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Tumour Survival Signals and Epigenetic Gene Silencing in Multiple Myeloma: Implications for Biology and Therapy
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis is focused on multiple myeloma (MM), a haematological malignancy that still remains incurable. The pathogenesis of MM is not fully understood and there is a large intra-tumour and interclonal genetic variation in MM patients. One of the most challenging areas in MM research is to find mechanisms for initiation and progression of MM, but also to overcome the arising resistance to therapy.

In paper I, a signature of under-expressed genes in MM was found to significantly correlate with already defined Polycomb target genes. In selected genes from the profile we found an enrichment of H3K27me3, a repressive mark catalysed by Polycomb repressive complex 2 (PRC2), in MM patients and MM cell lines. Treatment with LBH589 (HDAC inhibitor) and DZNep (methyltransferase inhibitor) reactivated the H3K27me3 target genes and induced apoptosis in MM cell lines. LBH589 reduced tumour load and increased overall survival in the 5T33MM mice. These results suggest an important role for Polycomb complex in MM development and highlight PRC2 as a drug target in MM.

In paper II, the insulin-like growth factor type 1 receptor tyrosine kinase (IGF-1RTK) inhibitor picropodophyllin (PPP) in combination with LBH589 synergistically inhibited cell proliferation and enhanced the apoptotic effect in MM. Since the bone marrow microenvironment has an important role in MM disease and also contributes to drug-resistance, we therefore evaluated the drug combination in the immunocompetent 5T33MM murine model. The drug combination significantly prolonged the survival of the 5T33MM mice compared to single drug treatment. We conclude that the combination of PPP and LBH589 has a therapeutic potential in MM.

In paper III, the role of the cellular inhibitor of apoptosis protein 2 (cIAP2) was evaluated in MM cells harbouring TRAF3 deletion/mutation. By overexpressing cIAP2 in these cells we found an increased resistance to proteasome inhibitors. cIAP2 over-expression by lentiviral constructs led to decreased caspase activation, activation of the canonical NF-κB pathway, and down-regulation of tumour suppressor genes and genes that contribute to apoptosis. Supporting the role of cIAP2 mediated drug-resistance, we here demonstrate that inhibiting cIAP2 using an IAP antagonist, increased the sensitivity to the proteasome inhibitor, bortezomib.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. , 45 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1071
Keyword [en]
Multiple myeloma, Polycomb, apoptosis, cIAP2, IAP-inbibitors, proteasome inhibitors, LBH589, PPP, DZNep
National Category
URN: urn:nbn:se:uu:diva-242571ISBN: 978-91-554-9159-8OAI: oai:DiVA.org:uu-242571DiVA: diva2:785793
Public defence
2015-03-25, Fåhraeussalen, Rudbeck Laboratory, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Available from: 2015-02-25 Created: 2015-01-28 Last updated: 2015-03-11
List of papers
1. Polycomb target genes are silenced in multiple myeloma
Open this publication in new window or tab >>Polycomb target genes are silenced in multiple myeloma
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2010 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 5, no 7, e11483- p.Article in journal (Refereed) Published
Abstract [en]

Multiple myeloma (MM) is a genetically heterogeneous disease, which to date remains fatal. Finding a common mechanism for initiation and progression of MM continues to be challenging. By means of integrative genomics, we identified an underexpressed gene signature in MM patient cells compared to normal counterpart plasma cells. This profile was enriched for previously defined H3K27-tri-methylated genes, targets of the Polycomb group (PcG) proteins in human embryonic fibroblasts. Additionally, the silenced gene signature was more pronounced in ISS stage III MM compared to stage I and II. Using chromatin immunoprecipitation (ChIP) assay on purified CD138+ cells from four MM patients and on two MM cell lines, we found enrichment of H3K27me3 at genes selected from the profile. As the data implied that the Polycomb-targeted gene profile would be highly relevant for pharmacological treatment of MM, we used two compounds to chemically revert the H3K27-tri-methylation mediated gene silencing. The S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin (DZNep) and the histone deacetylase inhibitor LBH589 (Panobinostat), reactivated the expression of genes repressed by H3K27me3, depleted cells from the PRC2 component EZH2 and induced apoptosis in human MM cell lines. In the immunocompetent 5T33MM in vivo model for MM, treatment with LBH589 resulted in gene upregulation, reduced tumor load and increased overall survival. Taken together, our results reveal a common gene signature in MM, mediated by gene silencing via the Polycomb repressor complex. The importance of the underexpressed gene profile in MM tumor initiation and progression should be subjected to further studies.

