Bartlomiej Przychodzen

Cleveland Clinic, Cleveland, Ohio, United States

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Publications (32)282.67 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Next generation sequencing technologies have provided insights into the molecular heterogeneity of various myeloid neoplasms, revealing previously unknown somatic genetic events. In our cohort of 1444 cases analyzed by next generation sequencing, somatic mutations in the gene BRCA1-BRCA2-containing complex 3 (BRCC3) were identified in 28 cases (1.9%). BRCC3 is a member of the JAMM/MPN+ family of zinc metalloproteases capable of cleaving Lys-63 linked polyubiquitin chains, and is implicated in DNA repair. The mutations were located throughout its coding region. The average variant allelic frequency of BRCC3 mutations was 30.1%, and by a serial sample analysis at two different time points a BRCC3 mutation was already identified in the initial stage of a myelodysplastic syndrome. BRCC3 mutations commonly occurred in nonsense (n=12), frameshift (n=4), and splice site (n=5) configurations. Due to the marginal male dominance (odds ratio; 2.00, 0.84-4.73) of BRCC3 mutations, the majority of mutations (n=23; 82%) were hemizygous. Phenotypically, BRCC3 mutations were frequently observed in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms and associated with -Y abnormality (odds ratio; 3.70, 1.25-11.0). Clinically, BRCC3 mutations were also related to higher age (p=0.01), although prognosis was not affected. Knock-down of Brcc3 gene expression in murine bone marrow lineage negative, Sca1 positive, c-kit positive cells resulted in 2-fold more colony formation and modest differentiation defect. Thus, BRCC3 likely plays a role as tumor-associated gene in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms. Copyright © 2015, Ferrata Storti Foundation.
    Haematologica 05/2015; DOI:10.3324/haematol.2014.111989 · 5.87 Impact Factor
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    ABSTRACT: Most cases of adult myeloid neoplasms are routinely assumed to be sporadic. Here, we describe an adult familial acute myeloid leukemia (AML) syndrome caused by germline mutations in the DEAD/H-box helicase gene DDX41. DDX41 was also found to be affected by somatic mutations in sporadic cases of myeloid neoplasms as well as in a biallelic fashion in 50% of patients with germline DDX41 mutations. Moreover, corresponding deletions on 5q35.3 present in 6% of cases led to haploinsufficient DDX41 expression. DDX41 lesions caused altered pre-mRNA splicing and RNA processing. DDX41 is exemplary of other RNA helicase genes also affected by somatic mutations, suggesting that they constitute a family of tumor suppressor genes. Copyright © 2015 Elsevier Inc. All rights reserved.
    Cancer cell 04/2015; 27(5). DOI:10.1016/j.ccell.2015.03.017 · 23.89 Impact Factor
  • Leukemia Research 04/2015; 39:S33. DOI:10.1016/S0145-2126(15)30068-0 · 2.69 Impact Factor
  • Leukemia Research 04/2015; 39. DOI:10.1016/S0145-2126(15)30021-7 · 2.69 Impact Factor
  • Leukemia Research 04/2015; 39:S135-S136. DOI:10.1016/S0145-2126(15)30271-X · 2.69 Impact Factor
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    Dataset: JCI78789sd
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    ABSTRACT: BACKGROUND. Mutational inactivation in cancer of key apoptotic pathway components, such as TP53/p53, undermines cytotoxic therapies that aim to increase apoptosis. Accordingly, TP53 mutations are reproducibly associated with poor treatment outcomes. Moreover, cytotoxic treatments destroy normal stem cells with intact p53 systems, a problem especially for myeloid neoplasms, as these cells reverse the low blood counts that cause morbidity and death. Preclinical studies suggest that noncytotoxic concentrations of the DNA methyltransferase 1 (DNMT1) inhibitor decitabine produce p53-independent cell-cycle exits by reversing aberrant epigenetic repression of proliferation-terminating (MYC-antagonizing) differentiation genes in cancer cells. METHODS. In this clinical trial, patients with myelodysplastic syndrome (n = 25) received reduced decitabine dosages (0.1–0.2 mg/kg/day compared with the FDA-approved 20–45 mg/m2/day dosage, a 75%–90% reduction) to avoid cytotoxicity. These well-tolerated doses were frequently administered 1–3 days per week, instead of pulse cycled for 3 to 5 days over a 4- to 6-week period, to increase the probability that cancer S-phase entries would coincide with drug exposure, which is required for S-phase–dependent DNMT1 depletion. RESULTS. The median subject age was 73 years (range, 46–85 years), 9 subjects had relapsed disease or were refractory to 5-azacytidine and/or lenalidomide, and 3 had received intensive chemoradiation to treat other cancers. Adverse events were related to neutropenia present at baseline: neutropenic fever (13 of 25 subjects) and septic death (1 of 25 subjects). Blood count improvements meeting the International Working Group criteria for response occurred in 11 of 25 (44%) subjects and were highly durable. Treatment-induced freedom from transfusion lasted a median of 1,025 days (range, 186–1,152 days; 3 ongoing), and 20% of subjects were treated for more than 3 years. Mutations and/or deletions of key apoptosis genes were frequent (present in 55% of responders and in 36% of nonresponders). Noncytotoxic DNMT1 depletion was confirmed by serial BM γ-H2AX (DNA repair/damage marker) and DNMT1 analyses. MYC master oncoprotein levels were markedly decreased. CONCLUSION. Decitabine regimens can be redesigned to minimize cytotoxicity and increase exposure time for DNMT1 depletion, to safely and effectively circumvent mutational apoptotic defects. TRIAL REGISTRATION. Clinicaltrials.gov NCT01165996. FUNDING. NIH (R01CA138858, CA043703); Department of Defense (PR081404); Clinical and Translational Science Award (CTSA) (UL1RR024989); and the Leukemia and Lymphoma Society (Translational Research Program).
