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Nguyen, Nhu, Kristbjorn Orri Gudmundsson, Anthony R. Soltis, Kevin Oakley, Yufen Han, Jaroslaw P. Maciejewski, Patricia Ernst, Clifton L. Dalgard, and Yang Du. "Recruitment of MLL1 Complex Is Essential for SETBP1 to Induce Myeloid Transformation." Blood 138, Supplement 1 (November 5, 2021): 1147. http://dx.doi.org/10.1182/blood-2021-152825.
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Abstract Abnormal activation of SETBP1 due to overexpression or missense mutations occurs frequently in various myeloid neoplasms and associates with poor prognosis. Direct activation of Hoxa9/Hoxa10/Myb transcription by SETBP1 and its missense mutants is essential for their transforming capability; however, the underlying mechanisms for such activation remain elusive. We found that knockdown of Mll1 in mouse myeloid progenitors immortalized by SETBP1 or its missense mutant SETBP1(D/N) caused significant reduction in the mRNA levels of Hoxa9/Hoxa10/Myb, suggesting that Mll1 is critical for their transcriptional activation induced by SETBP1 and its missense mutants. Physical association of MLL1 with SETBP1/SETBP1(D/N) was readily detected by co-immunoprecipitation in nuclear extracts of these cells, further suggesting that they may form a complex in myeloid cells to activate transcription. This complex formation is likely mediated by direct interactions between SETBP1/SETBP1(D/N) and MLL1 as both SETBP1 and SETBP1(D/N) are capable of interacting with multiple regions of MLL1 in binding assays using proteins synthesized by in vitro transcription and translation. To better understand the extent of SETBP1/SETBP1(D/N)-MLL1 interaction in regulating gene transcription, we carried out both ChIP-seq and RNA-seq analysis in mouse Lin -Sca-1 +c-Kit + (LSK) cells transduced by pMYs retrovirus expressing SETBP1 or SETBP1(D/N) or empty pMYs virus. These analyses revealed extensive overlap in genomic occupancy for MLL1 and SETBP1/SETBP1(D/N) and their cooperation in activating many oncogenic transcription factor genes in addition to Hoxa9/Hoxa10/Myb, including additional HoxA genes (Hoxa1, Hoxa3, Hoxa5, Hoxa6, and Hoxa7), Myc, Eya1, Mef2c, Meis1, Sox4, Mecom, and Lmo2. A large group of ribosomal protein genes were also found to be directly activated by MLL1 and SETBP1/SETBP1(D/N), identifying ribosomal biogenesis as another significant pathway induced by their cooperation. To further assess the requirement for MLL1 in SETBP1-induced transformation using a genetic approach, we also generated SETBP1/SETBP1(D/N)-induced immortalized myeloid progenitors and AMLs using LSK cells from Mll1 conditional knockout mice. Mll1 deletion in immortalized progenitors significantly decreased SETBP1/SETBP1(D/N)-induced transcriptional activation and their colony-forming potential. More importantly, Mll1 deletion significantly extended the survival of mice transplanted with SETBP1/SETBP1(D/N)-induced AMLs, indicating that Mll1 is essential for the maintenance of such leukemias in vivo. We further found that pharmacological inhibition of MLL1 complex using a WDR5 inhibitor OICR-9429 efficiently abrogated SETBP1/SETBP1(D/N)-induced transcriptional activation and transformation. Thus, MLL1 complex plays a critical role in Setbp1-induced transcriptional activation and transformation and represents a promising target for treating myeloid neoplasms with SETBP1 activation. Disclosures Maciejewski: Novartis: Consultancy; Regeneron: Consultancy; Alexion: Consultancy; Bristol Myers Squibb/Celgene: Consultancy.
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Pacharne, Suruchi, Oliver M. Dovey, Jonathan L. Cooper, Muxin Gu, Mathias J. Friedrich, Sandeep S. Rajan, Maxim Barenboim, et al. "SETBP1 overexpression acts in the place of class-defining mutations to drive FLT3-ITD–mutant AML." Blood Advances 5, no. 9 (May 6, 2021): 2412–25. http://dx.doi.org/10.1182/bloodadvances.2020003443.
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Abstract Advances in cancer genomics have revealed genomic classes of acute myeloid leukemia (AML) characterized by class-defining mutations, such as chimeric fusion genes or in genes such as NPM1, MLL, and CEBPA. These class-defining mutations frequently synergize with internal tandem duplications in FLT3 (FLT3-ITDs) to drive leukemogenesis. However, ∼20% of FLT3-ITD–positive AMLs bare no class-defining mutations, and mechanisms of leukemic transformation in these cases are unknown. To identify pathways that drive FLT3-ITD mutant AML in the absence of class-defining mutations, we performed an insertional mutagenesis (IM) screening in Flt3-ITD mice, using Sleeping Beauty transposons. All mice developed acute leukemia (predominantly AML) after a median of 73 days. Analysis of transposon insertions in 38 samples from Flt3-ITD/IM leukemic mice identified recurrent integrations at 22 loci, including Setbp1 (20/38), Ets1 (11/38), Ash1l (8/38), Notch1 (8/38), Erg (7/38), and Runx1 (5/38). Insertions at Setbp1 led exclusively to AML and activated a transcriptional program similar, but not identical, to those of NPM1-mutant and MLL-rearranged AMLs. Guide RNA targeting of Setbp1 was highly detrimental to Flt3ITD/+/Setbp1IM+, but not to Flt3ITD/+/Npm1cA/+, AMLs. Also, analysis of RNA-sequencing data from hundreds of human AMLs revealed that SETBP1 expression is significantly higher in FLT3-ITD AMLs lacking class-defining mutations. These findings propose that SETBP1 overexpression collaborates with FLT3-ITD to drive a subtype of human AML. To identify genetic vulnerabilities of these AMLs, we performed genome-wide CRISPR-Cas9 screening in Flt3ITD/+/Setbp1IM+ AMLs and identified potential therapeutic targets, including Kdm1a, Brd3, Ezh2, and Hmgcr. Our study gives new insights into epigenetic pathways that can drive AMLs lacking class-defining mutations and proposes therapeutic approaches against such cases.
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Kawashima, Nozomu, Yusuke Okuno, Yuko Sekiya, Xinan Wang, Yinyan Xu, Atsushi Narita, Sayoko Doisaki, et al. "Generation of Cell Lines Harboring SETBP1 Mutations By the Crispr/Cas9 System." Blood 124, no. 21 (December 6, 2014): 4622. http://dx.doi.org/10.1182/blood.v124.21.4622.4622.
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Abstract Introduction Recent advances in cancer genetics have led to the identification of somatic mutations in SET-binding protein 1 (SETBP1) in myeloid malignancies categorized as myeloproliferative neoplasm (MPN) and myelodysplastic syndromes (MDS). Heterozygous point mutations in SETBP1 are essentially found at a genomic level in myeloid malignancies, and the frequency of the mutated allele in cDNA suggests somatic heterozygosity without substantial imbalance in allelic expression. Thus, mutant SETBP1 presumably has a dominant altered biological activity. Most mutations in SETBP1 are located in the SKI homologous region. This region is suggested to include regions critical for ubiquitin binding and SETBP1 degradation. SETBP1 binds to SET, which protects against protease cleavage, and thus may result in PP2A inhibition and cell proliferation. Overexpression of SETBP1 resulting from a p.G870S alteration showed higher levels of the protein compared with wild-type (WT), indicating a prolonged halftime of SETBP1, which led to reduced PP2A activity and a higher cell proliferation rate. To date, however, our molecular biological understanding of SETBP1 mutations has been obtained only through observations of exogenous overexpression in cell lines. This may result in bias, considering the predicted dominant-negative function of SETBP1 mutations. Therefore, we used an RNA-guided endonuclease (RGEN), the CRISPR/Cas9 system, to generate a cell line harboring point mutations resulting in only relevant single amino acid substitutions in SETBP1. We analyzed cell signaling using the cell line thus established. Methods pSpCas9(BB) (PX330) was used to express humanized S. pyogenes Cas9 and gRNAs of interest. The gRNAs were designed by searching for NGG protospacer adjacent motif (PAM) sequences near the point mutation target sites. The candidate gRNAs were gRNA#1, 5′-TAGGGAGCCAATCTCGCAC-3′; gRNA#2, 5′-TGTCCCAATGCCGCTGTCGC-3′; gRNA#4, 5′-GTCCCAATGCCGCTGTCGCT-3′; and gRNA#7, 5′-GAGACGATCCCCAGCGACAG-3′. pCAG-EGxxFP harboring the 500 bp target region of WT SETBP1 was constructed for gRNA selection. For homology-dependent repair (HDR), we synthesized 70 mer single-stranded oligonucleotides (ssODN) having both the SETBP1 c.2602G>A, p.D868N mutation and synonymous mutation in the PAM. HEK293T cells were cultured in DMEM with 10% FBS. For cell signaling analysis, the cells were serum-depleted for 16 h prior to western blotting. Anti-SETBP1 antibody (ab98222), anti-phospho-Y307 PP2A antibody (E155), and anti phospho-p44/42 MAPK antibody (CST#4370) were used for cell signaling analysis. Results To validate an efficient sgRNA for DNA scission, we cotransfected pCAG-EGxxFP-SETBP1 and pSpCas9(BB)-SETBP1-gRNA plasmids into HEK293T cells. EGFP fluorescence, whose intensity is correlated with the efficacy of HDR, was observed 48 h later, and we determined that gRNA#2 was the most efficient. Next we cotransfected 293T cells with pCAG-EGxxFP-SETBP1, pSpCas9(BB)-SETBP1-gRNA#2, and ssODN for mutagenesis. Five days after transfection, single EGFP-positive clones were isolated using the FACSAria cell sorting system. Sanger sequencing confirmed that 293T cells harboring the SETBP1 p.D868N homozygous mutation were established. A clone with WT SETBP1 was also maintained as a control. To elucidate the effects of the SETBP1 mutation in 293T cells, we performed cell signaling analysis by western blotting. 293T-SETBP1 p.D868N cells showed higher levels of SETBP1 protein with lower molecular weight compared with WT, indicating a prolonged halftime, possibly due to loss of ubiquitination. In addition, 293T-SETBP1 p.D868N cells showed a higher phosphorylation level of PP2A (Y307, C subunit), a marker of PP2A inactivation. Finally, the phosphorylation level of p44/42 MAPK (ERK1/2) was increased in 293T-SETBP1 p.D868N cells. Conclusions We confirmed that the SETBP1 p.D868N mutation caused a prolonged halftime, resulting in PP2A inactivation and p44/42 MAPK activation in 293T cell lines. Our data suggest a potential therapy target for malignancies harboring SETBP1 mutations. More generally, this work illustrates the utility of RGEN technology for studying hematological malignancies. Disclosures No relevant conflicts of interest to declare.
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Pacharne, Suruchi, Oliver M. Dovey, Jonathan L. Cooper, Muxin Gu, Vijay Baskar, Mathias J. Friedrich, Malgorzata Gozdecka, et al. "Setbp1 Overexpression Acts in the Place of Class-Defining Somatic Mutations to Drive Mouse and Human FLT3-ITD-Mutant AMLs." Blood 136, Supplement 1 (November 5, 2020): 31–32. http://dx.doi.org/10.1182/blood-2020-141743.
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Setbp1 overexpression acts in the place of class-defining somatic mutations to drive mouse and human FLT3-ITD-mutant AMLs Suruchi Pacharne,1,2 Oliver M. Dovey,1 Jonathan L. Cooper,1 Muxin Gu,1,2 MS Vijaybaskar,1,2 Mathias J. Friedrich,1,5 Malgorzata Gozdecka,1,2 Sandeep S. Rajan,1,4, Etienne De Braekeleer,1,2 Maxim Barenboim,5,6 Grace Collord,1,2 Hannes Ponstingl,1 Ruben Bautista,1 Milena Mazan,1,8 Roland Rad,5,6 Konstantinos Tzelepis,1,7 Penny Wright,3 and George S. Vassiliou1,2,9* Abstract Internal tandem duplications in FLT3 (FLT3-ITD) are found in 30% of acute myeloid leukemia (AML) cases and impart a poor prognosis. FLT3-ITD commonly synergizes with class-defining mutations such as chimeric fusion genes or mutations in NPM1, RUNX1, CEBPA or MLL to drive AML. However, 20% of FLT3-ITD-mutant AMLs bare no class-defining mutations and the mechanisms of acute leukemic transformation in these cases are unknown. To identify pathways that can drive FLT3-ITD-mutant AML in the absence of class-defining mutations, we performed an insertional mutagenesis (IM) screen in Flt3-ITD mice using the Sleeping Beauty transposon system, activated by the Mx1-Cre recombinase in hematopoietic stem cells. All mice developed acute leukemia, predominantly AML, after a median latency of 73 days (Figure A). Analysis of transposon insertions in 38 Flt3-ITD/IM leukemias identified common integration sites (CISs) in 22 loci (Figure B). The most commonly "hit" genes were Setbp1 (20/38), Ets1 (11/38), Ash1l (8/38), Notch1 (8/38), Erg (7/38), Flt3 (6/38) and Runx1 (5/38) (Figure B). Of these, Setbp1 and Runx1 were unique to Flt3-ITD and not identified as CISs in insertional mutagenesis screens of wild type, Npm1c or BCR-ABL-expressing mice. Transposon insertions in Setbp1, primarily located upstream of its first coding exon, were associated with Setbp1 and Hoxa mRNA overexpression and were invariably associated with AML development (Figure B). These findings propose that overexpression of wild type SETBP1 may collaborate with FLT3-ITD to drive leukemogenesis in human AMLs lacking mutations known to collaborate with mutant FLT3. Corroborating this, we found that SETBP1 expression was higher in human FLT3-ITD-mutant AMLs lacking class-defining mutations and in those with RUNX1 mutations (Figure C). We go on to show that Setbp1 insertions activate a Hoxa gene signature such that shares significant similarities, but also specific differences to those driven by mutant Npm1 and MLL fusion genes. We go on to show, using CRISPR-gRNA, that whilst Flt3ITD/+/SETBP1IM+AMLs are entirely dependent on Setbp1 expression, Flt3ITD/+/Npm1cA/+AMLs are not, but do depend on the expression of the homebox gene Nkx2.3. Our findings propose that SETBP1 overexpression activates a gene expression pattern that collaborates with FLT3-ITD to drive many human AMLs and that this combination represents a specific subtype of AML amongst AMLs lacking class-defining mutations. To identify genetic vulnerabilities of this AML subtype, we performed genome-wide CRISPR-Cas9 recessive screens in primary murine Flt3ITD/+SETBP1IM+AMLs and identified more than 2000 genetic vulnerabilities, of which 677 were not required for the survival of HPC7 non-leukemic hematopoietic cells including >100 "druggable" genes such as Brd3, Ezh2 and Hmgcr (Figure D). Collectively our study: i) identifies SETBP1overexpression as a non-genetic alteration driving a subgroup of FLT3-ITD mutant AMLs lacking class-defining somatic mutations and ii) goes on to define the genetic vulnerabilities of such AMLs as a starting point for the development of targeted therapies. Figure Disclosures Vassiliou: Kymab Ltd - Monoclonal antibody company. Currently not working in myeloid cancers or clonal haematopoiesis.: Consultancy.
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Oakley, Kevin, Yufen Han, Bandana A. Vishwakarma, Su Chu, Ravi Bhatia, Kristbjorn O. Gudmundsson, Jonathan Keller, et al. "Setbp1 promotes the self-renewal of murine myeloid progenitors via activation of Hoxa9 and Hoxa10." Blood 119, no. 25 (June 21, 2012): 6099–108. http://dx.doi.org/10.1182/blood-2011-10-388710.
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Abstract Acquisition of self-renewal capability by myeloid progenitors to become leukemic stem cells during myeloid leukemia development is poorly understood. Here, we show that Setbp1 overexpression efficiently confers self-renewal capability to myeloid progenitors in vitro, causing their immortalization in the presence of stem cell factor and IL-3. Self-renewal after immortalization requires continuous Setbp1 expression. We also found that Hoxa9 and Hoxa10 mRNA are present at dramatically higher levels in Setbp1-immortalized cells compared with other immortalized cells, and are induced shortly after Setbp1 expression in primary myeloid progenitors. Suppression of either gene in Setbp1-immortalized cells drastically reduces their colony-forming capability. Interestingly, Setbp1 protein associates with Hoxa9 and Hoxa10 promoters in chromatin immunoprecipitation assays in these cells, suggesting that both are direct transcriptional targets of Setbp1. Setbp1 also promotes self-renewal of myeloid progenitors in vivo as its coexpression with BCR/ABL transforms primary mouse myeloid progenitors, generating aggressive leukemias in recipient mice resembling chronic myelogenous leukemia (CML) myeloid blast crisis. Increased SETBP1 mRNA levels were also detected in a subset of CML advanced phase/blast crisis patients with high levels of HOXA9 and HOXA10 expression. Thus, Setbp1 activation represents a novel mechanism conferring self-renewal capability to myeloid progenitors in myeloid leukemia development.
