This study consists of two pieces of work investigating haematological malignancies; Acute Lymphoblastic Leukaemia (ALL) and Diffuse Large B Cell Lymphoma (DLBCL). Firstly, Pre-B ALL represents the most common paediatric malignancy and despite increasingly improved outcomes for patients, ~ 20% of all patients diagnosed with ALL relapse. Activating mutations in the RAS pathway are common (~50%) and result in hyperactivation of the MAPK pathway. I identified Erk negative feedback control via DUSP6 to be crucial for NRASG12D-mediated pre-B cell transformation and investigated its potential as a therapeutic target. I showed that a small molecule inhibitor of DUSP6 (BCI) selectively induced cell death in patient-derived pre-B ALL cells; with a higher sensitivity observed in relapse pre-B ALL. I also discovered that a high level of Erk activity is required for proliferation of normal pre-B cells, but dispensable in leukemic pre-B ALL cells. In addition, I found that human B cell malignancies can be grouped into three categories that fundamentally differ in their ability to control Erk signalling strength. Secondly, DLBCL is the most common haematological malignancy and although potentially curable with chemotherapy, 40% of patients still succumb from their disease. Recent exome sequencing studies have identified hundreds of genetic alterations but, for most, their contribution to disease, or their importance as therapeutic targets, remains uncertain. I optimised a novel approach to screen the functional importance of these mutations. This was achieved by reconstituting non-malignant, primary, human germinal centre B cells (GC B cells) with combinations of wildtype and mutant genes to recapitulate the genetic events of DLBCL. When injected into immunodeficient mice, these oncogene-transduced GC B cells gave rise to tumours that closely resemble human DLBCL, reinforcing the biological relevance of this system. To screen potential tumour suppressor mutations in this system in a high throughput fashion, I developed a lymphoma-focused CRISPR library of 692 genes recurrently altered in B cell lymphomas. These experiments identified GNA13 as an unexpectedly potent tumour suppressor in human GC B cells and provided new understanding to its mechanism of action. These findings provide novel understanding of the complexity of oncogenic mechanisms in human B cell malignancies.

<p>Lymphomas are a heterogeneous group of neoplasias originating from B- or T-lymphocytes. In this thesis, we determined the genetic and immunophenotypic characterization of DLBCL and their prognostic impact. Moreover, genomic alterations associated with the transformation to DLBCL from Hodgkin lymphoma (HL) and follicular lymphoma (FL) were elucidated. </p><p>In order to outline the impact of cytogenetic as well as immunophenotypic prognostic markers in DLBCL, we firstly studied a series of 54 DLBCL tumors using comparative genomic hybridization (CGH) and we identified several frequently occurring chromosomal imbalances. Loss of 22q was more often found in the diagnostic tumors with a more advanced clinical stage, while gain of 18q21 was more commonly identified in relapses. Secondly, we correlated the expression patterns of CD10, bcl-6, IRF-4 and bcl-2 with clinical parameters in a series of 173 de novo DLBCL patients. Patients with a germinal center (GC) phenotype displayed a better survival than the non-GC group. Expression of bcl-6 and CD10 was correlated with a better survival while bcl-2 expression was associated with a poor prognosis.</p><p>In approaching the HL transformation, two novel B-cell lines (U-2932 and U-2940), derived from patients with DLBCL following HL, were characterized. Interestingly, a translocation with materials from 2q and 7q as well as loss of material on 6q was found in both cell lines. For FL transformation, we assessed chromosomal alterations in a panel of 28 DLBCL patients with a previous history of FL. The DLBCL tumors displayed more chromosomal imbalances compared to FL tumors. Loss of 6q16-21 and gain of 7pter-q22 were more commonly found in the DLBCL counterparts, suggesting the chromosomal location of putative genes that may be involved in the transformation process.</p>

