The high demand of novel peptide and peptidomimetics based on the amount of genomic and proteomic data need be matched by synthesis and screening. The design and synthesis of peptide and peptidomimetics are so important because the peptide and protein-protein interaction play a key role in molecule recognition and signaling. The modified peptides have better stability and pharmacokinetic properties which may be guided by rational design and molecular modeling. Now many organic and medicinal chemists have chosen peptide and peptidomimetics as potential drug candidates for many targets. In this dissertation, research efforts in design and synthesis of cyclic peptides with stabilized secondary structure have been investigated. Cyclization of linear peptides may restrict the number of available conformations which may improve the affinity attaching to the target. In this study, different beta turn linkers have been designed and synthesized to achieve more stable cyclic peptides with beta-sheet structures. Based on different beta turn linkers, analogs of cyclic peptides have been synthesized and screened. The structure activity relationships (SAR) of these cyclic peptide analogs have been studied. In chapter three, analogs of peptidomimetic inhibitors have been designed and synthesized. These peptide analogs are targetingHuman Rhinovirus (HRV) and Coronavirus (CoV) by inhibiting the cysteine protease. The docking and modeling studies have been shown. The structures of this kind of inhibitors include five fragments. The warhead provides the activity, which can covalently react with the thiol of cysteine protease and permanently eliminate its proteolytic activity. The warhead is linked to a peptide backbone including the other four parts that are designed to position the warhead where it can specially react with the critical thiol of the cysteine protease active site. The side chain of each amino acid has been optimized to achieve better solubility and permeability. We successfully synthesized some compounds with good potency.

The prognosis for multiple myeloma (MM) and the activated B-cell subtype of diffuse large B-cell lymphoma (ABC DLBCL) is poor. Gene expression profiling studies have identified that the transcription factor, nuclear factor kappa B (NF-κB) is overexpressed and confers a poor prognosis in MM and ABC DLBCL. NF-κB regulates the transcription of genes involved in cell proliferation and survival. Thus, several groups have tried to identify and/or develop agents that target NF-κB to improve therapy and patient prognosis for MM and ABC DLBCL. Our laboratory has shown that the manganese porphyrin MnTE-2-PyP5+ inhibits NF-κB in a murine lymphoma cell culture model and enhances tumor cell death in combination with dexamethasone and cyclophosphamide, two agents that are routinely used to treat these neoplasms. MnTE-2-PyP5+ inhibits NF-κB by glutathionylating p65, a member of the NF-κB family. The objective of the following studies was to determine whether MnTE-2-PyP5+ enhances the chemotherapeutic response in human MM and ABC DLBCL cells that overexpress and depend on NF-κB for survival. The following studies demonstrate that MnTE-2-PyP5+ glutathionylates and inhibits NF-κB in human MM and ABC DLBCL cells. MnTE-2-PyP5+ also synergizes with several MM and DLBCL chemotherapeutics, including dexamethasone, cyclophosphamide, vincristine and bortezomib to enhance cell death. The data from these human cell lines will provide the basis for future studies to test MnTE-2-PyP5+ in animal models and for translating MnTE-2-PyP5+ to the clinic.

Le myélome multiple (MM) est une néoplasie B caractérisée par l'accumulation d'un clone plasmocytaire dans la moelle osseuse. Cette pathologie demeure incurable d'où la nécessité d'identifier de nouvelles cibles thérapeutiques. L'utilisation des puces à ADN a permis d'identifier, de nombreux gènes dont l'expression par les cellules de MM est associée à un mauvais ou bon pronostic. La plupart des gènes pronostics identifiés dans le MM codent pour des protéines impliquées dans les processus de réplication, de réparation et de recombinaison de l'ADN. Nous avons voulu aller plus loin dans l'identification et la fonction biologique de ces gènes pronostics. D'une part, nous avons recherché les gènes présentant des pics d'expression très élevés ‘gènes spikés' chez une fraction des patients. Ces gènes sont généralement associés à des évènements oncogéniques. D'autre part, nous avons identifié des gènes pronostics