Immunomodulatory drugs (IMiDs) are a class of drugs, derived from the teratogenic compound thalidomide, that have made a major impact on treatment of many diseases, from multiple myeloma to assorted inflammatory diseases. From its dark beginnings as a teratogenic agent that caused phocomelia in newborn infants, thalidomide has resurged decades later as a potent immunomodulatory agent with widespread anti-inflammatory and anti-tumor effects. Research examining Thalidomide’s effects in vitro on malignant myeloma cells has led to the development of newer analogs, lenalidomide and pomalidomide, both of which are now available on the market. Clinically, these drugs have had a tremendous impact on patient progression-free survival, especially when administered in conjunction with other novel agents. Despite the numerous properties that have been reported for IMiDs, until recently, little was known about their mechanism of action. Knowledge of likely only one of IMiDs’ direct mechanism of action has not only opened up opportunities for additional discoveries, but also propelled research to better characterize genetic profiles of multiple myeloma patients and potential biomarkers of disease progression and response to treatment. This thesis will attempt to review the history and literature behind the biological mechanisms of IMiDs, the clinical risks and benefits of using such drugs as treatment for cancer, and future directions for areas of research.

BACKGROUND: Multiple myeloma is a rare form of cancer that affects the proper function of plasma cells in the immune system. Patients experience symptoms ranging from bone pain to otherwise avoidable infections that can have negative effects on quality of life. Despite advances in multiple myeloma treatment leading to longer patient survival, it is still an incurable form of blood cancer. As a result, it is important for researchers to constantly investigate new avenues of treatment in order to delay disease progression. This study investigated whether the next generation proteasome inhibitor, ixazomib, could safely delay disease progression in patients who failed a combination regimen that included either the proteasome inhibitor bortezomib or carfilzomib. METHODS: This study is a phase 1/2, 3+3 design, intra-patient, multicenter, open-label, and non-randomized clinical trial that recruited patients that were previously on one of ten combination treatments containing the proteasome inhibitors bortezomib or carfilzomib. Patients must have shown progressive disease while on this treatment in order to qualify. They were given the same drugs and doses they were previously taking except that the proteasome inhibitor was replaced with ixazomib. The safety and efficacy measurements were taken periodically to assess patients’ disease burden. To assess safety, adverse events (AEs) and serious adverse events (SAEs) were recorded, codified, and quantified for analysis. In addition, the maximum tolerated dose (MTS) of ixazomib for three regimens for which it was unknown was investigated through the analysis of dose limiting toxicities (DLTs). Clinical benefit rate (CBR) and overall response rate (ORR) using response data were also determined. Lastly, Kaplan-Meir statistical analysis was used to calculate the secondary efficacy endpoints such as progression free survival (PFS) using data collected throughout the trial. RESULTS: Safety: 24.4% of patients experienced at least one ≥ Grade 3 serious adverse event, 33.3% experienced at least one ≥ Grade 3 adverse event, and two experienced dose limiting toxicities. Efficacy: ORR was 13.2% and the CRR was 18.4%. Median PFS was 2.1 months, duration of response (DOR) was 2.0 months, and overall survival (OS) was 7.9 months. However, the MTD of ixazomib for the three regimens which it was unknown for was not found due to the nature of the data distribution. CONCLUSION: The results indicated that ixazomib is not an effective replacement for bortezomib or carfilzomib in combination treatments containing these drugs, which is apparent from low primary and secondary efficacy endpoints. However, due to a low occurrence of adverse events, serious adverse events, and dose limiting toxicities safety was confirmed. In addition, physicians should determine the MTD on a case by case basis through individual dose escalations if ixazomib is to be used in this context.

