ALBERT

Beschreibung: Identification of growth factors in neoplasias may be a target for future therapies by blocking either growth factor receptor interaction or the induced pathway. Using gene expression profiling, we identified overexpression of 2 receptors for a proliferation-inducing ligand (APRIL) and B-cell activating factor (BAFF) in malignant plasma cells compared with normal plasma cells. APRIL and BAFF are involved in a variety of tumor and autoimmune diseases, including B-cell malignancies. We confirmed the expression of BAFF and APRIL receptors (B-cell maturation antigen [BCMA], transmembrane activator and calcium modulator and cyclophilin ligand interactor [TACI], and BAFF-R) in a majority of 13 myeloma cell lines and in the purified primary myeloma cells of 11 patients. APRIL and BAFF were potent survival factors for exogenous cytokine-dependent myeloma cell lines and were autocrine growth factors for the RPMI8226 and L363 autonomously growing cell lines. These factors activated nuclear factor (NF)–κB, phosphatidylinositol-3 (PI-3) kinase/AKT, and mitogen-activated protein kinase (MAPK) kinase pathways and induced a strong up-regulation of the Mcl-1 and Bcl-2 antiapoptotic proteins in myeloma cells. BAFF or APRIL was also involved in the survival of primary myeloma cells cultured with their bone-marrow environment, and protected them from dexamethasone (DEX)–induced apoptosis. Finally, the serum levels of BAFF and APRIL were increased about 5-fold in patients with multiple myeloma (MM) as compared with healthy donors. Altogether, these data suggest that APRIL/BAFF inhibitors may be of clinical value in MM. (Blood. 2004;103:3148-3157)

Beschreibung: Multiple myeloma (MM) is a hematological malignancy characterized by a plasma cell accumulation in the bone marrow (BM), for which novel treatment options are urgently needed. Epigenetic modulating agents such as histone deacetylase inhibitors (HDACi) and DNA methyltransferase inhibitors (DNMTi) are under intense investigation for cancer therapy. As shown in numerous functional in vitro studies, HDACi and DNMTi affect various biological processes important for tumor control including tumor cell survival, proliferation, differentiation and DNA repair. Given the broad mechanisms of action of these agents, it remains important to continue pre-clinical evaluation to identify in vivo relevant mechanisms of action. This may lead to the identification of novel biologically relevant targets and predictive biomarkers allowing clinical trial optimization. Recently, we developed gene expression based risk scores after treating human MM cell lines with TSA (HA-score) or decitabine (DM-score). These scores were predictive for MM patient survival using two independent cohorts (Heidelberg-Montpellier (HM) and University of Arkansas for Medical Sciences-Total Therapy 2) and identified potential biomarkers predictive for drug sensitivity. However, the transcriptional response of MM cells in vivo may be influenced by the close contact with the BM-environment. Therefore, we here aimed to characterize the transcriptional response of MM cells after in vivo treatment with the HDACi JNJ-26481585 (quisinostat) or the DNMTi decitabine using the syngeneic immunocompetent murine 5T33MM model. 5T33MM mice (n=4/group) with established disease were treated with quisinostat or decitabine for 5 days after which tumor cells were isolated from the BM and subjected to microarray analysis. Using Significance Analysis of Microarray, we identified 574 and 180 probesets deregulated by respectively quisinostat and decitabine (of which 111 are in common). To assess the prognostic value of the deregulated genes, we performed MaxStat analysis in the HM cohort. JNJ-585 deregulated 31 genes associated with good prognosis and 31 associated with bad prognosis. Decitabine altered expression of 20 genes linked with poor prognosis while 5 genes were linked with good prognosis. The prognostic value of these genes was then implemented in a murine (Mu)-DM and Mu-HA score. The score values were significant higher in MM patients and human myeloma cell lines compared to MGUS and healthy bone marrow plasma cells. In addition, the scores were useful to separate patients of the 2 cohorts into a low risk and high risk group. Patients from the proliferation subgroup had a higher score compared to all other subgroups. In concordance, the scores were highest in patients with a high gene-expression based proliferation index. Using gene ontology (GO) tools (DAVID) and pathway tools (Reactome, STRING and Pathway-guide), we next explored the association of in vivo deregulated genes with biological processes and pathways. GO analysis showed that quisinostat-deregulated genes were mainly involved in immune modulation. Pathway analysis revealed associations with lymphocyte activation and proliferation, immune effector mechanisms and T-helper-1 development through processes like cytokine interactions, chemokine signaling and T-cell receptor- and NK-cell-signaling. In concordance, the signature represented elevated presence and signaling of interferon, tumor necrosis factor, interleukin-1 (IL-1), IL-2 and IL-12. The second most prominent alterations were genes linked with transcriptional misregulation in cancer. Pathways predicted to be affected by these alterations are linked with differentiation, resistance and cell survival. For decitabine, the gene list was substantially smaller and for more than half shared by JNJ-585. The pathway analysis also identified genes linked to immune system, gene expression regulation and metabolism pathways. In conclusion, in vivo treatment with epigenetic modulating agents identified a prognostic gene signature. In addition, HDACi (and to a lesser extent DNMTi) deregulated immunomodulatory genes and genes involved in transcriptional regulation. This indicates that immune regulation is an important in vivo anti-tumor property of HDACi and supports the rational to combine HDACi with immunomodulatory therapies such lenalidomide or cellular/peptide vaccination strategies. Disclosures Hose: Novartis: Research Funding. Seckinger:Novartis: Research Funding.