    The Journal of clinical investigation 01/2015; DOI:10.1172/JCI78789 · 13.77 Impact Factor
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    ABSTRACT: Interstitial deletion of the long arm of chromosome 5 (del(5q)) is the most common chromosomal abnormality in MDS. The extent of individual defects vary, which may account for observed clinical diversity. Del(5q) pathogenesis has been related to haploinsufficiency of genes contained in the common deleted regions (CDR), including RPS14, miR-145/146a and SPARC. Driver mutations or pathogenic microdeletions were not identified for these genes, suggesting that multiple genes must function in combination to promote clonal evolution and phenotypic heterogeneity. Hence, we performed a comprehensive analysis of somatic mutations in genes located on chromosome 5 (chr5), both in patients with diploid 5q and in those with del(5q), to clarify the role of germline and somatic mutations in disease pathogenesis. In parallel, expression analysis was performed to correlate haploinsufficiency with the frequency of mutational events, in particular for diploid 5q cases. Applying SNP-array karyotyping to samples from 146 patients with del(5q), the lesion was identified in 5q31.1q33.1. Two retained regions (CRRs) were also observed in q11.1q14.2 (CRR1) and q34qter (CRR2). Lower-risk MDS is frequently affected by CDR, while in higher-risk MDS and secondary AML CRR1/2 are commonly co-involved. Using whole exome sequencing, we identified 11 hemizygous mutations located within the deleted area in del(5q) (N=59), while in cases diploid for 5q (N=330), 243 heterozygous mutations were found. One of the mutations discovered on chr5q afflicted a gene G3BP1 (5q33.1), located within the CDR and present in 2 patients. Both were missense mutations (one heterozygous and the other homo/hemizygous). A mutant case showed good responses to lenalidomide even though diploid 5. In addition, other somatic mutations of driver genes including TET2, CUX1 and EZH2 were concomitantly observed. Whole transcriptome sequencing demonstrated hemizygous loss of G3BP1 resulting in haploinsufficiency. G3BP1 was haploinsufficient in 48% of RAEB as well as low-risk MDS cases with del(5q). Overall, defective G3BP1 is associated with shorter overall survival (P<.001) in AML, consistent with the reports that del(5q) is a worse prognostic factor in myeloid neoplasms with aggressive phenotype. G3BP1 is a nuclear RNA-binding protein and is ubiquitously expressed in bone marrow, CD34+ progenitors and leukemic cell lines. Furthermore, G3BP1 binds to TP53 and its expression leads to the redistribution of TP53 from the nucleus to the cytoplasm. Similar to RPS14, haploinsufficient of G3BP1 resulted in TP53 up-modulation. Moreover, low expression of G3BP1 in diploid 5q cases was indeed associated with higher TP53 expression. Next, we generated haploinsufficient G3BP1 cell lines using shRNA transduction. Decreased expression of G3BP1 led to growth inhibition and impaired colony formation by transduced cells lines and hematopoietic progenitor cells, respectively. Knockdown of G3BP1 in K562 cell line increased TP53 in the nucleus, and when treated with CPT11, DNA-damaged induced G1-arrest was more prominent in knockdown cells. Furthermore, after knockdown of G3BP1 in TP53-null HL60 cells, we observed increased aneuploidy, suggesting that the loss of function of G3BP1 and TP53 may result in chromosomal instability. Most significantly, G3bp1-/+ mice showed lower blood counts and defective, dysplastic hematopoiesis, similar to lower-risk MDS. As previously described, TP53 defects are associated with advanced disease but recently it became apparent that TP53 may be one of the most common somatic lesions found in the context of del(5q). We stipulate that loss of TP53 function might overcome TP53 tumor suppressor effects and induce leukemic evolution in the defective G3BP1 status. In our cohort, TP53 mutations were more frequently present in high-risk phenotype with G3BP1 haploinsufficient expression. In conclusion, novel somatic mutations of G3BP1 suggest that it could be a candidate gene associated with the clonal evolution of del(5q). Loss of function or low expression of G3BP1 has been shown to up-modulate TP53 and result in dysplasia and growth inhibition, hallmarks of early stages of MDS. Additional events constitute loss of function of TP53, resulting in chromosomal instability, which is associated with leukemogenesis.