Multiple myeloma is characterized by proliferation of plasma cells in the bone marrow, producing monoclonal immunoglobulin. It accounts for 17% of hematologic malignancies in the US. Diagnosis is often suspected in the setting of bone lytic lesions, anemia, hypercalcemia or renal failure. Rarely, multiple myeloma can present with soft tissue involvement which can be difficult to diagnose. Below we present one such presentation. Our patient is a 53-year-old who was initially diagnosed with multiple myeloma six years back when he presented to hospital with back and right leg pain. On admission he was found to have multiple lytic lesions involving the appendicular and axial skeleton. On further workup, bone marrow biopsy showed 30% plasma cells with IgG kappa monoclonal protein elevation. Patient was diagnosed with ISS stage II multiple myeloma. He was treated with standard regimen with Velcade, Revlimid and dexamethasone with excellent response. Patient was evaluated for stem cell transplant however did not qualify for it due to social challenges. Patient was continued on maintenance therapy with Velcade and Revlimid for 8 cycles prior to clinical relapse with lytic lesions in the C-spine. At this point patient was switched to different therapeutic regimen with pomalidomide, carfilzomib and dexamethasone and had excellent response for 35 cycles on this regimen. Patient had interruption in treatment for 3 months due to other medical comorbidities. A repeat bone marrow biopsy which was done in November of 2019 revealed extensive bone marrow involvement with 70% plasma cells concerning for relapse. Patient was started on single agent daratumumab in December 2019 however had a difficult course interrupted by right-sided abdominal pain, persistent nausea and decreased appetite requiring hospital admission. Further workup revealed a 2.7 cm lesion in the liver as well as a 4.9 x 7.3 cm T11 left paraspinal soft tissue mass. Biopsy of the liver lesion revealed sheets of kappa restricted abnormal plasma cells concerning for progression of disease. Given the involvement of the visceral organ and the extent of his disease, it was decided to switch patient's treatment from single agent daratumumab to a multi agent chemotherapy regimen with dexamethasone, cyclophosphamide, etoposide and cisplatin. Patient received his 1st cycle inpatient and had marked symptomatic improvement and was discharged home. His M-protein spike reduced from 3.9 to 1.8 g/dl post once cycle of treatment. Soft tissue involvement by multiple myeloma is rare event. Though malignant plasma cells may diffusely infiltrate the liver parenchyma, the nodular spread is unique. In review by Talamo et al, out of 2,584 patients with MM, only 11 had liver plasmacytomas. This phenomenon is driven by lack of expression of adhesion molecules, increased heparanase-1 expression and loss of chemokine receptors on myeloma cells. Such alterations in cell architecture lead to more aggressive disease behavior. At present time treatment for this unique patient population does not differ from other MM cases. It is important for clinicians to recognize the possibility of such event.

Multiple myeloma is a plasma cell cancer that progressively evolves to an aggressive, multi-drug resistant disease, which presents an unmet clinical need. In clinical trials, myeloma shows susceptibility to novel immunotherapeutic agents, particularly those targeting B-cell maturation antigen (BCMA). Among different classes of immunotherapies, T cell-based approaches have progressed the most due to their ability to induce durable responses in patients with advanced drug-resistant blood cancers. Most T cell engineering strategies rely on the use of chimeric antigen receptors (CARs), which although effective, can cause serious life-threatening toxicities. We created a new synthetic receptor, T cell antigen coupler (TAC), which recruits the endogenous T cell receptor and allows T cells to autoregulate their activity. Our experience in solid tumor models has shown that TAC-T cells are similarly efficacious and significantly less toxic than CAR-T cells. This thesis describes our optimization of BCMA-specific TAC-T cells and analysis of different anti-BCMA antigen-binding domains. TAC receptor functions by engaging endogenous TCR-CD3 complex and redirecting it to the target of interest. In Chapter 3, we characterize optimization and humanization of the CD3-recruitment domain in the TAC scaffold and provide evidence that TAC-T cells are effective against multiple myeloma, irrespective of receptor surface levels. In Chapter 4, we describe selection of the human BCMA-binding domain and the creation of a fully humanized TAC receptor against BCMA. Chapters 5 and 6 describe how a BCMA-targeting antigen-binding domain that cross-reacts with an unknown antigen in mice augments in vivo efficacy of TAC- and CAR-T cells, respectively. The work described in Chapters 3 and 4 presents an optimized, fully human BCMA-TAC that is being moved into clinical testing. The work in Chapters 5 and 6 improves our understanding of how antigen-targeting domains in synthetic receptors influence the functionality of engineered T cells. / Thesis / Doctor of Science (PhD) / Multiple myeloma is an incurable blood cancer that has a remarkable ability to develop resistance to different types of chemotherapy. In recent years, treatments redirecting immune cells against tumors have shown impressive clinical responses against different types of chemotherapy-resistant blood cancers, including multiple myeloma. Our lab has developed a new technology for redirecting T cells against tumors, called T cell antigen coupler (TAC) receptor. This thesis describes optimization of a fully human TAC receptor specific for a target on the surface of myeloma cells, known as BCMA. Durable remissions induced by TAC-engineered T cells in a preclinical mouse model of myeloma in the absence of toxicity warrant further testing of this therapeutic in a clinical trial.

