M. D'Anca
Università degli Studi di Milano, 2016-02-18

Multiple Myeloma (MM) is malignancy of terminally differentiated plasma cells characterized by a marked heterogeneity of genetic lesions and clinical course. Despite significant efforts towards the development of risk stratification strategies for patients with Multiple Myeloma (MM), we are still limited in our capacity to molecularly predict the natural history of these patients. Recent molecular analyses have illuminated many aspects of the pathogenesis of this heterogeneous disease, although there remains an elemental view of the compendium of genetic elements driving MM initiation and progression and how such genetic alterations functionally contribute to specific aspects of disease pathobiology, prognosis and treatment responses. Indeed, despite considerable progress in the management of MM patients, many studies have shown that some genetic alterations especially t(4;14) translocation, loss of the short arm of chromosome 17, loss of the long arm of chromosome 13 and amplification of chromosome 1q21 remain associated with a poor outcome and represent independent adverse predictors of shorter progression free survival (PFS) and overall survival (OS). The 1q21 amplicon is among the most frequent chromosomal aberrations in patients with MM (about 40% of de novo MM) and is considered a highly poor-risk genetic feature correlated with disease progression and drug resistance; it spans approximately a region of 10-15 Mb containing a large number of possible candidate genes. To date the relevant genes on 1q21 remain unclear and the absence of focal amplifications involving this region strongly suggests that more than a single candidate may represent the driver event responsible for poor outcome of this group of MM patients. Thus, the identification of critical 1q21 ‘Achilles heel’ vulnerabilities may yield a comprehensive catalog of the potential therapeutic targets for these high risk MM and provide a rationale for patient stratification. In an effort to accomplish this goal, we first identified a high-priority list of 78 copy number-driven 1q21 MM-relevant genes. Then, we have designed a high-throughput systematic shRNA screen approach in vitro to identify 1q21 genes whose loss of function results in selective death and/or growth inhibition of MM cells carrying the 1q21 amplification. After excluding shRNAs that display citotoxic activity regardless 1q21 amplification, we defined 1q21 “Achilles heel” vulnerabilities as shRNA target genes whose down-regulation decreases substantially the percentage of GFP-positive MM cells with 1q21 amplification over a time of 7 days based on a GFP-competion assay. This assay provided a list of candidate genes implicated in survival or proliferation of MM cells with 1q21 amplification; MCL1, UBAP2L, INTS3, LASS2, KRTCAP2, and ILF2. By targeting these six genes we performed secondary validation experiments in JJN3 and H929 MM cell lines, carrying 4 copies of 1q21 amplicon. The results of this secondary validation confirmed that the down-regulation of these genes caused an important decrease of proliferation and increase of apoptosis as well as growth cycle arrest. Further GEP analysis and clinical outcome studies revealed that only UBAP2L and ILF2 showed a significant prognostic value but in vivo validation studies on NOD-SCID mice identified only ILF2 correlated with in vivo survival. So our studies focused to investigate the role of ILF2 in 1q21 amplified MM. Nuclear Factor 45 (NF45) or ILF-2 is widely expressed in normal tissue with a predominant nuclear distribution. NF45/ILF2 associates with NF90/NF110 (ILF3) interacting with DNA and RNA. ILF2 and ILF3 contribute to gene regulation at different levels, transcription, splicing, nuclear exporting, but they are also involved in other important processes like mitotic control and DNA break repair. Down-regulation of ILF2 in MM cells with 1q21 amplification resulted in multinucleated phenotypes and abnormal nuclear morphologies that were associated with a significant accumulation of γH2AX foci and DNA damage response activation, increased sensitivity to Melphalan, DNA damaging agent, and impaired activation of DNA repair pathways. Experiments of immunoprecipitation combined with mass spectometry showed that ILF2 interacts with numerous RNA binding proteins directly implicated in DNA repair or regulation of DNA damage response by modulating alternative splicing and stability of specific pre-mRNAs. Accordingly, RNA-sequencing analysis of ILF2-depleted MM cells, when compared to cells carrying scrambled shRNAs, identified specific changes in RNA splicing patterns before and after treatment with Melphalan. Thus, our findings have raised a new tight correlation between 1q21 amplification and DNA damage response. We identified ILF2 as a key driver of this interaction, and our findings support the development of strategies designed to modulate ILF2 expression in patients with high-risk MM carrying 1q21 amplification providing personalized therapies for patients who do not benefit from recent treatment improvements.

diritti: info:eu-repo/semantics/openAccess
In relazione con info:eu-repo/semantics/altIdentifier/hdl/2434/362028
tutor: A. Neri ; correlatore: S. Colla ; coordinatore: P. Corradini
Settore MED/15 - - Malattie del Sangue

Tesi di dottorato. | Lingua: Inglese. | Paese: | BID: TD18002395