Molecular Defects In BRCC3 Complex, a Novel Pathogenic Pathway In MDS

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 264-264 ◽  
Author(s):  
Dayong Huang ◽  
Hideki Makishima ◽  
Yang Du ◽  
Naoko Hosono ◽  
Wenyi Shen ◽  
...  
Keyword(s):  
Deep Sequencing ◽  
Research Funding ◽  
Chromosomal Abnormalities ◽  
Snp Array ◽  
K562 Cells ◽  
Strand Break ◽  
Clinical Analysis ◽  
Single Strand ◽  
Myeloid Neoplasms

Abstract In addition to the known cytogenetic heterogeneity of MDS, systematic application of new generation sequencing technologies (NGS) and SNP-arrays have further unraveled the complexity of MDS, revealing previously unknown somatic mutational patterns and chromosomal abnormalities. While many of the mutational events are secondary and acquired during disease progression, some may be ancestral in nature. Discovery of novel somatic defects may contribute to the understanding of molecular pathogenesis of MDS and lead to the introduction of new prognostic biomarkers or new therapeutic targets. When we performed analysis of whole exome NGS in patients with MDS and other myeloid neoplasms, including 205 from our own MDS cohort as well as 201 primary AML cases from TCGA, we noted that the BRCC3 complex gene was recurrently mutated in 9 patients (2%). These somatic mutations were confirmed by both Sanger and targeted deep sequencing. UIMC1, FAM175A, BABAM1 and FAM175B gene mutations were each found in separate single patients. The most commonly affected gene was BRCC3, found in 5 patients (1%): 1 with primary AML, 2 with CMML and 2 with MDS. There were 3 canonical mutations at exon 4 (p.R81X) and 2 in exon 1 (p.Q7X). SNP-array analysis (N=682) showed deletion of the BRCC3 complex gene in 45 patients (7%). Deletion involving the UIMC1 locus (5q35.2) and the BRCC3 locus (Xq28) were found in 31 (4.5%) and 8 (1%) patients, respectively. Evaluation of deep sequencing results demonstrates that the variant allelic frequencies of BRCC3 mutations were more than 48% in the early MDS stage, suggesting that BRCC3 mutations are initial events. The BRCC3 complex is located in the nucleus and participates in DNA double-strand break (DSB) repair. BRCC3 is a member of the JAMM/MPN+ family of zinc metalloproteases and specifically cleaves Lys-63 linked polyubiquitin chains. BRCC3 is a component of two complexes, the BRCA1-A complex and the BRISC complex. The BRCA1-A complex consists of UIMC1, FAM175A, BABAM1, BRE, BARD1, BRCC3 and BRCA1. DSB defects may be deleterious to genomic stability and instigate tumorigenesis. Clinical analysis revealed that mutations and deletions of BRCC3 complex genes were more common in MDS than in pAML (P<.01). When 684 patients genotyped for these defects were analyzed, the presence of BRCC3 complex defects was associated with shorter survival (HR=2.44 95%CI; 1.73-3.34; P<.001). The presence of BRCC3 complex gene defects affected survival in all subgroups of MDS, MDS/MPN, sAML, and pAML when analyzed separately. Overall, analysis of the additional mutational spectrum of patients with BRCC3 complex defects showed that the most frequently mutated genes were KRAS/NRAS, TET2 and U2AF1, raising the possibility of synergistic leukemogenic effects of multiple mutations. However, multivariate analysis identifies a BRCC3 complex gene defect as an independent adverse prognostic factor (HR=2.3 95%CI; 1.63-3.14; P<.001). For functional studies, we first generated a BRCC3 shRNA lentivirus vector and used it in in vitro immortalization assays utilizing a serial replating principle. BRCC3 knockdown resulted in decreased colony formation and lacked any immortalization properties. To further elucidate the functional consequences of BRCC3 lesions in the pathogenesis of MDS we performed silencing studies targeting BRCC3 in K562 cells and normal human CD34+ cells. While depletion of BRCC3 alone was not lethal, it led to enhanced etoposide-induced apoptosis. Consistent with these results, overall cell viability was substantially lower in shRNA-BRCC3–treated cells following etoposide when compared with control cells (46% vs. 55%). RPA2 expression, a single-strand DNA-binding protein used as a marker, was higher in BRCC3 knockdown cells than control cells and was further enhanced by etoposide treatment, thus indicating an increased end resection of single strand breaks. We concluded that defects in BRCC3 decrease non-homologous end joining and increased homologous recombination. In sum, our study demonstrates for the first time detection of BRCC3 complex defects, leading to impairment of DSBs repair in patients with myeloid neoplasms. Furthermore, BRCC3 defects are associated with an aggressive phenotype and shorter survival. Disclosures: Makishima: AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding. Polprasert:MDS foundation: Research Funding. Maciejewski:NIH: Research Funding; AA/MDS foundation: Research Funding.