No description available.

Cheung Kin Fai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 87-105). / Abstracts in English and Chinese. / Abstract (English version) --- p.i / Abstract (Chinese version) --- p.iii / Acknowledgments --- p.vi / Table of Contents --- p.v / List of Tables --- p.viii / List of Figures --- p.iv / List of Abbreviations --- p.xi / Chapter CHAPTER 1 --- GENERAL INTRODUCTION --- p.1 / Chapter CHAPTER 2 --- LITERATURE REVIEW --- p.3 / Chapter 2.1 --- Multiple myeloma --- p.3 / Chapter 2.2 --- Epidemiology of MM --- p.3 / Chapter 2.3 --- Risk factors --- p.4 / Chapter 2.4 --- Pathophysiology of MM --- p.5 / Chapter 2.5 --- Clinical presentations and diagnosis --- p.6 / Chapter 2.5.1 --- Diagnosis --- p.6 / Chapter 2.5.1.1 --- Laboratory testing of blood and urine --- p.6 / Chapter 2.5.1.2 --- Radiographic evaluations --- p.1 / Chapter 2.5.1.3 --- Bone marrow biopsy --- p.7 / Chapter 2.6 --- Staging and classification --- p.9 / Chapter 2.6.1 --- Staging --- p.9 / Chapter 2.6.2 --- Classification --- p.11 / Chapter 2.6.2.1 --- Monoclonal gammopathy of undetermined significance --- p.11 / Chapter 2.6.2.2 --- Asymptomatic MM --- p.12 / Chapter 2.6.2.3 --- Smouldering MM --- p.12 / Chapter 2.6.2.4 --- Indolent MM --- p.12 / Chapter 2.6.2.5 --- Symptomatic MM --- p.12 / Chapter 2.7 --- Treatment --- p.14 / Chapter 2.8 --- Epigenetics: DNA methylation --- p.15 / Chapter 2.9 --- Fundamental aspects of DNA methylation --- p.16 / Chapter 2.9.1 --- CpG islands --- p.16 / Chapter 2.9.2 --- Roles of DNA methylation --- p.16 / Chapter 2.9.3 --- Proposed mechanisms of transcriptional repression mediated by methylation --- p.18 / Chapter 2.10 --- Possible mechanisms to initiate aberrant DNA methylation --- p.21 / Chapter 2.11 --- DNA methylation in tumorigenesis --- p.22 / Chapter 2.11.1 --- Oncogenic point C → T mutation --- p.22 / Chapter 2.11.2 --- Global DNA hypomethylation --- p.23 / Chapter 2.11.3 --- Regional DNA hypermethylation --- p.23 / Chapter 2.12 --- Aberrant DNA methylation in MM --- p.25 / Chapter 2.12.1 --- Self-sufficiency in growth signals --- p.25 / Chapter 2.12.2 --- Evading apoptosis --- p.26 / Chapter 2.12.3 --- Insensitivity to antigrowth signals --- p.26 / Chapter 2.12.4 --- Tissue invasion and metastasis --- p.27 / Chapter 2.12.5 --- Infinite replicative potential --- p.28 / Chapter 2.12.6 --- Genome instability --- p.30 / Chapter 2.13 --- Methodologies of DNA methylation analysis --- p.32 / Chapter 2.13.1 --- Genome wide screening method: MS.AP-PCR --- p.32 / Chapter 2.13.2 --- Combined bisulfite restriction analysis --- p.34 / Chapter 2.13.3 --- Cloned bisulfite genomic sequencing --- p.36 / Chapter 2.13.4 --- Treatment with demethylating agent --- p.36 / Chapter CHAPTER 3 --- MATERIALS AND METHODS --- p.38 / Chapter 3.1 --- MM specimens --- p.38 / Chapter 3.1.1 --- MM samples --- p.38 / Chapter 3.1.2 --- MM cell lines --- p.38 / Chapter 3.2 --- Magnetic cell sorting of CD138-positive plasma cells --- p.39 / Chapter 3.3 --- Isolation of nuclear pellet from PB --- p.41 / Chapter 3.4 --- "DNA extraction from MM cell lines, MM plasma cells and PB" --- p.41 / Chapter 3.5 --- MS.AP-PCR --- p.42 / Chapter 3.5.1 --- Restriction enzyme digestion of genomic DNA --- p.42 / Chapter 3.5.2 --- Arbitrarily