    The American Society of Hematology, San Francisco, CA; 12/2014
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    ABSTRACT: The splicing factor SF3B1 is the most commonly mutated gene in the myelodysplastic syndromes (MDS), particularly in patients with refractory anemia with ring sideroblasts (RARS). We investigated the functional effects of SF3B1 disruption in myeloid cell lines: SF3B1 knockdown resulted in growth inhibition, cell cycle arrest and impaired erythroid differentiation, and deregulation of many genes and pathways, including cell cycle regulation and RNA processing. MDS is a disorder of the hematopoietic stem cell and we thus studied the transcriptome of CD34(+) cells from MDS patients with SF3B1 mutations using RNA-sequencing. Genes significantly differentially expressed at the transcript and/or exon level in SF3B1 mutant compared to wildtype cases include genes involved in MDS pathogenesis (ASXL1, CBL), iron homeostasis and mitochondrial metabolism (ALAS2, ABCB7, SLC25A37) and RNA splicing/processing (PRPF8, HNRNPD). Many genes regulated by a DNA damage-induced BRCA1-BCLAF1-SF3B1 protein complex showed differential expression/splicing in SF3B1 mutant cases. This is the first study to determine the target genes of SF3B1 mutation in MDS CD34+ cells. Our data indicate that SF3B1 plays a critical role in MDS by affecting the expression and splicing of genes involved in specific cellular processes/pathways, many of which are relevant to the known RARS pathophysiology, suggesting a causal link.Leukemia accepted article preview online, 27 November 2014. doi:10.1038/leu.2014.331.
    Leukemia 11/2014; 29(5). DOI:10.1038/leu.2014.331 · 9.38 Impact Factor
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    ABSTRACT: Paroxysmal nocturnal hemoglobinuria (PNH) is a nonmalignant clonal disease of hematopoietic stem cells that is associated with hemolysis, marrow failure, and thrombophilia. PNH has been considered a monogenic disease that results from somatic mutations in the gene encoding PIGA, which is required for biosynthesis of glycosylphosphatidylinisotol-anchored (GPI-anchored) proteins. The loss of certain GPI-anchored proteins is hypothesized to provide the mutant clone with an extrinsic growth advantage, but some features of PNH argue that there are intrinsic drivers of clonal expansion. Here, we performed whole-exome sequencing of paired PNH+ and PNH- fractions on samples taken from 12 patients as well as targeted deep sequencing of an additional 36 PNH patients. We identified additional somatic mutations that resulted in a complex hierarchical clonal architecture, similar to that observed in myeloid neoplasms. In addition to mutations in PIGA, mutations were found in genes known to be involved in myeloid neoplasm pathogenesis, including TET2, SUZ12, U2AF1, and JAK2. Clonal analysis indicated that these additional mutations arose either as a subclone within the PIGA-mutant population, or prior to PIGA mutation. Together, our data indicate that in addition to PIGA mutations, accessory genetic events are frequent in PNH, suggesting a stepwise clonal evolution derived from a singular stem cell clone.
