Bortezomib resistance in mantle cell lymphoma is associated with plasmacytic differentiation.

Bortezomib induces remissions in 30%-50% of patients with relapsed mantle cell lymphoma (MCL). Conversely, more than half of patients' tumors are intrinsically resistant to bortezomib. The molecular mechanism of resistance has not been defined. We generated a model of bortezomib-adapted subclones of the MCL cell lines JEKO and HBL2 that were 40- to 80-fold less sensitive to bortezomib than the parental cells. Acquisition of bortezomib resistance was gradual and reversible. Bortezomib-adapted subclones showed increased proteasome activity and tolerated lower proteasome capacity than the parental lines. Using gene expression profiling, we discovered that bortezomib resistance was associated with plasmacytic differentiation, including up-regulation of IRF4 and CD38 and expression of CD138. In contrast to plasma cells, plasmacytic MCL cells did not increase immunoglobulin secretion. Intrinsically bortezomib-resistant MCL cell lines and primary tumor cells from MCL patients with inferior clinical response to bortezomib also expressed plasmacytic features. Knockdown of IRF4 was toxic for the subset of MCL cells with plasmacytic differentiation, but only slightly sensitized cells to bortezomib. We conclude that plasmacytic differentiation in the absence of an increased secretory load can enable cells to withstand the stress of proteasome inhibition. Expression of CD38 and IRF4 could serve as markers of bortezomib resistance in MCL. This study has been registered at http://clinicaltrials.gov as NCT00131976.

Expression of gp100 and CDK2 in melanoma cells is not co-regulated by a shared promoter region.

Expression of the pigmentation-associated gene PMel17 is regulated by a 1 kB promoter region shared between the PMel17 and CDK2 genes. The encoded melanosomal glycoprotein gp100 and the cell cycle regulatory protein CDK2 are transcribed in opposite directions. Luciferase reporter constructs were generated for subregions of the promoter containing 0, 1, 2 or 3 putative binding sites for transcription factors with basic helix-loop-helix (bHLH) motifs. The potential contribution of bHLH transcription factor microphthalmia transcription factor (MITF) to promoter activity was investigated by re-introducing microphthalmia into melanoma cells lacking expression. A bi-directional reporter construct was generated to investigate potential co-regulation of gp100 and CDK2 transcription. Promoter activity was assessed in presence and absence of phorbol ester tetradecanoyl phorbol 13-acetate (TPA). FACS analysis and immunohistology served to evaluate co-regulation of gp100 and CDK2 expression at the protein level. The full-length promoter, including a consensus binding site for MITF was found to contain sequences that suppressed gp100 expression. Introduction of MITF into non-expressing 1123 melanoma cells did not restore gp100 expression levels. A lack of coregulation for gp100 and CDK2 as suggested by immunostaining was supported by findings of dissimilar expression regulation by TPA for either gene. The current study provides insight into transcriptional regulation of the PMel17 and CDK2 genes, important to identify strategies for modulating expression of gp100 and CDK2 proteins by melanoma cells.

Interferon-gamma reduces melanosomal antigen expression and recognition of melanoma cells by cytotoxic T cells.

In malignant melanoma, tumor-infiltrating lymphocytes are frequently reactive with melanosomal antigens. Achieving complete remissions by peptide therapy is frequently hampered by metastases evading immune recognition. The tumor microenvironment seems to favor reduced expression of target antigens by melanoma cells. Among candidate factors, interferon-gamma (IFN-gamma) (10(2) to 10(3) U/ml) suppressed expression of antigens MART-1, TRP-1, and gp100 by M14 melanoma cells as shown by immunohistology and fluorescence-activated cell sorting analysis, reducing MART-1 expression by >65%. Northern blot analysis revealed that reduced expression was regulated at the transcriptional level, demonstrating a 79% reduction in MART-1 transcript abundance after 32 hours of IFN-gamma treatment. To evaluate consequences of IFN-gamma exposure for immune recognition, MART-1-responsive T cells were reacted with pretreated HLA-matched melanoma cells. Cytotoxicity was reduced up to 78% by IFN-gamma pretreatment, and was restored by addition of MART-1 peptide AAGIGILTV for 2 hours. Examination of melanoma lesions by quantitative reverse transcriptase-polymerase chain reaction revealed up to 188-fold more abundant IFN-gamma transcripts when compared to control skin. Laser capture microdissection and immunohistology localized most IFN-gamma-producing T cells to the tumor stroma. Reduced MART-1 expression was frequently observed in adjacent tumor cells. Consequently, IFN-gamma may enhance inflammatory responses yet hamper effective recognition of melanoma cells.