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Makishima, Hideki, Kenichi Yoshida, Nhu Nguyen, Masashi Sanada, Yusuke Okuno, Kwok Peng Ng, Bartlomiej P. Przychodzen, et al. "Somatic Mutations in Schinzel-Giedion Syndrome Gene SETBP1 Determine Progression in Myeloid Malignancies." Blood 120, no. 21 (November 16, 2012): 2. http://dx.doi.org/10.1182/blood.v120.21.2.2.
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Abstract Abstract 2 MDS and other chronic myeloid malignancies such as MDS/MPN are characterized by a frequent progression to secondary AML (sAML), a likely multistep process of acquisition of genetic abnormalities. Genes involved in congenital genetic cancer susceptibility syndromes are often targets of somatic mutations in various tumors. For instance, germ-line mutations of SETBP1 are associated with Schinzel-Giedion syndrome (SGS), which is characterized by skeletal malformations, mental retardation and frequent neuroepithelial tumors. While SETBP1 overexpression in myeloid malignancies links to poor prognosis, somatic mutations of SETBP1 were not previously identified in leukemias. When we performed whole exome sequencing of 20 cases with myeloid malignancies, in addition to detecting previously described lesions, such as TET2, CBL and ASXL1, we identified a somatic SETBP1 mutation (D868N) in 2 cases with RAEB. Analysis of DNA from CD3+ cells from these patients confirmed its somatic nature. Sanger sequencing was applied to all coding exons in an additional 48 cases, leading to detection of 2 additional somatic mutations (G870S and I871T) in 2 patients with CMML and sAML, respectively. These findings prompted us to further expand our screening cohort: targeted SETBP1 sequencing was performed in a total of 734 patients (283 with MDS, 106 with sAML, 167 with MDS/MPN, 138 MPN and 146 with primary AML): 52 mutations were detected in 52 patients (7.1%); D868N, G870S and I871T alterations were more frequently observed (N=27, N=16 and N=5, respectively), while D868Y, S869N, D880E and D880N were less prevalent. These mutations, of which 92% (48 out of 52) were identical to those in the SGS germ line, were detected in 15% with CMML (24/156), 15% with sAML (16/106) and 7% CML blast phase (2/28). Clinically, mutant cases were associated with higher age (p=.014), deletion of chromosome 7q (p=.0005) and shorter median survival (28 vs. 13 months, p<.0001). As shown in the analysis of 11 paired samples of progressing MDS patients, all SETBP1 mutations were acquired during leukemic evolution. In addition to mutations, SETBP1 overexpression can be found in 12% and 26% of cases of MDS and sAML, respectively, a finding linking higher activity of SETBP1 to leukemic progression. To directly test whether SETBP1 mutations represent gain-of-function, we performed retroviral transduction of murine Setbp1 engineered with two of the somatic mutations, D868N and I871T, and evaluated the ability of the mutants to immortalize normal murine myeloid progenitors. With a low viral titer of 1 x105 cfu, both Setbp1 mutants caused efficient immortalization of myeloid progenitors, similar to overexpressed WT Setbp1. In addition, cells immortalized with mutant Setbp1 proliferated faster than cells with WT Setbp1. These data suggest that mutations of SETBP1 in our study represent gain-of-function in leukemias. The in vitro immortalization effect of overexpressed WT Setbp1 was associated with and dependent on Hoxa9 and Hoxa10 overexpression. We performed quantitative RT-PCR and western blot experiments to evaluate expression of these genes in our mutant cases. Relative HOXA9 and HOXA10 mRNA expression values were higher in all mutant cases (N=7) than median of those in WT cases (N=4). Also, both HOXA9 and HOXA10 proteins were detected in all cases with SETBP1 mutations, suggesting that HOXA9 and HOXA10 induction is consistently associated with SETBP1 mutations similar to observations in forced expression of WT Setbp1. Moreover, in agreement with findings in primary cells showing that SETBP1 mutations or high SETBP1 expression share a common genetic association with RUNX1 mutations, Runx1 expression was reduced after in vitro immortalization of normal bone marrow cells by forced Setbp1 overexpression and two Runx1 promoter sequences were amplified after ChIP performed with antibody specific for exogenous Setbp1 protein. Moreover, Setbp1 shRNA knockdown resulted in enhanced Runx1 transcription consistent with the negative regulation of this gene by Setbp1. These results indicate that SETBP1 is associated with decreased activity of RUNX1 due to hypomorphic mutations or by direct down-modulation WT RUNX1 expression bypassing the need for mutations. In sum, somatic recurrent SETBP1 mutations are lead to gain of function and are associated with molecular pathogenesis of myeloid leukemic transformation of various primary myeloid subentities. Disclosures: Makishima: Scott Hamilton CARES Initiative: Research Funding. Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.
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Carratt, Sarah A., Zachary Schonrock, Theodore Braun, Cody Coblentz, Amy Foley, and Julia E. Maxson. "SETBP1 Mutations Accelerate NRAS-Mutant Leukemia." Blood 134, Supplement_1 (November 13, 2019): 1254. http://dx.doi.org/10.1182/blood-2019-125125.
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Juvenile myelomonocytic leukemia (JMML) is an aggressive, rare form of early childhood leukemia driven by Ras pathway mutations. Mutations in SET binding protein 1 (SETBP1) are a strong predictor of relapse in JMML, and are associated with reduced five-year event-free survival. Although some mechanisms of oncogenesis have been established for SETBP1 mutations, it remains unclear why they are associated with poor prognosis and relapse. The goal of this study was to understand how SETBP1 modulates the biology of Ras-driven leukemias and to determine whether there are therapeutic vulnerabilities of SETBP1-JMML that can be exploited. Here, we present novel findings on the synergy of SETBP1 and NRAS, and provide pre-clinical evidence for therapeutic intervention. To address our central question of how SETBP1 mutations modulate Ras pathway-driven leukemia, we first set out to determine whether mutant SETBP1 promotes the growth of hematopoietic progenitors in the context of a Ras pathway mutation. To this end, we performed mouse hematopoietic colony forming unit assays in the absence of exogenous cytokines. Both NRAS-G12D and PTPN11-E76K alone formed a modest number of colonies, and the addition of SETBP1-D868N significantly augmented colony number with either Ras pathway mutation. The combination of NRAS-G12D and SETBP1-D868Nconfer robust serial replating, indicating that the SETBP1-D868N promotes oncogenic transformation and progenitor self-renewal in the NRAS-G12D mutant cells. To understand how SETBP1 modulates therapeutic response, a novel NRAS/SETBP1-mutant cell line was generated and analyzed with a chemical screen of essential cell growth and survival pathways. This screen revealed dependencies on the mTOR/AKT/PI3K and Raf/MEK/ERK pathways. An immunoblot analysis revealed that mutant SETBP1 enhanced NRAS-driven ERK and mTOR pathway activation. Inhibitors of these pathways, such as rapamycin and trametinib were highly efficacious against our cell line. The combination of trametinib and rapamycin had sub-nanomolar efficacy in our NRAS/SETBP1-hematopoietic cell line and exhibited greater than bliss additivity at several time points. To evaluate the efficacy in vivo, our SETBP1/NRAS-mutant cell line was injected into mice. At the onset of disease, mice were given once-daily treatment of trametinib, rapamycin, combination treatment, or DMSO control. The median survival of mice receiving DMSO was 19.5-days post-transplant, compared to 21 days for rapamycin, 35 days for the combination treatment, and 42 days for trametinib. Treatment with trametinib significantly increased the median survival to beyond rapamycin or DMSO, doubling the survival time of the mice. Our data demonstrates that SETBP1 mutations accelerate NRAS-driven oncogenesis and enhance MAPK pathway activation by NRAS-G12D. Despite enhanced transforming potential, SETBP1-mutant cells are still sensitive to inhibitors of the RAS/ERK/MAPK pathway. Trametinib, an inhibitor of this pathway, doubles overall survival in our murine model of NRAS/SETBP1-mutant leukemia, thus providing encouraging pre-clinical data for the use of trametinib in SETBP1-mutant disease. Disclosures No relevant conflicts of interest to declare.
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Carratt, Sarah A., Theodore P. Braun, Zachary Schonrock, Brittany M. Smith, Daniel J. Coleman, Garth Kong, Joseph Estabrook, Adrian Baris, Lauren Maloney, and Julia E. Maxson. "Oncogenic SETBP1 Mutations Combine with Activating Mutations in CSF3R to Produce a Highly Proliferative, Lethal Leukemia through Aberrant Myc Signaling." Blood 136, Supplement 1 (November 5, 2020): 51–52. http://dx.doi.org/10.1182/blood-2020-143072.
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SETBP1 (SET Binding Protein 1) mutations are associated with exceptionally poor prognosis in myeloid neoplasms. Despite this, SETBP1's role in oncogenesis remains incompletely understood. In this study, we find that SETBP1 leads to a marked upregulation of the Myc oncogene and associated pro-stem and progenitor programs through epigenetic dysregulation. We further identify an epigenetic modulatory drug that normalizes SETBP1-driven Myc overexpression and synergizes with disease-relevant therapy. SETBP1 has documented roles in both the regulation of tumor suppressor pathways and modulation of transcription. To understand how SETBP1 modulates leukemia biology and leverage this mechanistic insight to develop novel therapeutic strategies, we turned to a genetically well-defined model system, chronic neutrophilic leukemia (CNL). SETBP1 is mutated in approximately half of all cases of CNL, a myeloproliferative neoplasm characterized by the presence of signaling-activating mutations in Colony Stimulating Factor 3 Receptor (CSF3R). By expressing SETBP1 and CSF3R mutations in mouse hematopoietic progenitors, we have generated models of CNL that can be leveraged for mechanistic studies and drug development. In a hematopoietic colony forming unit (CFU) assay, murine hematopoietic progenitors co-expressing mutant SETBP1 and mutant CSF3R have a high proliferation phenotype compared to those with mutant CSF3R alone. When cells co-expressing mutant SETBP1 and CSF3R are transplanted into lethally irradiated mice, they develop a rapidly lethal disease relative to the control mice. This is associated with an expansion of the granulocyte lineage, quantified by complete blood count, flow cytometry and histology. We find that SETBP1 is essential for the induction of a pro-proliferative transcriptional program. One of the most prominent SETBP1-associated signatures is that of Myc target genes. Myc itself is one of the top differentially expressed genes driven by SETBP1. Congruent with its increased expression, we also find higher Myc transcriptional activity in cells overexpressing SETBP1. To better understand the epigenetic regulation of Myc and progenitor pathways by SETBP1, we employed a low input profiling methods called CUT&Tag to assess the activation of regulatory elements. We began by profiling two epigenetic marks associated with active enhancers-H3K4me1 and H3K27Ac. We also assessed activation of the Myc promoter by measuring H3K27Ac and H3K4me3. Together this data helps us to understand the epigenetic underpinnings for dysregulation of Myc-driven programs by SETBP1. Therapeutic strategies that normalize aberrant Myc activity may be effective against SETBP1-driven disease. We found that SETBP1 and CSF3R transformed hematopoietic progenitors are highly sensitive to inhibitors of the epigenetic regulator lysine specific demethylase 1 (LSD1). LSD1 inhibition restores Myc expression to physiological levels and represses Myc promotor activity in vitro. Furthermore, LSD1 inhibitors are highly synergistic with JAK inhibitors, which block signaling downstream of CSF3R. Together these data establish the importance of Myc activation in SETBP1-driven malignancies and identify a therapeutic approach to normalize aberrant Myc activity. In future, we will use the insight gained in this genetically well defined disease to inform studies on the mechanistic basis and therapeutic vulnerabilities of other SETBP1-mutant myeloid malignancies. Disclosures Maxson: Gilead Sciences: Research Funding; Ionis Pharmaceuticals: Other: Joint oversight committee for a collaboration between OHSU and Ionis Pharmaceuticals.
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Wakamatsu, Manabu, Hideki Muramatsu, Norihiro Murakami, Yusuke Okuno, Hironobu Kitazawa, Seiji Kojima, and Yoshiyuki Takahashi. "Detection of Subclonal SETBP1 and JAK3 Mutations in Patients with Juvenile Myelomonocytic Leukemia Using Droplet Digital PCR." Blood 134, Supplement_1 (November 13, 2019): 4213. http://dx.doi.org/10.1182/blood-2019-125354.
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Background Juvenile myelomonocytic leukemia (JMML) is a rare pediatric myelodysplastic/myeloproliferative disease. Approximately 85% of patients with JMML harbor germline and/or somatic mutations in RAS pathway genes, such as PTPN11, NF1, CBL, NRAS, and KRAS. In a subset of patients with JMML, SETBP1 and JAK3 mutations were identified as secondary mutations in addition to primary RAS mutations. These secondary mutations are associated with the disease progression and poor clinical outcome. Recently, it has been reported that subclonal SETBP1 mutation also correlates with a dismal prognosis. Therefore, we hypothesized that subclonal JAK3 mutation is present in a higher than expected number of patients with JMML and associated with poor prognosis. The aim of this study is to identify patients with subclonal SETBP1 and/or JAK3 mutations at the diagnosis using droplet digital PCR (ddPCR) and to elucidate their clinical outcomes. Patients and Methods We enrolled 128 patients with JMML and 15 with Noonan syndrome-associated myeloproliferative disorder (NS/MPD). Using bone marrow (BM) or peripheral blood derived genomic DNA, ddPCR was performed in the 143 patients to detect SETBP1 p.D868N and JAK3 p.R657Q hotspot mutations with low variant allele frequencies (VAF). The study was approved by the institutional review board of Nagoya University Graduate School of Medicine. Results To assess the false-positive rate of the ddPCR assay for each mutation, the assay was also performed in 30 healthy volunteers. Among these, the false-positive rate (mean ± standard deviation) for SETBP1 and JAK3 mutations was 0.010% ± 0.010% and 0.013% ± 0.012%, respectively. Due to the presence of false-positive droplets, the sensitivity and the quantitative linearity was evaluated for >0.01% VAF. The significant correlation between the expected and the observed VAF in SETBP1 and JAK3 was observed (R-squared, 0.9923 and 0.9922, respectively). Therefore, 0.05% VAF was defined as the cut-off value in this assay. Among the 143 patients, ddPCR detected SETBP1 and JAK3 mutations in nine (6.3%) and fifteen (10.5%), respectively. SETBP1 and JAK3 mutations, including variants with low allele frequencies, were not detected in NS/MPD. Among patients with SETBP1 and/or JAK3 mutations, two and six patients harbored less than 1.0% VAF. Patients with less than 1.0% VAF in SETBP1 or JAK3 mutation exhibited a significantly poorer 2-year transplantation-free survival than those without SETBP1 and JAK3 mutations (P = 3.05 × 10-3). JMML is genetically characterized by an extremely small number of somatic mutations (an average of 0.8 mutations/exome/patient). However, we demonstrated that among 19 patients with SETBP1 and/or JAK3 mutations, five patients (26.3%) harbored both the mutations. This finding suggested a statistically significant co-occurrence of SETBP1 and JAK3 mutations in JMML. In order to determine whether SETBP1 and JAK3 mutations were present in the same clone or not, we performed colony formation assays using BM cells in one of the five patients with both SETBP1 and JAK3 mutations. This case harbored 0.9% VAF in JAK3 and 41.9% in SETBP1 in addition to 46.0% in PTPN11 mutation (c.227A>C, p.E76A), respectively. In total, 93 colonies were collected and individually analyzed by Sanger sequencing, of which two colonies (2.1%) were identified with both SETBP1 and JAK3 mutations. Conclusions ddPCR is a useful tool to assess subclonal SETBP1 and JAK3 hotspot mutations and to estimate the prognosis. It would be better to start preparing for hematopoietic stem cell transplantation when patients with JMML harbored subclonal SETBP1 and/or JAK3 mutations at the diagnosis. While JMML is characterized by a paucity of somatic mutations, clones harboring SETBP1 and JAK3 mutations were identified. This finding suggests that SETBP1 and JAK3 mutation are susceptible to each other. Furthermore, the serial acquisition of SETBP1 and JAK3 mutations might correlate with the disease progression. Disclosures No relevant conflicts of interest to declare.
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Cristóbal, Ion, Francisco J. Blanco, Laura Garcia-Orti, Nerea Marcotegui, Carmen Vicente, José Rifon, Francisco J. Novo, et al. "SETBP1 overexpression is a novel leukemogenic mechanism that predicts adverse outcome in elderly patients with acute myeloid leukemia." Blood 115, no. 3 (January 21, 2010): 615–25. http://dx.doi.org/10.1182/blood-2009-06-227363.