non associés à la machinerie du cycle cellulaire et qui sont fortement exprimés dans des cellules souches pluripotentes ou adultes. L'identification de ces gènes nous a permis de construire un score pronostic très puissant, éliminant les scores pronostics publiés à présent. Un autre aspect majeur est l'élucidation des mécanismes biologiques impliquant ces gènes et qui sont responsables de la résistance aux traitements et/ou de la courte durée de survie des patients, afin de pouvoir les reverser. Nous avons donc évalué le rôle biologique de DEPDC1A un gène fortement exprimée dans les cellules de MM en association avec un mauvais pronostic. Le knockdown conditionnel de DEPDC1A par l'utilisation d'un shRNA, inhibe la croissance des lignées de myélome avec une accumulation des cellules en phase G2/M du cycle cellulaire. Cette accumulation est associée à la phosphosphorylation et à la stabilisation de P53, et à l'accumulation de P21/WAF1. Le knockdown de DEPDC1A résulte également en l'expression de marqueurs de cellules plasmocytaires matures dans les lignées de MM : CD31, CD38, CD138, IL6R, CXCR4, CD9, VLA6. DEPDC1A contrôle donc le cycle cellulaire des plasmocytes tumoraux en interférant avec la voie P53 et bloque leur différenciation. Ces travaux montrent que DEPDC1A pourrait jouer un rôle essentiel dans la croissance des cellules de myélome et pourrait être une cible moléculaire prometteuse pour de nouvelles drogues ou de peptides-vaccins dans le MM. / Multiple myeloma (MM) is a B neoplasia characterized by the accumulation of a plasma cell clone in the bone marrow. This disease remains incurable, hence the need to identify new therapeutic targets. The use of DNA microarrays has identified many genes whose expression in MM cells is associated with poor or good prognosis. Most of the genes identified in the MM predictions encode proteins involved in DNA replication, repair and recombinaison processes. We wanted to go further in the identification and biological function of these prognostic genes.First, we looked for genes that have a spike expression, i.e. they are highly expressed in MMCs of a fraction of patients. These genes are generally associated with oncogenic events.On the other hand, we have identified pluripotent and adult stem cell genes unrelated to cell cycle and aberrantly expressed by human multiple myeloma cells in association with poor prognosis. The identification of these genes has allowed us to build a powerful prognostic score, stonger than already published scores.Another major aspect is the elucidation of biological mechanisms involving these genes that are responsible for drug resistance and/or short-term survival of patients, to revert them. We evaluated the biological role of DEPDC1A gene which are highly expressed in MM cells in association with a poor prognosis. The conditional knockdown of DEPDC1A by using an shRNA, inhibits the growth of myeloma cell lines with an accumulation of cells in G2/M phase of cell cycle. This accumulation is associated with phosphosphorylation and stabilization of p53, and accumulation of P21/WAF1. The knockdown of DEPDC1A also results in the expression of mature plasma cell in MM cell lines: CD31, CD38, CD138, IL6R, CXCR4, CD9, VLA6. DEPDC1A therefore controls the cell cycle of plasma cells by interfering with the p53 pathway and blocks their differentiation. This work shows that DEPDC1A could play a role in the growth of myeloma cells and could be a promising molecular target for new drugs or vaccine peptides in MM.

Le myélome multiple (MM) est une néoplasie lymphocytaire B qui se caractérise par l'accumulation d'un clone de cellules plasmocytaires tumorales dans la moelle osseuse interagissant de manière étroite avec le microenvironnement. L'étude du profil d'expression génique à l'aide des puces à ADN a permis de préciser l'hétérogénéité de cette pathologie et de découvrir de nouveaux acteurs susceptibles d'avoir un rôle importent dans sa physiopathologie. La surexpression de Oct-3/4, Sox2, c-Myc et KLF4 dans des cellules adultes les reprogramme à l'état de cellules souches. J'ai montré une surexpression significative de 3 de ces 4 gènes – KLF4, MYC, SOX2 - dans le MM. Ces gènes sont également fréquemment surexprimés dans 18 types de cancers sur 40 étudiés. Par ailleurs, leur expression peut y être associé à un mauvais pronostique ou un signe de progression tumorale, dus peut-être à leur capacité à induire des caractéristiques de cellules souches cancéreuses. Nous avons par