    Journal of Clinical Investigation 09/2014; 124(10). DOI:10.1172/JCI74747 · 13.77 Impact Factor
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    ABSTRACT: Somatic cohesin mutations have been reported in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). To account for the morphologic and cytogenetic diversity of these neoplasms, a well-annotated cohort of 1060 patients with myeloid malignancies including MDS (n=386), myeloproliferative neoplasms (MPN, n=55), MDS/MPN (n=169) and AML (n=450) were analyzed for cohesin gene mutational status, gene expression, therapeutic and survival outcomes. Somatic cohesin defects were detected in 12% of patients with myeloid malignancies, while low expression of these genes was present in an additional 15% of patients. Mutations of cohesin genes were mutually exclusive and mostly resulted in predicted loss-of-function. Patients with low cohesin gene expression showed similar expression signatures as those with somatic cohesin mutations. Cross-sectional deep sequencing analysis for clonal hierarchy demonstrated STAG2, SMC3, RAD21 mutations to be ancestral in 18%, 18% and 47% of cases, respectively, and each expanded to clonal dominance concordant with disease transformation. Cohesin mutations were significantly associated with RUNX1, Ras-family oncogenes, BCOR and ASXL1 mutations and were most prevalent in high-risk MDS and secondary AML. Cohesin defects were associated with poor overall survival (27.2 vs. 40 months; p=0.023) especially in STAG2 mutant MDS patients surviving >12 months (median survival 35 vs. 50 months; p=0.017).
    Blood 07/2014; 124(11). DOI:10.1182/blood-2014-04-567057 · 9.78 Impact Factor
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    ABSTRACT: Mutations of spliceosome components are common in myeloid neoplasms. One of the affected genes, PRPF8, encodes the most evolutionarily conserved spliceosomal protein. We identified either recurrent somatic PRPF8 mutations or hemizygous deletions in 15/447 and 24/450 cases, respectively. 50% of PRPF8 mutant and del(17p) cases were found in AML and conveyed poor prognosis. PRPF8 defects correlated with increased myeloblasts and ring sideroblasts in cases without SF3B1 mutations. Knockdown of PRPF8 in K562 and CD34+ primary bone marrow cells increased proliferative capacity. Whole RNA deep sequencing of primary cells from patients with PRPF8 abnormalities demonstrated consistent missplicing defects. In yeast models, homologous mutations introduced into Prp8 abrogated a block experimentally produced in the second step of the RNA splicing process suggesting that the mutants have defects in proof-reading functions. In sum, the exploration of clinical and functional consequences suggests that PRPF8 is a novel leukemogenic gene in myeloid neoplasms with a distinct phenotype likely manifested through aberrant splicing.Leukemia accepted article preview online, 30 April 2014; doi:10.1038/leu.2014.144.
    Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 04/2014; 29(1). DOI:10.1038/leu.2014.144 · 9.38 Impact Factor
  • Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 01/2014; DOI:10.1038/leu.2014.25 · 9.38 Impact Factor
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    ABSTRACT: Granulocyte colony stimulating factor (GCSF) drives the production of myeloid progenitor and precursor cells towards neutrophils via the GCSF Receptor (GCSFR, gene name CSF3R). Children with severe congenital neutropenia chronically receive pharmacologic doses of GCSF, and ~30% will develop myelodysplasia/acute myeloid leukemia (AML) associated with GCSFR truncation mutations. In addition to mutations, multiple isoforms of CSF3R have also been reported. We found elevated expression of the alternatively spliced isoform, Class IV CSF3R in adult MDS/AML patients. Aside from its association with monosomy 7 and higher rates of relapse in pediatric AML patients, little is known about the biology of the Class IV isoform. We found developmental regulation of CSF3R isoforms with the Class IV expression more representative of a progenitor cell stage. Striking differences were found in phosphoprotein signaling involving JAK-STAT and cell cycle gene expression. Enhanced proliferation by Class IV GCSFR was associated with diminished STAT3 and STAT5 activation, yet showed sensitivity to JAK2 inhibitors. Alterations in the C-terminal domain of the GCSFR result in leukemic properties of enhanced growth, impaired differentiation, and resistance to apoptosis, suggesting that they can behave as oncogenic drivers, sensitive to JAK2 inhibition.Leukemia accepted article preview online, 30 October 2013; doi:10.1038/leu.2013.321.
    Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 10/2013; DOI:10.1038/leu.2013.321 · 9.38 Impact Factor
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    ABSTRACT: New, massively parallel sequencing technology enables, through deep sequencing of rearranged TCR Vβ CDR3 regions, a previously inaccessible level of TCR repertoire analysis. The CDR3 repertoire diversity reflects clonal composition, the potential antigenic recognition spectrum, and quantity of available T cell responses. In this context, T cell large granular lymphocyte (T-LGL) leukemia is a chronic clonal lymphoproliferation of cytotoxic T cells (CTL) often associated with autoimmune diseases and various cytopenias. Using CD8+ T-LGL leukemia as a model disease, we set out to evaluate and compare the TCR deep sequencing spectra of both patients and healthy controls to better understand as to how deep TCR sequencing could be used in the diagnosis and monitoring not only of T-LGL leukemia, but also reactive processes such as autoimmune disease and infection. Our data demonstrate, with high resolution, significantly decreased diversity of the T cell repertoire in CD8+ T-LGL leukemia, and suggest that many T-LGL clonotypes may be private to the disease and not present in the general public, even at the basal level.
