slight growth advantage in the presence of Kit-KD. As demonstrated by pharmacological inhibition studies, Akt activation was necessary for the transformation mediated by Cbl-70Z and Kit-KD / Flt3-KD. Cbl mutants enhanced the Src family kinases (SFKs) activity. The pharmacological inhibition of SFK activity inhibited the proliferation and colonogenic growth. Interaction was found between Cbl-70Z, SFKs and Kit-KD. The SFK member Fyn was identified to bind to Cbl. In addition, kinase activity of SFKs was necessary for binding to Cbl, since SFKs inhibition by PP-2 abolished the binding between the complex-binding partners. Dasatinib and PP-2, both SFK inhibitors, inhibited the Cbl and Akt phosphorylation indicating that Fyn acts upstream of Akt. Inhibition of Kit with imatinib reduced the proliferation of cells overexpressing Kit WT and Cbl-70Z much stronger compared with cells expressing Kit-KD and Cbl-70Z, but much less than the dual KIT/SFK inhibitor dasatinib. This indicated that Kit kinase activity was required but not essential. The data presented in this thesis work implies that both RTK and SFK inhibition may have to be targeted, in order to effectively prevent transformation. In summary, the present thesis work indicates an important role of Cbl, Kit and SFKs in myeloid transformation and deregulated signal transduction.
• Die akute myeloische Leukämie (AML) ist eine Erkrankung hämatopoetischer Zellen, die durch einen Differenzierungsblock und die unkontrollierte Bildung unreifer hämatopoetischer Zellen oder Blasten charakterisiert ist. Dabei kommt es zu einer starken Einschränkung der normalen Hämatopoese, was letztlich ohne Behandlung zum Tod des Patienten führt. Die Entstehung einer AML wird grundsätzlich als Entwicklung angesehen, die in mehreren Schritten vonstatten geht. Genetische Veränderungen, wie Mutationen, Translokationen, Amplifikationen und Deletionen sind an der Pathogenese der AML beteiligt. Diese genetischen Veränderungen können in zwei Gruppen eingeteilt werden: solche die zu einem Proliferations- oder Überlebensvorteil führen (Klasse I Mutationen) und andere, die einen Differenzierungsblock hervorrufen (Klasse II Mutationen). Eine einzelne Mutation dieser Art ist nicht in der Lage alleine einer AML auszulösen. Während Kit-Mutationen relativ selten sind, wird bei der Mehrzahl der AML-Patienten eine Überexpression des Kit Rezeptors gefunden. Der Kit-Rezeptor wird durch seinen Liganden „Stem Cell Factor“ (SCF) aktiviert. Die Aktivierung von Kit führt zur Rekrutierung von Signalmolekülen. Sog. „Gain-of-function“-Mutationen von Kit führen zu einer konstitutiven Aktivierung des Rezeptors. Cbl-Proteine degradieren bekanntermaßen aktivierte Rezeptortyrosinkinasen (RTKs) durch Ubiquitinierung. Die Untersuchung der Signaltransduktion von RTKs wie Kit oder Flt3 und deren Degradation, ist für die Entwicklung neuer therapeutischer Strategien zu von enormer Wichtigkeit. Gegenstand der vorliegenden Untersuchung ist der Einfluss von Cbl-Mutanten (Cbl-R420Q und Cbl-70Z) auf die Kit–vermittelte Signaltransduktion. Cbl-R420Z wurde bei einem AML-Patienten gefunden, während Cbl-70Z in Maus-Myelom-Studien identifiziert wurde. Diese Cbl Mutanten haben die E3-Ligase Aktivität, die eine wichtige Rolle beim Transfer der Ubiquitin Moleküle zum Zielprotein spielt, verloren. ...