primed polymerase chain reaction --- p.42 / Chapter 3.5.3 --- Isolation of differentially methylated DNA fragments --- p.43 / Chapter 3.6 --- Cloning of differentially methylated DNA fragments --- p.46 / Chapter 3.6.1 --- TA cloning --- p.46 / Chapter 3.6.2 --- Heat shock transformation --- p.46 / Chapter 3.6.3 --- Screening of positive clones by PCR --- p.46 / Chapter 3.6.4 --- Alkaline lysis for plasmid DNA preparation --- p.47 / Chapter 3.7 --- MS.AP-PCR sequence analysis --- p.47 / Chapter 3.7.1 --- Nucleotide sequencing --- p.47 / Chapter 3.7.2 --- CpG islands analysis of differentially methylated sequences --- p.48 / Chapter 3.8 --- DNA methylation analysis --- p.48 / Chapter 3.8.1 --- Sodium bisulfite modification --- p.48 / Chapter 3.8.2 --- Combined bisulfite restriction analysis --- p.49 / Chapter 3.8.3 --- Cloned bisulfite genomic sequencing --- p.49 / Chapter 3.9 --- Gene expression analysis --- p.50 / Chapter 3.9.1 --- RNA extraction --- p.50 / Chapter 3.9.2 --- Reverse transcription PCR --- p.50 / Chapter 3.9.3 --- 5'-aza-2'-deoxycytidine treatment --- p.51 / Chapter CHAPTER 4 --- RESULTS --- p.53 / Chapter 4.1 --- Generation of DNA methylation patterns by MS.AP-PCR --- p.53 / Chapter 4.1.1. --- Global methylation content in MM samples and normal PB lymphocytes --- p.56 / Chapter 4.1.2. --- Differential methylation in MM --- p.56 / Chapter 4.2 --- UCSC BLAT analysis of differentially methylated DNA fragments --- p.60 / Chapter 4.3 --- Identification of two candidate genes with downregulated expression --- p.60 / Chapter 4.4 --- Zinc fingers and homeoboxes 2 (ZHX2) --- p.62 / Chapter 4.4.1 --- ZHX2 CpG islands BLAT search analysis --- p.62 / Chapter 4.4.2 --- Hypermethylation of ZHX2 in MM cell lines --- p.63 / Chapter 4.4.3 --- Downregulated expression of ZHX2 in methylated MM cell lines --- p.66 / Chapter 4.4.4 --- Restoration of ZHX2 expression by 5-Aza-dC treatment --- p.67 / Chapter 4.4.5 --- Unmethylation of ZHX2 in primary MM tumors --- p.68 / Chapter 4.5 --- Ring finger protein 180 (RNF180) --- p.69 / Chapter 4.5.1 --- RNF180 CpG islands BLAT search analysis --- p.69 / Chapter 4.5.2 --- Hypermethylation of RNF180 in MM cell lines --- p.70 / Chapter 4.5.3 --- Downregulated expression of RNF180 in methylated MM cell lines --- p.73 / Chapter 4.5.4 --- Restoration of RNF180 expression by 5-Aza-dC treatment --- p.74 / Chapter 4.5.5 --- Methylation of RNF180 in primary MM tumors --- p.75 / Chapter CHAPTER 5 --- DISCUSSION --- p.76 / Chapter 5.1 --- Importance of methylation in MM --- p.76 / Chapter 5.2 --- Genome-wide screening approach by MS.AP-PCR --- p.76 / Chapter 5.3 --- Sample selection in MS.AP-PCR --- p.78 / Chapter 5.4 --- Methylation patterns in MM --- p.79 / Chapter 5.5 --- Candidate genes selection strategies --- p.81 / Chapter 5.6 --- Zinc fingers and homeoboxes 2 --- p.81 / Chapter 5.7 --- Ring finger protein 180 --- p.83 / Chapter 5.8 --- Limitations --- p.84 / Chapter CHAPTER 6 --- CONCLUSION --- p.86 / REFERENCES --- p.87

