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Genetic Characterization of SF3B1 Mutations in Single Chronic Lymphocytic Leukemia Cells

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Genetic Characterization of SF3B1 Mutations in Single Chronic Lymphocytic Leukemia Cells Letters to the Editor 2264 Taken together, these findings identify a novel drug combina- 2 Oltersdorf T, Elmore SW, Shoemaker AR, Armstrong RC, Augeri DJ, Belli BA et al. tion, which is able to overcome resistance to a BCL2 inhibitor in a An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature clinically relevant CLL model. Importantly, normal lymphocytes 2005; 435: 677–681. and platelets are resistant to the combination of ABT-737 and 3 Tse C, Shoemaker AR, Adickes J, Anderson MG, Chen J, Jin S et al. ABT-263: a gossypol, which offers a therapeutic window to selectively kill CLL potent and orally bioavailable Bcl-2 family inhibitor. Cancer Res 2008; 68: cells. It is therefore plausible that this combination may kill 3421–3428. resistant CLL cells within the stromal niche in vivo, and may 4 Souers AJ, Leverson JD, Boghaert ER, Ackler SL, Catron ND, Chen J et al. ABT-199, a improve clinical outcome. As both the gossypol and navitoclax potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets. Nat Med 2013; 19: 202–208. have been tested in humans, this data support the notion of a 5 Roberts AW, Seymour JF, Brown JR, Wierda WG, Kipps TJ, Khaw SL et al. combination trial in patients with CLL. Both drugs have demon- Substantial susceptibility of chronic lymphocytic leukemia to BCL2 inhibition: 5,10 strated toxicity when administered on a chronic daily basis, but results of a phase I study of Navitoclax in patients with relapsed or refractory the data presented here suggest acute treatment might be disease. J Clin Oncol 2012; 30: 488–496. effective, and thereby avoid the undesirable toxicities. 6 Vogler M, Butterworth M, Majid A, Walewska RJ, Sun X-M, Dyer MJS et al. Concurrent up-regulation of BCL-XL and BCL2A1 induces approximately 1000-fold resistance to ABT-737 in chronic lymphocytic leukemia. Blood 2009; 113: 4403–4413. CONFLICT OF INTEREST 7Ge´linas C, White E. BH3-only proteins in control: specificity regulates MCL-1 and BAK-mediated apoptosis. Genes Dev 2005; 19: 1263–1268. The authors declare no conflict of interest. 8 Zhang L, Lopez H, George NM, Liu X, Pang X, Luo X. Selective involvement of BH3- only proteins and differential targets of Noxa in diverse apoptotic pathways. Cell Death Differ 2011; 18: 864–873. ACKNOWLEDGEMENTS 9 Albershardt TC, Salerni BL, Soderquist RS, Bates DJP, Pletnev AA, Kisselev AF et al. This research was supported by a Translational Research Grant from the Leukemia Multiple BH3 mimetics antagonize antiapoptotic MCL1 protein by inducing the and Lymphoma Society (AE) and a Cancer Center Support Grant to the Norris Cotton endoplasmic reticulum stress response and up-regulating BH3-only protein Cancer Center (NIH CA23108). NOXA. J Biol Chem 2011; 286: 24882–24895. 10 Liu G, Kelly WK, Wilding G, Leopold L, Brill K, Somer B. An open-label, multicenter, phase I/II study of single-agent AT-101 in men with castrate-resistant prostate R Soderquist1,2, DJP Bates1,2, AV Danilov2,3 and A Eastman1,2 1 cancer. Clin Cancer Res 2009; 15: 3172–3176. Department of Pharmacology and Toxicology, 11 Chen R, Guo L, Chen Y, Jiang Y, Wierda WG, Plunkett W. Homoharringtonine Giesel School of Medicine at Dartmouth, One Medical Center Drive, reduced Mcl-1 expression and induced apoptosis in chronic lymphocytic leuke- Lebanon, NH, USA; mia. Blood 2011; 117: 156–164. 2Norris Cotton Cancer Center, Giesel School of Medicine at 12 Neron S, Pelletier A, Chevrier M-C, Monier G, Lemieux R, Darveau A. Induction of Dartmouth, One Medical Center Drive, Lebanon, NH, USA and LFA-1 independent human B cell proliferation and differentiation by binding of 3Department of Medicine, Giesel School of Medicine at Dartmouth, CD40 with its ligand. Immunol Invest 1996; 25: 79–89. One Medical Center Drive, Lebanon, NH, USA 13 Me´rino D, Khaw SL, Glaser SP, Anderson DJ, Belmont LD, Wong C et al. Bcl-2, E-mail: [email protected] Bcl-xL, and Bcl-w are not equivalent targets of ABT-737 and navitoclax (ABT-263) in lymphoid and leukemic cells. Blood 2012; 119: 5807–5816. 14 Meng Y, Tang W, Dai Y, Wu X, Liu M, Ji Q et al. Natural BH3 mimetic (-)-gossypol chemosensitizes human prostate cancer via Bcl-xL inhibition REFERENCES accompanied by increase of Puma and Noxa. Mol Cancer Ther 2008; 7: 2192–2202. 