Beschreibung: Multiple myeloma (MM) is proposed to consist of two main pathogenetic groups. While hyperdiploidy (HD) is characterized by multiple trisomies of odd-numbered chromosomes (i.e. 3, 5, 9, 11, 15, and 19), non-hyperdiploid MM (NHD) show frequently one of the several recurrent IgH-translocations. The aim was to compare HD versus NHD by gene expression profiling (GEP). CD138-positive multiple myeloma cells from 74 newly diagnosed MM patients (42 GEP training group (TG), 32 GEP validation group (VG)) were purified by autoMACS-sorting. Sorted cells were analyzed by interphase-FISH with probes specific for 6q21, 8p21, 9q34, 11q23, 13q14, 15q22, 17p13, 19q13 and translocations t(4;14) and t(11;14). HD and NHD were defined by using a copy number score (CS), which was calculated by subtracting the number of probes indicating losses from the number of probes detecting additional copies (CS 〉0: HD; CS ≤0: NHD). GEP was performed with Affymetrix DNA-microarrays. Nearest shrunken centroid classification (NSCC) was used to discriminate the different groups, using GCRMA-normalized gene expression values. The prediction error was estimated by means of nested cross-validation using 10 repetitions of 10-fold cross-validation within the training set and separately calculated by use of the NSCC classifier of the training set to predict the validation set. Goeman’s global test was used to check the influence of ribosomal protein expression between HD and NHD. In the TG, both HD and NHD were found in 21 patients. The VG comprised 13 patients with NHD and 19 patients with HD. NSCC resulted in a predictor for HD versus NHD of 81 probe sets with a cross-validated misclassification rate of 14.2% for the TG and 26.5% for the VG. Three of the top ten genes were ribosomal proteins, overexpressed in patients with HD. Goeman’s global test further showed that ribosomal proteins are overexpressed in HD (TG: p

