Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 89-105).

Hematological malignancies, such as lymphomas and myelomas, have low cure rates or remain refractory to treatment, although advances have been made in treatment regimens for these patients. Questions still remain as to what is occurring in these cells on a molecular level, specifically at the level of gene transcription. The positive regulatory domain I binding factor 1 (PRDI-BF1) has been shown to directly repress genes required for cell proliferation and maintenance of the B cell phenotype, however very little is known as to its regulation. The first study presented in this dissertation demonstrates regulation of the PRDM1 gene occurs primarily at the level of transcription in B cell receptor (BCR)-stimulated lymphoma cells and myeloma cells. It also demonstrates PU.1 binding is involved in BCR-mediated activation of lymphoma cells. Most importantly, this study presents evidence of a promoter poised and primed for activation in lymphoma cells. These studies lay the groundwork for the second study which examines modulation of PRDM1 expression in lymphoma cells by chemotherapeutic agents. Induction of PRDI-BF1 in lymphoma cells negative for PRDM1 gene expression correlates with increased apoptosis, which has important therapeutic implications for treatment of lymphomas. One common problem that arises in treatment of cancer patients is the eventual emergence of a drug resistant population of cells. Identifying specific drug targets and whether they confer drug resistance is an important area of study, which is the focus of the third study presented in this dissertation. It demonstrates the response of myeloma cells to treatment with the farnesyltransferase inhibitor (FTI)-277 and examines whether known mechanisms of drug resistance in these cells are responsible for cross-resistance to FTI-277.

Cell proliferation is a process that is strictly regulated by a large number of proteins. An alteration in one of the encoding genes inserts an error into the regulative protein, which may result in uncontrolled cell growth and eventually tumor formation. Lymphoma is a cancer type originating in the lymphocytes, which are part of the body’s immune defence. In the present thesis, Znfn1a1, Notch1 and Bcl11b were studied; all involved in the differentiation of T lymphocytes. The three genes are located in chromosomal regions that have previously shown frequent loss of heterozygosity in tumor DNA. Ikaros is a protein involved in the early differentiation of T lymphocytes. In this thesis, mutation analysis of the Znfn1a1 gene in chemically induced murine lymphomas revealed point mutations and homozygous deletions in 13 % of the tumors. All of the detected deletions lead to amino acid substitutions or abrogation of the functional domains in the Ikaros protein. Our results support the role of Ikaros as a potential tumor suppressor in a subset of tumors. Notch1 is a protein involved in many differentiation processes in the body. In lymphocytes, Notch1 drives the differentiation towards a T-cell fate and activating alterations in the Notch1 gene have been suggested to be involved in T-cell lymphoma. We identified activating mutations in Notch1 in 39 % of the chemically induced murine lymphomas, supporting the involvement of activating Notch1 mutations in the development of T-cell lymphoma. Bcl11b has been suggested to be involved in the early T-cell specification, and mutations in the Bcl11b gene has been identified in T-cell lymphoma. In this thesis, point mutations and deletions were detected in the DNA-binding zinc finger regions of Bcl11b in 15 % of the chemically induced lymphomas in C57Bl/6×C3H/HeJ F1 mice. A mutational hotspot was identified, where four of the tumors carried the same mutation. Three of the identified alterations, including the hotspot mutation in Bcl11b, increased cell proliferation when introduced in a cell without endogenous Bcl11b, whereas cell proliferation was suppressed by wild-type Bcl11b in the same cell line. Mutations in Bcl11b may therefore be an important contributing factor to lymphomagenesis in a subset of tumors. A germ line point mutation was identified in BCL11B in one of 33 human B-cell lymphoma patients. Expression of BCL11B in infiltrating T cells was significantly lower in aggressive compared to indolent lymphomas, suggesting that the infiltrating T cells may affect the B-cell lymphomas.

As new therapeutic targets and drugs are discovered for B-cell lymphoma and other cancers, companion diagnostics are also needed to determine target engagement, therapeutic efficacy, and patient segmentation for clinical trials. We first employed synthetic chemistry to build a platform for modifying small molecule drugs into imaging probes, using the poly(ADP-ribose) polymerase 1 (PARP1) inhibitor AZD2281 (Olaparib) as a model for technology development. Our results showed that small-molecule companion imaging drugs can be used for fluorescence imaging in cells, as well as for pharmacokinetic studies and positron emission tomography (PET) imaging in vivo, without significantly perturbing their target binding properties or cellular uptake. To apply this approach to B-cell lymphoma drugs currently in clinical trials, we modified an irreversible inhibitor of Bruton's Tyrosine Kinase (BTK), PCI-32765 (Ibrutinib), with the fluorophore Bodipy FL (BFL), and used it for imaging in cells and in a mouse window-chamber xenograft model. The excellent co-localization of our probe (Ibrutinib-BFL) with BTK demonstrated its utility for studying additional BTK inhibitors and as a companion imaging probe. In parallel, we hypothesized that central nervous system (CNS) lymphoma diagnosis from paucicellular cerebrospinal fluid (CSF) samples could be improved with molecular profiling of putative lymphoma cells trapped in a customized microfluidic chip. Following fabrication and characterization of a polydimethylsiloxane (PDMS) diagnostic device containing an array of affinity-free single-cell capture sites, we were able to efficiently recover >90% of lymphocytes, perform immunostaining on chip, and apply an image-processing algorithm to group cells based on their molecular marker expression, such as kappa/lambda light chain restriction. Additionally, in combination with Ibrutinib-BFL or other imaging drugs, we demonstrated the potential for on-chip drug imaging for use in conjunction with drug development. Finally, we applied bioorthogonal conjugation chemistries on cellulose paper for potential applications in lowering the cost of drug screening. We anticipate that these approaches will enable direct, molecular information for personalized treatment decisions in B-cell lymphomas, as well as provide a roadmap for the development of companion diagnostic probes and devices for additional indications.