多發性骨髓瘤(Multiple myeloma) 為骨髓內漿細胞異常增生的惡性腫瘤，到目前為止仍然難以治癒。其發生發展是一個複雜的多步驟事件，涉及腫瘤細胞中遺傳和表觀遺傳的改變，以及骨髓微環境的支持。現已確定骨髓瘤細胞和骨髓微環境之間的相互作用對於骨髓瘤的病理發生，以及骨髓瘤細胞的生長，遷移和抗藥性起著關鍵作用。血小根因子四(Platelet factor 4, or PF4) 是一種抗血管生成的趨化因子。它不僅在體外抑制血管內皮細胞增殖和遷移，而且在體內抑制腫瘤的生長。此前，我們發現PF4 基因在多發性骨髓瘤中等位缺失以及DNA 高度甲基化，因而導致其在骨髓瘤病人及細胞系中的表達缺失或降低。在本研究中，我們利用體內和體外實驗鑒定了PF4 對骨髓瘤細胞以及血管生成的作用，並闡明了其作用機制。 / 首先，我們在體外鑒定了PF4 在骨髓瘤細胞中的功能。我們發現PF4 抑制骨髓瘤細胞系以及從病人骨髓中分離出來的骨髓瘤細胞的生長，以及促進其凋亡。其促凋亡活性與caspase-3 和PARP 的激活有關。我們也檢測了PF4 在骨髓瘤中對血管生成的作用。我們首先分離了病人骨髓中的內皮細胞。結果顯示PF4抑制骨髓瘤內皮細胞的生長和管狀物的形成。這些結果證明PF4 在骨髓瘤中可能是一個抑癌因子。 / 接下來我們進一步檢測了PF4 在體內的抑癌功能。在第一種模型中，骨髓瘤細胞被皮下移植到重症聯合兔疫缺陷型(NOD-SCID) 小鼠中。尾靜脈注射200ngPF4 明顯的抑制了腫瘤的生長，並延長了小鼠的成活率。第二種小鼠模型稱為兔鼠融合模型(SCID-rab model) 。在這一模型中，大白兔的腿骨先被皮下移植到(NOD-SCID) 小鼠中，再將骨髓瘤細胞注射入已植入的大白兔腿骨的骨腔中。兩周後，小鼠被尾靜脈注射入20 或200ng PF4 。結果顯示200ng PF4 顯著抑制了腫瘤的生長。通過兔疫組化分析大白兔腿骨切片，我們進一步證明了PF4 在腫瘤細胞中的增瘟，凋亡以及血管生成的作用。我們的發現因此證實了PF4 是骨髓瘤中的一個抑制因子。 / 為了鑒定PF4 在骨髓瘤中的作用機制，我們用Protein/DNA 微陣列(Protein/DNA array) 分析了PF4 參與的信號通路。結果顯示PF4 調節了若干個轉錄因子，其中包括STAT3 。凝膠遷移(EMSA) 和螢光素酪報告基因(luciferase reporter assay )檢測進一步證實PF4 抑制了STAT3 的DNA 結合能力以及轉錄活性。因此PF4 可能通過抑制STAT3 信號通路而抑制骨髓瘤的生長。我們進一步發現PF4 能抑制組成性的以及自介素6 (IL-6) 誘導的STAT3的激活。我們發現PF4 下調了STAT3 下游的靶基因，包括Mc1-1， Survivin 以及血管內皮細胞生長因子(VEGF)。而過表達組成性激活的STAT3 能逆轉PF4 所誘導的細胞凋亡。在兔鼠敵合模型中，通過兔疫組化分析大白兔腿骨切片，我們發現PF4 能抑制STAT3 的入核。SOCS3 是STAT3 其中的一個抑制因子，我們發現PF4 能誘導SOCS3 的表達。而干擾掉SOCS3 能使PF4 喪失其抑制STAT3 激活的能力。這些結果表明PF4 可能通過誘導SOCS3 的表達，從而抑制STAT3 信號通路，引起骨髓瘤的生長抑制以及抗血管生成。 / 總而言之，本研究表明PF4 是骨髓髓中一個重要調節因子。在體外和體內，PF4 通過抑制STAT3 信號通路，從而抑制腫瘤細胞的生長，促進凋亡以及抑制血管生成。本文為PF4 的臨床研究，作為一種新的治療骨髓瘤藥物，提高骨髓瘤病人的治療效果提供基礎。 / Multiple myeloma (MM) is an incurable hematological malignancy characterized by accumulation of clonal plasma cells in bone marrow (BM). The development and progression of MM is a complex multistep tumorigenic event involving both genetic and epigenetic changes in the tumor cell as well as the support by the BM microenvironment. It has been well established that the physical interaction of MM cells with the BM milieu are crucial for MM pathogenesis, MM cell growth, survival, migration and drug resistance. Platelet factor 4 (PF4), a potent antiangiogenic chemokine, not only inhibits endothelial cell proliferation and migration in vitro but also solid tumor growth in vivo. Our group previously demonstrated loss of PF4 expression in patient MM samples and MM cell lines due to concurrent allelic loss and DNA hypermethylation. In this study, we characterized the effects of PF4 on MM cells and angiogenesis in the BM milieu both in vitro and in vivo and elucidated the mechanism of PF4 effects on MM. / To characterize the effects of PF4 on MM cells in vitro, assays on cell growth, cell cycle arrest and apoptosis were performed and we found that PF4 inhibited growth and induced apoptosis in both MM cell lines and MM cells from patients. The proapoptotic activity of PF4 is associated with activation of caspase-3 and poly (ADP) ribose polymerase (PARP). We also investigated the effects of PF4 on angiogenesis in MM using endothelial cells isolated from patient's BM aspirates (MMECs). Our results showed that PF4 suppressed MMECs growth and tube formation on matrigel in a dose-dependent manner. / Given the ability of PF4 to suppress MM cell growth and angiogenesis in vitro, we evaluated its tumor suppressive function in vivo. In human subcutaneously matrigel xenograft mouse model, tail vein injection of 200ng PF4 significantly reduced MM tumor growth and prolonged survival. We next used the SCID-rab mouse model which recapitulates the human BM milieu in vivo. In this model, MM cells