Mutations in the splicing factor SF3B1 are found in several cancer types and have been associated with various splicing defects. Using transcriptome sequencing data from chronic lymphocytic leukemia, breast cancer and uveal melanoma tumor samples, we show that hundreds of cryptic 3’ splice sites (3’SSs) are used in cancers with SF3B1 mutations. We define the necessary sequence context for the observed cryptic 3’ SSs and propose that cryptic 3’SS selection is a result of SF3B1 mutations causing a shift in the sterically protected region downstream of the branch point. While most cryptic 3’SSs are present at low frequency (<10%) relative to nearby canonical 3’SSs, we identified ten genes that preferred out-of-frame cryptic 3’SSs. We show that cancers with mutations in the SF3B1 HEAT 5-9 repeats use cryptic 3’SSs downstream of the branch point and provide both a mechanistic model consistent with published experimental data and affected targets that will guide further research into the oncogenic effects of SF3B1 mutation.
Key Points Persistent CLL cells during ibrutinib therapy show evidence of biochemical activation, but inhibited BCR and no proliferation. Long lymphocytosis during ibrutinib therapy is not associated with adverse progression-free survival.
SUMMARY Mutations in SF3B1, which encodes a spliceosome component, are associated with poor outcome in chronic lymphocytic leukemia (CLL), but how these contribute to CLL progression remains poorly understood. We undertook a transcriptomic characterization of primary human CLL cells to identify transcripts and pathways affected by SF3B1 mutation. Splicing alterations, identified in the analysis of bulk cells, were confirmed in single SF3B1-mutated CLL cells and also found in cell lines ectopically expressing mutant SF3B1. SF3B1 mutation was found to dysregulate multiple cellular functions including DNA damage response, telomere maintenance, and Notch-signaling—mediated through KLF8 upregulation, increased TERC and TERT expression, or altered splicing of DVL2 transcript, respectively. SF3B1 mutation appears to be a mechanism by which changes in diverse cancer-related pathways are generated.
IntroductionAcute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disease. Cytogenetic and/or molecular studies are used to assign 30% to 40% of AML cases carrying specific genetic lesions (eg, t(15;17), t(8;21) or Inv(16)) to different prognostic subgroups in order to monitor minimal residual disease and to select patients who could benefit from targeted therapies. 1 However, they cannot be applied to 40% to 50% of patients with AML who at conventional cytogenetics exhibit a normal karyotype. 1,2 We recently found exon-12 nucleophosmin (NPM) gene mutations in approximately 60% of AMLs with normal karyotype (about one third of all adult AML) that are characterized by distinct morphologic, phenotypic, and molecular features, 3 as well as distinct gene expression profile signature. 4 NPM mutations predict good response to induction therapy and favorable prognosis, 5-8 and could serve to monitor minimal residual disease. 9 Thus, analysis of NPM mutations emerges as a major new step in the diagnostic/prognostic work-up of patients with AML with a normal karyotype.Reverse-transcriptase-polymerase chain reaction (RT-PCR), mutational analysis, or gene expression profiling is usually used for detecting genetic abnormalities or specifically overexpressed genes with diagnostic/prognostic significance. However, demand is growing for simple, inexpensive, and specific immunologic tests 10 that can serve as a surrogate to, or used in integration with, molecular studies. Examples include immunohistochemical detection of proteins such as anaplastic lymphoma kinase (ALK) in CD30 ϩ anaplastic large cell lymphoma, 10 Zap-70 in chronic lymphocytic leukemia, 11 annexin were involved in biochemical studies; I.N. and R.M. were involved in the confocal microscopic analysis of cells; R.P., A.T., and E.T. were involved in the immunohistochemical study of AML samples; C.M. was involved in the identification of NPM mutations; R.B., D.D., G.R., R.R., M.A., L.L., S.V., L.B., E.G., A.G., G.S., F.P., F.L.-C., and P.-G.P. were involved in the analysis of NPM gene mutations in the GIMEMA-EORTC trial; and F.M., S.A., G.S., and M.F.M. were involved in organizing the clinical trial.M.P.M. and N.B. contributed equally to this work.Reprints: Brunangelo Falini, Institute of Hematology, Policlinico Monteluce, 06122 Perugia, Italy; e-mail: faliniem@unipg.it.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 U.S.C. section 1734. For personal use only. on April 8, 2019. by guest www.bloodjournal.org From A1 in hairy cell leukemia, 12 myeloid leukemia factor-1 (MLF1) in myelodysplasia/AML with t(3;5), 13 and promyelocytic leukemia (PML) in acute promyelocytic leukemia carrying the t(15;17). 14 In a small group of patients with AML, we showed that aberrant cytoplasmic expression of NPM, a multifunctional 15-20 nucleocytoplasmic shuttling protein 21 with restricted nucleolar localization, 10...
• High-level miR-155 enhances BCR signaling, and is associated with poor prognosis in CLL.• Signals within the CLL microenvironment, such as CD154 or BAFF, can induce miR-155 and enhance BCR signaling.High-level leukemia cell expression of micro-RNA 155 (miR-155) is associated with more aggressive disease in patients with chronic lymphocytic leukemia (CLL), including those cases with a low-level expression of z-chain-associated protein of 70 kD. CLL with highlevel miR-155 expressed lower levels of Src homology-2 domain-containing inositol 5-phosphatase 1 and were more responsive to B-cell receptor (BCR) ligation than CLL with low-level miR-155. Transfection with miR-155 enhanced responsiveness to BCR ligation, whereas transfection with a miR-155 inhibitor had the opposite effect. CLL in lymphoid tissue expressed higher levels of miR155HG than CLL in the blood of the same patient. Also, isolated CD5 bright CXCR4 dim cells, representing CLL that had been newly released from the microenvironment, expressed higher levels of miR-155 and were more responsive to BCR ligation than isolated CD5 dim CXCR4 bright cells of the same patient.Treatment of CLL or normal B cells with CD40-ligand or B-cell-activating factor upregulated miR-155 and enhanced sensitivity to BCR ligation, effects that could be blocked by inhibitors to miR-155. This study demonstrates that the sensitivity to BCR ligation can be enhanced by high-level expression of miR-155, which in turn can be induced by crosstalk within the tissue microenvironment, potentially contributing to its association with adverse clinical outcome in patients with CLL. (Blood. 2014;124(4):546-554)
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