Acute myeloid leukemia (AML) is a clonal malignant disease characterized by a blockage in the differentiation of myeloid cells resulting in the accumulation of highly proliferating immature blast cells. With the success of All Trans Retinoic acid (ATRA) in acute promyelocytic leukemia (AML3), differentiation therapy has become a very attractive treatment option. Ligation of CD44 (a cell surface antigen) with anti-CD44 monoclonal antibodies (mAbs) is reported to reverse the blockage of differentiation and suppress the proliferation of blasts derived from most AML subtypes. However, the molecular mechanisms underlying this apparent ‘normalization’ (reversal) of AML cells induced by CD44 have not been fully elucidated. To expand our understanding of the cellular regulation and circuitry involved, we aimed to apply a quantitative phosphoproteomic approach using Stable Isotope Labeling with Amino acids in Cell culture (SILAC) to monitor dynamic changes of phosphorylation states in HL60 cells following treatment with CD44-mAbs. Phosphoproteomic analysis identified differentially phosphorylated proteins among CD44-mAb treated and control HL60 cells that are involved in a number of major signaling pathways as determined by the Ingenuity Pathway analysis (IPA®) platform. Among others, Rho signaling emerged as a major pathway significantly changed by CD44-mAb treatment. Rho GTPases are well-recognized regulators of the actin cytoskeleton but have also been implicated in diverse cellular events such as cell polarity, microtubule dynamics, membrane trafficking, transcriptional regulation, cell growth control and development. An interesting Rho family member, PAK2 was identified in our search. PAK2 is a ubiquitously expressed serine/threonine protein kinase, which is a direct target for small GTPases and has been identified as a switch between cell survival and cell death signaling depending on its mode of activation. Western-blot analyses of cell lysates of CD44-mAb treated and control HL60 cells confirmed that the phosphorylation of PAK2 ,as well as protein level,were altered as early as 5 minutes following treatment. PAK2 knockdown decreased the effect of CD44-mAb induction of proliferation and inhibition of proliferation proving its importance for mediating it’s signaling transduction. PAK1, a structural homologue of PAK2 had the opposite effect of augmenting CD44-mAb effects suggesting a different mechanism involved. This specificity is attributed to the specific mode of activation that PAK2 exhibits which is not shared with the rest of PAK group I members. Caspase-mediated cleavage of PAK2 producing pro-apoptotic fragments is hypothesized to be the signaling transduction mediated by CD44-mAb. In-Vivo experiments show that PAK2 is essential for leukemic cell migration to the spleen. Additionally, it proved essential for CD44-mAb inhibition of leukemic cells migration to the spleen. Further validation and characterization of PAK2’s activation mode, phosphorylation dynamics, subcellular localization as well as its role in invivo migration are essential in understanding its role in AML.

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by the accumulation of immature nonfunctional highly proliferative hematopoietic cells in the blood, due to a blockage in myeloid differentiation at various stages. Since the success of the differentiation agent, All-trans retinoic acid (ATRA), in the treatment of acute promyelocytic leukemia (APL), much effort has gone into trying to find agents that are able to differentiate AML cells and specifically the leukemic stem cell (LSC). CD44 is a cell surface receptor that is over-expressed on AML cells. When bound to anti-CD44 monoclonal antibodies (mAbs), this differentiation block is relieved in AML cells and their proliferation is reduced. The molecular mechanisms that AML cells undergo to achieve this reversal of their apparent phenotype is not fully understood. To this end, we designed a study using quantitative phosphoproteomics approaches aimed at identifying differences in phosphorylation found on proteins involved in signaling pathways post-treatment with CD44-mAbs. The Rho family of GTPases emerged as one of the most transformed pathways following the treatment with CD44-mAbs. The P21 activated kinase 2(PAK2), a target of the Rho family of GTPases, was found to be differentially phosphorylated in AML cells post-treatment with CD44-mAbs. This protein has been found to possess a role similar to that of a switch that determines whether the cell survives or undergoes apoptosis. Beyond confirming these results by various biochemical approaches, our study aimed to determine the effect of knock down of PAK2 on AML cell proliferation and differentiation. In addition, over-expression of PAK2 mutants using plasmid cloning was also explored to fully understand how levels of PAK2 as well as the alteration of specific phospohorylation sites could alter AML cell responses to CD44-mAbs. Results from this study will be important in determining whether PAK2 could be used as a potential therapeutic target for AML once its levels are altered.

