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.

Acute myeloid leukemia (AML) is a subset of leukemia that is characterized by the clonal expansion of cytogenetically and molecularly abnormal myeloid blasts. These blasts are highly proliferative accumulating in bone marrow and blood which leads to severe infections, anemia, and bone marrow failure. The poor prognosis of AML patients caused by the low tolerance to intensive chemotherapy has encouraged the pursuit of alternative therapeutic approaches. Differentiation therapy which involves the use of agents that can release the differentiation block in these leukemic blasts has emerged as a promising therapeutic approach. The use of All-trans retinoic acid (ATRA) represents a successful example of such an approach, nonetheless its efficacy is restricted to one subtype of AML. Efforts have been focused on finding differentiation agents which are effective for the other more common AML subtypes. Anti-CD44 targeted antibodies that activate the CD44 cell surface antigen are a promising candidate. Previous studies have shown that anti-CD44 treatment has been able to release the differentiation block in AML1 through AML5 subtypes. The exact mechanism by which anti-CD44 treatment is able to induce its effects has not been fully elucidated. Recent studies highlight the role that epigenetic mechanisms play during haematopoiesis and leukemogenesis and therefore, in this work we investigated the epigenetic mechanisms associated with anti-CD44 induced differentiation. Using AML cell lines from different subtypes, we demonstrated that anti-CD44-induced differentiation results in an extensive change of histone modification levels. We found that inhibiting enzymes responsible for the H3K9ac, H3K4me, H3K9me, and H3K27me modifications, attenuated the anti-proliferative and differentiation promoting effects of antic-CD44 treatment. Taken together, these data highlight the promising potential of using anti-CD44 as a therapeutic agent across multiple subtypes in AML

京都大学 / 新制・課程博士 / 博士(人間健康科学) / 甲第23124号 / 人健博第86号 / 新制||人健||6(附属図書館) / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 岡 昌吾, 教授 藤井 康友, 教授 髙折 晃史 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

In recent years, magnetic nanowires (NWs) have been widely used for their therapeutic potential in biomedical applications. The use of iron (Fe) NWs combines two important properties, biocompatibility and remote manipulation by magnetic fields. In addition the NWs can be coated and functionalized to target cells of interest and, upon exposure to an alternating magnetic field, have been shown to induce cell death on several types of adherent cells, including several cancer cell types. For suspension cells, however, using these NWs has been much less effective primarily due to the free-floating nature of the cells minimizing the interaction between them and the NWs. Leukemic cells express higher levels of the cell surface marker CD44 (Braumüller, Gansauge, Ramadani, & Gansauge, 2000), compared to normal blood cells. The goal of this study was to functionalize Fe NWs with a specific monoclonal antibody towards CD44 in order to target leukemic cells (HL-60 cells). This approach is expected to increase the probability of a specific binding to occur between HL-60 cells and Fe NWs. Fe NWs were fabricated with an average diameter of 30-40 nm and a length around 3-4 μm. Then, they were coated with both 3-Aminopropyl-triethoxysilane and bovine serum albumin (BSA) in order to conjugate them with an anti-CD44 antibody (i.e. anti-CD44-iron NWs). The antibody interacts with the amine group in the BSA via the 1-Ethyl-3-3-dimethylaminopropyl-carbodiimide and N-Hydroxysuccinimide coupling. The NWs functionalization was confirmed using a number of approaches including: infrared spectroscopy, Nanodrop to measure the concentration of CD44 antibody, as well as fluorescent-labeled secondary antibody staining to detect the primary CD44 antibody. To confirm that the anti-CD44-iron NWs and bare Fe NWs, in the absence of a magnetic field, were not toxic to HL-60 cells, cytotoxicity assays using XTT (2,3-Bis-2-Methoxy-4-Nitro-5-Sulfophenyl-2H-Tetrazolium-5-Carboxanilide) were performed and resulted in a high level of biocompatibility. In addition, the internalization of the coated NWs have been studied by coating them with a pH dependent dye (pHrodoTM Red) that showed a signal once the NWs were internalized by the cell.

Newer biological treatment strategies have been developed in the last decade with some promising outcomes. Their safety, however, has been questioned lately with multiple reports of increased risk for malignancies and infectious complications. These reports render their use suboptimal. We report a 44-year-old woman receiving adalimumab (Humira®) for advanced juvenile rheumatoid arthritis who then developed acute myelogenic leukemia.

