Abstract
Myeloma cells are highly dependent on the unfolded protein response to assemble folded immunoglobulins correctly. Therefore targeting protein handling within a myeloma cell by inhibiting the aminopeptidase enzyme system that catalyses the hydrolysis of amino acids from the N terminus of proteins may be a novel therapeutic approach. The effect of the aminopeptidase inhibitor CHR-2797 on myeloma cell proliferation and survival, gene expression, protein turnover, cell migration and myeloma-bone marrow stromal cell interactions was determined on a panel of myeloma cell lines and patient cells. CHR-2797 is able to inhibit the proliferation of myeloma cell lines and primary patient cells, whereas its derivative CHR-79888, an acid metabolite with low cell membrane permeability fails to induce myeloma cell death. This occurs though apoptosis as demonstrated by trypan blue exclusion and annexin V/PI staining, and is proceeded by G1 growth arrest. Western blot analysis demonstrates apoptosis occurs via a non-caspase dependant mechanism. Importantly CHR-2797 is able to induce apoptosis in cells known to be resistant to conventional chemotherapeutic agents. Analysis of the pathways involved using Affymetrix gene expression arrays demonstrates CHR-2797 causes an upregulation of many genes involved in the proteasome/ubiquitin pathway, as well as amino acid deprivation response genes and some aminopeptidases. A further mechanism contributing to cell death is activation of the unfolded protein response with activation of all three UPR pathways demonstrated by splicing of XBP1 to its active from XBP1s, an increase in CHOP with activation of the PERK pathway and cleavage of ATF6. Cytoplasmic inclusions are also present on light microscopy suggestive of the build up of misfolded proteins within the cytoplasm. CHR-2797 causes minimal inhibition of the proliferation of bone marrow stroma, but is able to overcome the protective effects of the micro-environment on myeloma cells, as the drug is still able to inhibit the proliferation of myeloma cells when they are bound to bone marrow stromal cells. Aminopeptidase inhibition is also able to inhibit the increase VEGF that occurs when myeloma cells and bone marrow stroma are bound together. Combination experiments of CHR-2797 with dexamethasone demonstrate synergy, in keeping with the different mechanisms of action the two drugs. CHR-2797 in combination with the proteasome inhibitor bortezomib demonstrates an additive effect. Although both drugs target intracellular protein turnover, gene expression studies of cells treated with CHR-2797 or bortezomib show deregulation of a number of genes specific to aminopeptidase inhibition, as well as a series of genes characteristic of protein turnover. These differences in the mechanism of action of the two drugs are also reflected in the Western blot analysis that demonstrates a predominately non-caspase mediated cell death in CHR2797 compared to a caspase mediated cell death with bortezomib. In summary inhibiting intracellular protein turnover using the aminopeptidase inhibitor CHR-2797 results in myeloma cell death and represents a novel therapeutic approach for the treatment of myeloma. A phase 1 clinical trial has been initiated in haematological malignancies and the results will also be presented at this meeting.
Loss of αA or αB-Crystallin Accelerates Photoreceptor Cell Death in a Mouse Model of P23H Autosomal Dominant Retinitis Pigmentosa