Somatic PHF6 Mutations In Myeloid Malignancies

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2514-2514
Author(s):  
Wenyi Shen ◽  
Bartlomiej P Przychodzen ◽  
Chantana Polprasert ◽  
Naoko Hosono ◽  
Brittney Dienes ◽  
...  
Keyword(s):  
Deep Sequencing ◽  
Research Funding ◽  
Somatic Mutations ◽  
Snp Array ◽  
Illustrative Case ◽  
Sequencing Analysis ◽  
Data Set ◽  
Myeloid Malignancies ◽  
Dysmorphic Features ◽  
Myeloid Neoplasms

Abstract X chromosome genomics is an important area of hematologic malignancy research because of frequent acquired X-abnormalities, location of important genes on this chromosome, and issues surrounding Lyonization (X-inactivation). For example, we previously described somatic mutations of UTX (KDM6A), a H3K27 demethylase located on chromosome Xp11.3, in aggressive myeloid neoplasms. In a companion abstract to the the results here, we also report loss of function somatic mutations of BRCC3 (Xq28), encoding a subunit of the BRCA1-BRCA2-containing complex. In an index young female case of a proliferative CMML with dysmorphic features, we have identified PHF6 mutation mosaisism (p.K44fs), confirmed by deep sequencing of bone marrow, skin and spleen tissues. Subsequently, we screened our MDS exome project data set, involving 206 patients with MDS and related neoplasms, and have detected and confirmed additional somatic PHF6mutations. Plant homeodomain finger protein 6 (PHF6) is a ubiquitously expressed 41 kDa protein that is conserved and vertebrate-specific. Human PHF6 is located on chrXq26.3. Germline mutations of PHF6 cause Borjeson−Forssman−Lehmann syndrome (BFLS), an X-linked mental retardation disorder characterized by truncal obesity, gynaecomastia, hypogonadism and other dysmorphic features. BFLS patients have been reported to develop leukemias. More recently, rare somatic PHF6mutations were detected in patients with T-ALL, but rarely also in AML. To assess the clinical associations and significance of PHF6 mutations, we analyzed NGS results in a total of 809 patients with MDS, MDS/MPN, MPN and AML. In addition we also investigated for the presence of PHF6 mutation in the TCGA AML data sets (n=199). All mutations in our patients were confirmed by Sanger sequencing and targeted deep NGS. In total, we identified 19/809 cases with PHF6 mutations; they were located throughout the gene including 15 SNVs and 4 indels. In addition TCGA pAML NGS results revealed PHF6 mutations in 6/199 cases, including 4 SNVs and 2 indels. Thus, PHF6 mutation occurs at a frequency of 2.5% in myeloid neoplasm and are most frequently observed in pAML (36%) together with sAML (32%) phenotypes. Gender distribution showed male predominance (84%), likely related to PHF6 locus on chrXq26.3. SNP-array karyotyping showed that deletions of Xq, involving PHF6locus (Xq26) were present in about 2% of myeloid neoplasms. Chromosome 7 abnormalities, including del(7q), were the most frequent lesions seen in conjunction with PHF6 mutations. Most commonly coinciding mutations were in RUNX1 (n=8), TET2 (n=4), ASXL1 (n=3) and U2AF1 (n=3) and unbiased statistical analysis confirmed the significant association between PHF6 and RUNX1 mutations (P=.002). Interestingly, all of 8 cases with concomitant RUNX1 and PHF6 mutations were diagnosed as high-risk diseases; 1 RAEB-2 and 7 AMLs. Deep sequencing analysis of 5 cases with coexisting PHF6 and RUNX1 mutations showed that PHF6 mutated clones were always significantly larger than RUNX1 mutated clones. Such a serial clonal acquisition pattern of ancestral PHF6 and secondary RUNX1 mutations was also observed clearly in an illustrative case with evolution from aplastic anemia (AA) to sAML, in which small clone of PHF6 was detected in AA sample and expanded during MDS stage, followed by secondary driver RUNX1 mutations at the stage. These findings suggest that RUNX1 mutations were acquired as a subclone of the main population with primary driver PHF6mutations. In conclusion, our results indicate that PHF6 mutations, as a recurrent genetic abnormality, were frequently mutated in more aggressive types of myeloid malignancies. Newly identified ancestral nature of PHF6 mutations specifically favor being followed by secondary driver RUNX1 mutations during leukemic evolution. Disclosures: Polprasert: MDS foundation: Research Funding. Maciejewski:Aplastic anemia&MDS International Foundation: Research Funding; NIH: Research Funding. Makishima:Scott Hamilton CARES grant: Research Funding; AA & MDS international foundation: Research Funding.

scholarly journals RecF and RecR Play Critical Roles in the Homologous Recombination and Single-Strand Annealing Pathways of Mycobacteria