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Abstract Acute myeloid leukemias (AMLs) result from multiple genetic alterations in hematopoietic stem cells. We describe a novel t(12;18)(p13;q12) involving ETV6 in a patient with AML. The translocation resulted in overexpression of SETBP1 (18q12), located close to the breakpoint. Overexpression of SETBP1 through retroviral insertion has been reported to confer growth advantage in hematopoietic progenitor cells. We show that SETBP1 overexpression protects SET from protease cleavage, increasing the amount of full-length SET protein and leading to the formation of a SETBP1–SET-PP2A complex that results in PP2A inhibition, promoting proliferation of the leukemic cells. The prevalence of SETBP1 overexpression in AML at diagnosis (n = 192) was 27.6% and was associated with unfavorable cytogenetic prognostic group, monosomy 7, and EVI1 overexpression (P < .01). Patients with SETBP1 overexpression had a significantly shorter overall survival, and the prognosis impact was remarkably poor in patients older than 60 years in both overall survival (P = .015) and event-free survival (P = .015). In summary, our data show a novel leukemogenic mechanism through SETBP1 overexpression; moreover, multivariate analysis confirms the negative prognostic impact of SETBP1 overexpression in AML, especially in elderly patients, where it could be used as a predictive factor in any future clinical trials with PP2A activators.
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Inoue, Daichi, Hirotaka Matsui, Hsin-An Hou, Wen-Chien Chou, Akiko Nagamachi, Jiro Kitaura, Kimihito Cojin Kawabata, et al. "SETBP1 Mutations Drive Leukemic Transformation in ASXL1-Mutated MDS." Blood 124, no. 21 (December 6, 2014): 525. http://dx.doi.org/10.1182/blood.v124.21.525.525.
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Abstract Mutations in a variety of genes have been identified in MDS patients. Among them, mutations of additional sex combs-like 1 (ASXL1), found in 15-20% of MDS patients, have been identified as an independent poor prognostic factor. We previously demonstrated that C-terminal–truncating ASXL1 mutations (ASXL1-MT) inhibited myeloid differentiation and induced an MDS-like disease in mice after 1~2 years by inhibiting polycomb repressive complex 2–mediated methylation of histone H3K27 (Inoue et al. J Clin Invest. 2013). Given that ASXL1 mutations have been shown to be related to high-risk MDS or leukemic transformation, it is not clear how ASXL1-mutated MDS clones can transform into advanced MDS or AML. First, we examined genetic alterations in 368 WHO-defined MDS patients; ASXL1 mutations were detected in 64 of them (17.39%). Intriguingly, the patients with ASXL1 mutations had a significantly higher incidence of the concurrent SET binding protein 1 (SETBP1) mutation than those with the wild-type ASXL1 (6 out of 64, 9.38% vs. 2 out of 304, 0.66%, P=0.0005). Moreover, among ASXL1-mutated MDS patients, those harboring SETBP1 mutations had a higher incidence of leukemic transformation than those without (P=0.042), and MDS patients with both mutations had a significantly shorter overall survival compared to those without SETBP1 mutations (median, 10.5 vs. 22.5 months, P=0.046). In addition, we demonstrated that most SETBP1 mutations, such as D868N, occur in the PEST domain of the SKI homology region, preventing ubiquitination and subsequent proteasomal degradation. These results prompted us to investigate whether SETBP1 mutations play a critical role in the leukemic transformation of ASXL1-mutated MDS cells. In in vitro experiments, the expression of SETBP1-D868N enhanced myeloid colony formation of ASXL1-MT-transduced LSK cells, augmenting ASXL1-MT-induced differentiation blocking of 32Dcl3 cells. Of note, SETBP1-D868N collaborated with ASXL1-MT to induce AML after a short latency (median survival, 73 days) in a murine BMT model, while all mice expressing either ASXL1-MT or SETBP1-D868N survived for 6 months after transplantation (P<0.0001). Mice with leukemia induced by the combination of ASXL1-MT and SETBP1-D868N exhibited remarkable leukocytosis, anemia, thrombocytopenia, macrocytosis, hematosplenomegaly and hypercellular BM when compared to control mice. To clarify the molecular mechanism leading to leukemic transformation, we first investigated the Pp2a-Akt pathway because SETBP1 protein has been shown to interact with SET oncoprotein, resulting in Pp2a phosphorylation and subsequent inhibition. Consistent with previous reports using overexpression systems of SETBP1 wild type protein (SETBP1-WT), BM cells of leukemic mice displayed phosphorylated Pp2a and Akt compared to those of the control mice. Administration of FTY720, a Pp2a activator, efficiently repressed the growth rate in vitro and slightly improved the survival of serially transplanted mice. Next, using RNA-seq and GSEA, we demonstrated that SETBP1-D868N enriched hematopoietic stem cell-related genes and posterior Hoxa genes. Chromatin immnoprecipitation assay showed that both SETBP1-WT and SETBP1-D868N interacted with the promoter regions of Hoxa9 and Hoxa10, raising the possibility that a gain-of-function mutant of SETBP1 enhances transcription of these genes, directly or indirectly. Moreover, GSEA indicated global repression of the TGF-β signaling pathway and reciprocal upregulation of the Myc pathway in leukemic mice. In conclusion, our data provide evidence for the role of SETBP1 mutations in leukemic transformation and suggest the resulting deregulated pathways as potential therapeutic targets to prevent disease progression in MDS. Disclosures Harada: Kyowa Hakko Kirin Co., Ltd.: Research Funding.
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Stieglitz, Elliot, Camille B. Troup, Laura C. Gelston, Eric D. Chow, Kristie B. Yu, Jon Akutagawa, Amaro N. Taylor-Weiner, et al. "Subclonal Mutations in SETBP1 Predict Relapse in Juvenile Myelomonocytic Leukemia." Blood 124, no. 21 (December 6, 2014): 410. http://dx.doi.org/10.1182/blood.v124.21.410.410.
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Abstract Juvenile Myelomonocytic Leukemia (JMML) is an aggressive myeloproliferative neoplasm of childhood with a 5-year event free survival of 52% after hematopoietic stem cell transplantation (HSCT). A hallmark of JMML is aberrant Ras pathway activation due to mutations in NF1, NRAS, KRAS, PTPN11 and CBL. However, robust predictors of response are lacking, as individual mutations are not reliably associated with outcome, and relapse remains the most common reason for treatment failure. Recently, massively parallel sequencing has identified recurrent mutations in the SKI domain of SETBP1 in a variety of myeloid disorders, including JMML (Piazza et al Nat Genet 2012, Makishima et al Nat Genet 2013, Sakaguchi et al Nat Genet, 2013). These mutations had a lower allelic frequency compared to Ras pathway mutations, but were associated with poor prognosis. These and other data suggested that SETBP1 mutations contribute to disease progression rather than initiation. We identified several patients with JMML who had clonal SETBP1 mutations detected at relapse. Analysis of mononuclear cell extracted DNA from serial samples of two patients who relapsed revealed an increase in the SETBP1 mutant allele frequency over time (Figure 1). Similarly, analysis of colonies plated in methylcellulose from serial time points indicated that the percentage of individual myeloid progenitor colonies that were heterozygous or homozygous for the SETBP1 mutation increased with each sequential sample despite intensive treatment. Based on these data, we tested the hypothesis that rare SETBP1 mutant clones exist at diagnosis in many patients who relapse, and that these rare cells undergo positive selection during treatment. Using a droplet digital PCR (ddPCR) technology with a detection threshold as low as 0.001% of mutant DNA, we identified SETBP1 mutations in 16/53 (30%) of diagnostic JMML specimens from children treated on Children's Oncology Group trial AAML0122. Of these mutations, 12 were subclonal and 4 were clonal. Event free survival (EFS) at 4 years in patients with SETBP1 mutations was 19% ± 10% compared to 51% ± 8% in those with wild type SETBP1 (p=0.006). While samples of patients who relapsed on the AAML0122 trial were not available for analysis, one patient recently undergoing treatment who had a subclonal SETBP1 mutation (0.45% allelic fraction) detected at diagnosis by ddPCR, demonstrated an overt SETBP1 mutation at relapse. Finally, we isolated and analyzed hematopoietic stem (HSC), multipotent progenitor (MPP), common myeloid progenitor (CMP), and granulocyte-monocyte progenitor (GMP) populations from a relapsed sample with a SETBP1 mutation. Sanger sequencing demonstrated that all four progenitor compartments were affected by the mutation. Analysis of additional samples is underway. We conclude that the presence of a subclonal mutation in SETBP1 is a novel biomarker of adverse outcome in JMML. Understanding the mechanisms underpinning SETBP1-mediated resistance and relapse, and further identifying therapeutic vulnerabilities of HSCs expressing these mutant proteins will be critical to improve outcomes for patients with JMML and other myeloid malignancies. Furthermore, the presence of a subclonal SETBP1 mutation at diagnosis might identify JMML patients who will benefit from more intensive conditioning prior to HSCT or from novel therapeutic strategies. Figure 1 Figure 1. Disclosures Troup: Bio-Rad Laboratories: Employment.
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Meggendorfer, Manja, Tamara Alpermann, Elisabeth Sirch, Claudia Haferlach, Wolfgang Kern, Torsten Haferlach, and Susanne Schnittger. "Mutations In SETBP1 Occur In 3.1% Of De Novo AML and Show a Distinct Genetic Pattern That Highly Resembles Atypical CML." Blood 122, no. 21 (November 15, 2013): 2560. http://dx.doi.org/10.1182/blood.v122.21.2560.2560.
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Abstract Introduction Recently, mutations in SETBP1 (SETBP1mut) have been identified in different myeloid malignancies. We previously determined mutation frequencies in the range of 5-10% in MPN and MDS/MPN overlap, while we found SETBP1 more frequently mutated in atypical CML (32%). SETBP1mut has been shown to associate with CBL and ASXL1 mutations, as well as the cytogenetic abnormalities -7 and i(17)(q10). While SETBP1 mutations have been detected in 3% of s-AML cases, so far no mutations of SETBP1 in de novo AML have been described. Aim To analyze the mutation frequency of SETBP1 mutations in de novo AML with corresponding cytogenetic abnormalities and their respective correlation to clinical data and other gene mutations. Patients and Methods We investigated 422 adult de novo AML patients, diagnosed by cytomorphology, immunophenotyping and genetic studies following WHO classification. SETBP1 was analyzed by Sanger sequencing of the coding region for amino acids 800 to 935. The cohort comprised 229 males and 193 females, the median age was 65.8 years (range: 19.3 – 89.0). Cytogenetics was available in all 422 cases. Based on the previously described association of SETBP1mut with -7 and i(17)(q10) in other myeloid malignancies there was a selection bias to these karyotypes. Cases were grouped according to cytogenetic abnormalities: normal karyotype (n=88) and aberrant karyotype (n=334), i.e. i(17)(q10) (n=15), +14 (n=20), -7 (n=100), other abnormalities (n=129), and complex karyotype (n=114; 44 contained i(17)(q10), +14 or -7). Within the SETBP1mut cases the following molecular markers were analyzed: ASXL1, CBL, CEBPA, FLT3-ITD, FLT3-TKD, IDH1/2, KRAS, NRAS, NPM1, MLL-PTD, RUNX1, SRSF2, TP53 and WT1 by Sanger sequencing, next generation sequencing, gene scan or melting curve analyses. These data were also available in sub-cohorts of SETBP1 negative cases. Results In the total cohort mutations in SETBP1 were detected in 3.1% (13/422) of all cases. SETBP1mut patients were older (median age: 73.5 vs. 65.7 years; p=0.05) and showed a slightly higher white blood cell count (14.5 vs. 13.8x109/L; p<0.001). There was no correlation to gender, hemoglobin level and platelet count. However, analyzing the cytogenetic groups SETBP1mut showed, like in other myeloid malignancies, a strong co-occurrence with -7 and i(17)(q10), since 4/13 SETBP1 positive cases carried a monosomy 7, and 7/13 an i(17)(q10). The two remaining cases showed a trisomy 14 or a complex karyotype that also contained a i(17)(q10). No SETBP1mut was found in any other cytogenetic subgroup. Therefore, SETBP1mut correlated significantly with i(17)(q10) (8/15 i(17)(q10) were SETBP1mut vs. 5/407 in all other karyotypes; p<0.001). Further, we analyzed the association of SETBP1 mutations with other molecular markers. SETBP1mut correlated with ASXL1mut, 9/33 (27%) ASXL1mut patients showed a mutation in SETBP1, while only 2 (1%) showed a SETBP1 mutation in 229 ASXL1 wild type (wt) patients (p<0.001). This was also true for CBLmut, where 4/8 (50%) CBLmut cases were SETBP1mut, while only 8/158 (5%) were SETBP1mut in the group of CBLwt (p=0.001). This was even more prominent in SRSF2mut patients, where all 9 SRSF2mut were also SETBP1mut, while only 4/8 (50%) patients carried a SETBP1 mutation within the SRSF2wt group (p=0.029). In contrast, SETBP1mut were mutually exclusive of mutations in TP53 (0/67 in TP53mut vs. 12/194 in TP53wt; p=0.04), possibly reflecting the exclusiveness of TP53mut in i(17)(q10) patients. There was no correlation to any other analyzed gene mutation. Remarkably, while there was a high coincidence of SETBP1mut, SRSF2mut (9/13) and ASXL1mut (9/11), none of these patients showed mutations in the typical AML markers NPM1, FLT3-ITD, CEBPA, MLL-PTD, or WT1. Comparing the mutational loads of SETBP1, ASXL1 and SRSF2 resulted in SRSF2 having in most cases the highest mutational loads (range: 30-70%) while ASXL1 and SETBP1 showed equal or lower mutational loads (15-50% and 10-50%, respectively), possibly indicating that SRSF2 mutation is a former event followed by ASXL1 and SETBP1 mutation. Conclusions 1) For the first time we describe, that SETBP1 mutations occur in de novo AML. 2) SETBP1 mutations are correlated with a distinct genetic pattern with high association to i(17)(q10), ASXL1 and SRSF2 mutations and are mutually exclusive of TP53mut. 3) Thus, the genetic pattern of SETBP1 mutated AML highly resembles that of atypical CML. Disclosures: Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Sirch:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.
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Yu, Justine, Giovannino Silvestri, Lorenzo Stramucci, Masashi Sanada, Tomoyuki Yamaguchi, Yang Du, Jukka Westermarck, et al. "Potential Targeting Ph+ Acute Lymphoblastic Leukemia Stem and Progenitor Cells By Modulating the CIP2A-SET-SETBP1 -Mediated Suppression of PP2A Activity." Blood 128, no. 22 (December 2, 2016): 2909. http://dx.doi.org/10.1182/blood.v128.22.2909.2909.
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Abstract Tyrosine kinase inhibitors (TKIs) combined with chemotherapy significantly improved outcomes in adult Philadelphia-chromosome-positive (Ph+) B-cell Acute Lymphoblastic Leukemia (B-ALL). However, high relapse rates due to development of TKI resistance or chemotherapy-induced adverse effects remain the major therapeutic challenges. Furthermore, all TKIs are not effective against Ph+leukemia-initiating cells (LICs). The tumor suppressor protein phosphatase 2A (PP2A) is inactive in almost all solid and hematopoietic tumors. Suppression of PP2A activity correlates with poor outcome and disease progression, and largely relies on the aberrant expression of CIP2A, SET and/or SETBP1. SETBP1 was discovered as a SET-interacting protein, and recently described as mutated or overexpressed in several myeloid malignancies where it acts as an independent negative prognostic factor and as an inhibitor of PP2A, however its role in lymphoid malignancies is still unknown. Thus, we postulated that SETBP1 regulates survival and self-renewal of Ph+B-ALL LICs and progenitors through inhibition of PP2A. We reported that BCR-ABL1 kinase-dependent and -independent mechanisms induce SET-dependent PP2A inhibition in Ph+ (CMLand B-ALL) progenitors and quiescent TKI-resistant CML LICs, respectively, and that SET downregulation or pharmacologic (i.e. SET-interacting PP2A-activating drugs; PADs) restoration of PP2A activity strongly impaired malignant but not normal hematopoiesis by selectively killing Ph+progenitors (CML and ALL) and TKI-resistant quiescent stem (CML) cells. Here, we show that wild type SETBP1 is markedly induced in an imatinib (IM)-insensitive manner in primary CD34+CD19+ Ph+ B-ALL progenitors and Ph+ B-ALL cell lines (BV173 and SUP-B15) and barely detectable in CD34+ cells from CML patients in chronic and blastic phase. Overexpression of SETBP1 was found essential for PP2A inhibition in Ph+ B-ALL blasts. Accordingly, shRNA-dependent SETBP1 downregulation impaired clonogenic potential and self-renewal of CD34+CD19+ Ph+ B-ALL cells in CFC and serial replating assays. Furthermore, we have evidence that a SETBP1-SET/CIP2A inhibitory complex may exist in Ph+ cells, suggesting that SETBP1 might serve to recruit SET and CIP2A to suppress PP2A activity. Indeed, we found that CIP2A, like SET and SETBP1, is also overexpressed in CD34+CD19+ Ph+ B-ALL compared to CD34+CD19+cell from BM of healthy donors. Because, SETBP1 stabilizes SET and augments PP2A inhibition, and ectopic SETBP1 expression confers self-renewal to mouse myeloid progenitors and cooperates with BCR-ABL1 to induce a CML blast crisis-like disease in mice, our data suggest that aberrant SETBP1 expression might significantly contributes to the development of Ph+ B-ALL and persistence of TKI-resistant Ph+ B-ALL LICs. Disclosures Milojkovic: Ariad: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Novartis: Honoraria. Roy:Paladin: Consultancy; Fate Therapeutics: Consultancy; Novartis: Consultancy; Kiadis Pharma: Research Funding.