Improved therapeutic strategies for patients with leukemia remain in great demand and beckon better understanding of the mechanisms underlying leukemic treatment resistance and relapse. Accordingly, discoveries in leukemic pathophysiology have been achieved in various animal models. Danio rerio—commonly known as the zebrafish—is a vertebrate organism well suited for the investigation of human leukemia. Zebrafish have a conserved hematopoietic program and unique experimental strengths. Recent technological advances in zebrafish research including efficient transgenesis, precise genome editing, and straightforward transplantation techniques have led to the generation of numerous zebrafish leukemia models. Additionally, improved imaging techniques, combined with the transparency of zebrafish, have revealed exquisite details of leukemic initiation, progression, and regression. Finally, advances in high-throughput drug screening in zebrafish are likely to hasten the discovery of novel anti-leukemic agents. Zebrafish provide a reliable experimental system for leukemic disease research and one in which investigators have accumulated knowledge concerning the genetic underpinnings of leukemic transformation and treatment resistance. Without doubt, zebrafish are rapidly expanding our understanding of disease mechanism and are helping to shape therapeutic strategy for improved patient outcomes.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

Acute myeloid leukemia (AML) is characterized by a blockage in the differentiation of myeloid cells at different stages. CD44-ligation using anti-CD44 monoclonal antibodies (mAbs) has been shown to reverse the blockage of differentiation and to inhibit the proliferation of blasts in most AML-subtypes. However, the molecular mechanisms underlying this property have not been fully elucidated. Here, we sought to I) analyze the effects of anti-CD44 mAbs on downstream signaling pathways, including the ERK1/2 (extracellular signal-regulated kinase 1 and 2) and mTOR (mammalian target of rapamycin) pathways and II) use state-of-the-art Nuclear Magnetic Resonance (NMR) technology to determine the global metabolic changes during differentiation induction of AML cells using anti-CD44 mAbs and other two previously reported differentiation agents. In the first objective (Chapter 4), our studies provide evidence that CD44-ligation with specific mAbs in AML cells induced an increase in ERK1/2 phosphorylation. The use of the MEK inhibitor (U0126) significantly inhibited the CD44-induced differentiation of HL60 cells, suggesting that ERK1/2 is critical for the CD44-triggered differentiation in AML. In addition, this was accompanied by a marked decrease in the phosphorylation of the mTORC1 and mTORC2 complexes, which are strongly correlated with the inhibition of the PI3K/Akt pathway. In the second objective (Chapter 5), 1H NMR experiments demonstrated that considerable changes in the metabolic profiles of HL60 cells were induced in response to each differentiation agent. These most notable metabolites that significantly changed upon CD44 ligation were involved in the tricarboxylic acid (TCA) cycle and glycolysis such as, succinate, fumarate and lactate. Therefore, we sought to analyze the mechanisms underlying their alterations. Our results revealed that anti-CD44 mAbs treatment induced upregulation in fumarate hydratase (FH) expression and its activity which was accompanied by a decrease in succinate dehydrogenase (SDH) activity. Interestingly, our results indicated that FH induced by anti-CD44 mAb is regulated through the activation of the ERK1/2 pathway. Therefore, our findings highlight new elements in support for the use of anti-CD44 mAbs in AML therapies and open new perspectives to use metabolic profiling as a tool to support the potential possibilities for the development of CD44-targeted therapy of AML.

This investigation is concerned with cell surface antigens present on murine AKR/J mice spleen and thymus cells which have been extracted with papain. Isolation of individual proteins was accomplished by granulated gel electrofocusing. Similar patterns recorded by both electrofocusing procedures identified several proteins limited to the AKR/J and C3Heb/FeJ spleen and thymus samples, which represent Murine Leukemia Virus-associated surface proteins.

The goal of part I of this thesis was to isolate pure anti cancer compounds from the Russian plant Sanguisorba officinalis. This plant was selected for investigation because it showed 100% activity against leukemia (L1210 mouse leukemia cells) during the preliminary screening of approximately 100 Russian plant extracts. This work has resulted in the isolation of novel compound 11 using traditional chromatography techniques. Compound 11 was characterized with spectroscopic techniques such as IR, 1H NMR, 13C NMR, DEPT, DQCOSY and MS. Compound 11 was assigned the structure dipentyl 2-(4-(pentan-3-yl) phenyl) malonate. The goal of part II of this thesis was to synthesize novel ethers of podocarpic acid. Ethers are of great interest in biological studies and pharmaceutical applications because of their wide variety of uses in the treatment of various diseases. A novel family of ether derivatives was synthesized using podocarpic acid (a natural tricyclic diterpene derived from podocarpus species) as a template. Novel ether derivatives of podocarpic acid were synthesized from podocarpic acid in three steps. The first step involved methylation of podocarpic acid with dimethyl sulfate to form methyl-o-methyl podocarpate. The second step was iodination of methyl-o-methyl podocarpate with iodine in presence of a mercury catalyst to form 13-iodo methyl-o-methyl podocarpate. This was followed by formation of novel aliphatic ethers using a copper catalyst. Thus this research had led to the discovery of new methodology for synthesis of three novel aliphatic ether derivatives of podocarpic acid. These ethers will be tested for their biological activity against various types of cancer, tuberculosis by National Institutes of Health.