2015 ◽  
Vol 197 (19) ◽  
pp. 3121-3132 ◽  
Author(s):  
Richa Gupta ◽  
Stewart Shuman ◽  
Michael S. Glickman
Keyword(s):  
Dna Repair ◽  
Homologous Recombination ◽  
Strand Break ◽  
Single Strand ◽  
Central Position ◽  
End Joining ◽  
Dna Double Strand Break ◽  
Single Strand Annealing ◽  
Strand Annealing

ABSTRACTMycobacteria encode three DNA double-strand break repair pathways: (i) RecA-dependent homologous recombination (HR), (ii) Ku-dependent nonhomologous end joining (NHEJ), and (iii) RecBCD-dependent single-strand annealing (SSA). Mycobacterial HR has two presynaptic pathway options that rely on the helicase-nuclease AdnAB and the strand annealing protein RecO, respectively. Ablation ofadnABorrecOindividually causes partial impairment of HR, but loss ofadnABandrecOin combination abolishes HR. RecO, which can accelerate annealing of single-stranded DNAin vitro, also participates in the SSA pathway. The functions of RecF and RecR, which, in other model bacteria, function in concert with RecO as mediators of RecA loading, have not been examined in mycobacteria. Here, we present a genetic analysis ofrecFandrecRin mycobacterial recombination. We find that RecF, like RecO, participates in the AdnAB-independent arm of the HR pathway and in SSA. In contrast, RecR is required for all HR in mycobacteria and for SSA. The essentiality of RecR as an agent of HR is yet another distinctive feature of mycobacterial DNA repair.IMPORTANCEThis study clarifies the molecular requirements for homologous recombination in mycobacteria. Specifically, we demonstrate that RecF and RecR play important roles in both the RecA-dependent homologous recombination and RecA-independent single-strand annealing pathways. Coupled with our previous findings (R. Gupta, M. Ryzhikov, O. Koroleva, M. Unciuleac, S. Shuman, S. Korolev, and M. S. Glickman, Nucleic Acids Res 41:2284–2295, 2013,http://dx.doi.org/10.1093/nar/gks1298), these results revise our view of mycobacterial recombination and place the RecFOR system in a central position in homology-dependent DNA repair.

SNP Array Karyotyping Improves Detection Rate of Clonal Chromosomal Abnormalities in Refractory Anemia with Ringed Sideroblasts.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4132-4132
Author(s):  
Theodore Ghazal ◽  
Lukasz P. Gondek ◽  
Abdo S. Haddad ◽  
Karl S. Theil ◽  
Mikkael A. Sekeres ◽  
...  
Keyword(s):  
Copy Number ◽  
Chromosomal Abnormalities ◽  
Neutral Loss ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Blood Analysis ◽  
Refractory Anemia ◽  
Ringed Sideroblasts ◽  
Normal Copy Number

Abstract Among WHO low-risk categories of MDS, refractory anemia with ringed sideroblasts (RARS) can be more accurately diagnosed by characteristic pathomorphology. Clonal hematopoiesis and chromosomal abnormalities exemplify a close pathogenetic relationship to other forms of MDS. RARS shows considerable clinical variability even for patients (pts) with identical cytogenetic defects. Due to the low resolution of metaphase cytogenetics (MC) and its dependence on cell growth in vitro, this test is often non-informative in MDS. High-density SNP arrays (SNP-A) allow for a precise identification of unbalanced genomic lesions and copy-neutral loss of heterozygozity. We hypothesize that cryptic chromosomal (chr) aberrations exist in most, if not all, pts with RARS. Their detection may help to improve prognostication, distinguish distinct phenotypes and point towards unifying pathogenic defects. Initially, we analyzed the results of MC in pts with MDS and MDS/MPD (N=455) and in a sub-cohort of RARS, RCMD-RS, RARSt and other MDS subtypes with >15% RS. When we compared pts with/without RS, chr defects were found at comparable frequencies (∼50%). The most commonly occurring defects associated with RS, compared to other forms of MDS, included those of chr 5 (9% vs. 16%, 7 (8% vs. 12%) and 20 (3% vs. 8%). DNA was available for 36 pts with RS and was subjected to 250K SNP-A karyotyping. Pathologic lesions were defined upon exclusion of normal copy number polymorphisms identified in 81 controls (O’Keefe at al ASH 2007), as well as the Database of Genomic Variants (http://projects.tcag.ca/variation). By MC, a defective karyotype was present in 16/36 pts (44%). Deletions involving chr 5, 7 and complex MC were found in 3, 5, and 2pts, respectively. However, when SNP-A was applied as a karyotyping tool (copy number and LOH analysis), all aberrations found by MC were confirmed, but also new lesions were detected so that an abnormal karyotype was established in 62% of pts. Several previously cryptic/recurrent lesions included losses of a portion of chr. 2 (N=2; 2p16.2, 2p16.3), and deletions (N=4; 7p11.1–14.1, 7p21.3, 7q11.23–21.11, 7q21.12-qter) as well as gains (N=1; 7q33) on chr 7. We have also detected segmental uniparental disomy (UPD) in chr 1 (N=2; 1p21.3–22.2, 1p). This type of lesion cannot be detected using MC and provides an additional mechanism leading to LOH. When both bone marrow and blood of 5 RARS patient were tested using SNP-A, blood analysis had 100% accuracy rate as compared to marrow; all defects seen in the marrow were also found in blood. We conclude that chromosomal defects are present in a majority of RARS patients and arrays with higher resolution will identify defects in most, if not all of the patients. Our study also demonstrates testing of peripheral blood by SNP-A can complement marrow MC, especially in cases in which marrow is not available. Detection of clonal marker aberrations in blood of RARS patients suggests that mostly clonal dysplastic progenitor cells contribute to blood production rather than residual “normal” progenitors.

ATP Dependent Efflux Transporters ABCB1 and ABCG2 Are Unlikely to Impact the Efficacy, or Mediate Resistance to the Tyrosine Kinase Inhibitor, Ponatinib

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2745-2745 ◽  
Author(s):  
Deborah L. White ◽  
Liu Lu ◽  
Timothy P. Clackson ◽  
Verity A Saunders ◽  
Timothy P Hughes
Keyword(s):  
Tyrosine Kinase ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Kinase Inhibitor ◽  
K562 Cells ◽  
Temperature Dependent ◽  
Advisory Committees ◽  
Significant Difference

Abstract Abstract 2745 Ponatinib is a potent pan-BCR-ABL tyrosine kinase inhibitor (TKI) currently in a pivotal phase 2 clinical trial. Ponatinib (PON) was specifically designed to target both native and all mutant forms of BCR-ABL, including T315I. The phase I study of oral ponatinib in patients with refractory CML/ALL or other hematologic malignancies recently reported that 66% and 53% of patients with CP-CML achieved MCyR and CCyR respectively (Cortes et al., ASH 2011 abstract #210). While extensive modelling experiments in BaF3 cells have been performed characterising in vitro response to ponatinib, little is known about the interactions of this drug and drug transporters that impact the response of other tyrosine kinase inhibitors (TKIs). To explore this we have examined both the degree of in vitro kinase inhibition mediated by ponatinib in BCR-ABL+ cell lines, and the intracellular uptake and retention (IUR) of ponatinib achieved. The IC50 was determined by assessing the reduction in %p-Crkl in response to increasing concentrations of ponatinib in vitro. The IUR assay was performed as previously using [14-C]-ponatinib. To determine the role of ABCB1 and ABCG2, both previously implicated in the transport of other TKIs, IC50 analysis was performed on K562 cells, and variants; ABCB1 overexpressing K562-DOX and ABCG2 overexpressing K562-ABCG2. As shown in Table 1, in contrast to the results previously observed with imatinib (IM), nilotinib (NIL) and dasatinib (DAS) there was no significant difference in the IC50ponatinib between these three cell lines, suggesting neither ABCB1 nor ABCG2 play a major role in ponatinib transport. Furthermore, the addition of either the ABCB1 and ABCG2 inhibitor pantoprazole, or the multidrug resistance (MDR) inhibitor cyclosporin did not result in a significant change in the IC50ponatinib in any of the cell lines tested. In contrast the addition of either pantoprazole or cyclosporin resulted in a significant reduction in IC50IM, IC50NIL. and IC50DAS of K562-DOX cells, supporting the notion that these TKIs interact with ABCB1.Table 1:The IC50 of ponatinib (compared to IM, NIL and DAS) in K562 cells and the over-expressing variants DOX and ABCG2 in the presence of the ABC inhibitors pantoprazole and cyclosporin. n=5. *p<0.05IC50% reduction in IC50+ pantoprazole+ cyclosporinPON (nM)IM (μM)NIL (nM)DAS (nM)PONIMNILDASPONIMNILDASK5627.793751111544*NA−107NA2DOX7.919*598*100*1018*63*1655*88*ABCG26.4730025*6NA To further examine the effect of ABC transporters on ponatinib efflux we have determined the IUR of [14-C]-ponatinib in K562, DOX and ABCG2 cell lines. We demonstrate no significant difference in the IUR between these cell lines at 37°C (n=6) (K562 vs DOX p=0.6; K562 vs ABCG2 p=0.37 and DOX vs ABCG2 p=0.667 at 2uM respectively). Temperature dependent IUR experiments reveal a significant reduction in the ponatinib IUR at 4°C compared to 37°C in K562 cells (n=6) (p=0.008), DOX cells (p=0.004) and ABCG2 cells (p=0.002) supporting the likely involvement of an ATP/temperature dependent, and yet to be determined, component of ponatinib influx. There was no significant difference in the IUR between these cell lines at 4°C (p=0.824, p=0.7 and p=0.803 respectively). Importantly, these data are consistent with the IC50ponatinib findings. If ATP dependent efflux pumps (ABCB1 and ABCG2) were actively transporting ponatinib, a significant decrease in IUR in DOX and ABCG2 at 37°C compared to K562 cells would be expected, but is not observed here. Analysis of ponatinib IUR in the prototypic ABCB1 over-expressing CEM-VBL100 cells, and their parental, ABCB1 null counterparts (CCRF-CEM) further confirmed these findings. The IUR in VBL100 cells was significantly higher than that observed in CEM's (p<0.001; n=5), providing further evidence that ponatinib was not being exported from the cell actively via ABCB1. These data suggest that the transport of ponatinib is, at least in part, temperature-dependent indicating a yet to be determined ATP transporter may be involved in the transport of ponatinib into leukaemic cells. Importantly, this data suggests that ponatinib is unlikely to be susceptible to resistance via the major ATP efflux transporters (ABCB1 or ABCG2) that have been previously demonstrated to significantly impact the transport of, and mediate resistance to other clinically available TKIs. Disclosures: White: BMS: Honoraria, Research Funding; Novartis Pharmaceuticals: Honoraria, Research Funding. Clackson:ARIAD: Employment. Hughes:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; ARIAD: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Whole Exome Sequencing Detecting Kinesin Family Gene Defects In Myeloid Neoplasm