were directly injected into the rabbit bone which was subcutaneously implanted into the NOD-SCID mice. Two weeks after injection, SCID mice were treated with various dose of PF4 (20 or 200ng per injection, three times per week) or PBS by tail vein injection. ELISA assay for hIg (lambda) showed that tumor growth in 200ng PF4-treated mice was markedly reduced by 58% compared with the control group, which was further confirmed by immunohistochemistry analysis of CD 138 staining on rabbit bone section. Consistent with the in vitro results, induction of apoptosis in MM cells and inhibition of angiogenesis by PF4 could also be demonstrated in vivo, as evidenced by the findings on ki67, Cleaved caspase-3, CD31 and VEGF staining on rabbit bone sections from treated versus control mice. Our findings thus confirmed that PF4 is a novel tumor suppressor in MM. / However, the molecular mechanism of how PF4 inhibits MM tumorigenesis is still unclear. To identify the signal pathway PF4 involved in MM, Protein/DNA array was performed. We found that PF4 regulated several transcription factors including STAT3 in U266 cells. EMSA and luciferase reporter assay further confirmed that PF4 suppressed STAT3 DNA binding and transcriptional activity. So it is possible that PF4 mediates its tumor suppressive function, through suppressing STAT3 pathway in MM cells. We further found that pre-treatment of PF4 blocked both constitutive and interleukin-6-induced STAT3 activation in a time-dependent manner in human MM cells. PF4 could also down-regulate the STAT3-regulated gene products including Mcl-I, Survivin and vascular endothelial growth factor (VEGF). Moreover, enforced expression of constitutively active STAT3 rescued cells from PF4-induced apoptosis. In SCID-rab mouse model, we also found that PF4 inhibited STAT3 nuclear translocation by immunostaining of rabbit bone sections. When examined further, we found that PF4 induced the expression of one of the STAT3 inhibitor SOCS3, and gene silencing of SOCS3 by small interfering RNA abolished the ability of PF4 to inhibit STAT3 activation, suggesting a critical role of SOCS3 in the action of PF4. Our findings therefore suggest that by inducing SOCS3 expression, PF4 abrogates STAT3 activity, thus induces tumor growth inhibition and anti-angiogenesis. / Together, these novel studies have shown that PF4 is an important regulator of MM tumorigenesis. By abrogating STAT3 signaling it targets cell growth, induces apoptosis, suppresses angiogenesis both in vitro and in vivo in MM. These scientific observations provide the framework for clinical studies of this chemokine, as a novel drug for treatment of MM to improve patient outcome in MM. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liang, Pei. / "November 2011." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 139-161). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract in English --- p.I / Abstract in Chinese --- p.IV / List of Publications --- p.VI / Acknowledgements --- p.VII / List of abbreviations --- p.IX / List of Tables --- p.XII / List of Figures --- p.xm / Table of Contents --- p.XV / Chapter Chapter1 --- Introduction and Literature Review --- p.1 / Chapter 1.1 --- Multiple myeloma-General description --- p.1 / Chapter 1.1.1 --- Epidemiology of MM --- p.1 / Chapter 1.1.2 --- Stages of MM --- p.1 / Chapter 1.2 --- The bone marrow (BM) microenvironment in MM --- p.3 / Chapter 1.3 --- Signal pathways in MM cells --- p.5 / Chapter 1.3.1 --- JAK/STAT3 in cancers and MM --- p.5 / Chapter 1.3.1.1 --- IL-6 and its receptor --- p.7 / Chapter 1.3.1.2 --- Activation of downstream signals-The "on" signals --- p.9 / Chapter 1.3.1.3 --- Inactivation of downstream signaling --- p.11 / Chapter 1.3.1.3.1 --- Phosphatases --- p.12 / Chapter 1.3.1.3.2 --- SOCS family --- p.13 / Chapter 1.3.1.3.3 --- The PIAS family --- p.14 / Chapter 1.3.2. --- NF-κB pathway --- p.15 / Chapter 1.3.3 --- RAS-MAPK pathway --- p.17 / Chapter 1.3.4 --- Phosphatidyl inositol-3 kinase (PI3K)/AKT --- p.18 / Chapter 1.4 --- Angiogenesis in MM --- p.18 / Chapter 1.4.1 --- The process of angiogenesis --- p.18 / Chapter 1.4.2 --- Angiogenesis in caner --- p.20 / Chapter 1.4.3 --- Angiogenesis in MM --- p.22 / Chapter 1.5 --- Animal models in MM --- p.24 / Chapter 1.6 --- Treatment of MM --- p.27 / Chapter 1.6.1 --- Chemotherapy --- p.27 / Chapter 1.6.2 --- Autologous stem cell transplantation --- p.28 / Chapter 1.6.3 --- Biologically based therapies --- p.28 / Chapter 1.7 --- Platelet factor 4 (PF4) --- p.30 / Chapter 1.8 --- Structure of PF 4 --- p.30 / Chapter 1.9 --- Role of PF4 in physiological process --- p.32 / Chapter 1.9.1 --- Inhibition of megakaryocytopoiesis --- p.32 / Chapter 1.9.2 --- PF4 and coagulation --- p.33 / Chapter 1.10 --- Role of PF4 in pathological process --- p.34 / Chapter 1.10.1 --- PF4 and cancer --- p.34 / Chapter 1.10.2 --- PF4 is an angiogenic inhibitor --- p.35 / Chapter 1.11 --- Clinical applications of PF4 --- p.37 / Chapter 1.12 --- Summary and project aims --- p.37 / Chapter Chapter 2 --- Materials and Methods --- p.40 / Chapter 2.1 --- Reagents and antibodies --- p.40 / Chapter 2.2 --- MM Cell lines --- p.40 / Chapter 2.3 --- CD138⁺ primary MM cells --- p.41 / Chapter 2.4 --- CD31⁺ MM endothelial cells (MMECs) --- p.42 / Chapter 2.5 --- WST-1 assay --- p.43 / Chapter 2.6 --- Trypan blue exclusion --- p.43 / Chapter 2.7 --- Cell cycle analysis --- p.44 / Chapter 2.8 --- Apoptosis analysis --- p.44 / Chapter 2.9 --- In vitro tube formation assay --- p.45 / Chapter 2.10 --- SCID-rab mice model --- p.45 / Chapter 2.10.1 --- Construction of SCID-rab mice --- p.45 / Chapter 2.10.2 --- Establishment and monitoring of myeloma in SCID-rab mice --- p.46 / Chapter 2.10.3 --- Enzyme-linked immunosorbent assay (ELISA) --- p.46 / Chapter 2.10.4 --- PF4 treatment --- p.47 / Chapter 2.10.5 --- Immunohistochemistry --- p.48 / Chapter 2.11 --- Protein/DNA arrays --- p.49 / Chapter 2.12 --- Electrophoretic mobility shift assay (EMSA) --- p.50 / Chapter 2.13 --- Luciferase reporter assay --- p.52 / Chapter 2.14 --- Western blotting --- p.53 / Chapter 2.15 --- RNA extraction --- p.54 / Chapter 2.16 --- Real-time Polymerase Chain Reaction (Real-time PCR) --- p.54 / Chapter 2.17 --- Nuclear transfection --- p.55 / Chapter 2.18 --- Statistical analysis --- p.55 / Chapter Chapter3 --- The role of PF4 in MM: in vitro studies --- p.58 / Chapter 3.1 --- Results --- p.58 / Chapter 3.1.1 --- PF4 inhibited growth of human MM cell lines --- p.58 / Chapter 3.1.2 --- PF4 did not cause cell