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Plasma cells are central to an effective immune response, being the sole producers of the antibodies, yet they can caqse severe disease in autoimmunity and multiple myeloma. Therefore their differentiation and survival must be tightly regulated. In this work I investigated some of the mechanisms regulating plasma cell differentiation and life span. The differentiation of a long lived B cell to a short lived plasma cell entails a profound structural and functional metamorphosis finalized to the massive production of immunoglobulins (Ig). Exuberant Ig synthesis causes several types of stress in differentiating plasma cells. My work deals with the characterization of. the C/EBP transcription factor CHOP in plasma cell differentiation. Comparing differentiation of B cells harvested from chop'!' mice to wt cells I found a mild phenotype, consisting in an increased accumulation of intracellular IgM aggregates and a decreased secretion of this antibody class, in vitro and in vivo. These findings' reveal a novel role for CHOP in ensuring optimal functionality of the secretory pathway in the course of plasma cell differentiation. CHOP is involved in the differentiation of various cell types, where it interacts with other members of the C/EBP family favoring or impeding differentiation and it is an important factor in the ER stress response named unfolded protein response (UPR), in which it plays a pro-apoptotic role in most of the systems tested. I extended my investigation on the functions of CHOP in B cells by examining the resistance to ER stress-induced apoptosis in wt and in chop-I' cells. Surprisingly, I observed that in B cells CHOP expression in the UPR plays an anti-apoptotic function. Altogether my data suggest a cell-type specific role for CHOP in B cells and add information on the multi-faceted role of this transcription factor. Most plasma cells exhibit a short life span. The mechanisms at the basis of plasma cell apoptosis are still obscure. I propose that multiple forms of stress, linked to the massive antibody production, contribute to plasma cell death.

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Cyclin-Dependent Kinases (CDKs) play a fundamental role in eukaryotic cell cycle progression, particularly at cell cycle checkpoints, and are therefore important targets for anticancer drug discovery. Activation of CDK2 in complex with Cyclin A regulates entry into S phase of the eukaryotic cell cycle. CDK7, a dual-function enzyme, acts both as a CDK-Activating Kinase (CAK) and as a component of the general transcription factor TFIIH. However, experiments with MAT1-knockdown mice have shown that cell cycle arrest by CAK inhibition would not be detrimental for transcriptional activity in non-dividing cells, as CDK9 in complex with Cyclin T can perform transcriptional duties in the absence of TFIIH. Previous studies have resulted in the identification of NU6102 (1, IC50 mM = 0.005 (CDK2), 4.4 (CDK7)) as a potent and selective CDK2 inhibitor, and NU6247 (2, IC50 mM = 0.12 (CDK2), 0.23 (CDK7)) as an equipotent CDK2/7 inhibitor. It was shown that the sulfonamide group of 1 confers potency and selectivity for CDK2, whereas the pendant piperazinyl substituent of 2 diminishes CDK2 actvity whilst improving activity versus CDK7. S NH N N NH N O O O 2 N N AccordinglyAs part of the work described in the present thesis, sulfonamide 3 (IC50 mM = 0.012 (CDK2), 0.67 (CDK7)) was synthesised and found to be is a potent CDK2 inhibitor, but with some CDK7-inhibitory activity (IC50 mM = 0.012 (CDK2), 0.67 (CDK7)). Further elaboration of the side-chain function has enabled the development of structure-activity relationships (SARs), and the identification of purines (e.g. 4, IC50 mM = 2.6 (CDK2), 0.56 (CDK7)) exhibiting some selectivity for CDK7, albeit with a loss of potency. 4 Subsequent SAR studies conducted on 2 have enabled the following observations to be made: firstly, the purine 6-cyclohexylmethoxy substituent is necessary for activity, with the corresponding 6-unsubstituted purine (5, IC50 mM = 46.9 (CDK2), 20.8 (CDK7)) exhibiting a 100-fold loss of potency against both CDK2 and CDK7. A terminal basic group (e.g. piperazinyl in 2) is required for activity, as replacement by a cyclohexyl substituent results in loss of activity against both kinases (6, 11% inhibition at 10 mM (CDK2), 13% inhibition at 100 mM (CDK7)). The sulfone linker is not a prerequisite for CDK7 activity, with the simple alkylpiperazine derivative (7, IC50 mM = 0.48 (CDK2), 0.51 (CDK7)) exhibiting comparable potency and selectivity. Finally, there appears to be some opportunity for expansion into the gatekeeper pocket of CDK7 by introducing small substituents at the purine C-8 position, with the potential for selectivity over CDK2 (8, 47% inhibition at 100 mM (CDK2), IC50 = 5 mM (CDK7)). Isolation and biological evaluation of the vinyl sulfone 9, an intermediate in the synthesis of 2, indicated a time-dependent inhibition of CDK2, suggesting that 9 is an irreversible inhibitor of CDK2. This would be the first reported irreversible inhibitor of a cyclin-dependent kinase, and therefore the activity of the compound against CDK2 was investigated using protein crystallography and site-directed mutagenesis 5 techniques. From these studies, encouraging evidence has emerged that 9 acts as an irreversible inhibitor of CDK2, covalently binding to a lysine residue within the ATP-binding pocket.