Beschreibung: Background: Atacicept (formerly referred to as TACI-Ig) is a soluble receptor fusion protein comprised of the extracellular domain of TACI and the Fc portion of a human IgG. Atacicept binds to APRIL (A Proliferation-Inducing Ligand) and BLyS (B Lymphocyte Stimulator), which are potent survival factors for normal B cells and are over-expressed in plasma cell malignancies. APRIL and BLyS are produced by MM cellsand other cells within the tumor environment, resulting in the enhanced survival of malignant cells via both autocrine and paracrine loops. The aim of this trial was to determine the tolerability, PK, PD and biological activity of atacicept in patients with MM or WM. Methods: This was an open-label, dose-escalation study to determine the maximum tolerated dose and the optimal biologic dose of atacicept in patients with refractory or relapsed MM or active, progressive WM. Eligible patients were enrolled in sequential cohorts to receive one cycle of five weekly subcutaneous injections of atacicept at 2, 4, 7 or 10 mg/kg. Patients who demonstrated at least stable disease after the first cycle were allowed to continue to the extension phase consisting either of two additional cycles separated by a 4-week wash-out period or 15 weekly injections at a dose of 10 mg/kg. PK was assessed after the 1st and 5th dose. Usual safety parameters were assessed, including measurement of potential anti-atacicept antibodies. The biological activity assessment included M-protein, beta 2-microglobulin, soluble syndecan-1, lymphocyte subpopulation counts (by flow cytometric analysis), polyclonal immunoglobulins, serum and urinary free light chains and CRP. Response was assessed using modified Bladé criteria. Results: A total of 16 patients (12 MM and 4 WM) entered the trial. Fourteen patients completed the study, one patient was withdrawn for progressive disease and one is still being treated. No dose limiting toxicity (DLT) and no SAE related to study drug were observed. Five MM patients and 3 WM patients had stable disease after the first treatment cycle; the rest of the patients progressed. There was no obvious correlation of response with the dose received. Eight patients entered the extension phase: 4 received two additional cycles and 4 received 15 weekly injections of atacicept. Seven of the eight patients have completed the extension: 4 patients with stable disease (3 MM and 1 WM), 2 patients with progressive disease and 1 WM patient with a minimal response (M-component decrease greater than 25%). Most of patients showed a decrease of polyclonal immunoglobulins and B lymphocytes. A marked decrease in the M component and syndecan-1 was observed in several patients, while CRP was not affected. Conclusions: Treatment with atacicept was well tolerated. No dose limiting toxicity was observed. A biological response in accordance with the expected atacicept mode of action was observed in this heavily treated refractory population. Disease stabilization was seen in several patients and one WM patient achieved a minimal response.

Beschreibung: BACKGROUND. The proliferation-rate of primary myeloma cells is a strong adverse prognostic factor in various trials, but not routinely assessed, partially due to effort in obtaining it. AIM. As gene-expression profiling is increasingly considered a standard diagnostics in myeloma, we investigated the possibility to develop a prognostically relevant gene-expression based proliferation index (GPI). PATIENTS AND METHODS. Gene expression was determined by Affymetrix DNA-microarrays in 784 samples including two independent sets of 233 and 345 CD138-purified myeloma cells from previously untreated patients. The GPI was derived by selecting genes associated with proliferation (in terms of gene ontology) differentially expressed in proliferating malignant (human myeloma cell lines) and benign (plasmablastic) cells compared to non-proliferating, non-malignant cells (normal plasma cells and memory B-cells). The GPI comprises the sum of the expression values of 50 genes (ASPM, AURKA, AURKB, BIRC5, BRCA1, BUB1, BUB1B, CCNA2, CCNB1, CCNB2, CDC2, CDC20, CDC25C, CDC6, CDCA8, CDKN3, CEP55, CHEK1, CKS1B, CKS2, DLG7, ESPL1, GINS1, GTSE1, KIAA1794, KIF11, KIF15, KIF20A, KIF2C, KNTC2, MAD2L1, MCM10, MCM6, MKI67, NCAPD3, NCAPG, NCAPG2, NEK2, NPM1, PAK3, PCNA, PGAM1, PLK4, PTTG1, RACGAP1, SMC2, SPAG5. STIL, TPX2, ZWINT). Proliferation of primary myeloma cells was assessed by propidium iodinestaining (n=67). Chromosomal aberrations were assessed by comprehensive iFISH using a set of probes for the chromosomal regions 1q21, 6q21, 8p21, 9q34, 11q23, 11q13, 13q14.3, 14q32, 15q22, 17p13, 19q13, 22q11, as well as the translocations t(4;14)(p16.3;q32.3) and t(11;14)(q13;q32.3). RESULTS. In the two groups, 39 and 32 percent of primary myeloma cells show a GPI above the median plus three standard deviations of normal bone marrow plasma cells, respectively. The GPI is significantly higher in advanced- compared to early-stage myeloma (P=.001) and in patients harboring a gain of 1q21 (n=95, P