cycle arrest --- p.59 / Chapter 3.1.3 --- PF4 induced apoptosis of myeloma cell lines --- p.63 / Chapter 3.1.4 --- PF4 caused cell apoptosis in primary MM cells cultured in vitro --- p.64 / Chapter 3.1.5 --- PF4 suppressed MMECs growth --- p.69 / Chapter 3.1.6 --- PF4 suppressed MMECs tube formation --- p.69 / Chapter 3.2 --- Discussion --- p.73 / Chapter 3.2.1 --- Negative regulation of PF4 in MM cells growth in vitro --- p.73 / Chapter 3.2.2 --- PF4 induces apoptosis in MM cell lines and primary MM cells --- p.74 / Chapter 3.2.3 --- PF4 inhibits angiogenesis in MM in vitro --- p.76 / Chapter 3.3 --- Summary --- p.79 / Chapter Chapter4 --- The role ofPF4 in MM tumorigenesis: in vivo studies --- p.82 / Chapter 4.1 --- Results --- p.82 / Chapter 4.1.1 --- PF4 inhibited MM tumor growth and prolonged survival in subcutaneous matrigel xenograft model --- p.82 / Chapter 4.1.2 --- PF4 inhibited MM tumor growth and prolonged survival in SCID-rab mouse model --- p.85 / Chapter 4.1.3 --- PF4 reduced human MM cell proliferation, angiogenesis and induced apoptosis in SCID-rab mice --- p.88 / Chapter 4.2 --- Discussion --- p.91 / Chapter 4.2.1 --- PF4 inhibited human tumor growth in subcutaneous matrigel xenograft mouse model --- p.91 / Chapter 4.2.2 --- SCID-rab mouse model was successfully established and PF4 inhibited human MM turnor growth in this model --- p.92 / Chapter 4.2.3 --- PF4 inhibited human MM cell proliferation, angiogenesis and induced apoptosis in SCID-rab mice --- p.95 / Chapter 4.3 --- Summary --- p.96 / Chapter Chapter 5 --- The molecular mechanisms of PF4 in MM tumorigenesis --- p.98 / Chapter 5.1 --- Results --- p.98 / Chapter 5.1.1 --- ProteinlDNA array hybridization and Quantification of protein/DNA array spots --- p.98 / Chapter 5.1.2 --- PF4 suppressed DNA binding and transcriptional activity of STAT3 --- p.102 / Chapter 5.1.3 --- PF4 inhibited constitutive STAT3 phosphorylation in MM cells --- p.104 / Chapter 5.1.4 --- PF4 inhibited IL-6-induced STAT3 activation --- p.105 / Chapter 5.1.5 --- PF4 suppressed STAT3 regulated gene expression --- p.107 / Chapter 5.1.6 --- Enforced expression of constitutively active STAT3 rescued cells from PF4-induced apoptosis --- p.109 / Chapter 5.1.7 --- PF4 induced the expression of SOCS3 --- p.111 / Chapter 5.1.8 --- PF4-induced inhibition of STAT3 activation was reversed by gene silencing of SOCS3 --- p.111 / Chapter 5.1.9 --- PF4 inhibited nuclear accumulation of STAT3 and induced expression of SOCS3 in vivo --- p.114 / Chapter 5.2 --- Discussion --- p.115 / Chapter 5.2.1 --- PF4 regulated several TFs --- p.115 / Chapter 5.2.2 --- PF4 inhibited constitutive activation of STAT3 --- p.118 / Chapter 5.2.3 --- PF4 inhibited IL-6 induced activation of STAT3 --- p.120 / Chapter 5.2.4 --- PF4 suppressed STAT3 regulated gene expression --- p.121 / Chapter 5.2.5 --- PF4 induced the expression of SOCS3 --- p.124 / Chapter 5.3 --- Summary --- p.125 / Chapter Chapter 6 --- Conclusion and future studies --- p.128 / Chapter 6.1 --- Conclusion --- p.128 / Chapter 6.2 --- Future studies --- p.135 / Appendices --- p.137 / References list --- p.139

Cheung, Hing Yau Coty. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 111-132). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.