la suite débuté l'étude de la fonction des protéines codées par ces gènes dans le MM, en commençant par KLF4, facteur de transcription activateur ou répresseur. KLF4 est exprimé dans les plasmocytes (PC) normaux, mais son expression est perdue dans les PC tumoraux (cellules myélomateuses, MMC) de 2 patients sur 3 atteints de MM au diagnostic. Parmi les patients pour lesquels les MMC expriment KLF4, se trouve un groupe de patients à haut risque, le groupe MMSET portant la translocation t(4 ;14). L'utilisation d'un modèle inductible d'expression de KLF4 dans des lignées de MM avec transduction lentivirale, a mis en évidence un arrêt du cycle associé à l'expression de P27/KIP1 pour les lignées avec des mutations inactivatrices de P53, mais également de P21/WAF1 pour des lignées avec P53 sauvage. Cette sortie du cycle due à l'expression de KLF4 pourrait protéger les plasmocytes tumoraux de l'apoptose induite par certaines drogues ciblant le cycle, comme nous l'observons in vitro avec le Melphalan.L'objectif majeur de notre équipe est de comprendre le fonctionnement du PC normal et celui du PC tumoral. Afin d'atteindre cet objectif, il est nécessaire d'obtenir un système efficace permettant d'introduire un gène dans un PC. Nous avons montré que des lentivirus pseudotypés avec des glycoprotéines tronquées (hemagglutinine et fusion) issus du virus de la rougeole, transduisent de façon stable et efficace des PC normaux et tumoraux. / Multiple myeloma (MM) is a B-cell neoplasia characterized by the accumulation of a clone of malignant plasma cells in bone marrow closely interacting with its microenvironment.Gene expression profiling using DNA microarrays has clarified the heterogeneity of this disease and has allowed the finding of new actors that may have an important function in MM pathophysiology.Overexpression of Oct-3/4, Sox2, c-Myc and KLF4 in adult cells causes their return to the state of stem cells, commonly called induced pluripotent stem cells (iPS). Our team has shown a significant overexpression of at least one of these four factors in 18 out of 40 cancers studied. Moreover, their expression may be associated with poor prognosis or may be a sign of tumor progression, perhaps due to their ability to induce characteristics of cancer stem cells.We therefore began the study of the function of these genes in MM, starting with KLF4, which can either be an activator or a repressor of transcription, depending on the promoter. KLF4 is expressed in normal plasma cells (PC), but its expression is lost in 2 out of 3 patients with MM at diagnosis. Among patients for whom the PCs express KLF4, is a group of high-risk patients, the MMSET group, bearing the t(4;14) translocation.An inducible model of KLF4's expression in MM cell lines was obtained using lentiviral transduction. Our model revealed a KLF4 induced cycle arrest, associated with the expression of P27/KIP1 when P53 is mutated, but also P21/WAF1 in case of wild type P53. This cell cycle blockade due to the expression of KLF4 could protect malignant plasma cells from the apoptosis induced by certain drugs targeting the cell cycle, as shown by our in vitro observations using melphalan.The main goal of our team is to understand the normal function of PCs and the PC tumor. To achieve this, it is necessary to obtain an effective system for introducing a gene in a given PC. We have shown that lentiviruses pseudotyped with truncated glycoproteins (Hemagglutinin and Fusion) from measles virus, can a stably and efficiently transduce normal and malignant PCs.

abstract: Novel biological strategies for cancer therapy have recently been able to generate improved anti-tumor effects in the clinic. Of these new advancements, oncolytic virotherapy is a promising strategy through a dual mechanism of oncolysis and stimulation of tumor immunogenicity against the target cancer cells. Myxoma virus (MYXV) is an oncolytic poxvirus that has a natural tropism for Leporids, being nonpathogenic in humans and all other known vertebrates. MYXV is able to infect cancer cells due to mutations and defects in many innate signaling pathways, such as those involved in anti-viral responses. While MYXV alone infects and kills many classes of human cancer cells, recombinant techniques allow for the implementation of therapeutic transgenes, which have the potential of ‘arming’ the virus to enhance its potential as an oncolytic virus. The implementation of certain transgenes allows improved cancer cell killing and/or promotion