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2762-2762
Author(s):  
Chantana Polprasert ◽  
Hideki Makishima ◽  
Bartlomiej P Przychodzen ◽  
Naoko Hosono ◽  
Wenyi Shen ◽  
...  
Keyword(s):  
Research Funding ◽  
Somatic Mutations ◽  
Snp Array ◽  
Chromosome 17 ◽  
Therapeutic Window ◽  
Myeloid Malignancies ◽  
Myeloid Neoplasm ◽  
Myeloid Neoplasms ◽  
Whole Exome ◽  
Kinesin Family

Abstract Clinical and pathomorphologic diversity in MDS is a reflection of heterogeneity of molecular lesions. Somatic mutations and chromosomal deletions/amplifications affect various pathways in a convergent and divergent fashion, generate phenocopy and can occur in a variety of combinations. Recent technological advances, including high density arrays and the new generation sequencing (NGS) led to the discovery of novel pathway mutations or gene families affected by somatic defects, e.g., cohesin or spliceosomal mutations. We have performed whole exome NGS of paired (tumor/germ line) samples in 222 patients with myeloid neoplasms from the Cleveland Clinic and University of Tokyo. Clinical parameters were studied including age, gender, overall survival (OS), bone marrow blast count, and metaphase cytogenetics. Additionally, we also used in our analysis data sets from 197 AML included in the Cancer Genome Atlas (TCGA). We found 1.4% (6/419) of non-canonical somatic mutations of KIF2Bwhich is a member of kinesin13 family located on the long arm of chromosome 17; 3 cases from our cohort (p.V32M (c.G94A), p.T113M (c.C338T), p.R163C (c.C487T)) and 3 cases from TCGA database (p.T47M (c.C140T), p.T310M (c.C929T), p.H551N (c.C1651A)). By analyzing clonal architecture and intra-tumor heterogeneity in 2 cases (RCMD and RAEB) by targeted deep sequencing, allelic frequencies of KIF2B mutations were more than 45% and larger than for any other concomitant mutations, suggesting that KIF2B mutations might consequently constitute ancestral events followed by subclonal acquisitions of the other mutations. Of note is that 6 non-sense mutations were also reported in lung cancer. Based on SNP-array mapping of chromosomal abnormalities, deletions of 17q involving the KIF2B locus (17q22) was present about 3% (6/215) of myeloid neoplasm. KIF2B defects were frequently detected in higher-risk MDS and AML phenotypes (9%). KIF2B performed an important role in regulation of kinetochore-microtubule attachment. Previous studies showed that the velocity of chromosomes’ movement in KIF2B-deficient cells is reduced 80% comparing to control and fail to perform cytokinesis. In our series, 56% of myeloid neoplasms with KI2B defects had complex cytogenetics and 67% cases of them were also UPD, suggesting that KIF2B defects might lead to inducing abnormal chromosomal movements and segregations. We then, expanded our study to the whole kinesin gene family: 17 somatic mutations and 57 deletions were identified in KIF1A (n=6), KIF23 (n=1), KIF26A (n=1), KIF27 (n=7), KIF1C (n=9), KIF21B (n=2), KIF13A (n=10), KIF14 (n=2), KIF17 (n=15), KIF25 (n=1), KIF3C (n=8), KIF6 (n=2) and CENPE (n=10). All mutations were heterozygous and mutually exclusive. By survival analysis of such mutated cases, a tendency towards worse prognosis was observed (HR; 1.72, 95%CI 0.86-3.37). Analysis of concomitant mutations associated with whole kinesin family mutations or deletions showed that most frequently affected genes are TET2 (n=14), DNMT3A (n=8), IDH1/2 (n=8) and MLL (n=5), all involved in epigenetic regulation. In conclusion, somatic mutations in kinesin family genes are found in myeloid malignancies and might be responsible for another pathogenesis of the disease. KIF2B is most frequently found in myeloid malignancies and associated with aggressive type of MDS. Since knockout mice of multiple kinesin family genes (KIF5A, KIF16B and EG5) were lethal in embryo and all the mutations occur in a heterozygous configuration, it is likely synthetic lethal approach might create therapeutic window between defective malignant cells and healthy controls. Kinesin family of motor proteins may be an emerging novel therapeutic target. In fact some kinesins have been already successfully targeted in solid tumors. Disclosures: Polprasert: MDS foundation: Research Funding. Makishima:AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding. Maciejewski:NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding.

