Acute myeloid leukemia (AML) often remains refractory to current chemotherapy and transplantation approaches despite many advances in our understanding of mechanisms in leukemogenesis. The bone marrow “niche” or microenvironment, however, may be permissive to leukemia development and studying interactions between the microenvironment and leukemia cells may provide new insight for therapeutic advances. Mesenchymal stem cells (MSCs) are central to the development and maintenance of the bone marrow niche and have been shown to have important functional alterations derived from patients with different hematological disorders. The extent to which MSCs derived from AML patients are altered remains unclear. The aim of this study was to detect changes occurring in MSCs obtained from human bone marrow in patients with AML by comparing their function and gene expression pattern with normal age-matched controls. MSCs expanded from patients diagnosed with acute leukemia were observed to have heterogeneous morphological characteristics compared to the healthy controls. Immunohistochemistry and flow data confirmed the typical cell surface immunophenotype of CD90+ CD105+ CD73+ CD34- CD45-, although MSCs from two patients with AML revealed reduced surface expression of CD105 and CD90 antigens respectively. Differentiation assays demonstrated the potential of MSCs from AML patients and healthy donors to differentiate into bone, fat and cartilage. However, the ability of MSCs from AML samples to support hematopoietic function of CD34+ progenitors was found to be impaired while the key hematopoietic genes were found to be differentially expressed on AML-MSCs compared to nMSCs. These studies indicate that there exist differences in the biologic profile of MSCs from AML patients compared to MSCs derived from healthy donors. The results described in the thesis provide a formulation for additional studies that may allow us to identify new targets for improved treatment of AML.

Previous studies had demonstrated the presence of the p70 (CAMAL) molecule in human myeloid leukemia cells and the promyelocytic leukemia cell line HL60, but not in equivalent preparations of normal cells (Malcolm et al., 1982, 1984; Shipman et al., 1983; Logan et al., 1984). Subsequent studies demonstrated that the p70 (CAMAL) protein was detectable and expressed in human myeloid leukemia cells and the leukemic cell lines HL60, KG1, K562 and U937. The association of p70 (CAMAL) expression with human myeloid leukemia cells prompted its consideration as a candidate leukemia-associated antigen. The demonstration, following CAMAL purification and peptide sequencing, that two tryptic peptides (tp27, tp31) displayed significant homology to sequences present in human serum albumin (HSA) and human alpha-1-fetoprotein (AFP), while one tryptic peptide (tp20) displayed unique peptide sequence, suggested that CAMAL might represent a protein that was structurally and functionally related to the albumins. Consequently, a comparative biochemical analysis of CAMAL and HSA was initiated. The results of the comparative studies demonstrated that although CAMAL and HSA shared conformational antigenic determinants, both proteins were also shown to be distinct molecules by a number of other criteria. The possibility that the CAMAL preparation, used for protein sequencing and comparative studies, was contaminated with HSA was thought likely, in view of the HSA/AFP-related peptide sequences from the CAMAL tryptic peptide sequence analysis. However, other results, particularly the antibody reactivity and ligand binding studies, showed that the CAMAL preparation was not contaminated with HSA. The unique CAMAL tryptic peptide (tp20) sequence supported further the contention that CAMAL was a distinct protein with regions homologous to HSA and AFP. Further analysis of the CAMAL molecule, through extensive protein sequencing, will be, in all likelihood, the only means by which to establish the degree of relatedness between CAMAL, HSA and AFP. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

京都大学 / 0048 / 新制・課程博士 / 博士(社会健康医学) / 甲第22152号 / 社医博第100号 / 新制||社医||10(附属図書館) / 京都大学大学院医学研究科社会健康医学系専攻 / (主査)教授 川上 浩司, 教授 武藤 学, 教授 髙折 晃史 / 学位規則第4条第1項該当 / Doctor of Public Health / Kyoto University / DFAM

Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by great heterogeneity and clonal nature. In recent years, rapidly evolving next-generation sequencing methods provided a deep insight into the mutational background of AML. It was shown that ~ 44 % of AML patients harbor mutations in genes that regulate DNA methylation. So far, many researchers have tried to evaluate the prognostic significance of DNA methylation changes in AML, however, due to a great inconsistency in these studies, none of the reported markers were implemented into clinical practice. The aim of this work was to further investigate the DNA methylation changes in AML patients with specific mutations and their prognostic effect. Next, we wanted to develop a new approach for a complex evaluation of prognostically significant DNA methylation aberrations. In our first project, we assessed the overall DNA methylation, hydroxymethylation, and gene expression in AML patients with mutations in either DNMT3A or IDH1/2 or their combinations. We discovered that each genetic aberration is connected with a distinct pattern of DNA hydroxy-/methylation changes that are not entirely reflected in altered gene expression. Patients with mutations in both genes exhibited a mixed DNA methylation profile most similar to healthy...

Acute myeloid leukemia(AML) is the most common acute leukemia among adults in the United states with approximately 19,940 people being diagnosed of this disease in 2020 and 11,180 deaths. It is a heterogenous group of malignancy characterized by clonal expansion of blast with myeloid lineage in the bone marrow, peripheral blood and/or other tissues. Our patient is a 79-year-old male who presented to the hospital with reports of sharp, throbbing low back pain for one month, moderately controlled with pain medications. He reported 5 lb. weight loss with decreased appetite over one month but denied other constitutional symptoms. MRI Lumbar spine revealed multiple foci of marrow signal abnormality compatible with extensive metastatic disease. CT chest, abdomen and pelvis did not show any lesions concerning for primary or metastatic malignancy. CBC revealed normal WBC count, platelet count and hemoglobin level (with macrocytosis, MCV 104.7). Initial work up including Vitamin B12 and folic acid level, TSH, SPEP/IFE, serum light chain ratio and quantitative immunoglobulins were within normal limits. Pathology from a CT guided bone biopsy of the L spine lesion was concerning for high grade myeloid neoplasm. Patient had a bone marrow biopsy done at another hospital which was read as most consistent with acute myeloid leukemia (AML) with monocytic differentiation, with findings of hypocellular marrow, extensive fibrosis with focal areas of large clusters of immature cells, positive for MPO, CD33, CD43 and CD 56, Ki-67 of 60-80%. Cytogenetics showed an abnormal male karyotype with trisomy 8. FISH was negative for other AML or MDS related abnormalities. Given the above findings of AML and advanced age, patient was started on treatment with hypomethylating agent Decitabine along with BCL-2 inhibitor, Venetoclax. A repeat bone marrow biopsy after two cycles of the above regimen revealed progressive disease with extensive fibrosis and 80-90% blast on a core biopsy sample. Due to poor response to above regimen, lack of effective treatment options in older patients with AML and declining functional status, decision was made to pursue best supportive care. AML usually presents with symptoms of fevers, fatigue, dyspnea or bleeding. Skeletal lesions are usually associated with a diagnosis of multiple myeloma, or other solid organ malignancies and rare in AML. Extra medullary involvement of AML is known to happen in 2.5%-9% of patients and is termed as Myeloid Sarcoma. Due to the low incidence, prospective study data is limited. This entity is treated similarly to AML, depending on risk stratification by cytogenetics, age and targetable mutations which also govern its prognosis. This case highlights the importance of increased awareness and high index of suspicion among medical providers regarding this atypical presentation of AML since if missed or misdiagnosed could delay treatment and lead to poor outcomes.

No description available.