of more robust anti-tumor immune responses. To investigate the potential of immune-inducing transgenes in MYXV, in vitro screening experiments were performed with several single transgene-armed recombinant MYXVs. As recent studies have shown the ability of MYXV to uniquely target malignant human hematopoietic stem cells, the potential of oncolytic MYXV armed with individual immune-enhancing transgenes was investigated through in vitro killing analysis using human acute myeloid leukemia (AML) and multiple myeloma (MM) cell lines. Additionally, in vitro experiments were performed using primary bone marrow (BM) cells obtained from human patients diagnosed with MM. Furthermore, the action of an engineered bispecific killer engager (huBIKE) was investigated through co-culture studies between the CD138 surface marker of target MM cells and the CD16 surface marker of primary effector peripheral blood mononuclear cells (PBMCs), particularly NK cells and neutrophils. In this study, several of the test armed MYXV-infected human AML and MM cell lines resulted in increased cell death compared to unarmed MYXV-infected cells. Additionally, increased killing of CD138+ MM cells from primary human BM samples was observed following infection with huBIKE-armed MYXV relative to infection with unarmed MYXV. Furthermore, analysis of co-culture studies performed suggests enhanced killing of target MM cells via engagement of NK cells with U266 MM cells by huBIKE. / Dissertation/Thesis / Masters Thesis Biology 2020

Amyloidosis involves extracellular deposition of abnormal proteins/fibrils with potential end organ damage. AL type amyloidosis is one subtype and a clonal plasma cell disorder. A 74-year-old completely asymptomatic male presented with progressive renal dysfunction. Work up with serum protein electrophoresis (SPEP) and immunofixation revealed monoclonal IgG Lamda spike of 1.1 g/dL. Urine protein electrophoresis noted Bence Jones proteins. Notable labs, hemoglobin 10.6 g/dL, calcium was 8.2 mg/dL, and creatinine 2.4 mg/dL. Quantitative immunoglobulins IgA, IgG, and IgM, were 59 mg/dL, 1,939 mg/dL, and 23 mg/dL, respectively. Lambda and Kappa free light chains 26.37 mg/L and 127.87 mg/L, respectively, with a ratio of 0.21. Skeletal survey noted a 4 mm lucency of the left frontal bone. Bone marrow biopsy confirmed 21% plasma cells. Renal biopsy revealed AL lambda light chain confirming final diagnosis AL Lambda light chain Amyloidosis and IgG Lambda Multiple Myeloma. Treatment with daratumumab, cyclophosphamide, bortezomib, and dexamethasone initiated. His clinical course was complicated by COVID 19 infection prior to treatment initiation and with congestive heart failure secondary to cardiac amyloidosis (elevated Troponin and Brain Natriuretic Peptide level) during induction therapy requiring hospitalization, diuresis and optimization of cardiac medications. Very Good Partial Response (VGPR) noted after 2 cycles and near Complete Response (CR) after 4 cycles. Patient was evaluated and approved for Stem cell transplant (SCT) but decided against SCT and has now proceeded to single agent daratumumab maintenance. Amyloidosis is an uncommon disease seen in older adults (median age 64) with deposition of fibrils composed of low molecular weight subunits derived from normal proteins. Various subtypes and protien/fibrils include AL amyloidosis (immunoglobulin light chain), hereditary/ familial transthyretin amyloidosis (mutated transthyretin, apolipoprotien, fibrinogen A, lysozyme), wildtype transthyretin Amyoidosis/senile amyloidosis (unmutated transthyretin) and AA amyloidosis (serum amyloid A fibril). AL amyloidosis is a systemic disorder that presents with nephrotic syndrome or restrictive cardiomyopathy (as in this case). Other presentations involve peripheral neuropathy, hepatomegaly, macroglossia, arthropathy with “shoulder pad” sign, bleeding diathesis, purpura including “racoon eyes”. Biopsy of the affected organ (kidney, liver, fat pad aspirate, bone marrow) with Congo red staining confirms the histologic diagnosis. Amyloid light chains can be confirmed with proteomic analysis (mass spectrometry or immuno-electron-microscopy). AL amyloidosis treatment entails high dose chemotherapy and autologous SCT. Long term prognosis in advanced stage is poor. Survival can be short (4-6 months), heart failure causing about 50% of deaths. Daratumumab-regimens offer a 40-55% CR and with SCT data (83% five year and 50% ten-year survival) the outlook is improving.