Dasatinib-Induced Reduction of Tumor Growth Is Accompanied By the Changes in the Immune Profile in a Melanoma B16.OVA Mouse Model

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1408-1408
Author(s):  
Mette Matilda Ilander ◽  
Can Hekim ◽  
Markus Vähä-Koskela ◽  
Paula Savola ◽  
Siri Tähtinen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Cd8 T Cells ◽  
Research Funding ◽  
K562 Cells ◽  
Cell Cytotoxicity ◽  
Cd8 Cells

Abstract Background: Dasatinib is a 2nd generation tyrosine kinase inhibitor (TKI) used in the treatment of chronic myeloid leukemia (CML). Its kinase inhibition profile is broad and includes several kinases important in the immune cell function such as SRC kinases. Furthermore, it is known that dasatinib has immunomodulatory effects in vivo. Recently, we observed that dasatinib induces a rapid and marked mobilization of lymphocytes, which closely follows the drug plasma concentration. The phenomenon is accompanied by an increase of NK-cell cytotoxicity. In addition, we have shown that dasatinib alters T-cell responses long-term favoring Th1 type of responses. Interestingly, the dasatinib induced immune effects have been associated with better treatment responses. We now aimed to characterize the dasatinib-induced antitumor immune responses in a syngeneic murine melanoma model to address whether dasatinib-induced immunoactivation affects tumor growth. Methods: Direct cytotoxic effect of dasatinib on B16.OVA melanoma cells in vitro was assessed with an MTS cell viability assay. T-cell cytotoxicity was assessed by preincubating splenocytes isolated from naïve and OT-I mouse spleen with 100 nM dasatinib and measured their cytotoxic capacity against B16.OVA cells. To further evaluate the dasatinib induced antitumor immune effects in vivo, B16.OVA cells were implanted subcutaneously in C57BL/6J mice. The mice (n=6/group) were treated daily i.g. either with 30 mg/kg dasatinib or vehicle only. Blood was collected before tumor transplantation, before treatment, and on treatment days 4, 7 and 11. Tumor volumes were measured manually and specific growth rate was calculated based on the first and the last day of the treatment. In addition to white blood cell differential counts, immunophenotyping of blood and tumor homogenate was performed by flow cytometry using antibodies against CD45.1, CD3, CD4, CD8b, NK1.1, CTLA4, PD-1 and CD107. Immunohistochemical staining of CD8+ T-cells was performed from the paraffin embedded tumor samples. Results: In vitro incubation of B16.OVA cells with dasatinib showed only a moderate unspecific cytotoxicity with the two highest concentrations of dasatinib (1- and 10 µM), whereas in K562 cells (a CML blast crisis cell line) almost complete killing was observed already with the 100nM concentration. The cell viability of B16.OVA cells was 90% with at 100 nM of dasatinib concentration (as compared to 21% of K562 cells) suggesting that there was no direct dasatinib sensitive target oncokinase in this cell line. In contrast, a significant enhancement in the cytotoxic capacity of splenocytes was observed when they were pretreated with 100nM dasatinib (60% of target cells were alive when incubated with dasatinib pretreated naïve splenocytes compared to 100% with control treated splenocytes, p=0.004). The in vivo tumor experiments demonstrated that the tumor volumes were smaller in dasatinib group, and there was a significant decrease in the specific tumor growth rate (0.06 vs. 0.18, p=0.01) on the 11th day of treatment. Interestingly, dasatinib treated mice had increased proportion of CD8+cells in the circulation (17.9% vs. 14.4%, p=0.005) and the CD4/CD8 ratio was significantly decreased (1.39 vs. 1.52, p= 0.04). During the tumor growth the mean CTLA-4 expression on CD8+ cells in PB increased from 1.2% to 9% in the control group, whereas, in dasatinib group the increase was more modest (1.2% to 5.7%). When the tumor content was analyzed, dasatinib treated mice had significantly more tumor infiltrated CD8+ T-cells (median 17 vs. 4/counted fields, p=0.03). In dasatinib group 80% of the tumor infiltrating CD8+ cells expressed PD-1 antigen compared to <5% of PD1 positive CD8+ cells in the peripheral blood suggesting either tumor induced CD8 T-cell exhaustion or the presence of tumor-reactive effector cells. Lastly, when CD4 and CD8 cells were depleted before tumor inoculation, dasatinib was no longer able to slow down the tumor growth. Conclusions: Dasatinib treatment slowed the tumor growth in a B16.OVA mouse model. The growth retardation was due to immunomodulatory properties of dasatinib as the drug was not directly cytotoxic and depletion of T-cells abolished the effect. Dasatinib may be a therapeutically useful immunomodulatory agent for targeting tumor-associated anergy, particularly in combination with novel checkpoint inhibitors and tumor-targeting drugs. Disclosures Hemminki: Oncos Therapeutics Ltd: Shareholder Other; TILT BioTherapeutics Ltd: Employment, Shareholder, Shareholder Other. Porkka:BMS and Novartis: Honoraria, Research Funding; Pfizer: Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