<p>The interaction of human tumor viruses with host growth suppressive pathways is a fine balance between controlled latent infection and virus-induced oncogenesis. This dissertation elucidates how Epstein-Barr virus interacts with the host growth suppressive DNA damage response signaling pathways (DDR) in order to transform infected human B lymphocytes. </p><p> Here I report that the activation of the ATM/Chk2 branch of the DDR in hyper-proliferating infected B cells results in G1/S cell cycle arrest and limits viral-mediated transformation. Similar growth arrest was found in mitogen-driven proliferating of B cells that sets the DDR as a default growth suppressive mechanism in human B cells. Hence, the viral protein EBNA3C functions to attenuate the host DDR and to promote immortalization of a small portion of infected B cells. Additionally, the pharmacological inhibition of the DDR in vitro increases viral immortalization of memory B cells that facilitates the isolation of broadly neutralizing antibodies to various infectious agents. Overall, this work defines early EBV-infected hyper-proliferating B cells as a new stage in viral infection that determines subsequent viral-mediated tumorigenesis.</p> / Dissertation

The Neisseria genus includes pathogens, Neisseria gonorrhoeae (Ngo) and Neisseria meningitidis, as well as commensals. Ngo, the cause of gonorrhea, induces massive inflammation but a surprising lack of adaptive immune responses. We have observed that Ngo can inhibit both T cell activation and dendritic cell maturation through interaction with the host expressed co-inhibitory receptor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Therefore, I wondered whether B cells may also be affected in this manner. Herein, I examine primary human B cell responses to infection with Ngo, as well as the other Neisseria species. B cells infected with Ngo show no sign of inhibition, regardless of their ability to bind CEACAM1, instead responding to gonococci with robust activation and proliferation. There are distinct subsets of B cells found in the periphery and, intriguingly, the IgM memory B cell subset expand and produce polyreactive IgM in response to goncoccal infection. These cells are innate in function, producing low affinity, polyclonal IgM that is protective against bacterial and fungal dissemination. This effect was broadly specific for Neisseria sp., as B cell infection with all commensal Neisseria species examined induced innate B cell responses. Curiously, meningococcal strains avoid inducing the innate B cell responses, making it enticing to hypothesize that its avoidance of such an ancient immune response may contribute to its ability to cause disease in humans. Finally, I tested whether gonococcal Opa protein binding to CEACAM1 affects primary human B cell activation, and show that no inhibition was observed. This absence of co-inhibitory function of neisserial-bound CEACAM1 may reflect inherent differences between distinctive cell types. Combined, the results in this thesis contribute new insight regarding the poorly characterized human IgM memory B cells, as well as to the function of CEACAM1 in lymphocytes.