    Blood 10/2013; 122(25). DOI:10.1182/blood-2013-05-506386 · 9.78 Impact Factor
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    ABSTRACT: Large granular lymphocyte leukemia (LGL) is often associated with immune cytopenias and can co-occur in the context of aplastic anemia (AA) and myelodysplastic syndromes (MDS). We took advantage of the recent description of STAT3 mutations in LGL clonal expansions to test, using sensitive methods, for the presence of these mutations in a large cohort of 367 MDS and 140 AA cases. STAT3 clones can be found not only in known LGL concomitant cases, but in a small proportion of unsuspected ones (7% AA and 2.5% MDS). In STAT3 mutated AA patients, an interesting trend toward better responses of immunosuppressive therapy and an association with the presence of HLADR15 were found. MDSs harboring a STAT3 mutant clone showed lower degree of BM cellularity and a higher frequency of developing chromosome 7 abnormalities. STAT3-mutant LGL clones may facilitate a persistently-dysregulated autoimmune activation, responsible for the primary induction of bone marrow failure in a subset of AA and MDS patients.
    Blood 08/2013; 122(14). DOI:10.1182/blood-2013-04-494930 · 9.78 Impact Factor
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    ABSTRACT: Here we report whole-exome sequencing of individuals with various myeloid malignancies and identify recurrent somatic mutations in SETBP1, consistent with a recent report on atypical chronic myeloid leukemia (aCML). Closely positioned somatic SETBP1 mutations encoding changes in Asp868, Ser869, Gly870, Ile871 and Asp880, which match germline mutations in Schinzel-Giedion syndrome (SGS), were detected in 17% of secondary acute myeloid leukemias (sAML) and 15% of chronic myelomonocytic leukemia (CMML) cases. These results from deep sequencing demonstrate a higher mutational detection rate than reported with conventional sequencing methodology. Mutant cases were associated with advanced age and monosomy 7/deletion 7q (-7/del(7q)) constituting poor prognostic factors. Analysis of serially collected samples indicated that SETBP1 mutations were acquired during leukemic evolution. Transduction with mutant Setbp1 led to the immortalization of mouse myeloid progenitors that showed enhanced proliferative capacity compared to cells transduced with wild-type Setbp1. Somatic mutations of SETBP1 seem to cause gain of function, are associated with myeloid leukemic transformation and convey poor prognosis in myelodysplastic syndromes (MDS) and CMML.
    Nature Genetics 07/2013; DOI:10.1038/ng.2696 · 29.65 Impact Factor
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    ABSTRACT: Recently, recurrent mutations of spliceosomal genes were frequently identified in myeloid malignancies, as well as other types of cancers. One of these spliceosomal genes, U2AF1, was affected by canonical somatic mutations in aggressive type of myeloid malignancies. We hypothesized that U2AF1 mutations causes defects of splicing (missplicing) in specific genes and that such misspliced genes might be important in leukemogenesis. We analyzed RNA deep-sequencing to compare splicing patterns of 201,837 exons between the cases with U2AF1 mutations (N=6) and wild-type (N=14). We identified different alternative splicing patterns in 35 genes comparing cells with mutant and wild-type U2AF1. U2AF1 mutations are associated with abnormal splicing of genes involved in functionally important pathways, such as cell cycle progression and RNA processing. In addition, many of these genes are somatically mutated or deleted in various cancers. Of note is that the alternative splicing patterns associated with U2AF1 mutations were associated with specific sequence signals at the affected splice sites. These novel observations support the hypothesis that U2AF1 mutations play a significant role in myeloid leukemogenesis due to selective missplicing of tumor-associated genes.
    Blood 06/2013; 122(6). DOI:10.1182/blood-2013-01-480970 · 9.78 Impact Factor
  • Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 06/2013; DOI:10.1038/leu.2013.179 · 9.38 Impact Factor
  • Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 06/2013; DOI:10.1038/leu.2013.164 · 9.38 Impact Factor

Publication Stats

549 Citations
282.67 Total Impact Points

Institutions

  • 2015
    • Cleveland Clinic
      Cleveland, Ohio, United States
  • 2013
    • e.t.s.v. Thor
      Cleveland, Ohio, United States
  • 2012
    • ICL
      Londinium, England, United Kingdom
  • 2010
    • Royal Holloway, University of London
      Эгхем, England, United Kingdom