scholarly journals Interleukin 1 Beta As a Promising Therapeutic Target in ABL Tyrosine Kinase Inhibitor Resistant Chronic Myeloid Leukemia Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4249-4249
Author(s):  
Seiichi Okabe ◽  
Tetsuzo Tauchi ◽  
Yuko Tanaka ◽  
Kazuma Ohyashiki
Keyword(s):  
Gene Expression ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Kinase Inhibitor ◽  
Interleukin 1 ◽  
K562 Cells ◽  
Abl Tyrosine Kinase ◽  
Abl Tyrosine Kinase Inhibitor ◽  
Resistant Cells

Abstract Introduction: Although ABL tyrosine kinase inhibitor (TKI), imatinib, nilotinib and dasatinib have demonstrated the potency against chronic myeloid leukemia (CML) and Philadelphia chromosome (Ph) positive acute lymphoblastic leukemia (Ph+ALL) patients, resistance to ABL TKI can develop in the many patients. It has already reported that ABL kinase domain mutations have been implicated in the pathogenesis of ABL TKI resistance, however, it is fully not known the molecular mechanism of drug resistance ABL TKIs. Therefore, new approach against ABL TKI resistant cells may improve the outcome of Ph-positive leukemia patients. Interleukin-1 (IL-1) is a proinflammatory cytokine and central mediator of innate immunity. IL-1β also controls essential cell responses. Because enhanced IL-1β signaling is reported in patients of hematological malignancies, IL-1β may be the promising therapeutic value in ABL TKI resistant CML patients. Materials and methods: In this study, we established ABL TKI-resistant in vitro cell line models (K562 imatinib-R, K562 nilotinib-R, K562 dasatinib-R, K562 ponatinib-R). We also investigated whether IL-1β was involved in ABL TKI resistant Ph-positive leukemia cells and cytokines were induced by IL-1β in human umbilical vein endothelial cells (HUVEC). Results: We analyzed the relationship of IL-1β signaling pathways and ABL TKI sensitivity by microarray gene expression data from the online Gene Expression Omnibus (GEO). IL-1β is related to imatinib sensitivity and resistant in CML patients from the public microarray datasets of GSE14671. We next examined ABL TKI resistant cell lines (K562 imatinib-R, K562 nilotinib-R, K562 dasatinib-R, K562 ponatinib-R) in this study. BCR-ABL point mutation was not found in ABL TKI resistant cells. BCR-ABL expression levels were not increased in ABL TKI resistant K562 cells. These cells were highly resistant to ABL TKIs compare to K562 cells (K562 imatinib-R: imatinib 2μM, nilotinib-R: nilotinib 2μM, dasatinib-R: dasatinib 100nM, ponatinib-R: ponatinib 50nM). We investigated gene expression profiles in cultured ABL TKI resistant K562 cells by DNA microarray. We found gene expression of IL-1β and IL-1β mediated signaling pathway was increased ABL TKI resistant K562 cells. IL-1β gene amplification was confirmed by RT-PCR analysis. Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-kB) acts as a central mediator of inflammatory responses. Because bortezomib is a proteasome inhibitor whose anti-cancer action is partly mediated through inhibition of NF-κB, we examined the bortezomib in ABL TKI resistant cells. Combined treatment of ABL TKI resistant cells with ponatinib or imatinib and bortezomib caused more cytotoxicity than each drug alone. Caspase 3/7 activity and cellular cytotoxicity was also increased. ABL TKIs are also associated with vascular adverse events (VAEs) in CML, we next investigated the in vitro effects of ABL TKIs on cultured HUVEC. We found gene expression of IL-1β was increased after ABL TKI especially dasatinib and ponatinib treatment. IL-1β was increased in the cell culture supernatant after ABL TKIs treatment. In the immunoblot analysis, p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) phosphorylation was increased by IL-1β in the time dependent manner. We also found that gene expression of IL-6, IL-8, intercellular adhesion molecule 1 (ICAM1) and monocyte chemotactic protein-1 (MCP-1) was enhanced by IL-1β stimulation. Conclusion: The IL-1β signaling pathway is involved in ABL TKI sensitivity and drug resistant in CML cells and plays a key role in cytokine production of the HUVEC. We also provide the promising clinical relevance as a candidate drug for treatment of ABL TKI resistant leukemia patients. Disclosures Ohyashiki: Asahikase: Research Funding; Taiho Pharmaceutical KK: Honoraria, Research Funding; Dainippon Sumitomo KK,: Honoraria, Research Funding; MSD,: Honoraria, Research Funding; Bristol Meyer Squibb KK,: Honoraria, Research Funding; Ono Pharmaceutical KK,: Honoraria, Research Funding; Celegene KK,: Honoraria, Research Funding; Pfizer KK,: Honoraria, Research Funding; Kyowakko Kirin KK,: Research Funding; Nihon-Seiyaku,: Research Funding; Eizai,: Research Funding; Chugai KK,: Honoraria, Research Funding; Takeda Pharmaceutical KK,: Honoraria, Research Funding; Asteras KK,: Research Funding; Jansen Pharma KK,: Research Funding; Nippon-shinyaku,: Honoraria, Research Funding; Novartis KK,: Honoraria, Research Funding.