The presence of AML1-ETO (RUNX1-CBF2T1), a fusion oncoprotein resulting from a t(8;21) chromosomal translocation, is a necessary but insufficient event in the development of a subset of acute myeloid leukemias (AML). Although AML1-ETO is able to block differentiation and immortalize hematopoietic stem cells, other contributory events are required for cell proliferation and leukemogenesis, suggesting that specific tumor suppressor genes may counteract the leukemic potential of AML1-ETO. In studying del(9q), one of the most common concomitant chromosomal abnormalities with t(8;21), we identified the loss of TLE4 as a key cooperating event in the development of AML1-ETO AML, leading to increased cell proliferation, blocked apoptosis and differentiation, as well as cytarabine resistance in leukemic cells. This suggested TLE4 functions as a tumor suppressor gene in AML.We found these effects are mediated by the loss of TLE4 regulation of a COX-Wnt inflammatory axis. These effects were consequently reversible by Wnt signaling and cyclooxygenase inhibition, pointing towards anti-inflammatory agents as potentially new therapeutic and adjuvant strategies for AML. While studies in Drosophila implicate TLE/Groucho as a key mediator of various signaling pathways, including receptor tyrosine kinase/Ras/MAPK, Notch, Myc, and Wnt pathways, there is surprisingly little known about the role of TLE in mammalian development. Using a Tle4 knockout (T4KO) mouse, we identified previously unknown roles for Tle4 in regulating vertebrate mammalian hematopoietic and bone development. T4KO mice manifest leukocytopenia and defective hematopoietic stem cell populations. Using serial transplantation and stromal co-culture, we find that these hematopoietic deficiencies arise due to both intrinsic dysfunction of hematopoietic stem cells as well as defective extrinsic regulation of hematopoiesis by the stem cell niche. Additionally, T4KO mice are severely runted and exhibit markedly decreased bone mineralization concomitant with defective osteoblast function and decreased mineral apposition rates. Many of the pathways regulated by TLE are aberrant in cancer, which has led to increasing studies connecting TLE with various malignancies, including synovial cell sarcoma and glioblastoma. Our findings have great implications for current understanding of TLE function in not only cancer, but also bone and hematopoietic development.

Yes / The HOX genes encode a family of transcription factors that have key roles in both development and malignancy. Disrupting the interaction between HOX proteins and their binding partner, PBX, has been shown to cause apoptotic cell death in a range of solid tumors. However, despite HOX proteins playing a particularly significant role in acute myeloid leukemia (AML), the relationship between HOX gene expression and patient survival has not been evaluated (with the exception of HOXA9), and the mechanism by which HOX/PBX inhibition induces cell death in this malignancy is not well understood. In this study, we show that the expression of HOXA5, HOXB2, HOXB4, HOXB9, and HOXC9, but not HOXA9, in primary AML samples is significantly related to survival. Furthermore, the previously described inhibitor of HOX/PBX dimerization, HXR9, is cytotoxic to both AML-derived cell lines and primary AML cells from patients. The mechanism of cell death is not dependent on apoptosis but instead involves a regulated form of necrosis referred to as necroptosis. HXR9-induced necroptosis is enhanced by inhibitors of protein kinase C (PKC) signaling, and HXR9 combined with the PKC inhibitor Ro31 causes a significantly greater reduction in tumor growth compared to either reagent alone. / Funded in part through a grant to RA from the Cultural Bureau of the Kingdom of Saudi Arabia.

In patients with acute myeloid leukemia (AML), cytarabine-based chemotherapy usually achieves remission, but this is commonly followed by relapse and chemo-resistance. In this study, we aim to establish next-generation sequencing (NGS)-based microRNA expression profiling and pathway analysis to identify pathways regulated differentially between chemo-sensitive and -resistant AML as potential therapeutic targets. MicroRNA expression profiles differ significantly between chemo-sensitive and chemo-resistant AML cells and reflect differences in the activity of intracellular signaling cascades. Alterations in signaling pathway activities contribute to treatment resistance and thus represent potential drug targets. Our microRNA-led approach indicates a role for activin receptor type 2A in ARA-C resistance of AML cells and suggests activin receptor signaling to be a candidate pathway for targeted therapy.

No description available.