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京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第22812号 / 生博第446号 / 新制||生||59(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 垣塚 彰, 教授 原田 浩, 教授 井垣 達吏 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

The c-Myc transcription factor is a key player in cell homeostasis, being commonly deregulated in human carcinogenesis. In this PhD thesis we have addressed the question how regulatory mechanisms restrain the oncogenic activity of c-Myc and its impact on cell differentiation. In the first half, we report that PML promotes destabilization of c-Myc protein and re-activation of c-Myc-repressed target genes. The consequent re-expression of the cell cycle inhibitor CDKN1A/p21 mediates differentiation of leukemic cells. In the second half of the thesis we identified a novel mechanism of gene regulation by c-Myc, which is mediated through its interaction with DNA-bound RARα. In undifferentiated cells, c-Myc/Max dimers cooperate with RARα in the repression of genes required for differentiation. Upon phosphorylation of c-Myc by the previously identified Pak2, the complex switches from a repressive to an activating function by releasing Max and recruiting transcriptional coactivators. These findings add a new and at least partially Max-independent mechanism for transcriptional regulation by c-Myc and also discover an unexpected function of c-Myc in inhibiting and promoting cellular differentiation. Taken together, our results describe two new mechanisms that counteract the oncogenic activity of c-Myc. Both PML and Pak2 can be considered as tumor suppressors since they modulate c-Myc function in a way that ultimately promotes differentiation of leukemic cells. This knowledge provides the basis for novel approaches to be exploited for the development of c-Myc-targeted therapies. / El factor de transcripció c-Myc juga un paper clau en l’homeòstasi cel·lular, essent freqüentment desregulat en la carcinogènesi humana. En aquesta tesi s’ha estudiat com diferents mecanismes reguladors poden frenar l’activitat oncogènica de c-Myc i el subsegüent impacte en la diferenciació cel·lular. A la primera meitat de la tesi, es demostra que PML promou la desestabilització de la proteïna c-Myc i, en conseqüència, la reactivació dels genes diana reprimits per c-Myc. Entre aquests gens diana es troba l’inhibidor del cicle cel·lular CDKN1A/p21, la reexpressió del qual provoca la diferenciació de cèl·lules leucèmiques induïda per PML. En la segona meitat, s’identifica un nou mecanisme de regulació transcripcional per part de c-Myc a través de la interacció amb RARα, el qual està unit a l’ADN. En cèl·lules indiferenciades, els dimers c-Myc/Max cooperen amb RARα en la repressió de gens essencials per a la diferenciació. Un cop c-Myc és fosforil·lat per la kinasa Pak2, el complex de c-Myc amb RARα esdevé activador mitjançant la pèrdua de Max i el reclutament de coactivadors transcripcionals. Aquest descobriment suposa un nou mecanisme mitjançant el qual c-Myc pot exercicir la regulació gènica almenys en part independentment de Max, i també revela una funció desconeguda de c-Myc en la inhibició i promoció de la diferenciació cel·lular. En conjunt, aquests resultats descriuen dos nous mecanismes que contrarestren l’activitat oncogènica de c-Myc. PML i Pak2 poden ser considerats supressors de tumors ja que modulen la funció de c-Myc per a promoure la diferenciació de les cèl·lules leucèmiques. Aquests descobriments poden utilitzar-se com a base pel desenvolupament de noves teràpies anti-tumorals que tinguin com a diana la proteïna c-Myc.