Phosphoinositide 3-kinase is a family of lipid kinases that function by phosphorylating the D3 position of phosphoinositide (PI) lipids generating PI(3)P, PI(3,4)P2 and PI(3,4,5)P3. These D3 phosphoinositides regulate various cellular processes through the recruitment of effector proteins containing lipid specific pleckstrin homology (PH) domains. PI phosphatases such as PTEN and SHIP function to restrain PI3K signaling by limiting the amount of D3 PI available for binding. Deletion of either PTEN or SHIP significantly alters B cell function and humoral immune responses. TAPP1 and TAPP2 are dual PH domain containing adaptors which selectively bind the phosphoinositide PI(3,4)P2 via their C-terminal PH domains. PI(3,4)P2 is a lipid messenger generated by PI3K and through the inositol phosphatase activity of SHIP. The function of PI(3,4)P2 remains incompletely understood. To identify the functional role of TAPP-PI(3,4)P2 interactions, we utilized a knock-in (KI) mouse bearing mutations within the PI-binding pocket of both TAPPs. Our study assessed the effect of PI3K dependent KI mutation on B lymphocyte development, activation and antibody production. Flow cytometry analyses of lymphoid tissues found that TAPP KI mice develop relatively normal frequencies of mature B cell populations with the exception of peritoneal B1 cells, which are increased by approximately 50%. Strikingly, TAPP KI mice developed substantially elevated serum antibody levels. TAPP KI mice were able to generate high affinity antigen-binding antibodies upon immunization with NP-OVA in alum adjuvant; however, total immunoglobulin production was markedly increased under this immunization condition. We further assessed the germinal centre (GC) response, which are known to require PI3K signaling and a hallmark of T cell dependent (TD) antibody responses. TAPP KI mice generated larger germinal centers (GC) upon immunization, which was associated with increased GC B cell survival. We further assessed whether uncoupling of TAPPs from PI(3,4)P2 alters B cell signaling and functional responses in vitro. B cells purified from TAPP KI mice were found to have altered functional responses in vitro, with significantly increased survival and cell division following antigen receptor cross-linking. Consistent with increased cell survival, TAPP KI B cells show increased Akt phosphorylation on Ser473 and Thr308 after antigen receptor cross-linking. However, reconstitution of B cell deficient mice with either WT or TAPP KI B cells was found to generate similar GC responses, suggesting that activation of other cells may contribute to the enhanced in vivo responses. Consistently, when we examined the CD4+ T follicular helper cells, a subset providing critical cues to GC responses, we found increased expression of ICOS activation marker. Our results indicate the interactions of TAPP adapters with PI(3,4)P2 serve to restrain lymphocyte activation and limit antibody production, providing the first in vivo evidence that this interaction is important for immune function.

The Neisseria genus includes pathogens, Neisseria gonorrhoeae (Ngo) and Neisseria meningitidis, as well as commensals. Ngo, the cause of gonorrhea, induces massive inflammation but a surprising lack of adaptive immune responses. We have observed that Ngo can inhibit both T cell activation and dendritic cell maturation through interaction with the host expressed co-inhibitory receptor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Therefore, I wondered whether B cells may also be affected in this manner. Herein, I examine primary human B cell responses to infection with Ngo, as well as the other Neisseria species. B cells infected with Ngo show no sign of inhibition, regardless of their ability to bind CEACAM1, instead responding to gonococci with robust activation and proliferation. There are distinct subsets of B cells found in the periphery and, intriguingly, the IgM memory B cell subset expand and produce polyreactive IgM in response to goncoccal infection. These cells are innate in function, producing low affinity, polyclonal IgM that is protective against bacterial and fungal dissemination. This effect was broadly specific for Neisseria sp., as B cell infection with all commensal Neisseria species examined induced innate B cell responses. Curiously, meningococcal strains avoid inducing the innate B cell responses, making it enticing to hypothesize that its avoidance of such an ancient immune response may contribute to its ability to cause disease in humans. Finally, I tested whether gonococcal Opa protein binding to CEACAM1 affects primary human B cell activation, and show that no inhibition was observed. This absence of co-inhibitory function of neisserial-bound CEACAM1 may reflect inherent differences between distinctive cell types. Combined, the results in this thesis contribute new insight regarding the poorly characterized human IgM memory B cells, as well as to the function of CEACAM1 in lymphocytes.

<p>S. 1-80: sammanfattning, s. 81-159: 7 uppsatser</p> / digitalisering@umu

The B cell repertoire in the neonatal immune system (IS) is characterised by reactivity towards self-components, including other immunoglobulin (Ig) V-regions. These properties have been suggested to be a requirement for the development of a normal immune system. DNA sequencing of two interacting Ig idiotypes, derived from neonatal, preimmune mice, demonstrated that such idiotypic connectivity is germ- line encoded and devoid of VDJ junctional diversity. The serum levels of the same Ig idiotypes were studied in normal mice and demonstrated that the expression in serum fluctuated over time in a pattern compatible with a complex dynamic system. In contrast, similar analyses in autoimmune mice or humans demonstrated fluctuations in Ig titers that differed significantly from the healthy individuals. These findings suggested that pathological autoimmunity may be associated with fundamental alterations in the dynamics of natural antibody (ab) expression. This was further investigated in the nonobese diabetic (NOD) mouse, an animal model for human Type I diabetes. Suppression of the early B cell development in the NOD mouse prevented the development of diabetes, suggesting a role for B cells/Igs in the development of diabetes in these mice. Furthermore, neonatal injections of polyclonal Ig preparations or single, monoclonal natural abs inhibited disease induction. The prevention of diabetes development by one such natural ab was demonstrated to be dependent on both the dose injected and the timing of administration. Studies of the B cell repertoire development in NOD mice, compared to normal mice, by DNA-sequence analyses of IgVH rearrangements utilising genes from the most D-proximal Vh family, Vh7183, supported the idea of an aberrant B cell repertoire in this mouse model. Thus, the adult NOD mouse retained a neonatal pattern of Vh7183 rearrangements. This pattern could, however, be "normalised" by neonatal injection of a natural antibody, previously demonstrated to prevent the development of T cell dependent autoimmunity in the NOD mouse. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1993, härtill 6 uppsatser</p> / digitalisering@umu