Beschreibung: Epigenetics is characterized by a wide range of changes that are reversible and orchestrate gene expression. Recent studies have shown that epigenetic modifications play a role in multiple myeloma (MM) by silencing various cancer-related genes. We investigated the epigenetic genes differentially expressed between normal bone marrow plasma cells (BMPC ; N=5) and MM plasma cells from patients (N=206). Using SAM (Significance Analysis of Microarrays) analysis, only 12 genes significantly differentially expressed between BMPC and MM cells (ratio 〉 2 and FDR (false discovery rate) 〈 5%) were identified, including the SUV39H1 histone methyltransferase. SUV39H1 and SUV39H2 are regulators of chromatin organization. SUV39H1-dependent trimethylation of H3K9 is essential for maintenance of both pericentromeric and telomeric heterochromatin. SUV39H1 deficiency reduced cell viability severely and is associated to heterochromatin decompaction, loss of silencing, genome instability, and a wide range of defects in cell cycle, cell growth, and meiosis. SUV39H1-mediated H3K9me has been linked to gene silencing of the tumor suppressor genes, such as p15INK4B and E-cadherin, in acute myeloid leukemia (AML). Therefore, it is highly possible that the default function of SUV39H1 is to maintain genome stability by limiting the acute activation of oncogenes while its dysregulation could cause tumor formation. We reported that high SUV39H1 expression, in MM cells, is associated with a poor prognosis in two independent cohorts of patients (Heidelberg-Montpellier cohort - N=206 and UAMS-TT2 cohort - N=345). SUV39H1 expression was downregulated by conditional shRNA expression through lentiviral delivery. SUV39H1 knock down significantly inhibits H3K9me3, growth of myeloma cells, induces apoptosis, cell cycle deregulation, reactive oxygen species production and spontaneous accumulation of DNA double strand breaks. According to these results, SUV39H1 depletion sensitizes myeloma cells to melphalan. Chaetocin is a selective inhibitor of SUV39H1. We identified that chaetocin has anti-myeloma effects at low nanomolar doses (range: 4 to 17 nM), on 11 different human myeloma cell lines, that are representative of the molecular heterogeneity of the patients, in association with H3K9 trimethylation inhibition. Furthermore, this significant toxicity of chaetocin in MM was confirmed on primary myeloma cells of 5 patients cocultured with their bone marrow microenvironment without significant toxicity on normal bone marrow cells and hematopoietic stem cells. Interestingly, the IC50 doses of chaetocin in MM were 50 fold lower compared to results published in AML, suggesting H3K9 histone methyltransferases could be a potent therapeutic target in MM. Disclosures Seckinger: EngMab AG: Research Funding; Takeda: Other: Travel grant. Goldschmidt:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millenium: Honoraria, Research Funding, Speakers Bureau; Onyx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Chugai: Honoraria, Research Funding, Speakers Bureau. Hose:EngMab AG: Research Funding; Takeda: Other: Travel grant.

Beschreibung: The NF-κB pathway is involved in the physiological regulation of cell proliferation in many cell types as well as in the resistance of several malignancies to cell death. The pathophysiologic basis for multiple myeloma (MM) has been attributed to the dysregulation of various paracrine or autocrine growth factor loops and to perturbations in several signal transduction pathways including IKK/NF-κB. The aim of the present study was to investigate the effect of a pharmaceutical IKK2 inhibitor (IKK2-I), the anilinopyrimidine derivative AS602868 (Serono International SA), on the in vitro growth of human MM cell lines (HMCL) and primary MM cells. We evaluated the effect of AS602868 on the proliferation and the survival of 12 IL-6-dependent HCML and 2 autonomously growing HCML as well as on the survival of total bone marrow mononuclear cells from patients with newly diagnosed MM (n = 6) or with relapsing MM (n = 7). Results show that using HMCL or primary MM cells, AS602868 induces a clear dose-dependent inhibition of MM cell growth (the 50% inhibitory concentration (IC50) ranging from 0.28 to 8.3 μM, mean IC50 = 2.6 μM on HCML). It was shown using HMCL that the growth inhibition induced by AS602868 is the result of a simultaneous induction of apoptosis and inhibition of the cell cycle progression. Importantly, AS602868 does not alter the survival of other bone marrow mononuclear cells (CD138−) co-cultured with primary MM (CD138+) cells except on CD34+ hematopoietic stem cells. Interestingly, using gene expression profiling with Affymetrix microarrays on 13 HMCL, we show that the resistance (high IC50) to AS602868 inhibitor is strongly correlated to APRIL gene expression (r =.7603, p