INTRODUCTION Multiple Myeloma is characterized by the neoplastic proliferation of plasma cells producing a monoclonal immunoglobulin which can be detected by analysis of the protein electrophoresis of serum (SPEP) and/or urine(UPEP) and/or serum free light chain analysis. Here ,we present a rare case of non-secretory multiple myeloma in which no measurable monoclonal heavy or light chains can be identified. CASE 68 year-old female presented to the hospital with symptoms of intractable back pain and rib pain along with 20 lb weight loss over the past 2 months. Labs were significant for mild anemia with a hemoglobin of 11mg/dl, WBC count 5.6 K/ul , platelet 199 K/ul, Chemistry panel showed mild hypernatremia with serum sodium 149, serum K 4.1, BUN 23mg/dl, serum creatinine 1.20 mg/dl, serum calcium levels elevated to 13.7 mg/dl and normal liver function tests. Imaging with Computed Tomography of chest, abdomen, pelvis showed extensive lytic lesions in axial and appendicular skeleton, with pathologic fractures involving T3 and T9 vertebrae. Skeletal bone survey showed innumerable lytic lesions involving the calvarium, C- spine, humerus, scapula, pelvis and bilateral femurs. A core needle biopsy of the right sacral iliac crest bone lesion was performed with pathology showing evidence of plasma cell neoplasm. SPEP showed hypogammaglobulinemia, normal serum kappa and lambda light chains and normal free light chain ratio. Serum immunofixation, UPEP and urine IFE did not show any evidence of monoclonal protein. Serum beta-2 microglobulin was elevated to 13.8 mg/L. Subsequent bone marrow biopsy revealed hyper cellular marrow with 95% cellularity, with 80% involvement by plasma cells, congo red stain negative, findings consistent with plasma cell myeloma. Cytogenetics were normal with FISH showing duplication of 1q, but negative for 17p deletion, t(4,14) and t(14,16). Hypercalcemia was managed with pamidronate. She was diagnosed with Revised International Staging System(R-ISS) Stage II non secretory multiple myeloma. Patient received palliative radiation therapy for lytic lesions. Treatment was initiated with standard of care regimen Velcade, Revlimid and Dexamethasone along with bisphosphonates. DISCUSSION Non secretory myeloma(NSMM) constitutes about 1-2% of all MM cases. It is classically defined as clonal bone marrow plasma cells ≥10% or biopsy proven plasmacytoma, evidence of end-organ damage with anemia, renal insufficiency likely secondary to hypercalcemia, bone lesions and lack of serum and urinary monoclonal protein on electrophoresis and immunofixation. Majority of these patients have M protein detectable in the cytoplasm of plasma cells by Immunohistochemistry, but have impaired secretion of the protein, or they can be non-producer MM. The overall survival (OS) and progression free survival( PFS) of NSMM is similar or superior to patients with secretory myeloma phenotype. Monitoring of the these patients for treatment response is challenging and is mainly on the basis of imaging studies like PET-CT or MRI and bone marrow evaluation.

Multiple myeloma is an agressive hemato-oncological disease the diagnosis and treatment possibilities of which have been developing for the last 15 years. The diagnostic methods include flow cytometry which uses antigen detection to distinguish between pathological and physiological plasmocytes. One of the monitored markers is marker CD45 which could be, according to our monitoring, of a negative prognostic value. 71 patients in our group were divided according to the intensity of CD45 antigen expression and the group with a lower expression showed a statistically higher risk of relapse within 12 months 62% vs. 25 % (p=0,0011). We did not find any connection with the influences of induction therapy or cytogenetics which are otherwise considered the most important prognostic markers. Multiple myeloma treatment involves a lot of combined protocols; and the repeated autologous transplantation is still considered to be the most efficient. We observed more positive results in planning the second autologous transplant early in patients who reached just partial remission after the 1st autologous transplant - significantly better TFS (treatment free survival) and overall survival (OS) were in the group of patients with early tandem transplantation: 18 months vs. 10 months (p=0.04) and the OS median was not reached...

Multiple Myeloma is cancer of plasma cells and is known to be highly invasive. Multiple Myeloma makes up 1% of cancer diagnosis in western countries and affects men more predominantly than women. The American Cancer Association estimates that 32,110 new cases will be diagnosed in the United States in 2019. Lenalidomide is one of the main therapies used for multiple myeloma patients, but it has toxic side effects such as thrombocytopenia, neutropenia, and anemia. The purpose of the study is to investigate new cytotoxic agents for the treatment of multiple myeloma. In addition to lenalidomide alone, this study examined the effects of doxycycline alone and in combination with lenalidomide. Lenalidomide cell cultures were treated at concentrations from 0.5μM to 10μM on untreated 24 well plates and doxycycline concentration ranging from 10μM-80μM. Following incubation, cell viability was tested using MTT assay and the samples were analyzed using spectrophotometry. When compared to lenalidomide, doxycycline monotherapy showed a greater decrease in overall cell viability in preliminary results. Our results show that there is benefit of using 10μM of Doxycycline at higher concentration of 5μM and 10μM of lenalidomide. The potential decrease in the concentration of lenalidomide used by adding doxycycline, may reduce the toxic side effects of lenalidomide. Further studies are necessary to confirm these preliminary results and investigate the mechanism of action in order to determine optimal combinations of these drugs.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24187号 / 医博第4881号 / 新制||医||1060(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 伊藤 貴浩, 教授 滝田 順子, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM