Intrinsic and extrinsic mechanisms for B-cell lymphoma development and progression studied by global gene expression profiling
Sammanfattning: Mantle cell lymphoma (MCL) is a rare hematopoietic malignancy characterized by frequent relapses and poor survival, partly due to minimal residual disease, whereby a subset of malignant cells, harbored in protective niches, survive treatment. In vitro and ex vivo experiments have shown that MCL cells can be rescued from apoptosis through interactions with non- malignant cells such as stromal cells. The present thesis investigates the effect that extrinsic microenvironmental interactions may exert on MCL cells and discuss the presumptive role of these as well as intrinsic mechanisms in the development and progression of lymphomas. MCL cells commonly grow in suspension but when co-cultured with stromal cells a fraction of the MCL cells strongly attached to the stromal cell monolayer. Analysis of transcript levels by next generation sequencing and species-specific read separation we identified sets of genes with altered transcript levels between the adherent MCL cells and those that remained in suspension. These genes could broadly be divided into four functional themes of which three exhibited increased transcript levels in the adherent fraction: B-cell signaling, antiapoptosis and cell adhesion/migration, and one, early mitosis for which the associated genes exhibited lower transcript levels in the adherent MCL fraction. Additionally, we observed a significant overlap between the changed genes in the present co-culture model system and genes that change at the mRNA level between lymph node and cells in circulation in material from MCL and chronic lymphocytic leukemia patients. Suggesting that the model system faithfully represent aspects of microenvironment-promoted changes to the lymphoma cells also observed in vivo. As different MCL cell lines were subject to similar co-culture conditions we observed differences in engagement of and dependency on cell surface molecules, including the B cell receptor, for adhesion to stromal cells. Different responses to microenvironmental interactions for different MCL cells may therefore affect how different patients or different subsets of MCL cells within a patient will respond to different therapy regimens. The transcription factor SOX11 is a key diagnostic marker in MCL. Commonly not expressed in hematological malignancies it is expressed in 90% of MCL cases and most importantly identifies cyclin D1 negative MCL. Its role in MCL development and progression is however debated. Here we show that in the context of the non-malignant pre-B-cell-like cell line Ba/F3 SOX11 does not exhibit oncogenic properties. MYC is a transcription factor that is capable of regulating the expression of a vast and diverse set of genes and has a role in lymphoma development, progression and disease prognosis for many lymphoma types, including MCL. Here we show that progressively increasing MYC levels in B-cells lead to the gradual change in expression in a large set of genes and importantly the gradual increase of two lymphoma-associated MYC mutants significantly altered transcript levels differently from wild type MYC. Gene set enrichment analysis identified functions previously associated with regulation by MYC such as ribosome biogenesis and purine metabolism, while other, novel functions such as those related to Bcell identity and chemotaxis were observed. The MYC regulated genes overlapped with previous gene signatures observed in the Eµ-Myc mouse model and also with recently identified direct MYC targets, collectively indicating the utility of the present model system with inducible MYC for the identification of MYC dependent functions in lymphomagenesis. Two studied lymphoma-associated MYC mutants differentially affect largely distinct subsets on Myc-regulated genes via different mechanisms.
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