National Category
urn:nbn:se:uu:diva-133207 (URN)10.1371/journal.pone.0011483 (DOI)000279715300003 ()20634887 (PubMedID)
Available from: 2010-11-03 Created: 2010-11-03 Last updated: 2015-03-11Bibliographically approved
2. The HDAC Inhibitor LBH589 Enhances the Antimyeloma Effects of the IGF-1RTK Inhibitor Picropodophyllin
Open this publication in new window or tab >>The HDAC Inhibitor LBH589 Enhances the Antimyeloma Effects of the IGF-1RTK Inhibitor Picropodophyllin
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2012 (English)In: Clinical Cancer Research, ISSN 1078-0432, E-ISSN 1557-3265, Vol. 18, no 8, 2230-2239 p.Article in journal (Refereed) Published
Abstract [en]

Purpose: We have previously shown the use of the insulin-like growth factor type 1 receptor tyrosine kinase (IGF-1RTK) inhibitor picropodophyllin (PPP) as an attractive strategy to combat multiple myeloma (MM) in vitro and in vivo. After a combinatorial drug screening, the histone deacetylase inhibitor LBH589 was shown to act in synergy with PPP reducing survival of MM cells. In this study, we tried to elucidate the molecular mechanisms underlying this combinatorial effect.

Experimental Design: The in vitro anti-MM effects of PPP and LBH589 alone and in combination were evaluated by studying apoptosis, cell cycle distribution, and downstream transcriptome using both human MM cell lines and cells from the murine 5T3MM model. In vivo the effect on survival of 5T33MM-inoculated mice was evaluated.

Results: In the human MM cell line RPMI8226, treatment with PPP and LBH589 in combination resulted in a five-fold increase of apoptosis, and an additive effect on the cleavage of the active forms of caspase-8 was observed as compared with the single drug treatments. Cell cycle analysis revealed an accumulation of cells in the G2-M phase and subsequent downregulation of cell cycle regulating proteins. These data were also confirmed in the 5T33MM cells in vitro. Also, the transcriptome was analyzed by Affymetrix arrays showing gene expression alterations mainly in categories of genes regulating apoptosis and cell adhesion. Combined treatment in vivo resulted in a significantly prolonged survival of 5T33MM-inoculated mice.

Conclusions: The results indicate an improved MM treatment opportunity in using a combination of PPP and LBH589.

National Category
Medical and Health Sciences
urn:nbn:se:uu:diva-174374 (URN)10.1158/1078-0432.CCR-11-1764 (DOI)000302907300014 ()
Available from: 2012-05-22 Created: 2012-05-15 Last updated: 2015-03-11Bibliographically approved
3. Increased resistance to proteaome inhibitors in multiple myeloma mediated by cIAP2: implications for a combinatorial treatment
Open this publication in new window or tab >>Increased resistance to proteaome inhibitors in multiple myeloma mediated by cIAP2: implications for a combinatorial treatment
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2015 (English)In: OncoTarget, ISSN 1949-2553, Vol. 6, no 24, 20621-20635 p.Article in journal (Refereed) Published
Abstract [en]

Despite the introduction of new treatment options for multiple myeloma (MM), a majority of patients relapse due to the development of resistance. Unraveling new mechanisms underlying resistance could lead to identification of possible targets for combinatorial treatment. Using TRAF3 deleted/mutated MM cell lines, we evaluated the role of the cellular inhibitor of apoptosis 2 (cIAP2) in drug resistance and uncovered the plausible mechanisms underlying this resistance and possible strategies to overcome this by combinatorial treatment. In MM, cIAP2 is part of the gene signature of aberrant NF-kappa B signaling and is heterogeneously expressed amongst MM patients. In cIAP2 overexpressing cells a decreased sensitivity to the proteasome inhibitors bortezomib, MG132 and carfilzomib was observed. Gene expression analysis revealed that 440 genes were differentially expressed due to cIAP2 overexpression. Importantly, the data imply that cIAPs are rational targets for combinatorial treatment in the population of MM with deleted/mutated TRAF3. Indeed, we found that treatment with the IAP inhibitor AT-406 enhanced the anti-MM effect of bortezomib in the investigated cell lines. Taken together, our results show that cIAP2 is an important factor mediating bortezomib resistance in MM cells harboring TRAF3 deletion/mutation and therefore should be considered as a target for combinatorial treatment.

National Category
urn:nbn:se:uu:diva-242568 (URN)000360138200076 ()
Swedish Research CouncilSwedish Cancer SocietyEU, FP7, Seventh Framework Programme, 259796
Available from: 2015-01-28 Created: 2015-01-28 Last updated: 2015-09-23Bibliographically approved

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