Project description:We investigated at two time points a longitudinal cohort of 27 untreated Chronic Lymphocytic Leukemia (CLL) patients with either stable or progressive disease. The sequenced genes included BCOR, EGR2, HIST1H1E, ITPKB, KRAS, MED12, NRAS, RIPK1, SAMHD1, ATM, BIRC3, BRAF, CHD2, DDX3X, DDX3Y, FBXW7, KIT, KLHL6, MAPK1, MYD88, NOTCH1, PIK3CA, POT1, SF3B1, TP53, XPO1 and ZMYM3, which were previously identified as mutated in CLL studies.
Project description:Chronic Lymphocytic Leukemia (CLL) is a heterogeneous disease with a variable clinical course strictly dependent on cytogenetic and molecular features. However, in 15-20% of cases both conventional cytogenetic and FISH analyses do not show any kind of abnormality. With the aim to identify dependable molecular prognostic factors in this subgroup, we evaluated 171 CLL patients, without aberrations detected by chromosome banding and FISH analysis. A comprehensive analysis was performed including genomic arrays (CGH+SNP), IGHV status, flow cytometry and a targeted sequencing. By genomic arrays, we detected 73 aberrations in 39 patients (23%). Most frequently, patients had 1 aberration (25/171; 15%), while 14 patients (8%) had at least 2 aberrations. IGHV unmutated status was present in 53/171 (31%) patients. SF3B1 was the most frequently mutated gene (26/171 patients; 15%), followed by NOTCH1 (n=15, 9%), ATM (n=5; 3%); TP53 (n=5; 3%); KLHL6 (n=5; 3%); MYD88 (n=5; 3%) and XPO1 (n=5; 3%). At univariate analysis, an adverse impact on time to treatment (TTT) was evident for SF3B1 mutations, higher white blood cell count, higher CLL cells percentage by flow cytometry, CD38 positivity, IGHV unmutated status and at least 2 genomic array abnormalities. Of them, SF3B1 mutations, CLL cells percentage, IGHV unmutated status and number of genomic array aberrations maintained their impact in multivariate analysis. In conclusion, integrating genomic and molecular data, we identified patients at higher risk for treatment need. Therefore, we suggest to evaluate these factors for a better prognostic stratification of normal karyotype CLL subset.
Project description:Mutations of SF3B1 in CLL induce alternative splicing in multiple transcripts, including DVL2. DVL2 in turn can act as a negative regulator of NOTCH1 signaling. Gene Expression Profile (GEP) was used to investigate the activation of the NOTCH1 pathway in presence of alternatively spliced DVL2.
Project description:To identify genomic alterations contributing to the pathogenesis of high‑risk chronic lymphocytic leukemia (CLL) beyond the well‑established role of TP53 aberrations, we comprehensively analyzed 146 high‑risk CLL cases by single‑nucleotide polymorphism (SNP)‑arrays and targeted next‑generation sequencing including 75 relapsed/refractory and 71 treatment‑naïve high‑risk cases from prospective clinical trials. Increased genomic complexity was a hallmark of relapsed/refractory and treatment‑naïve high‑risk CLL, and was associated with TP53 and ATM dysfunction. In relapsed/refractory cases previously exposed to the selective pressure of chemo(immuno)therapy, gain(8)(q24.21) and del(9)(p21.3) were found particularly enriched. Both of these copy number alterations (CNAs) affected key regulators of cell cycle progression, namely c‑MYC and CDKN2A/B. Gains in 8q24.21 were either focal gains in a c‑MYC enhancer region or larger gains directly affecting the c‑MYC locus, but only the latter type was highly enriched in relapsed/refractory CLL (17%). Loss of CDKN2A/B was found frequently to co‑occur with gain of c‑MYC and in this combination it was likely associated with Richter transformation. In addition to a high frequency of NOTCH1 mutations (23%), we found recurrent genetic alterations in SPEN (4% mutated), RBPJ (8% deleted) and SNW1 (8% deleted), all affecting a protein complex that represses transcription of NOTCH1 target genes. We investigated the functional impact of these alterations on HES1, DTX1 and c‑MYC gene transcription and found de‑repression of these NOTCH1 target genes particularly with SPEN mutations. In summary, we provide new insights into the pathogenesis of high‑risk CLL by defining novel recurrent CNAs and identifying alterations that likely contribute to disease refractoriness.
Project description:The RNA splicing factor SF3B1 is recurrently mutated in chronic lymphocytic leukemia (CLL), but its functional role in the pathogenesis of this disease has not been firmly established. Here, we show that conditional expression of heterozygous Sf3b1-K700E mutation in mouse B lineage cells disrupts pre-mRNA splicing, alters B-cell development and function, and induces a state of cellular senescence. B-cell restricted expression of this mutation combined with Atm deletion led to the overcoming of cellular senescence, together with enhanced genome instability and the development of clonal B220+CD5+ CLL cells in elderly mice at low penetrance. Mice with CLL-like disease were found to have amplifications of chromosomes 15 and 17. Integrated transcriptome and proteome analysis of the CLL-like cells revealed coordinated dysregulation of multiple CLL-associated cellular processes. This included an unexpected signature of deregulated B-cell receptor (BCR) signaling, which we could also identify in SF3B1-mutated CLL samples from two independent patient cohorts. Notably, human CLLs harboring SF3B1 mutations exhibited greater sensitivity and altered response kinetics to BTK kinase ibrutinib. Our genetically faithful murine model of CLL thus reveals fresh insights regarding the impact of SF3B1 mutation on CLL pathogenesis and suggests a system for identifying vulnerabilities related to this mutation that can be further exploited for the treatment of CLLs with this common mutation.
Project description:Richter's syndrome (RS) is an aggressive transformation of Chronic Lymphocytic Leukaemia (CLL) frequently due to TP53, CDKN2, MYC or NOTCH1 mutations. whereas a significant proportion displays no specifically acquired driver mutation. We observe constitutive AKT phosphorylation not only in high-risk CLL patients harbouring p53 and NOTCH mutations but also in numerous RS patients. Consistently, genetic over-activation of AKT within the Eµ-TCL1 CLL mouse model results in a high-grade lymphoma phenotype of Richters syndrome. Multiomics assessment of our novel mouse model revealed a S100 defined subcluster of highly proliferative lymphoma cells developing from indolent CLL-like B-cells as a consequence of sudden NOTCH activation being fueled by enhanced NOTCH ligand exposure from T-cells in the microenvironment. Our data link AKT and NOTCH signaling in patient samples, genomic alterations, phosphoproteome and single-cell transcriptome profiles. Collectively, we have identified active AKT as a causative transforming pathway of indolent CLL towards aggressive RS thus providing novel mechanistic insights into the molecular understanding of RS.
Project description:RNA splicing factor SF3B1 is recurrently mutated in various cancers, particularly in hematological malig-nancies. We previously reported that co-expression of Sf3b1 mutation and Atm deletion in B cells, but not either lesion alone, leads to the onset of chronic lymphocytic leukemia (CLL) with CLL cells harboring chromosome amplification. However, the exact role of Sf3b1 mutation and Atm deletion in chromosomal instability (CIN) remains unclear. Here, we demonstrate that SF3B1 mutation promotes centromeric R-loop (cen-R-loop) accumulation, leading to increased chromosome oscillation, impaired chromosome segrega-tion, altered spindle architecture and aneuploidy, which can be alleviated by removal of cen-R-loop and exaggerated by deletion of ATM. Aberrant splicing of key genes involved in R-loop processing underlies augmentation of cen-R-loop as overexpression of the normal isoform, but not the altered form, mitigates mitotic stress in SF3B1 mutant cells. Our study underscores the critical role of novel splice variants in link-ing RNA splicing dysregulation and CIN, and highlights cen-R-loop augmentation as a key mechanism for leukemogenesis.
Project description:NOTCH1 is mutationally activated in ~15% of cases of chronic lymphocytic leukaemia (CLL), but its role in B-cell development and leukemogenesis is not known. Here, we report that the active intracellular portion of NOTCH1 (ICN1) is detectable in ~50% of peripheral blood CLL cases lacking gene mutations. We identify a ‘NOTCH1 CLL gene expression signature’ in CLL cells, and show that this signature is significantly enriched in primary CLL cases expressing ICN1, independent of NOTCH1 mutation. NOTCH1 target genes include key regulators of B-cell proliferation, survival and signal transduction physiology. In particular, we show that MYC is a direct target of NOTCH1 via B-cell specific distal regulatory elements, thus implicating this oncogene in the pathogenesis of the disease.
Project description:Stabilizing mutations of NOTCH1 have been identified in about 10% of chronic lymphocytic leukemia (CLL) cases at diagnosis, with a higher frequency in unmutated IGHV (IGHV-UM) CLL, chemorefractory CLL and CLL in advanced disease phases. Clinically, the presence of NOTCH1 mutations is an independent predictor of overall survival in CLL and associates with resistance to anti-Cd20 immunotherapy. The Gene Expression Profile was generated to identify the peculiar molecular signatures of NOTCH1 mutated CLL in the context of IGHV-UM CLL.