Monica Messina

Analysis of the coding genome of diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common form of human lymphoma. Although a number of structural alterations have been associated with the pathogenesis of this malignancy, the full spectrum of genetic lesions that are present in the DLBCL genome, and therefore the identity of dysregulated cellular pathways, remains unknown. By combining next-generation sequencing and copy number analysis, we show that the DLBCL coding genome contains, on average, more than 30 clonally represented gene alterations per case. This analysis also revealed mutations in genes not previously implicated in DLBCL pathogenesis, including those regulating chromatin methylation (MLL2; 24% of samples) and immune recognition by T cells. These results provide initial data on the complexity of the DLBCL coding genome and identify novel dysregulated pathways underlying its pathogenesis.

Somatically acquired JAK1 mutations in adult acute lymphoblastic leukemia

Aberrant signal transduction contributes substantially to leukemogenesis. The Janus kinase 1 (JAK1) gene encodes a cytoplasmic tyrosine kinase that noncovalently associates with a variety of cytokine receptors and plays a nonredundant role in lymphoid cell precursor proliferation, survival, and differentiation. We report that somatic mutations in JAK1 occur in individuals with acute lymphoblastic leukemia (ALL). JAK1 mutations were more prevalent among adult subjects with the T cell precursor ALL, where they accounted for 18% of cases, and were associated with advanced age at diagnosis, poor response to therapy, and overall prognosis. All mutations were missense, and some were predicted to destabilize interdomain interactions controlling the activity of the kinase. Three mutations that were studied promoted JAK1 gain of function and conferred interleukin (IL)-3–independent growth in Ba/F3 cells and/or IL-9–independent resistance to dexamethasone-induced apoptosis in T cell lymphoma BW5147 cells. Such effects were associated with variably enhanced activation of multiple downstream signaling pathways. Leukemic cells with mutated JAK1 alleles shared a gene expression signature characterized by transcriptional up-regulation of genes positively controlled by JAK signaling. Our findings implicate dysregulated JAK1 function in ALL, particularly of T cell origin, and point to this kinase as a target for the development of novel antileukemic drugs.

Mutations of the SF3B1 splicing factor in chronic lymphocytic leukemia: association with progression and fludarabine-refractoriness

The genetic lesions identified in chronic lymphocytic leukemia (CLL) do not entirely recapitulate the disease pathogenesis and the development of serious complications, such as chemorefractoriness. While investigating the coding genome of fludarabine-refractory CLL, we observed that mutations of SF3B1, encoding a splicing factor and representing a critical component of the cell spliceosome, were recurrent in 10 of 59 (17%) fludarabine-refractory cases, with a frequency significantly greater than that observed in a consecutive CLL cohort sampled at diagnosis (17/301, 5%; P = .002). Mutations were somatically acquired, were generally represented by missense nucleotide changes, clustered in selected HEAT repeats of the SF3B1 protein, recurrently targeted 3 hotspots (codons 662, 666, and 700), and were predictive of a poor prognosis. In fludarabine-refractory CLL, SF3B1 mutations and TP53 disruption distributed in a mutually exclusive fashion (P = .046). The identification of SF3B1 mutations points to splicing regulation as a novel pathogenetic mechanism of potential clinical relevance in CLL.

The coding genome of splenic marginal zone lymphoma: activation of NOTCH2 and other pathways regulating marginal zone development

Notch2 mutations represent the most frequent lesion in splenic marginal zone lymphoma.

BCR ligation induced by IgM stimulation results in gene expression and functional changes only in IgVH unmutated chronic lymphocytic leukemia (CLL) cells

Chronic lymphocytic leukemia (CLL) patients exhibit a variable clinical course. To investigate the association between clinicobiologic features and responsiveness of CLL cells to anti-IgM stimulation, we evaluated gene expression changes and modifications in cell-cycle distribution, proliferation, and apoptosis of IgV(H) mutated (M) and unmutated (UM) samples upon BCR cross-linking. Unsupervised analysis highlighted a different response profile to BCR stimulation between UM and M samples. Supervised analysis identified several genes modulated exclusively in the UM cases upon BCR cross-linking. Functional gene groups, including signal transduction, transcription, cell-cycle regulation, and cytoskeleton organization, were up-regulated upon stimulation in UM cases. Cell-cycle and proliferation analyses confirmed that IgM cross-linking induced a significant progression into the G(1) phase and a moderate increase of proliferative activity exclusively in UM patients. Moreover, we observed only a small reduction in the percentage of subG(0/1) cells, without changes in apoptosis, in UM cases; contrariwise, a significant increase of apoptotic levels was observed in stimulated cells from M cases. These results document that a differential genotypic and functional response to BCR ligation between IgV(H) M and UM cases is operational in CLL, indicating that response to antigenic stimulation plays a pivotal role in disease progression.

Genetic lesions associated with chronic lymphocytic leukemia transformation to Richter syndrome

Characterization of the pattern of clonal evolution from CLL to RS, the genetic determinants of CLL transformation to RS, and the pathogenetic relationship between RS and classical non–CLL-associated de novo DLBCL.

