Fabio Mirabella

Mutational Spectrum, Copy Number Changes, and Outcome: Results of a Sequencing Study of Patients With Newly Diagnosed Myeloma

PURPOSE At the molecular level, myeloma is characterized by copy number abnormalities and recurrent translocations into the immunoglobulin heavy chain locus. Novel methods, such as massively parallel sequencing, have begun to describe the pattern of tumor-acquired mutations, but their clinical relevance has yet to be established. METHODS We performed whole-exome sequencing for 463 patients who presented with myeloma and were enrolled onto the National Cancer Research Institute Myeloma XI trial, for whom complete molecular cytogenetic and clinical outcome data were available. RESULTS We identified 15 significantly mutated genes: IRF4, KRAS, NRAS, MAX, HIST1H1E, RB1, EGR1, TP53, TRAF3, FAM46C, DIS3, BRAF, LTB, CYLD, and FGFR3. The mutational spectrum is dominated by mutations in the RAS (43%) and nuclear factor-κB (17%) pathways, but although they are prognostically neutral, they could be targeted therapeutically. Mutations in CCND1 and DNA repair pathway alterations (TP53, ATM, ATR, and ZNFHX4 mutations) are associated with a negative impact on survival. In contrast, those in IRF4 and EGR1 are associated with a favorable overall survival. We combined these novel mutation risk factors with the recurrent molecular adverse features and international staging system to generate an international staging system mutation score that can identify a high-risk population of patients who experience relapse and die prematurely. CONCLUSION We have refined our understanding of genetic events in myeloma and identified clinically relevant mutations that may be used to better stratify patients at presentation.

Intraclonal heterogeneity is a critical early event in the development of myeloma and precedes the development of clinical symptoms

The mechanisms involved in the progression from monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM) to malignant multiple myeloma (MM) and plasma cell leukemia (PCL) are poorly understood but believed to involve the sequential acquisition of genetic hits. We performed exome and whole-genome sequencing on a series of MGUS (n=4), high-risk (HR)SMM (n=4), MM (n=26) and PCL (n=2) samples, including four cases who transformed from HR-SMM to MM, to determine the genetic factors that drive progression of disease. The pattern and number of non-synonymous mutations show that the MGUS disease stage is less genetically complex than MM, and HR-SMM is similar to presenting MM. Intraclonal heterogeneity is present at all stages and using cases of HR-SMM, which transformed to MM, we show that intraclonal heterogeneity is a typical feature of the disease. At the HR-SMM stage of disease, the majority of the genetic changes necessary to give rise to MM are already present. These data suggest that clonal progression is the key feature of transformation of HR-SMM to MM and as such the invasive clinically predominant clone typical of MM is already present at the SMM stage and would be amenable to therapeutic intervention at that stage.

Global methylation analysis identifies prognostically important epigenetically inactivated tumor suppressor genes in multiple myeloma

Outcome in multiple myeloma is highly variable and a better understanding of the factors that influence disease biology is essential to understand and predict behavior in individual patients. In the present study, we analyzed combined genomewide DNA methylation and gene expression data of patients treated in the Medical Research Council Myeloma IX trial. We used these data to identify epigenetically repressed tumor suppressor genes with prognostic relevance in myeloma. We identified 195 genes with changes in methylation status that were significantly associated with prognosis. Combining DNA methylation and gene expression data led to the identification of the epigenetically regulated tumor modulating genes GPX3, RBP1, SPARC, and TGFBI. Hypermethylation of these genes was associated with significantly shorter overall survival, independent of age, International Staging System score, and adverse cytogenetics. The 4 differentially methylated and expressed genes are known to mediate important tumor suppressive functions including response to chemotherapy (TGFBI), interaction with the microenvironment (SPARC), retinoic acid signaling (RBP1), and the response to oxidative stress (GPX3), which could explain the prognostic impact of their differential methylation. Assessment of the DNA methylation status of the identified genes could contribute to the molecular characterization of myeloma, which is prerequisite for an individualized treatment approach.

Selective FLT3 inhibition of FLT3-ITD+ acute myeloid leukaemia resulting in secondary D835Y mutation: A model for emerging clinical resistance patterns

Acquired resistance to selective FLT3 inhibitors is an emerging clinical problem in the treatment of FLT3-ITD+ acute myeloid leukaemia (AML). The paucity of valid pre-clinical models has restricted investigations to determine the mechanism of acquired therapeutic resistance, thereby limiting the development of effective treatments. We generated selective FLT3 inhibitor-resistant cells by treating the FLT3-ITD+ human AML cell line MOLM-13 in vitro with the FLT3-selective inhibitor MLN518, and validated the resistant phenotype in vivo and in vitro. The resistant cells, MOLM-13-RES, harboured a new D835Y tyrosine kinase domain (TKD) mutation on the FLT3-ITD+ allele. Acquired TKD mutations, including D835Y, have recently been identified in FLT3-ITD+ patients relapsing after treatment with the novel FLT3 inhibitor, AC220. Consistent with this clinical pattern of resistance, MOLM-13-RES cells displayed high relative resistance to AC220 and Sorafenib. Furthermore, treatment of MOLM-13-RES cells with AC220 lead to loss of the FLT3 wild-type allele and the duplication of the FLT3-ITD-D835Y allele. Our FLT3-Aurora kinase inhibitor, CCT137690, successfully inhibited growth of FLT3-ITD-D835Y cells in vitro and in vivo, suggesting that dual FLT3-Aurora inhibition may overcome selective FLT3 inhibitor resistance, in part due to inhibition of Aurora kinase, and may benefit patients with FLT3-mutated AML.

