The role of insulin like growth factor binding protein 7 (IGFBP7) in multiple myeloma

Insulin like growth factor binding protein 7 (IGFBP7) ist ein sekretiertes Protein mit Bindungsstellen für insulin like growth factor 1 (IGF-1), insulin, bone morphogenetic proteins (BMPs), activin A und vascular endothelial growth factor A (VEGFA). Im Zusammenhang mit der Tumorgenese wurde IGFBP7 eine Rolle in der Pathophysiologie unterschiedlichster solider und hämatologischer Tumore zugeschrieben. In der vorliegenden Studie wurde mittels Genexpressions-Analyse die Expression von BMP-Antagonisten im Multiplen Myelom untersucht. Dabei zeigte sich eine verminderte Expression von IGFBP7 in aufgereinigten Tumorzellen von neu-diagnostizierten Myelompatienten. Das Stilllegen der IGFBP7 Expression erfolgt mittels Methylierung. Dies wurde durch Behandlung von Myelomzellen mit 5-aza-2-deoxycytidine und Pyrosequenzierung von Primärproben verifiziert. Hinsichtlich der prognostischen Relevanz, beobachteten wir eine signifikant schlechtere Überlebensrate in Patienten mit hoher IGFBP7 Expression in zwei voneinander unabhängigen Kohorten (n=948). Eine hohe Expression von IGFBP7 ist des Weiteren mit Hochrisikofaktoren wie Translokation t(4;14) und Amplifikation 1q21 assoziiert. Mittels in-silico Analyse wurde der starke Zusammenhang zwischen IGFBP7 und der t(4;14) assoziierten Expression von MMSET weiter bekräftigt. In Knochenmarks-Stromazellen wiesen wir eine verminderte Expression von IGFBP7 nach direktem Kontakt mit Myelomzellen nach. Exogenes IGFBP7 verstärkte hingegen die Osteoblasten-Aktivität und überkam die suppressive Wirkung von Activin A. Diese Daten wurden durch klinische Beobachtungen bestätigt. Patienten mit einer hohen IGFBP7 Expressionsrate zeigten demnach ein verringertes Risiko für das Auftreten der myelom-assoziierten Knochenerkrankung im fortgeschrittenen Stadium. Die im Rahmen dieser Studie erhobenen Daten deuten darauf hin, dass IGFBP7 einen MMSET regulierten Marker für Myelompatienten mit Hochrisikoerkrankung und das verminderte Risiko von osteolytischen Läsionen darstellt.submitted by Arnold BolomskyAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersWien, Med. Univ., Diss., 2014OeBB(VLID)171421

Targeting of BMI-1 with PTC-209 shows potent anti-myeloma activity and impairs the tumour microenvironment

Abstract Background The polycomb complex protein BMI-1 (BMI-1) is a putative oncogene reported to be overexpressed in multiple myeloma (MM). Silencing of BMI-1 was shown to impair the growth and survival of MM cells. However, therapeutic agents specifically targeting BMI-1 were not available so far. Here, we investigated PTC-209, a novel small molecule inhibitor of BMI-1, for its activity in MM. Methods BMI-1 expression was analysed in human MM cell lines and primary MM cells by using publically available gene expression profiling (GEP) data. The anti-MM activity of PTC-209 was investigated by viability testing, cell cycle analysis, annexin V and 7-AAD staining, quantification of cleaved poly(ADP-ribose) polymerase (PARP), JC-1 as well as colony formation assays. Deregulation of central myeloma growth and survival genes was studied by quantitative PCR and flow cytometry, respectively. In addition, the impact of PTC-209 on in vitro osteoclast, osteoblast and tube formation was analysed. Results We confirmed overexpression of BMI-1 in MM patients by using publically available GEP datasets. Of note, BMI-1 expression was further increased at relapse which translated into significantly shorter overall survival in relapsed/refractory patients treated with bortezomib or dexamethasone. Treatment with PTC-209 significantly decreased viable cell numbers in human MM cell lines, induced a G1 cell cycle arrest, promoted apoptosis and demonstrated synergistic activity with pomalidomide and carfilzomib. The anti-MM activity of PTC-209 was accompanied by a significant decrease of cyclin D1 (CCND1) and v-myc avian myelocytomatosis viral oncogene homolog (MYC) expression as well as upregulation of cyclin-dependent kinase inhibitor 1A (CDKN1A) and cyclin-dependent kinase inhibitor 1B (CDKN1B). We also observed upregulation of NOXA (up to 3.6 ± 1.2-fold induction, P = 0.009) and subsequent downregulation of myeloid cell leukemia 1 (MCL-1) protein levels, which likely mediates the apoptotic effects of PTC-209. Importantly, the anti-MM activity was upheld in the presence of stromal support or myeloma growth factors insulin-like growth factor 1 (IGF-1) and interleukin 6 (IL-6). In the MM microenvironment, PTC-209 impaired tube formation, impaired osteoclast development and decreased osteoblast formation in a dose-dependent manner (P < 0.01 at 1 μM, respectively). The latter might be attributed to an induction of DKK1 and was reversed by concurrent anti-DKK1 antibody treatment. Conclusions We confirmed overexpression of BMI-1 in MM highlighting its role as an attractive drug target and reveal therapeutic targeting of BMI-1 by PTC-209 as a promising novel therapeutic intervention for MM

Maternal embryonic leucine zipper kinase is a novel target for proliferation associated high-risk myeloma.