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Piazza, Rocco, Sara Redaelli, Simona Valletta, Alessandra Pirola, Roberta Spinelli, Vera Magistroni, Dong-Wook Kim, Nicholas C. P. Cross, and Carlo Gambacorti-Passerini. "SETBP1 and CSF3R Mutations In Atypical Chronic Myeloid Leukemia." Blood 122, no. 21 (November 15, 2013): 2598. http://dx.doi.org/10.1182/blood.v122.21.2598.2598.
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Abstract Atypical Chronic Myeloid Leukemia (aCML) is a clonal disorder belonging to the group of myelodysplastic/myeloproliferative (MDS/MPN) syndromes. In aCML many clinical features suggest the diagnosis of CML, however the lack of the BCR-ABL1 fusion point to a different pathogenetic process. Recently, we identified the presence of clonal somatic mutations occurring in the SETBP1 gene in approximately 25% of aCML samples (Piazza R. et al., Nat Genet. 2013 Jan;45(1):18-24). A subsequent study (Maxson J. et al., N Engl J Med. 2013 May 9;368(19):1781-90) demonstrated the presence of somatic mutations of the CSF3R gene in Chronic Neutrophilic Leukemia (CNL) and, with lower frequency, in aCML. In a recent follow-up of the first study (Gotlib J. et al., Blood. 2013 Jul 29), the presence of both CSF3R and SETBP1 variants was tested in a cohort of 9 CNL and 20 aCML cases, demonstrating the presence of CSF3R somatic mutations in 40% of the aCML patients. Of these mutations, 20% were membrane, 5% truncating and 15% compound variants. Interestingly, 5% of the aCML patients showed coexistence of CSF3R and SETBP1 mutations, suggesting that variants occurring in these genes are not mutually exclusive. To gain further insight into the relationship between CSF3R and SETBP1 in aCML, we extended our initial study by analyzing an expanded cohort of 65 aCML plus a total of 230 AML, ALL, CLL, CML, PV, TE, MMM, CMML and MDS cases for the presence of CSF3R and SETBP1 mutations. In line with previous findings (Piazza R. et al., Nat Genet. 2013 Jan;45(1):18-24; Maxson J. et al., N Engl J Med. 2013 May 9;368(19):1781-90), we found evidence of SETBP1 and/or CSF3R mutations only in MDS/MPN disorders. In aCML we identified a total of 18 (27.7%) mutations occurring in SETBP1 and 8 (12.3%) in the CSF3R gene. A large fraction (94.4 %) of the SETBP1 mutations was clustered in a 14 amino acid stretch that is also mutated in the Schinzel-Giedion syndrome, as previously reported (Piazza R. et al., Nat Genet. 2013 Jan;45(1):18-24). Of the 8 CSF3R mutations 5 were membrane proximal (4 T618I and 1 T615A) and 3 were truncating (2 Q776X and 1 Q781X). In 2 aCML samples we detected the coexistence of CSF3R and SETBP1 mutations. In both cases the CSF3R variant was a membrane proximal mutation; CSF3R and SETBP1 mutations were at comparable levels at the time of detection, therefore no conclusion can be drawn about the timing of the two mutational events. Taken globally these data indicate that somatic mutations occurring in SETBP1 and CSF3R are present in aCML and can coexist. Interestingly, the frequency of the CSF3R mutations in our aCML cohort is largely different from that of Maxson and colleagues (12.3 vs 40%), although the frequency of the combined CSF3R/SETBP1 mutations is similar (3.1 vs 5%): the reasons for this discrepancy are actually unclear. Previously we demonstrated that the presence of SETBP1 mutations in aCML is an independent negative prognostic factor (Piazza R. et al., Nat Genet. 2013 Jan;45(1):18-24). Further studies with larger cohorts will be required to assess the prognostic impact of concurrent SETBP1 and CSF3R mutations. Disclosures: Cross: Novartis: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria. Gambacorti-Passerini:Pfizer, BMS: Consultancy, Consultancy Other; Pfizer: Research Funding.
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Cui, Yajuan, Bing Li, Robert Peter Gale, Qian Jiang, Zefeng Xu, Tiejun Qin, Peihong Zhang, Yue Zhang, and Zhijian Xiao. "Molecular Aberrations of Chronic Neutrophilic Leukemia: The CSF3R and SETBP1 Mutations." Blood 124, no. 21 (December 6, 2014): 5578. http://dx.doi.org/10.1182/blood.v124.21.5578.5578.
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Abstract Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative neoplasm (MPN) characterized by sustained elevated neutrophil levels with <10% immature cells according to WHO criteria. Recently, oncogenic mutations affecting the granulocyte-colony stimulating factor receptor (CSF3R) were reported to be highly prevalent in CNL. SET binding protein 1(SETBP1) mutations were also reported in some CNL cases. We reviewed clinical suspected 12 “CNL” patients and performed CSF3R and SETBP1 sequencing. All the 6 who met WHO criteria for CNL carried CSF3R T618I mutation. Four of the 6 also had SETBP1 mutations and a 5th had a CALR mutation (c.1154-1155insTTGTC). Two other who met these criteria but also had monoclonal gammopathy with uncertain significance (MGUS) had no CSF3R, SETBP1 or CALR mutation. The rest 4 carried infection or tumor and thus were excluded. We conclude including CSF3R and SETBP1 mutations into WHO criteria for CNL may improve diagnostic accuracy and may have therapeutic implications. Disclosures No relevant conflicts of interest to declare.
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Choi, Hyun-Woo, Hye-Ran Kim, Hwan-Young Kim, Ju-Heon Park, Jae-Sook Ahn, Duck Cho, Seung-Jung Kee, et al. "Prevalence and Clinical Impacts Of SETBP1 Mutation In East Asian Patients With MDS/MPN." Blood 122, no. 21 (November 15, 2013): 2629. http://dx.doi.org/10.1182/blood.v122.21.2629.2629.
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Abstract Introduction Recently, recurrent somatic SET-binding protein 1 (SETBP1) mutations were found in atypical chronic myeloid leukemia (aCML) and other related myeloid neoplasms. According to reports so far, SETBP1 mutations occur in 9% of myelodysplastic/myeloproliferative neoplasms (MDS/MPN), especially in high frequency (24∼30%) of aCML. SETBP1 mutations were associated with worse prognosis and higher white blood cell (WBC) counts. Most of the reports came from western countries and there was a need to further study its clinicopathological impacts in East Asian patients because of paucity of reports. Therefore, this study investigated the prevalence and clinical implications of SETBP1 mutations in MDS/MPN patients at a single medical center in South Korea. Patients and methods We analyzed a cohort of 34 MDS/MPN patients (10 aCML, 7 CMML-1, 9 CMML-2, 5 JMML, 3 MDS/MPN unclassifiable) who were diagnosed and treated in Chonnam National University Hwasun Hospital (Hwasun, Korea) from October 2004 to June 2013. The mononuclear cells from bone marrow of the patients were separated and the total DNA was extracted by commercial kit (QIAGEN, Hilden, Germany). PCR and sequencing reaction were performed by targeting the hot spot (exon 4, codon 778-979) of the SETBP1 gene. The PCR mixture consisted of 50 to 100 ng of total DNA, 20 pmol of each forward (5'-CCACTTTCAACACAGTTAGGTG-3') and reverse (5'-TCTCGTGGTAGAAGGTGTAACTC-3') primer, 0.4 mM of each dNTP, 5 μL 10X F-taq reaction buffer, 5 U of DNA Polymerase (Solgent, Daejeon, Korea) and H2O in a final reaction volume of 50 μL. Direct sequencing was performed using the ABI Prism 3130XL Genetic Analyzer with the BigDye Terminator v3.1 Ready Reaction Kit (Applied Biosystems). Clinical information about patients was obtained from our electronic medical record database. All statistical computations were performed using PASW 18.0 (SPSS Inc., Chicago, Illinois, USA). Results In this analysis, 4 (11.7%) of 34 MDS/MPN patients showed SETBP1 mutations. 3 of them were aCML patients and 1 was CMML-2 patient. The frequencies in aCML and CMML-2 were 30% and 11.1%, respectively. All of the aCML patients with SETBP1 mutation showed mutation encoding c.2898G>A (p.Asp868Asn) and the CMML-2 patient displayed c.2903C>T synonymous mutation (Ser869).The mutated SETBP1 patients showed a tendency of higher mean WBC counts, lower mean hemoglobin, lower mean platelet counts and lower mean BM blasts percentage than the wild-type patients, but they were not statistically significant. One of the mutated SETBP1 patients showed a i(17)(q10) cytogenetic abnormality. We found no statistical difference in overall survival (OS) between mutated SETBP1 patients and wild-type patients. Conclusions Alteration of SETBP1 gene was a common genetic event in aCML with an impact as a diagnostic marker for MDS/MPN. Disclosures: No relevant conflicts of interest to declare.
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Bresolin, Silvia, Paola De Filippi, Francesca Vendemini, Riccardo Masetti, Franco Locatelli, and Geertruy te Kronnie. "Secondary Mutations of JAK3 and SETBP1 in Juvenile Myelomonocytic Leukemia and Their Propagating Capacity; A Report from the AIEOP Study Group." Blood 124, no. 21 (December 6, 2014): 4625. http://dx.doi.org/10.1182/blood.v124.21.4625.4625.
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Abstract INTRODUCTION Juvenile myelomonocytic leukemia is a rare early childhood leukemia, characterized by excessive proliferation of granulocytic and monocytic cells. About 95% of JMML patients harbor driver mutations in the RAS signaling pathway. Recently, secondary hits in SETBP1 and JAK3 have been reported in a Japanese cohort of JMML patients showing an adverse clinical outcome of patients carrying these mutations. Here we report the mutational analysis of SETBP1 and JAK3 and clinical implications in a cohort of Italian JMML patients. METHODS Samples collected at diagnosis of 65 patients with JMML were analyzed by Sanger sequencing. Mutations were found in RAS (NRAS-KRAS) 31%, PTPN11 35%, CBL 5%, whereas in 29% of patients none of the above cited mutations was present. Mutation hot spot regions of SETBP1 (SKI domain) and of JAK3 (PTK domains) were sequenced. A xenografted murine model was used to assess the in vivo competitive repopulation advantage of clones carrying mutations of JAK3 and SETBP1. Mononuclear cells from a patient with JMML at diagnosis harboring PTPN11, SETBP1 and JAK3 mutations were transplanted in NSG mice and assessed for mutational status in the bone marrow and spleen after engraftment of JMML cells. RESULTS Screening for JAK3 and SETBP1 mutations in patients revealed 9 mutations in 8 out of 65 patients at diagnosis of JMML. All of the identified secondary mutations were associated with known driver mutations, more frequent with mutated PTPN11 and RAS (p=0.036 and p= 0.01 respectively) than with CBL or in cases without known driver mutations. Seventy-five percent of secondary mutations were found in SETBP1 and only 1 patient harbored a mutation in JAK3. Remarkably one patient carried mutations in JAK3 (L857P and L857Q, both predicted to damaging protein function), PTPN11 (G503A) and SETBP1 (D868N). All variants were identified as heterozygous mutations, confirmed bi-allelic expression at the transcriptome level. The only patient carrying JAK3 as secondary mutation at E958K showed wild-type expression of JAK3 pointing to absence of a functional role at the protein level. Univariate analysis revealed association between the presence of secondary mutations and patient’s age at diagnosis, with older patients carrying JAK3 and SETBP1 mutations (p=0.0067); no other clinical and biological characteristics (i.e. WBC count, percentage of monocyte, HbF level and platelet count) being significantly associated with the presence of secondary hits in bone marrow of JMML cases. Patients with secondary mutations showed a trend to shorter survival compared to those without secondary events in JAK3 and SETBP1 (5-years OS= 0% vs 54.01%, SE=8.1; p=0.41, respectively). Interestingly, the in vivo assay using xenografted mice revealed a different propagating capacity of JAK3 clones of patients carrying JAK3 (2 different clones), SETBP1 and PTPN11 mutations. Indeed, for JAK3 only the clone with the L857Q mutation engrafted in BM and spleen of the mouse, together with SETBP1 and PTPN11 mutations. Moreover, a second mouse engrafted with mononuclear cells of the same patients showed that only cells carrying the PTPN11 mutation had engrafted. CONCLUSIONS In conclusion we identified secondary mutations in JAK3 and SETBP1 in 12% of patients of a representative cohort of Italian JMML patients, showing a trend of adverse outcome for patients carrying these mutations. These secondary events in JMML patients showed to have distinct propagating capacities upon engraftment in NSG mice pointing to a different functional impact of these mutations. Disclosures No relevant conflicts of interest to declare.
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Borges Ferreira, Viviane. "Sobreposição da mutação ganho-de-função* do gen SETBP1 na Síndrome de Schinzel-Giedion e em doenças hematológicas malignas." Revista Científica Hospital Santa Izabel 2, no. 1 (May 14, 2020): 48–51. http://dx.doi.org/10.35753/rchsi.v2i1.86.
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A Síndrome de Schinzel-Giedion (SGS) é um raro distúrbio do desenvolvimento, caracterizado por múltiplas malformações, alterações neurológicas severas e elevação do risco de ocorrência de neoplasias malignas. SGS é causada por mutação de novo no hotspot (região do material genético mais propensa a mutação) do braço curto do cromossomo 12, no exon4, no gen SETBP1. Mutações nesse hotspot interrompem a degradação e regulação proteica, promovendo o acúmulo da proteína SETBP1. A sobreposição de mutações no hotspot do gen da proteína SETBP1 tem sido observada, de maneira recorrente em fenótipos como a leucemia. Foram coletadas informações clínicas de 47 pacientes com SGS (incluindo 26 casos novos), com mutação de células germinativas de SETBP1 e em quatro indivíduos com fenótipo moderado causado por mutação de novo adjacente ao hotspot SETBP1.
*Mutações de ganho de função são aquelas que causam a superexpressão de um produto gênico (hipermorfismo).
Publicado no PLOS Genetics. Volume 3, Issue 1, February 2017. Com o mesmo título. Autores: Rocio Acuna-Hidalgo, Pelagia Deriziotis, MarloesSteehouwer, Christian Gilissen, Sarah A. Graham, Sipko van Dam, Julie Hoover-Fong, Viviane Ferreira, et. al.
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Neupauerová, Jana, Katalin Štěrbová, Vladimír Komárek, Andrea Gřegořová, Markéta Vlčková, David Staněk, Pavel Seeman, Petra Laššuthová, and Markéta Havlovicová. "Schinzel—Giedion Syndrome: First Czech Patients Confirmed by Molecular Genetic Analysis." Journal of Pediatric Neurology 17, no. 03 (May 18, 2018): 125–27. http://dx.doi.org/10.1055/s-0038-1651520.
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AbstractSchinzel–Giedion syndrome (SGS) is a very rare genetic disorder characterized by distinctive facial features, severe developmental delay, seizures, and skeletal abnormalities. Whole exome sequencing, Sanger sequencing, and correlation with already published variants and cases allowed us to identify two different de novo mutations in the SETBP1 gene: NM_015559.2 (SETBP1): c.2601C > G (p.Ser867Arg) and c. 2608 G > A (p.Gly870Ser) in two Czech patients presenting with SGS features. Both mutations are within exon 4 of SETBP1, supporting the notion that exon 4 represents the mutation hotspot of the gene in patients with SGS.
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Gambacorti-Passerini, Carlo, Simona Valletta, Nils Winkelmann, Sara Redaelli, Roberta Spinelli, Alessandra Pirola, Laura Antolini, et al. "Recurrent SETBP1 Mutations in Atypical Chronic Myeloid Leukemia Abrogate an Ubiquitination Site and Dysregulate SETBP1 Protein Levels." Blood 120, no. 21 (November 16, 2012): LBA—2—LBA—2. http://dx.doi.org/10.1182/blood.v120.21.lba-2.lba-2.
