The lymph node microenvironment promotes B-cell receptor signaling, NF-kappaB activation, and tumor proliferation in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL), an incurable malignancy of mature B lymphocytes, involves blood, bone marrow, and secondary lymphoid organs such as the lymph nodes (LN). A role of the tissue microenvironment in the pathogenesis of CLL is hypothesized based on in vitro observations, but its contribution in vivo remains ill-defined. To elucidate the effects of tumor-host interactions in vivo, we purified tumor cells from 24 treatment-naive patients. Samples were obtained concurrently from blood, bone marrow, and/or LN and analyzed by gene expression profiling. We identified the LN as a key site in CLL pathogenesis. CLL cells in the LN showed up-regulation of gene signatures, indicating B-cell receptor (BCR) and nuclear factor-κB activation. Consistent with antigen-dependent BCR signaling and canonical nuclear factor-κB activation, we detected phosphorylation of SYK and IκBα, respectively. Expression of BCR target genes was stronger in clinically more aggressive CLL, indicating more effective BCR signaling in this subtype in vivo. Tumor proliferation, quantified by the expression of the E2F and c-MYC target genes and verified with Ki67 staining by flow cytometry, was highest in the LN and was correlated with clinical disease progression. These data identify the disruption of tumor microenvironment interactions and the inhibition of BCR signaling as promising therapeutic strategies in CLL. This study is registered at http://clinicaltrials.gov as NCT00019370.

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.

Proteasome inhibitors trigger NOXA-mediated apoptosis in melanoma and myeloma cells.

Patients with metastatic melanoma or multiple myeloma have a dismal prognosis because these aggressive malignancies resist conventional treatment. A promising new oncologic approach uses molecularly targeted therapeutics that overcomes apoptotic resistance and, at the same time, achieves tumor selectivity. The unexpected selectivity of proteasome inhibition for inducing apoptosis in cancer cells, but not in normal cells, prompted us to define the mechanism of action for this class of drugs, including Food and Drug Administration-approved bortezomib. In this report, five melanoma cell lines and a myeloma cell line are treated with three different proteasome inhibitors (MG-132, lactacystin, and bortezomib), and the mechanism underlying the apoptotic pathway is defined. Following exposure to proteasome inhibitors, effective killing of human melanoma and myeloma cells, but not of normal proliferating melanocytes, was shown to involve p53-independent induction of the BH3-only protein NOXA. Induction of NOXA at the protein level was preceded by enhanced transcription of NOXA mRNA. Engagement of mitochondrial-based apoptotic pathway involved release of cytochrome c, second mitochondria-derived activator of caspases, and apoptosis-inducing factor, accompanied by a proteolytic cascade with processing of caspases 9, 3, and 8 and poly(ADP)-ribose polymerase. Blocking NOXA induction using an antisense (but not control) oligonucleotide reduced the apoptotic response by 30% to 50%, indicating a NOXA-dependent component in the overall killing of melanoma cells. These results provide a novel mechanism for overcoming the apoptotic resistance of tumor cells, and validate agents triggering NOXA induction as potential selective cancer therapeutics for life-threatening malignancies such as melanoma and multiple myeloma.

p53-independent NOXA induction overcomes apoptotic resistance of malignant melanomas.

Once melanoma metastasizes, no effective treatment modalities prolong survival in most patients. This notorious refractoriness to therapy challenges investigators to identify agents that overcome melanoma resistance to apoptosis. Whereas many survival pathways contribute to the death-defying phenotype in melanoma, a defect in apoptotic machinery previously highlighted inactivation of Apaf-1, an apoptosome component engaged after mitochondrial damage. During studies involving Notch signaling in melanoma, we observed a gamma-secretase tripeptide inhibitor (GSI; z-Leu-Leu-Nle-CHO), selected from a group of compounds originally used in Alzheimer's disease, induced apoptosis in nine of nine melanoma lines. GSI only induced G2-M growth arrest (but not killing) in five of five normal melanocyte cultures tested. Effective killing of melanoma cells by GSI involved new protein synthesis and a mitochondrial-based pathway mediated by up-regulation of BH3-only members (Bim and NOXA). p53 activation was not necessary for up-regulation of NOXA in melanoma cells. Blocking GSI-induced NOXA using an antisense (but not control) oligonucleotide significantly reduced the apoptotic response. GSI also killed melanoma cell lines with low Apaf-1 levels. We conclude that GSI is highly effective in killing melanoma cells while sparing normal melanocytes. Direct enhancement of BH3-only proteins executes an apoptotic program overcoming resistance of this lethal tumor. Identification of a p53-independent apoptotic pathway in melanoma cells, including cells with low Apaf-1, bypasses an impediment to current cytotoxic therapy and provides new targets for future therapeutic trials involving chemoresistant tumors.

ON 01910.Na is selectively cytotoxic for chronic lymphocytic leukemia cells through a dual mechanism of action involving PI3K/AKT inhibition and induction of oxidative stress.

PURPOSE: Chronic lymphocytic leukemia (CLL), a malignancy of mature B cells, is incurable with chemotherapy. Signals from the microenvironment support leukemic cell survival and proliferation and may confer chemotherapy resistance. ON 01910.Na (Rigosertib), a multikinase phosphoinositide 3-kinase (PI3K) inhibitor, is entering phase III trials for myelodysplastic syndrome. Our aim was to analyze the efficacy of ON 01910.Na against CLL cells in vitro and investigate the molecular effects of this drug on tumor biology. EXPERIMENTAL DESIGN: Cytotoxicity of ON 01910.Na against CLL cells from 34 patients was determined in vitro with flow cytometry of cells stained with Annexin V and CD19. Global gene expression profiling on Affymetrix microarrays, flow cytometry, Western blotting, and cocultures with stroma cells were used to delineate ON 01910.Na mechanism of action. RESULTS: ON 01910.Na induced apoptosis in CLL B cells without significant toxicity against T cells or normal B cells. ON 01910.Na was equally active against leukemic cells associated with a more aggressive disease course [immunoglobulin heavy-chain variable region unmutated, adverse cytogenetics] than against cells without these features. Gene expression profiling revealed two main mechanisms of action: PI3K/AKT inhibition and induction of ROS that resulted in an oxidative stress response through activating protein 1 (AP-1), c-jun-NH(2)-terminal kinase, and ATF3 culminating in the upregulation of NOXA. ROS scavengers and shRNA mediated knockdown of ATF3- and NOXA-protected cells from drug-induced apoptosis. ON 01910.Na also abrogated the prosurvival effect of follicular dendritic cells on CLL cells and reduced SDF-1-induced migration of leukemic cells. CONCLUSIONS: These data support the clinical development of ON 01910.Na in CLL.

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.