Treatment of high-risk patients is a major challenge in multiple myeloma. This is especially true for patients assigned to the gene-expression-profiling defined proliferation subgroup. Although recent efforts have identified some key players of proliferative myeloma, genetic interactions and players that can be targeted with clinically effective drugs have to be identified to overcome the poor prognosis of these patients. We therefore examined maternal embryonic leucine zipper kinase (MELK) for its implications in hyper-proliferative myeloma and analysed the activity of the MELK inhibitor OTSSP167 in vitro and in vivo. MELK was found to be significantly overexpressed in the proliferative subgroup of myeloma. This finding translated into poor overall survival in patients with high vs. low MELK expression. Enrichment analysis of upregulated genes in myeloma cells of MELKhigh patients confirmed the strong implications in myeloma cell proliferation. Targeting of MELK with OTSSP167 impaired the growth and survival of myeloma cells, thereby affecting central survival factors such as MCL-1 and IRF4. This activity was also observed in the 5TGM.1 murine model of myeloma. OTSSP167 reduced bone marrow infiltration and serum paraprotein levels in a dose-dependent manner. In addition, we revealed a strong link between MELK and other proliferation associated high-risk genes (PLK-1, EZH2, FOXM1, DEPDC1) and MELK inhibition also impaired the expression of those genes. We therefore conclude that MELK is an essential component of a proliferative gene signature and that pharmacological inhibition of MELK represents an attractive novel approach to overcome the poor prognosis of high-risk patients with a proliferative expression pattern

Defining clonal plasticity using whole exome sequencing in single cells in an index case of amp1q21 multiple myeloma

Introduction: Genomic-wide analysis of multiple myeloma (MM) reveals subclonal evolution as a major mechanism in disease evolution and progression, and interrogation of the entire functional genome at the single cell (SC) level is essential to fully map intra- clonal variation (ICV) and competition as the driver mechanism. To progress this, we have sought to establish whole exome sequencing (WES) in SCs in MM, initially in an index case of amp1q21 MM. Methods: CD138+ tumour cells and CD3+ T-cells were isolated from a presentation case of amp1q21 MM. Single MM and T cells were isolated for single cell WES. Whole genome amplification was performed using Qiagen REPLI-g Mini kit, and exome capture was performed using Agilent SureSelect. Libraries were 90 bp PE sequenced on an Illumina HiSeq2000 (BGI, China). Data was produced for bulk (1000 cells) MM and T cells, 20 MM SCs and 5 T cell SCs. Fastq were aligned to hg19 ref sequence using NovoAlignMPI (v3.02.03). Variant calling was performed using VarScan (v2.3.6) and variants were annotated using ANNOVAR. High confidence variants were called in the bulk tumour WES by pairwise comparison with bulk germline WES and by annotation against variant databases to exclude germline variants. Multiple quality control measures were employed to minimise false positives. Results and Discussion: SC WES generated raw data that were similar to bulk WES, with comparable mapping to target (69-76% SC vs. 70% bulk) and mean fold coverage (56.8-59.1x vs. 59.7x bulk). On average, 82% of the exome was covered sufficiently for somatic variant (SV) calling (! 5x), matching seminal published SC WES studies (70-80%) (Hou et al., Cell, 2012; Xu et al., Cell, 2012). We called 33 high confidence potentially deleterious SVs in the bulk tumour, 21 of which were also called in ! 1 SC exomes. SVs include mutations in genes involved in plasma cell differentiation, the MAPK pathway and known pathways in origins of B cellmalignancy. Random SVs were validated by Sanger sequencing with 100% concordance in the bulk (15/15) and SCs (55/55). ICV was apparent from the SC exome variant data. Total variant counts varied considerably among SCs and most variant positions had at least several cells where no evidence of the variant existed. Bulk WES lacks crucial information: We called 23 variants in ! 2 SCs that were absent in the bulk. Of these, 7/7 amplifiable variants were re-sequenced to obtain 100% concordance. These variants are of high interest as they reveal a marked occurrence of subclonal mu- tations in the MM tumour that are not identified by bulk WES. They indicate that the mutational status of the MM genome may be substantially underestimated by bulk WES. Conclusion: We establish the feasibility of SC WES as a method for defining true intraclonal genetic variation as a driver of cancer plasticity in MM

Correction to: MCL-1 inhibitors, fast-lane development of a new class of anti-cancer agents

An amendment to this paper has been published and can be accessed via the original article

Additional file 1: of Targeting of BMI-1 with PTC-209 shows potent anti-myeloma activity and impairs the tumour microenvironment

PTC-209 displays additive and synergistic activity with dexamethasone. Additive/synergistic activity of drug combinations was confirmed by concurrent treatment of MM cell lines with PTC-209 and dexamethasone for 96 h at varying concentrations. Graphs for MM.1S and U266 are representative for the panel of HMCLs analysed. Combination index (CI) values were determined with CompuSyn. CI values <0.8, 0.8–1.2 or >1.2 indicate synergistic, additive or antagonistic drug activities, respectively. SK-MM-1 cells did not respond to dexamethasone at the concentrations used (viability >100 % at the end of the incubation period); determination of CI values was thus not possible. NA not applicable. (PDF 631 kb