1 Kitada S, Andersen J, Akar S, Zapata JM, Takayama S, Krajewski S et al. Expression 15 Tromp JM, Geest CR, Breij ECW, Elias JA, van Laar J, Luijks DM et al. Tipping of apoptosis-regulating proteins in chronic lymphocytic leukemia: correlations the Noxa/Mcl-1 balance overcomes ABT-737 resistance in chronic lymphocytic with in vitro and in vivo chemoresponses. Blood 1998; 91: 3379–3389. leukemia. Clin Cancer Res 2012; 18: 487–498. Genetic characterization of SF3B1 mutations in single chronic lymphocytic leukemia cells Leukemia (2013) 27, 2264–2267; doi:10.1038/leu.2013.155 gene has a dominant role in clonal evolution. We also provide evidence that SF3B1 mutations are potentially oncogenic, supporting the possibility that mutant SF3B1 is an attractive druggable therapeutic target. Like other cancers, chronic lymphocytic leukemia (CLL) is initiated Wang et al.1 reported that SF3B1 mutations are more prevalent and/or progresses as a consequence of concurrent chromosomal in CLL patients with 11q deletion. We randomly picked 73 abnormalities and recurrent somatic mutations. Using next- cryopreserved peripheral blood mononuclear cell (PBMC) samples generation sequencing, a short list of recurrent mutations in with 11q deletion from our CLL patient cohort, and screened various genes has been identified in CLL.1,2 One of these genes is for SF3B1 mutations (Supplemental Methods). We identified eight SF3B1 that encodes a key component of the mRNA splicing patients with various missense mutations including five patients machinery. SF3B1 mutations were initially discovered in with K700E (2098A4G), one with K649E (1945A4G), one with myelodysplastic syndromes (MDSs) and in some other solid K622E (1866G4T) and one with K666E (1996A4G). These tumors, suggesting it is having an important role in cancer mutations have been observed by others in CLL, MDS and other biology. In CLL, SF3B1 mutations are associated with disease cancers.1,2,7 We also found that SF3B1 mutations are only present subtype,1 progression,3,4 chemotherapy resistance4,5 and overall in a sub-allelic-fraction (ranging from 10 to 45%) of bulk DNA patient survival.6 However, the biological consequences of SF3B1 samples (Figure 1a). mutations in CLL pathogenesis are largely unknown. Here, we find Cancer progression is typically characterized by the emer- that acquisition of mutations in SF3B1 eventually leads to the loss gence and outgrowth of newly evolved subclones. By analyzing of the wild-type copy of this gene, suggesting the mutant SF3B1 the allelic burden of SF3B1 mutations in CLL using the Sanger Accepted article preview online 20 May 2013; advance online publication, 11 June 2013 Leukemia (2013) 2242 – 2267 & 2013 Macmillan Publishers Limited Letters to the Editor 2265 a G/T A/G A/G A/G Pat-24: 1866G>T Pat-35: 2098A>G Pat-38: 2098A>G Pat-59: 1996A>G b Semi-nested PCR Initial PCR (on DNA/RNA) Exon14 Exon 15 CD5-APC K700 E622 K666 Limit dilution (0.2 cells/well) CD19-FITC c A A/G G DNA Single cells Pat-35 Wt only Mu/Wt Mu only A A/G G RNA d Figure 1. SF3B1 genotyping in bulk and in single CLL cells. (a) Sanger DNA sequencing chromatograms of SF3B1 sequences amplified from four representative CLL patient samples. Purple- and yellow-color-filled peaks show allelic burdens of mutant and wild-type nucleotide sequences, respectively. (b) Schematic flowchart of single CLL cell preparation for SF3B1 mutation analysis. CD19 þ /CD5 þ leukemic cells are sorted from patient PBMCs, and plated by limiting dilution in 96-well PCR plates for two rounds of PCR amplification for SF3B1 sequences. (c) Representative chromatograms of the Sanger DNA sequencing of SF3B1 sequences amplified from single CLL cells from patient 35. Purple- and yellow-color-filled peaks are mutant and wild-type nucleotide sequences, respectively. (d) Summary table of all four patient samples analyzed using DNA or RNA at the single-cell level using a limiting dilution approach. sequencing in serial patient samples, Schwaederle et al.3 PCR (Figure 1b). As expected, many single cells exhibited showed that the weight of mutant SF3B1 increasesasthe either wild-type only (wt/wt), or wild-type plus mutant SF3B1 disease progresses. However, the size of SF3B1 DNA allelic sequences (heterozygous, wt/mu). To our surprise owing to fractions does not necessarily reflect the size of the subclone, previous predictions, in all four CLL samples we detected as it remains unknown whether the observed mutant SF3B1 multiple single cells possessing solely SF3B1 mutant sequences allelic increase at the bulk cell population level reflects a resembling ‘homozygous’ genotypes (mu/mu-like). This observa- change in size of the mutant SF3B1 subclone or instead whether tion suggests that a prominent CLL subclone in these patients there is a change in zygosity of SF3B1 mutations of the subclone. exclusively carries mutant SF3B1. Figures 1c and d depict In fact, it has been postulated that SF3B1 mutations are representative Sanger sequencing data of single cells from patient heterozygous in MDS and CLL7–9 largely based on the 35 and the summary of all four patients, respectively. Similar data observation that allelic burdens of mutant SF3B1 are typically were also obtained when single cells were collected directly into o50%. To ascertain the zygosity of SF3B1 mutations in CLL, we the 96-well PCR plates using fluorescence-activated cell sorting analyzed SF3B1 mutations at the single-cell level by DNA-based (results not shown).
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