Characterization of B‐ and T‐lineage acute lymphoblastic leukemia by integrated analysis of MicroRNA and mRNA expression profiles

Acute lymphoblastic leukemia (ALL) is an heterogeneous disease comprising several subentities that differ for both immunophenotypic and molecular characteristics. Over the years, the biological understanding of this neoplasm has largely increased. Gene expression profiling has allowed to identify specific signatures for the different ALL subsets and permitted the identification of pathways deregulated by a given lesion. MicroRNAs (miRNAs) are small noncoding RNAs, which play a pivotal role in several cellular functions. In this study, we investigated miRNAs expression profiles in a series of adult ALL cases by microarray analysis. Unsupervised hierarchical clustering largely recapitulated ALL subgroups. Furthermore, we identified miR‐148, miR‐151, and miR‐424 as discriminative of T‐lineage versus B‐lineage ALL; ANOVA highlighted a set of six miRNAs—namely miR‐425‐5p, miR‐191, miR‐146b, miR‐128, miR‐629, and miR‐126—that can discriminate B‐lineage ALL subgroups harboring specific molecular lesions. These results were confirmed and extended by quantitative‐PCR on a further cohort of cases. Finally, we used Pearson correlation analysis to combine miRNA and gene expression profiles. The distribution of correlation coefficients generated by comparing the expression of every miRNA/gene pair in our data set shows enrichment of both positively and negatively correlated pairs over background distributions obtained using randomized data. Moreover, a clear enrichment for predicted miRNA:target pairs is observed at negative correlation coefficient intervals. Signal‐to‐noise ratio highlighted several miRNA/gene pairs with a possible role in the disease. In fact, gene set enrichment analysis of genes composing the selected miRNA/gene pairs displays over‐representation of functional categories related to cancer and cell‐cycle regulation.

Genetic lesions associated with chronic lymphocytic leukemia chemo-refractoriness.

Fludarabine refractoriness (FR) represents an unsolved clinical problem of chronic lymphocytic leukemia (CLL) management. Although next-generation sequencing studies have led to the identification of a number of genes frequently mutated in FR-CLL, a comprehensive evaluation of the FR-CLL genome has not been reported. Toward this end, we studied 10 FR-CLLs by combining whole-exome sequencing and copy number aberration (CNA) analysis, which showed an average of 16.3 somatic mutations and 4 CNAs per sample. Screening of recurrently mutated genes in 48 additional FR-CLLs revealed that ~70% of FR-CLLs carry ≥1 mutation in genes previously associated with CLL clinical course, including TP53 (27.5%), NOTCH1 (24.1%), SF3B1 (18.9%), and BIRC3 (15.5%). In addition, this analysis showed that 10.3% of FR-CLL cases display mutations of the FAT1 gene, which encodes for a cadherin-like protein that negatively regulates Wnt signaling, consistent with a tumor suppressor role. The frequency of FAT1-mutated cases was significantly higher in FR-CLL than in unselected CLLs at diagnosis (10.3% vs 1.1%, P = .004), suggesting a role in the development of a high-risk phenotype. These findings have general implications for the mechanisms leading to FR and point to Wnt signaling as a potential therapeutic target in FR-CLL.

Critical Role of c-Myc in Acute Myeloid Leukemia Involving Direct Regulation of miR-26a and Histone Methyltransferase EZH2

Increased expression or aberrant activation of c-Myc plays an important role in leukemogenesis. Here, we show that in acute myeloid leukemia (AML), c-Myc directly controls the expression of EZH2, a component of the Polycomb repressive complex 2, and miR-26a. miR-26a is downregulated in primary blasts from AML patients and, during myeloid differentiation of AML cells, is induced together with a decrease in c-Myc and Ezh2 levels. Previously, EZH2 was shown to be regulated by miR-26a at the translational levels in lymphomas. However, we demonstrate that in AML, the variation of EZH2 mainly depends on c-Myc transcriptional control. We also show that enforced expression of miR-26a in AML cells is able to inhibit cell cycle progression by downregulating cyclin E2 expression. In addition, increased levels of miR-26a potentiate the antiproliferative effects of 1,25-dihydroxyvitamin D(3) (VitD) and stimulate myeloid differentiation. Our results identify new molecular targets of c-Myc in AML and highlight miR-26a attractiveness as a therapeutic target in leukemia.

IKAROS deletions dictate a unique gene expression signature in patients with adult B-cell acute lymphoblastic leukemia.

Background Deletions of IKAROS (IKZF1) frequently occur in B-cell precursor acute lymphoblastic leukemia (B-ALL) but the mechanisms by which they influence pathogenesis are unclear. To address this issue, a cohort of 144 adult B-ALL patients (106 BCR-ABL1-positive and 38 B-ALL negative for known molecular rearrangements) was screened for IKZF1 deletions by single nucleotide polymorphism (SNP) arrays; a sub-cohort of these patients (44%) was then analyzed for gene expression profiling. Principal Findings Total or partial deletions of IKZF1 were more frequent in BCR-ABL1-positive than in BCR-ABL1-negative B-ALL cases (75% vs 58%, respectively, p = 0.04). Comparison of the gene expression signatures of patients carrying IKZF1 deletion vs those without showed a unique signature featured by down-regulation of B-cell lineage and DNA repair genes and up-regulation of genes involved in cell cycle, JAK-STAT signalling and stem cell self-renewal. Through chromatin immunoprecipitation and luciferase reporter assays we corroborated these findings both in vivo and in vitro, showing that Ikaros deleted isoforms lacked the ability to directly regulate a large group of the genes in the signature, such as IGLL1, BLK, EBF1, MSH2, BUB3, ETV6, YES1, CDKN1A (p21), CDKN2C (p18) and MCL1. Conclusions Here we identified and validated for the first time molecular pathways specifically controlled by IKZF1, shedding light into IKZF1 role in B-ALL pathogenesis.