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Abstract Abstract LBA-2 The SETBP1 gene codes for a predominantly nuclear protein with a predicted MW of 170 kD. Germline mutations of SETBP1 were described in patients affected by the Schinzel-Giedion syndrome (SGS), a rare disease characterized by bone, muscle and cardiac abnormalities, and presenting neuroepithelial neoplasms. In an effort to investigate the molecular pathogenesis of myeloid malignancies we applied a HTS strategy, including both exome sequencing and RNA-SEQ, to atypical Chronic Myeloid Leukemia (aCML), as defined by WHO criteria, with the aim of identifying novel recurrent driver mutations. aCML shares clinical and laboratory features with CML, but it lacks the pathognomonic BCR-ABL1fusion. Since no specific recurrent genomic or karyotypic abnormalities have been identified in aCML, the molecular pathogenesis of this disease has remained elusive and the outcome dismal (median survival 37 months) with no improvement over the last 20 years. This sharply contrasts with the outcome for CML, for which the prognosis was dramatically improved by the development of imatinib as a specific inhibitor of the BCR/ABL protein. Whole-exome sequencing of 9 aCML patients revealed the presence of 62 unique mutations (range 5–14 per patient), including a recurrent alteration of SETBP1 (G870S and D868N) in three cases. Targeted resequencing performed in 70 aCMLs, 574 patients with different hematological malignancies and 344 cell lines, identified SETBP1 mutations in 17 of 70 aCML patients (24.3%; 95% CI: 16–35%), 4 of 30 (13%) MDS/MPN-u and 3 of 82 (3.6%) CMML patients. Patients with mutations had higher white blood cell counts (p=0.008) and worse prognosis (p=0.01) when tested in multivariate analysis. TF1 cells transfected with SETBP1G870S showed increased SET levels, decreased PP2A activity and increased proliferation rates. The vast majority of mutations (85%) was located between residues 858 and 871, in the SKI homologous region of SETBP1, and were identical to germline changes seen in patients with SGS. This region may be critical for ubiquitin binding and for subsequent protein degradation, since the Eukaryotic Linear Motif (ELM) identified with high probability score a putative functional site (aa. 868–873) for beta-TrCP, the substrate recognition subunit of the E3 ubiquitin ligase. This prediction was experimentally validated using biotinylated, phosphorylated peptides encompassing this region (aa 859–879): while the wild type peptide could efficiently bind beta-TrCP as predicted, a peptide presenting the G870S mutation was incapable of binding this E3 ligase subunit, indicating a possible alteration in SETBP1 protein stability caused by this mutation. In agreement with these findings, cells transfected with SETBP1G870Sshowed increased levels of SETBP1 protein when compared to cells with similar expression levels of the wild type gene. Finally, RNA-SEQ yielded gene expression profiles with overrepresentation of genes under the control of Transforming Growth Factor Beta 1 (TGFβ1) among genes differentially expressed between SETBP1-mutated and unmutated aCML patients. Mutated SETBP1 represents a novel type of oncogene which is specifically present in aCML and closely related diseases. These data allow for a better understanding of the molecular pathogenesis of this disease; they provide evidence that SETBP1 mutations might be a new biomarker for future diagnosis and classification of aCML and related diseases, and indicate a potential strategy to develop new treatment modalities for malignancies caused by mutated SETBP1. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.
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Stieglitz, Elliot, Camille B. Troup, Laura C. Gelston, John Haliburton, Eric D. Chow, Kristie B. Yu, Jon Akutagawa, et al. "Subclonal mutations in SETBP1 confer a poor prognosis in juvenile myelomonocytic leukemia." Blood 125, no. 3 (January 15, 2015): 516–24. http://dx.doi.org/10.1182/blood-2014-09-601690.
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Key Points Mutations in SETBP1 can be detected using droplet digital polymerase chain reaction in at least 30% of patients with JMML and are associated with a dismal prognosis. Patients harboring rare cells with mutant SETBP1 at diagnosis should be considered candidates for swift hematopoietic stem cell transplant.
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Niro, Antonio, Rocco Piazza, Gabriele Merati, Alessandra Pirola, Carla Donadoni, Diletta Fontana, Sara Redaelli, et al. "ETNK1 Is an Early Event and SETBP1 a Late Event in Atypical Chronic Myeloid Leukemia." Blood 126, no. 23 (December 3, 2015): 3652. http://dx.doi.org/10.1182/blood.v126.23.3652.3652.
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Abstract Atypical Chronic Myeloid Leukemia (aCML) is a clonal disorder belonging to the myelodisplastic-myeloproliferative neoplasms, according to the WHO-2008 classification. From a clinical point of view it closely resembles the classical Chronic Myeloid Leukemia (CML), however it lacks the presence of the Philadelphia chromosome and of the BCR-ABL1 fusion gene. In recent works, we and others characterized the somatic lesions present in the aCML genome, mainly by using Next Generation Sequencing (NGS) technologies, demonstrating the presence of a large set of recurrent somatic mutations involving, among the others, SETBP1, ETNK1, ASXL1, EZH2, CBL, TET2, NRAS and U2AF1 genes. The identification of somatic variants occurring in a large number of genes clearly indicates that the genetic bases of aCML are very heterogeneous, in striking contrast with classical CML. This heterogeneity poses a great challenge to the dissection of the molecular steps required for aCML leukemogenesis. The hierarchical reconstruction of the different mutations occurring in a clonal disorder can have important biological, prognostic and therapeutic repercussions; therefore we started a project focused on the dissection of the aCML clonal evolution steps through the analysis of individual leukemic clones by methylcellulose assays in samples whose mutational status has been previously characterized by matched whole-exome sequencing. Patient CMLPh-019 was characterized by the presence of a complex mutational status, with somatic variants occurring in SETBP1, ETNK1, ASXL1 and CBL genes (Fig. 1a). Targeted resequencing analysis of individual clones revealed the presence of all the 4 variants in 44/60 (73.3%) clones; in 15/60 (25%) we detected the presence of mutated ETNK1, ASXL1 and CBL and wild-type (WT) SETBP1. Of these 15 clones, 33% carried heterozygous and 67% homozygous CBL mutations. In one clone (1.7%) we detected heterozygous ETNK1, homozygous CBL and WT sequences for ASXL1 and SETBP1, suggesting a strong selective pressure towards the acquisition of homozygous CBL mutations. Identification of homozygous CBL mutations in all the main clonal phases suggests that a significant positive selective pressure is associated with this event. Allelic imbalance analysis of CMLPh-019 exome using CEQer revealed that CBL homozygosity is caused by a somatic uniparental disomy event occurring in the telomeric region of the long arm of chromosome 11. Patient CMLPh-005 (Fig. 1b) was mutated in ASXL1, CBL and SETBP1. Targeted analysis done on 68 clones revealed a complex, branching evolution, with 63 clones carrying all the 3 variants. Of them, 47 (74.6%) had a heterozygous and 16 (25.4%) a homozygous CBL variant. Four clones (4.2%) carried ASXL1 and SETBP1 but not CBL mutations, while 1 clone was mutated in ASXL1 and CBL in absence of SETBP1 mutations, which suggests that CBL mutations occurred independently in two different subclones. Also in this case, allelic imbalance analysis of exome data revealed that CBL homozygosity was caused by a telomeric somatic uniparental disomy event. According to exome sequencing, patient CMLPh-003 carried SETBP1 mutation G870S and NRAS variant G12R. Clonal analysis confirmed the presence of SETBP1 G870S in all the clones analyzed, while heterozygous NRAS G12R mutation was detected in 67% (Fig. 1c). Notably in the remaining 33% another heterozygous NRAS variant, G12D, was detected. Retrospective reanalysis of exome data confirmed the presence of the newly identified variant, which had been previously filtered-out from exome data because of the low frequency. Patient CMLPh-013 was mutated in ASXL1, ETNK1, NRAS and SETBP1. Of the 39 clones analyzed, 34 (82.9%) showed the coexistence of ASXL1, ETNK1, NRAS and SETBP1, 4 were mutated in ASXL1, ETNK1 and NRAS and 1 in ETNK1 and NRAS, suggesting that ETNK1 and NRAS were early events, ASXL1 an intermediate one and SETBP1 a late variant (Fig. 1d). Taken globally, these data indicate that ETNK1 variants occur very early in the clonal evolution history of aCML, while ASXL1 represents an early/intermediate event and SETBP1 is often a late event. They also suggest that, in the context of aCML, there is a strong selective pressure towards the accumulation of homozygous CBL variants, as already shown in other leukemias. Figure 1. Clonal analysis of four aCML cases. The asterisks indicate hypothetical clones. Figure 1. Clonal analysis of four aCML cases. The asterisks indicate hypothetical clones. Disclosures Rea: Novartis: Honoraria; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Ariad: Honoraria.
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Qiao, Chun, Yuan Ouyang, and Sujiang Zhang. "Clinical Significance of CSF3R, SRSF2 and SETBP1 mutation in Chronic Neutrophilic Leukemia and Chronic Myelomonocytic Leukemia." Blood 126, no. 23 (December 3, 2015): 1617. http://dx.doi.org/10.1182/blood.v126.23.1617.1617.
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Abstract Objective: To investigate the gene mutation and the clinical features of CSF3R, SETBP1 and SRSF2 in chronic neutrophilic leukemia (CNL) and chronic myelomonocytic leukemia (CMML) patients. Method: Sequence analysis of CSF3R, SETBP1 and SRSF2 were performed in 10 CNL and 56 CMML patients whose clinical features were also studied. Result: Among 10 CNL patients, 8(8/10, 80%) patients had CSF3R mutations and 7(7/8, 87.5%) of them were with CSF3R T618I. In 56 cases of patients with CMML, SRSF2 mutations were found in 14(14/56,25%), CSF3R in 4(4/56,7.1%) and SETBP1 in 3(3/56, 5.3%) patients. In CMML, compared to wild-type(wt) SRSF2 patients, SRSF2 mutated patients appeared to be more possible with SETBP1 mutations [1/14(7.1%) vs. 2/42(4.8%), P>0.05], less possible with CSF3R mutation [0/14(0%) vs. 4/42(9.5%), P<0.001]. The clinical characteristics such as age, gender, WHO category, FAB category, karyotype and blood cell counts did not reveal any difference between SRSF2 mutated and wtSRSF2 patients. Either SRSF2 mutated patients or SETBP1 mutated patients both had shorter overall survival (OS) and progression-free survival(PFS) when compared with those with wtSRSF2 (P<0.001 both) and wtSETBP1 (P<0.001 and P=0.02, respectively). No significant difference of OS and PFS between CSF3R mutated and wtCSF3R patients were observed. In multivariate analysis, SRSF2 mutation was an independent negative predictor for OS (HR, 3.307; 95% CI, 1.137 to 9.614; P=0.028) and PFS(HR, 15.431; 95% CI, 3.041 to 78.312; P = 0.001). What's more, SETBP1 mutation was also an independent negative predictor for OS(HR, 9.492; 95% CI, 1.183 to 76.128; P = 0.034). Conclusion: The majority of patients with WHO-defined CNL have oncogenic mutations in CSF3R and the T618I mutation type is a highly sensitive and specific molecular marker of the disease. While mutations of SRSF2 are common in CMML and may be of prognostic significance. As a non-specific molecular marker, SETBP1 was found in CNL, CMML and other blood cancers, which have poor prognosis. Disclosures No relevant conflicts of interest to declare.
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Qian, Yi, Yan Chen, and Xiaoming Li. "CSF3R T618I, SETBP1 G870S, SRSF2 P95H, and ASXL1 Q780* tetramutation co-contribute to myeloblast transformation in a chronic neutrophilic leukemia." Annals of Hematology 100, no. 6 (April 6, 2021): 1459–61. http://dx.doi.org/10.1007/s00277-021-04491-2.
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AbstractChronic neutrophilic leukemia (CNL) is a rare but serious myeloid malignancy. In a review of reported cases for WHO-defined CNL, CSF3R mutation is found in about 90% cases and confirmed as the molecular basis of CNL. Concurrent mutations are observed in CSF3R-mutated CNL patients, including ASXL1, SETBP1, SRSF2, JAK2, CALR, TET2, NRAS, U2AF1, and CBL. Both ASXL1 and SETBP1 mutations in CNL have been associated with a poor prognosis, whereas, SRSF2 mutation was undetermined. Our patient was a 77-year-old man and had no significant past medical history and symptoms with leukocytosis. Bone marrow (BM) aspirate and biopsy revealed a markedly hypercellular marrow with prominent left-shifted granulopoiesis. Next-generation sequencing (NGS) of DNA from the BM aspirate of a panel of 28 genes, known to be pathogenic in MDS/MPN, detected mutations in CSF3R, SETBP1, and SRSF2, and a diagnosis of CNL was made. The patient did not use a JAK-STAT pathway inhibitor (ruxolitinib) but started on hydroxyurea and alpha-interferon and developed pruritus after 4 months of diagnosis and nasal hemorrhage 1 month later. Then, the patient was diagnosed with CNL with AML transformation and developed intracranial hemorrhage and died. We repeated NGS and found that three additional mutations were detected: ASXL1, PRKDC, MYOM2; variant allele frequency (VAF) of the prior mutations in CSF3R, SETBP1, and SRSF2 increased. The concurrence of CSF3RT618I, ASXL1, SETBP1, and SRSF2 mutation may be a mutationally detrimental combination and contribute to disease progression and AML transformation, as well as the nonspecific treatment of hydroxyurea and alpha-interferon, but the significance and role of PRKDC and MYOM2 mutations were not undetermined.
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Bulut, Ozgul, Zeynep Ince, Umut Altunoglu, Sukran Yildirim, and Asuman Coban. "Schinzel-Giedion Syndrome with Congenital Megacalycosis in a Turkish Patient: Report of SETBP1 Mutation and Literature Review of the Clinical Features." Case Reports in Genetics 2017 (2017): 1–4. http://dx.doi.org/10.1155/2017/3740524.
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Schinzel-Giedion syndrome (SGS) is a rare autosomal dominant disorder that results in facial dysmorphism, multiple congenital anomalies, and an increased risk of malignancy. Recently, using exome sequencing, de novo heterozygous mutations in the SETBP1 gene have been identified in patients with SGS. Most affected individuals do not survive after childhood because of the severity of this disorder. Here, we report SETBP1 mutation confirmed by molecular analysis in a case of SGS with congenital megacalycosis.
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Kohyanagi, Naoki, Nao Kitamura, Keiko Tanaka, Takuya Mizuno, Nobuyuki Fujiwara, Takashi Ohama, and Koichi Sato. "The protein level of the tumour-promoting factor SET is regulated by cell density." Journal of Biochemistry 171, no. 3 (January 25, 2022): 295–303. http://dx.doi.org/10.1093/jb/mvab125.
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Abstract SET/I2PP2A is a multifunctional protein that acts as an intrinsic inhibitor of the tumour suppressor protein phosphatase 2A and as a histone chaperone. Increased SET levels have been observed in various cancers; however, the underlying molecular mechanisms remain unclear. In this study, we found that SET protein accumulates with the increasing density of cultured cells. This phenomenon was observed not only in cancer cell lines but also in non-cancer cell lines. The mRNA levels of SET were not affected by the cell density. Proteasome inhibition decreased SET levels, whereas autophagy inhibition led to SET accumulation, indicating the involvement of autophagy. The mRNA and protein expression of SETBP1, which stabilizes the SET protein, increased with cell density. The decrease in SET level due to the loss of SETBP1 was more pronounced in wild-type cells than that in autophagy-deficient cells. These results have revealed a mechanism underlying the regulation of SET level, wherein increased cell density induces SETBP1 expression and protects SET from autophagy.
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Hills, Robert K., Claire M. Lucas, Laura J. Scott, Natasha Carmell, Alison K. Holcroft, and Richard E. Clark. "PP2A Inhibition By CIP2A or SETBP1 Leads to Elevated Levels of AKT S473 Which Can be Used As a Biomarker of Outcome in Acute Myeloid Leukaemia." Blood 126, no. 23 (December 3, 2015): 1396. http://dx.doi.org/10.1182/blood.v126.23.1396.1396.
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Abstract Many cells utilise reversible phosphorylation as a mechanism of post-translational modification for activating and deactivating key regulatory molecules involved in cell signalling. Many malignancies are characterised by overactive kinases e.g. BCR-ABL or FLT-3 in myeloid malignancy, or ERK or ErbB2 in solid tumours. A major phosphatase working in opposition to kinases is protein phosphatase 2A (PP2A) but why the cellular phosphatases such as PP2A simply do not counteract the overactive kinase activity is unclear. PP2A activity is regulated by inhibitor proteins SET, (which is stabilised by a binding protein SETBP1) and cancerous inhibitor of PP2A (CIP2A). In CML, we have shown that high levels of CIP2A at diagnosis is a prospective biomarker for future progression to blast crisis. High levels of CIP2A in other malignancies have also been reported to confer a poor prognosis. In CML, high CIP2A levels lead to the stabilisation of c-Myc and elevation of E2F1, as well as PP2A inactivation. In AML there are data suggesting that high SETBP1 levels may confer a poor outcome in AML, by PP2A inhibition via increased action of SET, though little is known of CIP2A in AML. The aim of the study was to investigate if CIP2A or network related proteins could predict clinical outcome in AML patients. CIP2A and network related proteins were investigated in 119 AML patient samples collected in the UK MRC/NCRI trials AML15, 16 and 17. These were stratified into intermediate or adverse risk, based on cytogenetics as previously defined (Grimwade et al, Blood. 1998: 92; 2322-33). Protein levels were studied in diagnostic mono-nuclear cell samples by flow cytometry. The diagnostic CIP2A protein level (mean fluorescence intensity (MFI)) for all patients was calculated. This range was 0-13.69, the interquartile range was 1.47-5.11, the median was 3 and the mean was 3.5. High CIP2A patients are defined as those patients with a CIP2A level greater than or equal to 3. In all AML samples studied high CIP2A was associated with inactive PP2A as shown by high levels of PP2AY307 (p=0.05), as well as elevated levels of c-Myc p=<0.0001 and E2F1 p=<0.0001, a similar finding to what we have observed in CML. We also found that AKTS473 and STAT5 were also elevated in high CIP2A AML patients (p=0.002 and p=0.006 respectively). Unlike CML, we found in AML patients high CIP2A was associated with high levels of SET (p=<0.0001) and SETBP1 (p=0.01). Younger intermediate AML patients had significantly higher diagnostic CIP2A levels than adverse risk patients (p=0.004). In the intermediate risk group, overall survival was dominated by the survival from relapse. For younger intermediate risk patients with high and low CIP2A levels, median survival from relapse (censored at transplant) was 56 days and 303 days respectively. Multivariate analysis adjusted for FLT3-ITD showed that a high CIP2A level predicted inferior survival from relapse (p=0.04, hazard ratio 4.02). Interestingly, high diagnostic CIP2A was strongly associated with the presence of a FLT3-ITD mutation (p=0.03). In adverse risk patients SETBP1 levels were elevated but CIP2A levels were lower. SETBP1 acts via SET as an alternative inhibitor of PP2A. In younger patients with adverse cytogenetics, high SETBP1 levels predict for an inferior overall survival (p=0.02). For all 119 AML patients those with detectable SETBP1 protein levels at diagnosis had an inferior overall survival (p=0.01). High SETBP1 was also associated with secondary AML. In univariate analysis, high levels of AKTphosphorylation at seine 473 (which is inversely correlated with PP2A activity)were associated with poor survival in all patients. Multivariate analysis of known predictors of outcome (age, cytogenetics, white count, secondary leukaemia, FLT3-ITD) together with CIP2A and related proteins (PP2AY307, SET, SETBP1, c-Myc E2F1, AKTS473, STAT5, and E2F1) was used to derive a prognostic model for survival. This ranked high levels of AKTS473 as the third most important predictor of outcome after age and cytogenetics. Our data suggest that AKTS473 levels are elevated as a result of PP2A inactivation (p=>0.0001) caused by high level of PP2A inhibitors, either CIP2A (p=0.003) or SETBP1 (p=0.03). In summary, AKT phosphorylation at S473 is a biomarker of PP2A inhibition by either CIP2A (intermediate risk) or SETBP1 (adverse risk patients). The diagnostic AKTS473 level appears to be a novel biomarker for survival in AML. Disclosures No relevant conflicts of interest to declare.
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Adema, Vera, Larrayoz Maria Jose, Calasanz Maria Jose, Laura Palomo, Ana Patiño-Garcia, Xabier Aguirre, Jesús María Hernández-Rivas, et al. "Myelodysplastic Syndromes with I(17)(q10) and Prognostic Implications of Mutations of TP53 and SETBP1." Blood 124, no. 21 (December 6, 2014): 1910. http://dx.doi.org/10.1182/blood.v124.21.1910.1910.
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Abstract INTRODUCTION Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal myeloid stem cell disorders highly prevalent in elderly populations. MDS are characterized by inefficient hematopoiesis, peripheral blood (PB) cytopenias, and increased risk of transformation to acute myeloid leukemia (AML; 20–30%). Around 50% of MDS patients carry at least one karyotoypic aberration, the most common being 5q-, -7/7q-, +8, 20q-, and isochromosome 17(q10) [i(17q)]. Isochromosome 17(q10) according to cytogenetic risk stratification is of intermediate prognostic significance when is observed as a single abnormality. As i(17q) has be postulated to be associated to recurrent mutational patterns we investigated the TP53 and SETBP1 mutational status in 31 untreated MDS patients harboring i(17q). METHODS Genomic DNA was isolated from fixed cells from bone marrow samples using QIAamp DNA mini-kit Qiagen. TP53 exons 5–9 and SETBP1 exon 3 were amplified using PCR. Amplification products were all purified and sequenced in an automated sequencer. Additionally, we studied the methylation status of TP53 in 21 of the 31 patients. Sequence analyses were performed with PyroQ-CpG analysis software. RESULTS SETBP1 mutational spectrum was characterized by the presence of non-synonmous point mutations, mainly located in residues 868-871 in 13 out of the 31 analyzed patients (41.9 %). In seven of the 13 positive cases, the mutations corresponded to heterozygous D868N, in 5 cases associated with an isolated i(17q) and with 1 additional abnormality in the remaining samples. Three of the 13 SETBP1 mutations were heterozygous G870S associated to i(17q) as a single abnormality. Another three patients had single heterozygous mutations S869G, and I871T along with an i(17q) as a single abnormality or D868Y in the context of a complex karyotype. Regarding TP53 mutations six of the 31 had non-synonymous point mutations. Two patients had mutations in exon 7, three had mutations in exons 5, 6, and 8, and one patient had an intronic mutation at the splicing recognition site. A statistically significant correlation was found between TP53 mutation and a complex karyotype (P=0.001), and between SETBP1 mutation and isolated i(17q) (P=0.001). Univariate analysis for overall survival (OS) found a statistically significant difference between non-mutated and TP53-mutated patients (14.1 months vs. 2.9 months, respectively; P=0.001), and between SETBP1-mutated and TP53-mutated patients (14.5 months vs. 2.9 months, respectively; P=0.002). Only one patient, with isolated i(17q), was found to have an aberrant TP53 methylation status. CONCLUSIONS MDS patients with i(17q) as a sole abnormality presented a higher incidence of SETBP1 mutations, whereas a higher incidence of TP53 mutations, were found in the presence of complex karyotypes. These data allowed us to conclude that a better informed and more accurate prognosis can be achieved in MDS patients with isolated i(17q) or i(17q) plus one additional abnormality, by studying the mutational status of SETBP1 as a first approach. ACKNOWLEDGEMENTS: This work has been partially supported by grants from Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain (PI 11/02010); by Red Temática de Investigación Cooperativa en Cáncer (RTICC, FEDER) (RD07/0020/2004; RD12/0036/0044); 2014 SGR225 (GRE) Generalitat de Catalunya; Fundació Internacional Josep Carreras; Obra Social “la Caixa”; Sociedad Española de Hematología y Hemoterapia (SEHH) and Celgene Spain. Disclosures Sole: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.
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Zhou, Yaqing, Yan Quan, Yijun Wu, and Yinxing Zhang. "Prenatal diagnosis and molecular cytogenetic characterization of an inherited microdeletion of 18q12.3 encompassing SETBP1." Journal of International Medical Research 50, no. 9 (September 2022): 030006052211219. http://dx.doi.org/10.1177/03000605221121955.
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The 18q12.3 region contains the SET binding protein 1 (SETBP1) gene. SETBP1 mutations or deletions are associated with Schinzel–Giedion syndrome or intellectual developmental disorder, autosomal dominant 29. We report the prenatal diagnosis and genetic counseling of a patient with a maternally inherited 18q12.3 microdeletion. In this family, the mother and son carried the same microdeletion. Chromosomal microdeletions and microduplications are difficult to detect using conventional cytogenetics, whereas the combination of prenatal ultrasound, karyotype analysis, chromosomal microarray analysis, and genetic counseling is helpful for the prenatal diagnosis of chromosomal microdeletions/microduplications.
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Jansen, Nadieh A., Ruth O. Braden, Siddharth Srivastava, Erin F. Otness, Gaetan Lesca, Massimiliano Rossi, Mathilde Nizon, et al. "Clinical delineation of SETBP1 haploinsufficiency disorder." European Journal of Human Genetics 29, no. 8 (April 19, 2021): 1198–205. http://dx.doi.org/10.1038/s41431-021-00888-9.
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Makishima, Hideki, Kenichi Yoshida, Nhu Nguyen, Bartlomiej Przychodzen, Masashi Sanada, Yusuke Okuno, Kwok Peng Ng, et al. "Somatic SETBP1 mutations in myeloid malignancies." Nature Genetics 45, no. 8 (July 7, 2013): 942–46. http://dx.doi.org/10.1038/ng.2696.
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Makishima, Hideki. "Somatic SETBP1 mutations in myeloid neoplasms." International Journal of Hematology 105, no. 6 (April 26, 2017): 732–42. http://dx.doi.org/10.1007/s12185-017-2241-1.
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Meggendorfer, Manja, Niroshan Nadarajah, Claudia Haferlach, Wolfgang Kern, and Torsten Haferlach. "Analyzing the Transcriptome Discovers up-Regulation of HOXA Genes in Patients with Myeloid Neoplasms and Isochromosome 17q and Mutations in ASXL1, SETBP1 and SRSF2." Blood 128, no. 22 (December 2, 2016): 2703. http://dx.doi.org/10.1182/blood.v128.22.2703.2703.
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Abstract Introduction: Isochromosome 17 (i(17q)) is a rare cytogenetic abnormality reported in different myeloid neoplasms. I(17q) has been described as primary and as secondary chromosomal aberration, often acquired in the disease course. Recently, we have shown that patients with i(17q) show a distinct mutation profile with mutations in ASXL1, SETBP1 and SRSF2. Further data suggested a parallel acquisition of SETBP1 mutation and i(17q) (Meggendorfer et al., Leukemia, 2016). Of note, i(17q) results in three copies of the splicing factor SRSF2, potentially further influencing the transcriptome of affected cells. Aim: To characterize the transcriptome in myeloid neoplasms with i(17q) and the typical mutations in ASXL1, SETBP1 and SRSF2 (A/S/S/i17pos). Patients and Methods: In total 18 patients were selected based on the cytogenetic profile and the molecular mutations. All had the diagnosis of a myeloid neoplasm by cytomorphology according to the WHO. Chromosome banding and FISH analysis and mutation status of ASXL1, SETBP1 and SRSF2 was available in all cases. Three patient groups were defined: 1) ASXL1, SETBP1, SRSF2 mutated and a normal karyotype (A/S/S/-pos; n=5) and gain of i(17q) during follow up, 2) ASXL1, SETBP1, SRSF2 mutated and i(17q) as sole cytogenetic abnormality (A/S/S/i17pos; n=8), 3) ASXL1, SRSF2 mutated and i(17q) as sole cytogenetic abnormality (A/-/S/i17pos; n=5). In all cases RNA sequencing was performed (TruSeq RNA Sample Preparation V2, Illumina, San Diego, CA). A control RNA (Universal Human Reference RNA, Agilent Technologies, Santa Clara, CA) was investigated in triplicate for normalization and comparison. Expression analyses were performed with BaseSpace RNA Express app (Illumina, San Diego, CA). Results: In total 23,710 genes were annotated by RNA sequencing and at least 14,773 analyzed for differential expression, showing that all three patient groups show aberrant expression in comparison to the control RNA. In mean 4,930 genes/group (range: 3,603-6,711) were differentially expressed. The differential expression ranged from -12.7 to 11.2 log2(fold change). Comparing the two groups with all three gene mutations but different i(17q) status (A/S/S/-pos and A/S/S/i17pos) showed that the presence of i(17q) changes the expression pattern with 1,596/13,218 assessed genes differentially expressed. In detail, in A/S/S/i17pos cases 790 genes were significantly over expressed while 806 genes showed reduced expression compared to A/S/S/-pos, ranging from -2.84 to 2.78 log2(fold change). The expression of SRSF2 was not affected by i(17q), although i(17q) cases show three SRSF2 gene copies. Analyzing the most strongly affected genes (2<log2(fold change)<-2, n=48) showed that differential gene expression affected mostly the homeobox (HOX) genes clustering on chromosome 7p15 (HOXA1, HOXA5 and HOXA7) as well as MEIS1, an important cofactor of HOX genes. HOX genes represent a family of transcription factors, shown to be involved in hematopoiesis. Addressing specifically the differential expression of HOX genes showed that further 4 HOXA genes (HOXA2, HOXA4, HOXA9, HOXA10) and 3 HOXB genes (HOXB2, HOXB3, HOXB4) were significantly dysregulated, with HOXB clustering on 17q21. The expression of all HOX genes was up-regulated in cases with i(17q). Interestingly, the molecular mutation pattern A/S/Spos has also been shown to associate with patients having a monosomy 7 (Meggendorfer, #1364, ASH 2013), where the HOXA gene cluster is located. Comparing the HOXA and MEIS1 gene expression in all 18 samples to human control RNA revealed a significant lower expression level in A/S/S/-pos and an increased one in A/S/S/i17pos and A/-/S/i17pos patients, clearly differentiating i(17q) carrying patients. However, the additional SETBP1 mutation did not influence the expression pattern as seen by comparing A/S/S/i17pos and A/-/S/i17pos patients (0/13,398 assessed genes differentially expressed). Conclusion: 1) Transcriptome analysis of patients with myeloid malignancies, i(17q) and mutations in ASXL1, SETBP1 and SRSF2 show an up-regulation of HOXA genes. 2) Accompanying SETBP1 mutation does not further influence the transcriptome of A/-/S/i17pos patients. 3) The up-regulation of HOXA genes might indicate a pathogenic mechanism in patients with ASXL1, SETBP1, SRSF2 and i(17q). However, this finding has to be validated in a larger cohort and with an independent method. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.
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Pastor Loyola, Victor, Pritam Kumar Panda, Sushree Sangita Sahoo, Enikoe Amina Szvetnik, Emilia J. Kozyra, Rebecca K. Voss, Dirk Lebrecht, et al. "Monosomy 7 As the Initial Hit Followed By Sequential Acquisition of SETBP1 and ASXL1 Driver Mutations in Childhood Myelodysplastic Syndromes." Blood 132, Supplement 1 (November 29, 2018): 105. http://dx.doi.org/10.1182/blood-2018-99-118910.
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Abstract Childhood myelodysplastic syndromes (MDS) account for less than 5% of pediatric hematologic malignancies and differ from their adult counterpart in terms of biology, genetics, and cure rates. Complete (-7) or partial loss (del7q) of chromosome 7 constitutes the most common cytogenetic abnormality and is associated with more advanced disease typically requiring timely hematopoietic stem cell transplantation (HSCT). Previously, we and others established a link between -7 and germline GATA2 mutations in pediatric MDS (37% of MDS/-7 cases are GATA2-deficient) as well as constitutional SAMD9/9L disorders where -7 is utilized as an escape mechanism from the growth-restrictive effect of SAMD9/9L mutations. To date, comprehensive sequencing studies have been performed in 96 children with primary MDS, as reported by Pastor et al, Leukemia 2017 and Schwartz et al, Nature Comm 2017. This work established mutations in SETBP1, ASXL1, PTPN11, RUNX1 and RAS pathway genes as common somatic drivers. However, little is known about the clonal development of -7 and the role of additional somatic mutations. The knowledge about clonal hierarchies is essential for the understanding of disease progression on molecular level and for mapping potential drug targets. The rationale for the current study was to i) define the most common somatic drivers in a large cohort of patients with childhood MDS, ii) identify clonal/subclonal mutations, iii) infer clonal architecture of monosomy 7 and track the changes over time. We studied a cohort of 576 children and adolescents with primary MDS diagnosed between 1998 and 2016 in Germany, consisting of 482 (83%) patients with refractory cytopenia of childhood (RCC) and 94 (17%) MDS with excess blasts (EB). All patients underwent deep sequencing for 30 genes relevant to pediatric MDS and additional WES was performed in 150/576 patients. Using 20 computational predictors (including CADD and REVEL), population databases and germline testing, we identified the most likely pathogenic mutations. First, we excluded germline predisposing mutations in GATA2, SAMD9/SAMD9L and RUNX1 detected in 7% (38/576), 8% (43 of 548 evaluable) and 0.7% (4/576) of patients, respectively. Then we focused on the exploration of somatic aberrations. Most common karyotype abnormalities were monosomy 7 (13%, 77/576) and trisomy 8 (3%, 17/576). A total of 104 patients carried somatic mutations, expectedly more prevalent in the MDS-EB group as compared to RCC (56%, 53/94 vs 10.6%, 51/482; p<0.0001). The most recurrent somatic hits (≥ 1% frequency within 576 cases) were in SETBP1 (4.2%), ASXL1 (3.8%), RUNX1 (3.3%), NRAS (2.9%), KRAS (1.6%), PTPN11 (1.4%) and STAG2 (1%). We next focused on the -7 karyotype as a common denominator for the mutated group. Mutations were found in 54% (43/79), and the mutational load was significantly higher in -7 vs. non-7 (1.1 vs. 0.1 mutations per patient; p<0.001). In 11 patients with -7 and concomitant SETBP1/ASXL1 driver mutations, SETBP1 surpassed ASXL1 hits (median allelic frequency: 38% vs. 24%, p<0.05), while mutations in other genes were subclonal. Notably, these clonal patterns were independent of the underlying hereditary predisposition (4/11 GATA2; 3/11 SAMD9L). To explore the clonal hierarchy in MDS/-7 we performed targeted sequencing of several hundreds of single bone marrow derived colony forming cells (CFC) in 7 patients with MDS/-7. In all cases, the -7 clone was the founding clone followed by stepwise acquisition of mutations (i.e. -7>SETBP1>ASXL1; -7>SETBP1>ASXL1>PTPN11; -7>SETBP1>ASXL1>CBL, -7>EZH2>PTPN11). Finally, we tracked clonal evolution over time in 12 cases with 2-12 available serial samples using deep sequencing complemented by serial CFC-analysis. This confirmed that SETBP1 clones are rapidly expanding, while ASXL1 subclones exhibit an unstable pattern with clonal sweeping, while additional minor clones are acquired as late events. In 2 of 11 transplanted patients who experienced relapse, the original clonal architecture reappeared after HSCT. In summary, the hierarchy of clonal evolution in pediatric MDS with -7 follows a defined pattern with -7 aberrations arising as ancestral event followed by the acquisition of somatic hits. SETBP1 mutations are the dominant driver while co-dominant ASXL1 mutations are unstable. The functional interdependence and potential pharmacologic targetability of such somatic lesions warrants further studies. Disclosures Niemeyer: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees.
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Donadoni, Carla, Rocco Piazza, Diletta Fontana, Andrea Parmiani, Alessandra Pirola, Sara Redaelli, Giovanni Signore, et al. "Evidence of ETNK1 Somatic Variants in Atypical Chronic Myeloid Leukemia." Blood 124, no. 21 (December 6, 2014): 2212. http://dx.doi.org/10.1182/blood.v124.21.2212.2212.
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Abstract Atypical Chronic Myeloid Leukemia (aCML) is a clonal disorder belonging to the Myeloproliferative/Myelodysplastic (MPN/MDS) group. The molecular lesions responsible for the onset of aCML remained unknown until 2013 when recurrent somatic mutations of SETBP1 were identified. However, the frequency of SETBP1 mutations in aCML does not exceed 25-30%, which suggests that other lesions may play a role in the remaining cases. To gain further insight into the somatic variants responsible for the onset of aCML, we generated whole-exome and transcriptome sequencing data on 15 matched case/control aCML samples. A total of 151 non-synonymous and 42 synonymous single-nucleotide somatic variants were identified. Of these, 140 were transitions and 53 transversions. Of the non-synonymous mutations, 141 were missense and 10 nonsense mutations. In 2/15 (13.3%) samples we identified the presence of missense, single-nucleotide somatic variants occurring in the ETNK1 gene affecting two contiguous residues: H243Y and N244S. Sanger sequencing confirmed the presence and the somatic nature of the variants. Targeted resequencing of 383 clonal hematological disorders showed evidence of mutated ETNK1 in 7/70 aCML (10.0%, 95% C.I. 4.6-19.5%) and in 2/77 chronic myelomonocytic leukemia samples (CMML; 2.6%, 95% C.I. 0.2-9.5%) %), while no ETNK1 mutations were identified in the remaining hematological disorders. All the variants were heterozygous and clustered in the same, highly conserved region within the kinase domain (1/9 H243Y and 8/9 N244S). Somatic, heterozygous ETNK1 variants have been also recently reported in 10% of Systemic Mastocytosis (SM) cases and in 22% of SM with associated hypereosinophilia (Lasho T et al., Abstract 4062, EHA2014); strikingly, there is a large overlap between the variants that we identified in aCML and CMML and those described for SM (3 N244S and 2 G245A), which suggests that the common hotspot region may play a critical and yet unknown functional role. The hitherto described data suggest that ETNK1 variants are restricted to a limited subset of hematological disorders. This is further supported by the lack of somatic ETNK1 mutations in 60 paired whole-genome and over 600 exomes, comprising 276 paired tumor/germline primary samples and 344 cancer cell lines (http://cancer.sanger.ac.uk/cancergenome/projects/cell_lines/). In 2/6 ETNK1 mutated aCML cases (33%, 95% C.I. 9%-70%), we detected the presence of a coexisting somatic SETBP1 variant. The fraction of SETBP1 mutations identified in this group is perfectly in line with the overall frequency of SETBP1 mutations in aCML, suggesting that mutations occurring in ETNK1 and SETBP1 are not mutually exclusive. To discriminate if ETNK1 and SETBP1 mutations occur in different or in the same clone, we performed colony assay experiments, revealing the coexistence of the two somatic mutations within the same clone. Liquid Chromatography – Mass Spectrometry experiments revealed that in ETNK1 mutated cells the intracellular levels of phosphoethanolamine are over 5-fold lower than in the wild-type counterpart (p < 0.05), suggesting that ETNK1 mutations may impair the physiological catalytic activity of the kinase. Taken globally these data identify ETNK1 somatic mutations as a new oncogenic lesion in aCML and CMML, two overlapping MDS/MPN neoplasms. They also show that ETNK1 variants apparently cause a loss-of-function effect, leading to a decrease in the intracellular levels of phosphoethanolamine. Disclosures Campbell: 14M Genomics Limited: Consultancy, Equity Ownership.
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Meggendorfer, Manja, Claudia Haferlach, Wolfgang Kern, Susanne Schnittger, and Torsten Haferlach. "The Landscape of Myeloid Neoplasms with Isochromosome 17q Discloses a Specific Mutation Profile and Is Characterized By an Accumulation of Prognostically Adverse Molecular Markers." Blood 126, no. 23 (December 3, 2015): 1656. http://dx.doi.org/10.1182/blood.v126.23.1656.1656.
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Abstract Introduction: Isochromosome 17 (i(17q)) is a rare cytogenetic abnormality resulting in the loss of the short arm and the duplication of the long arm of chromosome 17. i(17q) has been reported in different myeloid neoplasms like acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), MDS/MPN overlap, as well as in Hodgkin- and non-Hodgkin-lymphoma. i(17q) has been described both as primary and as secondary chromosomal aberration. In myeloid neoplasms i(17q) as sole abnormality is suggested to define a distinctive clinicopathological entity with a high risk to leukemic progression and poor prognosis. So far, however, it is only briefly mentioned in the WHO classification. Aim: To comprehensively characterize the molecular features of patients with myeloid neoplasms and i(17q). Patients and Methods: Patients were selected by the presence of i(17q) and diagnosis of a myeloid neoplasm. Philadelphia positive CML were excluded. The cohort comprised 62 cases, 47 males and 15 females with a median age of 69 years (range: 30 - 87 years). Classification of all cases was performed by cytomorphology on peripheral blood and/or bone marrow smears according to the WHO. Chromosome banding and FISH analysis were performed in all cases. 19/27 cases with sole i(17q) were additionally analyzed by array CGH. All 62 samples were analyzed by next generation sequencing using a 29-gene panel targeting ASXL1, BCOR, BRAF, CALR, CBL, CSF3R, DNMT3A, ETV6, EZH2, FLT3-TKD, GATA1, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NPM1, NRAS, PTPN11, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1, and WT1. Variants of unknown significance were excluded from statistical analyses (n=14). Results: Following WHO classification four of the 62 patients were diagnosed as MPN, 13 as MDS/MPN overlap, 24 as MDS, and 21 as AML. 27 cases showed i(17q) as sole abnormality, while 23 cases showed additional chromosome aberrations, and eight even a complex karyotype (>3 aberrations). Further four cases had two independent cell clones, with one harboring the sole i(17q) abnormality. Array CGH revealed that in 15/19 cases i(17q) was the only abnormality, while four patients showed additional aberrations (1-3 per patient): loss of 7p, 7q, 12p, gain of 13q, and CN-LOH 19p and 22q (n=2). The comprehensive mutational analyses revealed only 3/62 patients carrying no mutation, while a median of 3 mutations per patient was observed (range 0-6). The three most frequently mutated genes were ASXL1 (66%, 41/62), SRSF2 (65%, 40/62), and SETBP1 (48%, 30/62) with no association to any WHO entity, indicating the presence of this genetic profile also across entities beyond the expected overlap between different neoplasms. Following genes showed mutation frequencies >10%: TET2 (24%), ETV6 (16%), CBL (13%), TP53 (15%), RUNX1 (11%), and NRAS (10%), all genes known for adverse prognostic impact. Interestingly, mutations in the three most frequently mutated genes ASXL1, SRSF2, and SETBP1 often co-occurred (n=21) and ASXL1 and SRSF2 were rarely mutated alone (n=9; n=5), while SETBP1 was even never mutated solely, indicating acquirement of SETBP1 mutations during disease course. Therefore, SETBP1 mutations associated significantly with mutations in ASXL1 as well as SRSF2 (24/41 vs 6/21 in ASXL1 wild type (wt), p=0.033; 27/40 vs 3/22 in SRSF2 wt, p<0.001). Furthermore, mutations in ASXL1 associated significantly with i(17q) sole in comparison to cases with additional chromosomal aberrations (22/27 vs 19/35, p=0.032). Therefore, also cases harboring mutations in all three genes ASXL1, SRSF2, and SETBP1 associated with sole i(17q) (13/27 vs 8/35, p=0.058), indicating that these three mutations might be drivers of disease pathogenesis in this cytogenetic background. Reviewing the bone marrow morphology showed characteristic pseudo-Pelger-Huet anomaly in 36 of 59 (61%) analyzed smears. These changes were not associated with the cytogenetic profile, but showed a trend towards co-occurrence with ASXL1 mutations (27/39 vs 9/20 ASXL1 wt p=0.094). Conclusion: 1) Myeloid neoplasms with i(17q) show a distinct molecular mutation pattern, accumulating prognostically adverse mutations. 2) Patients with sole i(17q) show co-occurring mutations in ASXL1, SRSF2, and SETBP1. 3) Frequency and co-existence of ASXL1, SRSF2, and SETBP1 mutations predispose these as driver mutations. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.
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Zhao, Helong, and Michael W. Deininger. "CSF3R and SETBP1 getting high on LSD1." Blood 140, no. 6 (August 11, 2022): 529–30. http://dx.doi.org/10.1182/blood.2022016740.
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López-González, V., M. R. Domingo-Jiménez, L. Burglen, M. J. Ballesta-Martínez, S. Whalen, J. A. Piñero-Fernández, and E. Guillén-Navarro. "Síndrome Schinzel-Giedion: nueva mutación en SETBP1." Anales de Pediatría 82, no. 1 (January 2015): e12-e16. http://dx.doi.org/10.1016/j.anpedi.2014.06.017.
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Lasho, Terra L., Alice Mims, Rebecca R. Laborde, Christy Finke, Animesh Pardanani, and Ayalew Tefferi. "Chronic Neutrophilic Leukemia With Concurrent CSF3R and SETBP1 Mutations: Single Colony Clonality Studies, In Vitro Sensitivity To JAK Inhibitors and Lack Of Treatment Response To Ruxolitinib." Blood 122, no. 21 (November 15, 2013): 2830. http://dx.doi.org/10.1182/blood.v122.21.2830.2830.
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Abstract Background High frequency mutations in both the colony-stimulating factor 3 receptor (CSF3R) and SET binding protein-1 (SETBP1) have recently been described in World Health Organization (WHO)-defined chronic neutrophilic leukemia (CNL) (NEJM 2013;368:1781; Leukemia. 2013. Prepublished on 2013/04/23). Response to treatment with ruxolitinib (10-15 mg twice-daily) was also described in one patient with CSF3RT618I mutation (NEJM 2013;368:1781). Primary cells from this patient were reportedly sensitive to inhibition by ruxolitinib (IC50, 127 nM). In a report of 12 patients with WHO-defined CNL, CSF3RT618I was seen in 10 (83%) patients, four (40%) of whom also expressed SETBP1 mutations (Leukemia. 2013. Prepublished on 2013/04/23). In the current study, we investigated the clonal distribution of mutant CSF3R and SETBP1in a CNL patient expressing both mutations and describe our observations regarding the effect of JAK inhibitors, both in vitro and in vivo. Methods Under institutional review board approved protocol, peripheral blood and buccal cells were collected and density gradient centrifugation was applied to enrich for mononuclear and granulocyte cell fractions. Antibody-labeled magnetic bead separation was used to obtain CD3+ and CD34+ cell fractions. DNA sequencing was used to screen for CSF3R and SETBP1 mutations. Single colonies were obtained by plating mononuclear cells in duplicate with cytokine enriched methylcellulose with and without the addition of fedratinib (a JAK2 inhibitor) or a commercially available JAK1 inhibitor. On day 11, erythroid and granulocyte individual colonies were counted and collected at each concentration. Single colonies were screened for both SETBP1 and CSF3Rmutations. Results The study patient was a 66-year-old lady with history of radiation therapy after lumpectomy for breast cancer in 1997. In October, 2012 the discovery of three synchronous lesions in the left breast necessitated mastectomy. White blood cell count (WBC) was approximately 13 x 10(9)/L at the time. Subsequently, her WBC gradually increased to 180 x 10(9)/L. Bone marrow examination on March 15, 2013 showed predominantly granulocytic proliferation with over 95% cellularity and no dysplastic features or monocytosis. Cytogenetic studies and mutation screening for JAK2V617F and BCR-ABL1were negative. A diagnosis of CNL was made and the patient was placed on hydroxyurea therapy. i) In vitro studies The patient harbored both CSF3RT618I and SETBP1D868N mutations in granulocytes, mononuclear cells and CD34+ myeloid progenitors; neither mutation was present in buccal or CD3+ cells. Colony forming unit (CFU) assay-derived 30 single colonies (15 erythroid and 15 granulocyte) were analyzed for the presence of CSF3R and SETBP1 mutations and all (100%) harbored heterozygous SETBP1D868N and 27 (90%) heterozygous CSF3RT618I; two granulocyte colonies were wild-type and one homozygous for CSF3RT618I. Fedratinib and the JAK1 inhibitor revealed activity in suppressing colony formation by >50% at a drug concentration of ≥600 nM. Mutation analysis in post-treatment residual single colonies revealed persistence of both mutations, even under conditions of higher ambient drug concentrations. However, CSF3R unmutated colonies were more likely to emerge than SETBP1unmutated colonies after in vitro drug exposure, which resulted in the appearance of occasional colonies that were negative for both mutations. ii) Treatment with ruxolitinib Ruxolitinib treatment at 10 mg twice-daily was started on 6/10/13, in addition to 500 mg/day of hydroxyurea. At the time, WBC was 86.5 x 10(9)/L, Hgb 11.8 g/dL and platelets 122 x 10(9)/L. On 6/17/13, WBC decreased to 43.9 and hydroxyurea was held. On 6/25/13, WBC increased to 89.6 x 10(9)/L and ruxolitinib was increased to 15 mg twice-daily. On 7/15/13, WBC had further increased to 190 x 10(9)/L and hydroxyurea was added to the treatment regimen at 500 mg/day. On 8/2/13, WBC was recorded at 91.3 x 10(9)/L. Conclusions In a double mutated CNL patient, we found CSF3R and SETBP1 mutations to be myeloid cell-restricted and co-expressed in erythroid and granulocytic cells; the latter antedated the former in order of acquisition. In vitro JAK inhibition had non-selective activity in suppressing myeloid colony formation. Treatment of the study patient with single agent ruxolitinib was ineffective. Disclosures: No relevant conflicts of interest to declare.
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Makishima, Hideki. "Correction to: Somatic SETBP1 mutations in myeloid neoplasms." International Journal of Hematology 114, no. 6 (October 23, 2021): 742. http://dx.doi.org/10.1007/s12185-021-03236-1.
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Coccaro, Nicoletta, Giuseppina Tota, Antonella Zagaria, Luisa Anelli, Giorgina Specchia, and Francesco Albano. "SETBP1 dysregulation in congenital disorders and myeloid neoplasms." Oncotarget 8, no. 31 (April 19, 2017): 51920–35. http://dx.doi.org/10.18632/oncotarget.17231.
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Thol, F., K. J. Suchanek, C. Koenecke, M. Stadler, U. Platzbecker, C. Thiede, T. Schroeder, et al. "P-114 SETBP1 mutations in MDS and sAML." Leukemia Research 37 (May 2013): S75. http://dx.doi.org/10.1016/s0145-2126(13)70162-0.
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Piazza, Rocco, Simona Valletta, Nils Winkelmann, Sara Redaelli, Roberta Spinelli, Alessandra Pirola, Laura Antolini, et al. "Recurrent SETBP1 mutations in atypical chronic myeloid leukemia." Nature Genetics 45, no. 1 (December 9, 2012): 18–24. http://dx.doi.org/10.1038/ng.2495.
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Lucas, Claire M., Laura J. Scott, Natasha Carmell, Alison K. Holcroft, Robert K. Hills, Alan K. Burnett, and Richard E. Clark. "CIP2A- and SETBP1-mediated PP2A inhibition reveals AKT S473 phosphorylation to be a new biomarker in AML." Blood Advances 2, no. 9 (April 27, 2018): 964–68. http://dx.doi.org/10.1182/bloodadvances.2017013615.
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Key Points PP2A inhibition occurs in AML by 2 different pathways: CIP2A in normal karyotype patients and SETBP1 in adverse karyotype patients. AKTS473 phosphorylation is a predictor of survival, and diagnostic levels of AKTS473 could be a novel biomarker in AML.
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Meggendorfer, Manja, Tamara Alpermann, Torsten Haferlach, Carina Schrauder, Rabea Konietschke, Claudia Haferlach, Wolfgang Kern, and Susanne Schnittger. "Mutational Screening Of CSF3R, ASXL1, SETBP1, and SRSF2 In Chronic Neutrophilic Leukemia (CNL), Atypical CML and CMML Cases." Blood 122, no. 21 (November 15, 2013): 105. http://dx.doi.org/10.1182/blood.v122.21.105.105.
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Abstract Introduction Chronic neutrophilic leukemia (CNL) and atypical chronic myeloid leukemia (aCML) are rare myeloproliferative and myelodysplastic/myeloproliferative neoplasms. So far, the diagnosis of CNL and aCML has been based on cytomorphology and the absence of JAK2V617F and PDGFR rearrangements. Recently, mutations in CSF3R and SETBP1 were identified and associated with CNL and aCML, respectively. Chronic myelomonocytic leukemia (CMML) and aCML also share several characteristics and need to be discriminated especially by the absolute number of monocytes in the peripheral blood. Aim To determine the frequency of CSF3R mutations (CSF3Rmut) in CNL, aCML, and CMML and to investigate a mutation pattern, cytogenetics and clinical data in all three entities. Patients and Methods To first delineate patients with potential CNL, we investigated blood and bone marrow smears and depicted patients with a white blood cell count >25x109/L, neutrophils >80%, immature granulocytes <10%, <1% myeloblasts and hypercellular bone marrow (according to WHO 2008). BCR-ABL1 fusion transcript, JAK2 and MPL mutations were excluded in all cases by RT-PCR and melting curve analyses. Indication for PDGFR rearrangements was precluded by over-expression analyses of PDGFRA and PDGFRB by quantitative real-time PCR, resulting in a final cohort of 20 cases declared as CNL patients. Additional 60 aCML and 252 CMML patients were included. Cytogenetics was available in 330/332 cases. Mutations in CSF3R exons 14 and 17 (n=332), in ASXL1 exon 13 (n=321), and the mutational hot spots in SETBP1 (n=331) and SRSF2 (n=320) were analyzed by Sanger sequencing. Results In the total cohort of 332 patients we detected CSF3R mutations in 11 cases (3.3%). 8/11 cases showed a p.Thr618Ile mutation in exon 14, four of them carried an additional nonsense/frame-shift mutation in exon 17. One additional patient was mutated in p.Thr615Ala and showed a nonsense mutation in exon 17. Two cases showed a mutation in exon 17 only, one a nonsense the other a frame-shift mutation, respectively. Analyzing the mutation frequencies within the different entities revealed a clustering of CSF3Rmut within CNL cases with 7 of 20 (35%) mutated cases in contrast to 2 of 60 (3.3%; p=0.001) aCML and 2 of 252 (0.8%; p<0.001) CMML cases. Cytogenetics in CSF3Rmut cases showed that 9/11 cases had a normal karyotype and only one aCML patient harbored a del(3q) and one CMML patient a complex karyotype. Mutations in the three other analyzed genes ASXL1, SETBP1 and SRSF2 were detected in the total cohort in 156/321 (49%), 34/331 (10%), and 149/330 (45%) patients, respectively. Analyses regarding concomitant mutations of CSF3R with ASXL1, SETBP1 or SRSF2 revealed no additional mutation in two cases. In 8 of 11 parallel analyzed CSF3Rmut patients an ASXL1mut was identified, SETBP1 as well as SRSF2 were mutated in 3 of the 11 cases. Notably, the 7 CSF3Rmut within the CNL group had no mutation in SETBP1. Analysis of mutational loads in CNL showed that 6/7 CSF3Rmut had a higher mutational load than the second mutated gene (range: 25-50% vs. 10-30%). In one case both mutated genes had equal mutational loads (40%). In contrast, in CMML and aCML 3/4 patients had lower mutational loads in CSF3Rmut than in the additional mutated genes (20-50% vs. 40-50%), while also one case showed equal mutational loads in the mutated genes (50%). Combining the mutational results of these four genes indicate a specific and individual molecular pattern for these three different entities. While ASXL1 is frequently mutated in all entities (CNL: 8/11 (73%); aCML: 38/59 (64%); CMML: 110/251 (44%)), SRSF2 shows the highest mutation frequency in CMML cases (121/251; 48%), followed by aCML (24/60; 40%) and CNL (4/19; 21%). In contrast, SETBP1 is often mutated in aCML (19/60; 32%) and rarely in CMML (13/252; 5%) and CNL (2/19; 10.5%) patients. In addition, CSF3R is much more associated with the CNL cases (35%) and less frequently found in aCML (2%) and CMML (1%). Conclusion 1) CNL, aCML and CMML are related diseases and difficult to distinguish by cytomorphology alone and therefore require additional diagnostic criteria, i.e. molecular mutations. 2) ASXL1 is the most frequently mutated gene in these entities and thus can help to prove clonality. 3) SETBP1 much more closely relates to aCML and SRSF2 to CMML. 4) Mutations in the novel marker CSF3R are closely related to CNL and thus qualify as a new molecular marker for diagnosis of CNL. Disclosures: Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schrauder:MLL Munich Leukemia Laboratory: Employment. Konietschke:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.
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Meggendorfer, Manja, Tamara Alpermann, Claudia Haferlach, Elisabeth Sirch, Wolfgang Kern, Torsten Haferlach, and Susanne Schnittger. "Myeloid Malignancies With Isochromosome 17q Harbor Frequently Mutations In ASXL1, SETBP1, and SRSF2 - This Distinct Genotype Presents With Various Morphological Phenotypes." Blood 122, no. 21 (November 15, 2013): 1364. http://dx.doi.org/10.1182/blood.v122.21.1364.1364.
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Abstract Introduction The identification of mutations (mut) in SETBP1 recently shed light on a molecular marker in atypical chronic myeloid leukemia (aCML), a disease previously defined by exclusion criteria. SETBP1mut have been identified in different myeloid malignancies. We previously reported mutation frequencies in the range of 5-10% in MPN and MDS/MPN overlap, 32% in aCML, while we found SETBP1 less frequently mutated in AML (3%). SETBP1mut has been shown to associate with ASXL1, CBL and SRSF2 mutations, as well as the cytogenetic abnormalities -7 and i(17)(q10). Aim To investigate the mutation frequency of ASXL1, SETBP1, and SRSF2 in different myeloid entities in correlation to the cytogenetic abnormalities -7 and i(17)(q10). Patients and Methods A cohort of 451 patients (pts) with different myeloid entities was analyzed. Diagnoses according to cytomorphology followed the WHO classification from 2008 (n=439, for n=12 cases no cytomorphology was available): AML (n=29), aCML (n=62), MDS/MPN overlap (n=16), CMML (n=283), MDS (n=5), MPN (n=43), CML (n=1). The cohort consisted of 303 males and 148 females; cytogenetics was available in 445 cases. Patients were grouped by normal karyotype (n=291), i(17)(q10) (n=16), -7 (n=22), and other cytogenetic aberrations (n=117); one case carried both a i(17)(q10) and a -7. ASXL1 exon 13, the mutational hotspot regions of SETBP1 and SRSF2 were analyzed by Sanger sequencing in all cases. Results In the total cohort ASXL1 was mutated in 222/451 (49%), SETBP1 in 61/451 (14%), and SRSF2 in 209/451 (46%) cases. 137 pts showed no mutation in any of these three genes. 171 pts carried one mutation, thereof 84 a sole ASXL1mut, 82 a sole SRSF2mut and only 5 cases showed sole SETBP1mut. In 108 pts two, and in 35 pts all three analyzed genes were mutated. The most frequent combination within the group with two mutations was ASXL1 and SRSF2 (n=78), followed by ASXL1 and SETBP1 (n=16), only 5 cases were mutated in SRSF2 and SETBP1. Addressing the association with cytogenetics revealed that in cases with only one mutation SRSF2mut associated as sole mutation with a normal karyotype (68/124 (55%) SRSF2mut in the normal karyotype group vs. 12/42 (28%) SRSF2mut in all other karyotypes; p=0.003). In contrast, ASXL1mut and SETBP1mut as sole mutations showed no correlation to any addressed karyotype. However, addressing the cases with two mutations the combination of SRSF2mut and ASXL1mut correlated with a normal karyotype (67/291 (23%) SRSF2mut/ASXL1mut in the normal karyotype group vs. 19/154 (12%) SRSF2mut/ASXL1mut in all other karyotypes; p=0.008), while SRSF2mut and SETBP1mut occurred more frequently in i(17)(q10) pts (2/16 (13%) SRSF2mut/SETBP1mut in i(17)(q10) vs. 2/429 (1%) SRSF2mut/SETBP1mut in all other karyotypes; p=0.007). Remarkably, cases with mutations in all three analyzed genes (ASXL1mut, SETBP1mut, and SRSF2mut) highly associated with i(17)(q10) and -7. 11 of 16 cases with i(17)(q10) (69%) showed all three mutations (vs. 24/429 (6%) in all other karyotypes; p<0.001). Furthermore, 6 of 22 cases with -7 (27%) showed mutations in all three genes (vs. 29/423 (7%); p=0.005). Therefore, 15 pts carried all three mutated genes as well as i(17)(q10) or -7. Interestingly, there was no case with only i(17)(q10) and no additional mutation, and only one case with i(17)(q10) and only one additional molecular mutation, 4 cases with two additional molecular mutations and 11 cases carrying all three mutations, possibly indicating that i(17)(q10) appear during clonal evolution. Therefore one might assume that this represents a specific genetic phenotype that is driven by the accumulation of molecular events, since addition of SETBP1mut shifts the association from a normal karyotype to i(17)(q10) or -7. Analyzing the distribution of these cases for mutations in all three analyzed genes +/- additional cytogenetic aberration i(17)(q10) or -7 in the different myeloid entities showed that AML, aCML, CMML, MDS as well as MPN showed this genetic phenotype (AML: n=7 (24%); atypical CML: n=12 (19%); CMML: n=8 (3%); MDS: n=1 (20%); MPN: n=6 (14%)). Conclusions Mutations in SETBP1 associate with ASXL1mut and SRSF2mut and are frequently found in patients with i(17)(q10) or -7. This combination of genetic lesions occurs in different myeloid entities and might therefore define a specific genetically defined subtype of myeloid malignancy. Disclosures: Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Sirch:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.
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Visconte, Valeria, Ali Tabarroki, Li Zhang, Edy Hasrouni, Chris Gerace, Robyn Frum, Anjali S. Advani, et al. "Molecular Characterization Of Myeloid Neoplasms Harboring Isochromosome 17q Abnormality." Blood 122, no. 21 (November 15, 2013): 2596. http://dx.doi.org/10.1182/blood.v122.21.2596.2596.
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Abstract Isochromosome 17q [i(17q)], a poor prognostic cytogenetic abnormality is a product of the breakage or inappropriate division of the pericentromere leading to the duplication of the long and loss of the short arm of chromosome 17. The region of the breakpoints maps at 17p11, a region encompassing a key tumor suppressor gene: TP53. I(17q) are detected in myelodysplastic/ myeloproliferative neoplasms (MDS/ MPN), chronic myeloid leukemia (CML), and acute myeloid leukemia (AML). This abnormality can occur as a sole structural abnormality or in combination with other chromosomal defects. The presence of i(17q) is associated with poor therapeutic response, disease progression, and an unfavorable clinical outcome. Elucidation of the molecular architecture of patients (pts) carrying i(17q) may lead to better understanding of disease biology and development of novel compounds that can target this disease. We selected 11 pts with i(17q) to characterize their genomic differences. We applied whole exome sequencing (WES) in order to define latent molecular defects explaining the clinical phenotype of this disease. The index case was a male MDS/MPN pt with isolated i(17q), 27% RS, hypercellular bone marrow (BM), mild splenomegaly, and atypical megakaryocytes. The pt developed 7% BM blasts without clinical response to growth factors. Molecularly this pt was a wild type SF3B1, a gene frequently mutated in RARS-T and associated with lower transformation rate to leukemia, better survival, and good/intermediate risk cytogenetic abnormalities. WES was performed on 2 ug of total DNA extracted from BM cells. Non-clonal CD3+ cells were used as source of germ-line control. Twenty-millions reads were run on an Illumina HiSeq2000 sequencer. Using a stringent bio-informatic algorithm developed in house, all variants were filtered based on a variation score (>=30) and a coverage (30X) and the tumor nucleotide variation analysis was performed for each pair (tumor vs. germ-line), where only the variants unique to the tumor were retained. Variants were ultimately filtered in order to exclude SNPs by an in-house annotation and importing the hg19 SNP135. We detected 65 unique candidate genes. Four genes were confirmed to be somatic: 3 were novel: ZFP42 (4q35.2), P4HTM (3p21.31), and VPRBP (3p21.2) and 1 includes the newly discovered SETBP1 (18q12.3) gene. Three variants detected on the chromosome 17 had a wild type configuration. The subsequently genotyped all the pts (MDS/MPN/-U 3; AML 4; RCMD 1; CML 1; RAEB-1 2; mean age: 68 years; male/female: 8/3; i(17q)/other abnormalities:3/8) for the above genes and for a panel of genes known to be mutated in MDS/MPN and other diseases in order to find any genetic association explaining the disase phenotype. We applied Sanger sequencing to DNA derived from BM/peripheral blood cells (BM/PB:7/4) for the following genes and respective exons: TP53 (all exons), SF3B1 (13-16), SRSF2 (1-2), U2AF1 (2 and 6), TET2 (all exons), DNMT3A (18-23), IDH1/2 (4), CBL (8-9), N/KRAS (1-2), ASXL1 (12), JAK2 (12 and 14), EZH2 (16, 18 and 19), MPL (exon 10), BCAS3 (12, 15 and 16), FLT3 (11 and 17), and CSF3R (13,14, and 17). In total, we found 16 heterozygous missense mutations and 1 tandem duplication. We found somatic mutations in ZFP42, P4HTM, and VPRBP in 1 pt. The index case reported a mutation in SETBP1 and SRSF2. SF3B1 was detected as a sole abnormality in 1 patient. Of note, the patient with SF3B1 mutation (K700E) had 50% RS and achieved a complete hematologic remission after decitabine therapy. The most frequent mutations were found in SETBP1 and SRSF2. SETBP1 was found to be mutated in 4/11 (36.3%) pts (D868N, I871T, and G870S was common in 2 pts) while SRSF2 mutations (P95H/R) were found in 3/11 (27.2%) pts. Three pts showed concomitant SRSF2 and SETBP1 mutations. NRAS (G12D) was mutated in 1 pt and associated with SRSF2 and SETBP1 mutations. One pt showed mutations in TET2, JAK2, and TP53. Of note, this pt did not respond to treatment. One pt with MDS/MPN showed a mutation in CSF3R (Q741X), a novel gene discovered in chronic neutrophilic leukemia and atypical CML. The pt also has monosomy 7 and i(17q) abnormality. FLT3-ITD was found in 1 pt. As of last follow-up, only 2 pts remain alive. In sum, we found that poor risk molecular mutations in SRSF2 and SETBP1 are frequently found in i(17q) myeloid malignancies and may be the drivers of poor outcomes in this disease. Disclosures: No relevant conflicts of interest to declare.
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Hoischen, Alexander, Bregje W. M. van Bon, Christian Gilissen, Peer Arts, Bart van Lier, Marloes Steehouwer, Petra de Vries, et al. "De novo mutations of SETBP1 cause Schinzel-Giedion syndrome." Nature Genetics 42, no. 6 (May 2, 2010): 483–85. http://dx.doi.org/10.1038/ng.581.
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Inoue, D., J. Kitaura, H. Matsui, H.-A. Hou, W.-C. Chou, A. Nagamachi, K. C. Kawabata, et al. "SETBP1 mutations drive leukemic transformation in ASXL1-mutated MDS." Leukemia 29, no. 4 (October 13, 2014): 847–57. http://dx.doi.org/10.1038/leu.2014.301.
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