<p>B cell lymphoma (BCL) comprises a biologically and clinically heterogeneous group of tumors deriving from different stages of B cell development. The immunoglobulin (Ig) variable heavy chain (V_H) gene rearrangement is unique for each BCL and can be used to reveal cellular origin, to study signs of antigen selection and to quantify tumor cell load.</p><p>The normal counterpart of mantle cell lymphoma (MCL) has been postulated to be a naïve B cell and in hairy cell leukemia (HCL) it is considered to be a post-germinal centre B cell. We analyzed the V_H gene rearrangements in 110 MCLs and 32 HCLs by PCR amplification and sequencing. Most MCLs (84%) displayed V_H genes lacking somatic hypermutation (SHM), thus correlating to a naïve cell origin, whereas a subgroup (16%) showed SHM, implying derivation from a more differentiated B cell. In HCL, a majority of cases (84%) displayed SHM with signs of intraclonal heterogeneity and 16% had unmutated V_H genes, thus questioning the cell of origin in HCL. Biased usage of particular V_H genes was detected in both HCL (V_H3-30) and MCL (V_H3-21 and V_H4-34), which indicates that antigen selection may be involved in lymphoma development. Furthermore, V_H3-21⁺ MCLs showed a highly restricted V_λ3-19 gene use and they also had a superior outcome compared to other MCLs.</p><p>Rearrangement analysis of 67 V_H3-21⁺ chronic lymphocytic leukemia (CLL) cases from three different countries verified, regardless of geographical origin, the short and highly homologous complementarity determining region 3s and the strikingly biased usage of the V_λ2-14 gene (75%), as previously reported in CLL. This further supports that antigen selection by a common antigenic epitope may have occurred in V_H3-21⁺ CLLs. </p><p>In an autologous transplantation study of 30 multiple myeloma patients, we quantified the tumor content in the autografts before and after stem cell selection using clone-specific PCR. We conclude that stem cell selection reduced the number of clonal cells linearly, but purging could not totally eliminate the tumor cells from the graft, thus increasing the risk of a relapse.</p><p>Altogether, our data allowed us to define new BCL subsets and to gain insights into the potential role of antigen selection in BCL development as well as the monitoring of tumor cell load using Ig gene rearrangements analysis. </p>

Lymphomas are a heterogeneous group of neoplasias originating from B- or T-lymphocytes. In this thesis, we determined the genetic and immunophenotypic characterization of DLBCL and their prognostic impact. Moreover, genomic alterations associated with the transformation to DLBCL from Hodgkin lymphoma (HL) and follicular lymphoma (FL) were elucidated. In order to outline the impact of cytogenetic as well as immunophenotypic prognostic markers in DLBCL, we firstly studied a series of 54 DLBCL tumors using comparative genomic hybridization (CGH) and we identified several frequently occurring chromosomal imbalances. Loss of 22q was more often found in the diagnostic tumors with a more advanced clinical stage, while gain of 18q21 was more commonly identified in relapses. Secondly, we correlated the expression patterns of CD10, bcl-6, IRF-4 and bcl-2 with clinical parameters in a series of 173 de novo DLBCL patients. Patients with a germinal center (GC) phenotype displayed a better survival than the non-GC group. Expression of bcl-6 and CD10 was correlated with a better survival while bcl-2 expression was associated with a poor prognosis. In approaching the HL transformation, two novel B-cell lines (U-2932 and U-2940), derived from patients with DLBCL following HL, were characterized. Interestingly, a translocation with materials from 2q and 7q as well as loss of material on 6q was found in both cell lines. For FL transformation, we assessed chromosomal alterations in a panel of 28 DLBCL patients with a previous history of FL. The DLBCL tumors displayed more chromosomal imbalances compared to FL tumors. Loss of 6q16-21 and gain of 7pter-q22 were more commonly found in the DLBCL counterparts, suggesting the chromosomal location of putative genes that may be involved in the transformation process.

B cell lymphoma (BCL) comprises a biologically and clinically heterogeneous group of tumors deriving from different stages of B cell development. The immunoglobulin (Ig) variable heavy chain (VH) gene rearrangement is unique for each BCL and can be used to reveal cellular origin, to study signs of antigen selection and to quantify tumor cell load. The normal counterpart of mantle cell lymphoma (MCL) has been postulated to be a naïve B cell and in hairy cell leukemia (HCL) it is considered to be a post-germinal centre B cell. We analyzed the VH gene rearrangements in 110 MCLs and 32 HCLs by PCR amplification and sequencing. Most MCLs (84%) displayed VH genes lacking somatic hypermutation (SHM), thus correlating to a naïve cell origin, whereas a subgroup (16%) showed SHM, implying derivation from a more differentiated B cell. In HCL, a majority of cases (84%) displayed SHM with signs of intraclonal heterogeneity and 16% had unmutated VH genes, thus questioning the cell of origin in HCL. Biased usage of particular VH genes was detected in both HCL (VH3-30) and MCL (VH3-21 and VH4-34), which indicates that antigen selection may be involved in lymphoma development. Furthermore, VH3-21+ MCLs showed a highly restricted Vλ3-19 gene use and they also had a superior outcome compared to other MCLs. Rearrangement analysis of 67 VH3-21+ chronic lymphocytic leukemia (CLL) cases from three different countries verified, regardless of geographical origin, the short and highly homologous complementarity determining region 3s and the strikingly biased usage of the Vλ2-14 gene (75%), as previously reported in CLL. This further supports that antigen selection by a common antigenic epitope may have occurred in VH3-21+ CLLs. In an autologous transplantation study of 30 multiple myeloma patients, we quantified the tumor content in the autografts before and after stem cell selection using clone-specific PCR. We conclude that stem cell selection reduced the number of clonal cells linearly, but purging could not totally eliminate the tumor cells from the graft, thus increasing the risk of a relapse. Altogether, our data allowed us to define new BCL subsets and to gain insights into the potential role of antigen selection in BCL development as well as the monitoring of tumor cell load using Ig gene rearrangements analysis.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009. / Härtill 4 uppsatser.

The most widely transfused blood component is red blood cells (RBCs), and voluntary donation is the main resource for RBC transfusion. In the UK, 7,000 units of RBCs are transfused daily but this life-saving cell therapy is completely dependent on donors and there are persistent problems associated with transfusion transmitted infections and in blood group compatibility. Furthermore, the quality, safety and efficiency of donated RBCs gradually decrease with storage time. A number of novel sources of RBCs are being explored including the production of RBCs from adult haematopoietic progenitor cells, erythroid progenitor cell lines and induced pluripotent stem cells (iPSCs). The iPSC source could essentially provide a limitless supply and a route to producing cells that are matched to the recipient. A number of protocols have been described to produce mature RBCs from human pluripotent stem cells but they are relatively inefficient and would be difficult to scale up to the levels required for clinical translation. We tested and evaluated a defined feeder- and serum-free differentiation protocol for deriving erythroid cells from hiPSCs. RBC production was not efficient, the cells that were produced did not enucleate efficiently and they expressed embryonic rather than adult globin. We hypothesised that the production of RBCs from iPSCs could be enhanced by enforced expression of erythroid-specific transcription factors (TFs). Previous studies had demonstrated that Krüppel-like factor 1 (KLF1) plays an important role in RBC development and maturation so we generated iPSC lines expressing a tamoxifen-inducible KLF1-ERT2 fusion protein. Using zinc finger nuclease technology, we targeted the expression cassette to the AAVS1 locus to ensure consistent expression levels and to avoid integration site specific effects and/or silencing. These iKLF1 iPSCs were applied to our defined RBC differentiation protocol and the activity of KLF1 was induced by adding tamoxifen. Activation of KLF1 from day 10 accelerated erythroid differentiation and maturation with an increase in the proportion of erythroblasts, a higher level of expression of erythroid genes associated with maturation and an apparently more robust morphology. However, KLF1 activation had an anti-proliferation effect resulting in significantly less cell generated overall and HPLC analysis demonstrated that KLF1-activated cells expressed higher levels of embryonic globin compared to control iPSCs-derived cells. Many of the effects that were observed when KLF1 was activated from day 10 were not observed when activated from day 18. We therefore concluded that activation of exogenous KLF1 is able to promote erythroid cell production and maturation in progenitors (day 10) but not at the later stage of erythropoiesis (day 18). We hypothesised that KLF1 might require a co-factor to regulate RBC maturation and adult globin expression at the later stage of erythropoiesis. The TF, B-cell lymphoma/leukaemia 11a (BCL11A), plays a key role in the suppression of foetal globin expression, thereby completing globin switching to adult globin. Preliminary data showed that iPSC-derived erythroid cells were able to express adult globin when transduced with a BCL11A-expressing lentiviral-vector. Based on that finding we then generated an iPSC line expressing tamoxifen-inducible BCL11AERT2 and KLF1-ERT2 fusion proteins, applied this iBK iPSC line to our differentiation protocol. Activation of both TFs from day 18 slightly increased the expression of genes associated with RBC maturation and the inclusion of BCL11A appeared to eliminate the anti-proliferation effect of KLF1. Most importantly, activation of both BCL11A and KLF1 from day 18 of the differentiation protocol increased the production of α- globin (foetal / adult globin) indicating that some definitive-like erythroid cells might be generated by activation of both TFs at the later stage of erythroid differentiation. Collectively, these findings demonstrate that enforced expression of erythroid TFs could be a useful strategy to enhance RBC maturation from iPSCs.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21451号 / 医博第4418号 / 新制||医||1032(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 生田 宏一, 教授 椛島 健治, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Primary gastrointestinal lymphoma is the most common type of extra-nodal lymphoma, representing about 30%-50% of all extra-nodal involvement. The stomach is the most common site, with the colon and rectum accounting for a minority of occurrences. Primary colorectal lymphoma is uncommon, representing only 0.3% of all large intestinal malignancies and approximately 3% of gastrointestinal (GI) lymphomas, with the majority of these being B-cell non-Hodgkin lymphoma and diffuse large B-cell lymphoma (DLBCL) being the most common subtype. We present a case of an 85-year-old male who presented with symptoms suggestive of bowel obstruction, who, after further evaluation, was diagnosed with primary non-Hodgkin lymphoma of the colon, DLBCL subtype.

Lymphomas are malignant tumours arising from cells in the lymphatic system. They are classified as B-cell lymphomas, T-cell lymphomas and Hodgkin lymphoma (HL). Of the B-cell lymphomas, one of the most common is diffuse large B-cell lymphoma (DLBCL). Many patients with lymphomas can be successfully treated however patients who relapse or are refractory have a poor prognosis, warranting further investigations to identify potential targets and develop novel drugs. Picropodophyllin (PPP), a potent and selective inhibitor of IGF-1R, inhibits malignant cell growth with low or no toxicity on normal cells in preclinical models. In paper I, we investigated the potential benefits of using PPP against DLBCL and found that the anti-tumor effects of PPP might possibly be explained by IGF-1R-unrelated mechanism(s). However, the inhibitory effects of PPP on lymphoma cells together with its low toxicity in vivo makes it a promising drug candidate for treatment. Melflufen, a derivative of melphalan, is currently being evaluated in a clinical phase I/II trial in relapsed or refractory multiple myeloma. In paper II, we confirmed previous reports of superior potency of melflufen over melphalan. Being active in cell lines and primary cultures of lymphoma cells as well as in a xenograft model in mice, melflufen considered being a candidate for further evaluation in treatment. bAP-15, a novel inhibitor of proteasome activity, inhibits ubiquitin specific peptidase 14 (USP14) and ubiquitin carboxyl-terminal hydrolase L5 (UCHL5). In paper III, we investigated the activity of b-AP15 in DLBCL and HL cell lines and compared the results to standard drugs used in treatment. Results showed inhibition of the proteasome and growth inhibition/cytotoxicity with IC50-values in the micromolar range. Treatment failure and lack of clinical benefit of proteasome inhibitors like bortezomib in DLBCL patients inspired us investigating for possible new targets, with major focus on proteasome inhibitors in DLBCL. In paper IV, we suggested that UCHL5 and/or USP14, as new targets for proteasome inhibitors in DLBCL, be further evaluated. The findings in this thesis suggest that PPP, Melflufen and b-AP15 are potential candidates for clinical drug development and UCHL5 and/or USP14 are new potential targets for proteasome inhibitors in DLBCL.