Improved risk stratification in myeloma using a microRNA-based classifier

Multiple myeloma (MM) is a heterogeneous disease. International Staging System/fluorescence hybridization (ISS/FISH)‐based model and gene expression profiles (GEP) are effective approaches to define clinical outcome, although yet to be improved. The discovery of a class of small non‐coding RNAs (micro RNAs, miRNAs) has revealed a new level of biological complexity underlying the regulation of gene expression. In this work, 163 presenting samples from MM patients were analysed by global miRNA profiling, and distinct miRNA expression characteristics in molecular subgroups with prognostic relevance (4p16, MAF and 11q13 translocations) were identified. Furthermore we developed an “outcome classifier”, based on the expression of two miRNAs (MIR17 and MIR886‐5p), which is able to stratify patients into three risk groups (median OS 19·4, 40·6 and 65·3 months, P = 0·001). The miRNA‐based classifier significantly improved the predictive power of the ISS/FISH approach (P = 0·0004), and was independent of GEP‐derived prognostic signatures (P < 0·002). Through integrative genomics analysis, we outlined the potential biological relevance of the miRNAs included in the classifier and their putative roles in regulating a large number of genes involved in MM biology. This is the first report showing that miRNAs can be built into molecular diagnostic strategies for risk stratification in MM.

Understanding the interplay between the proteasome pathway and autophagy in response to dual PI3K/mTOR inhibition in myeloma cells is essential for their effective clinical application

Understanding the interplay between the proteasome pathway and autophagy in response to dual PI3K/mTOR inhibition in myeloma cells is essential for their effective clinical application

MMSET is the key molecular target in t(4;14) myeloma

The t(4;14)(p16.3;q32.3) is found in 15% of presenting multiple myeloma (MM) cases and is associated with a significantly worse prognosis than other biological subgroups. As a consequence of the translocation, two genes are aberrantly expressed, the fibroblast growth factor receptor 3 (FGFR3) and a multiple myeloma SET domain containing protein, MMSET (WHSC1/NSD2), both of which have potential oncogenic activity.1 Importantly, FGFR3 shows only weak transforming activity and is eventually lost in 30% of patients,2 suggesting that it is not the main oncogenic factor. In contrast, MMSET gene overexpression is universal, and when it is knocked down experimentally, there is inhibition of proliferation, induction of apoptosis and alteration of cell adhesion,3, 4, 5 suggesting it is central to the pathogenesis of this subtype of MM. MMSET is known to have histone methyl transferase activity6 and is deregulated early on in the genesis of developing myeloma, and could therefore constitute a good therapeutic target. The MMSET locus in t(4;14) myeloma patients has a complicated genomic structure and after translocation events and RNA splicing, a number of different transcripts are generated (Figure 1). This genetic complexity of MMSET has been added to recently by the discovery of the H/ACA box RNA ACA11 (SCARNA22), that has been found within intron 20 of MMSET and is also overexpressed in the t(4;14) subgroup.7 This small RNA has been suggested to be key to the pathogenesis of t(4;14) MM, raising the question that it may constitute the main therapeutic target.

Hsp70 inhibition induces myeloma cell death via the intracellular accumulation of immunoglobulin and the generation of proteotoxic stress

Multiple myeloma (MM) cells rely on protein homeostatic mechanisms for survival. These mechanisms could be therapeutically targeted via modulation of the heat shock response. We studied the roles of Hsp72 and Hsc70, and show that the two major cytoplasmic Hsp70s play a key role in regulating protein homeostasis and controlling multiple oncogenic pathways in MM, and their inhibition can lead to myeloma cell death. Our study provides further evidence that targeting Hsp70 represents a novel therapeutic approach which may be effective in the treatment of MM.

Overexpression of EZH2 in multiple myeloma is associated with poor prognosis and dysregulation of cell cycle control

Myeloma is heterogeneous at the molecular level with subgroups of patients characterised by features of epigenetic dysregulation. Outcomes for myeloma patients have improved over the past few decades except for molecularly defined high-risk patients who continue to do badly. Novel therapeutic approaches are, therefore, required. A growing number of epigenetic inhibitors are now available including EZH2 inhibitors that are in early-stage clinical trials for treatment of haematological and other cancers with EZH2 mutations or in which overexpression has been correlated with poor outcomes. For the first time, we have identified and validated a robust and independent deleterious effect of high EZH2 expression on outcomes in myeloma patients. Using two chemically distinct small-molecule inhibitors, we demonstrate a reduction in myeloma cell proliferation with EZH2 inhibition, which leads to cell cycle arrest followed by apoptosis. This is mediated via upregulation of cyclin-dependent kinase inhibitors associated with removal of the inhibitory H3K27me3 mark at their gene loci. Our results suggest that EZH2 inhibition may be a potential therapeutic strategy for the treatment of myeloma and should be investigated in clinical studies.

Multiple myeloma risk variant at 7p15.3 creates an IRF4-binding site and interferes with CDCA7L expression

Genome-wide association studies have identified several risk loci for multiple myeloma (MM); however, the mechanisms by which they influence MM are unknown. Here by using genetic association data and functional characterization, we demonstrate that rs4487645 G>T, the most highly associated variant (P = 5.30 × 10−25), resides in an enhancer element 47 kb upstream of the transcription start site of c-Myc-interacting CDCA7L. The G-risk allele, associated with increased CDCA7L expression (P=1.95 × 10−36), increases IRF4 binding and the enhancer interacts with the CDCA7L promoter. We show that suppression of CDCA7L limits MM proliferation through apoptosis, and increased CDCA7L expression is associated with adverse patient survival. These findings implicate IRF4-mediated CDCA7L expression in MM biology and indicate how germline variation might confer susceptibility to MM.