scholarly journals BCL-2 Inhibitor Venetoclax Enhances Temozolomide Sensitivity in AML

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2646-2646
Author(s):  
Joseph M Brandwein ◽  
Asmaa Basonbul
Keyword(s):  
Western Blot ◽  
Cell Lines ◽  
Drug Combination ◽  
Research Funding ◽  
Annexin V ◽  
Complete Response ◽  
Strand Break ◽  
Single Strand Break ◽  
Mgmt Expression

Introduction: Many older patients with acute myeloid leukemia (AML) are ineligible for intensive chemotherapy due to frailty and co-morbidities; for such patients, existing treatments are often ineffective and new treatments are needed. Temozolomide (TMZ) is an alkylating agent that causes DNA methylation at O6 guanine, generating single strand break leading to apoptosis. However, the efficacy of TMZ depends on the DNA repair protein O6-methylguanine methyltransferase (MGMT), that maintains the genomic integrity by removing the O6-methyl group and restoring guanine nucleobase, thereby enhancing resistance to TMZ. Previous clinical trials in AML found that responses to TMZ correlated with low MGMT expression; however, even in those with low MGMT expression complete response rates were only in the 25% range. BCL-2, an anti-apoptotic protein, is overexpressed in AML cells. Direct inhibition by the selective BCL-2 inhibitor venetoclax (Venet) promotes apoptosis. This study evaluated the ability of Venet to enhance TMZ sensitivity in AML cells, including those with MGMT overexpression. Methods: KG1, MV4-11 and MOLM13 AML cell lines were studied, as well as bone marrow blast cells collected from AML patients. Western blot was used to measure MGMT and BCL-2 expression. The cells were incubated with TMZ at varying concentrations in combination with a fixed concentration of Venet. After 48 hours, cell viability and apoptosis assays were performed using spectrophotometry and flow cytometry, respectively. Synergy was evaluated by the Chou-Talalay method. Cleaved-PARP was measured by Western blot in selected combination doses after 3 hours in MV4-11 and MOLM13 and after 6 hours in KG1. Results: KG1 cells expressing high MGMT demonstrated strong resistance to TMZ; however, co-incubation with 1 uM Venet resulted in a marked enhancement of sensitivity to TMZ. Similarly, in MV4-11 and MOLM13 cell lines, which demonstrated very low or absent MGMT expression. Venet 2.5 nM in combination with TMZ markedly increased the cytotoxicity to TMZ. A synergistic effect was demonstrated in all cell lines with combination index (CI) < 1. Cells overexpressed annexin V and propidium iodide (PI) apoptotic marker after drug combination in all cell lines. Apoptotic effect with the drug combination was verified by cleaved-PARP expression. Most (6/8) AML patient samples which were resistant to TMZ in vitro became sensitized to TMZ in combination with 1 uM Venet, including those with moderate to high MGMT expression. Conclusion: Venetoclax synergizes with TMZ and induces cytotoxicity in all AML cell lines and in most AML patient samples, including those in whom MGMT was highly expressed, by activating apoptotic pathways to trigger cell death. This combination represents a potentially promising new treatment. Further studies evaluating this combination in animal models are in progress. Disclosures Brandwein: Roche: Research Funding; Novartis: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Otsuka: Honoraria; Jazz Pharma: Consultancy, Honoraria. OffLabel Disclosure: Temozolomide for AML

Novel Pathogenic Defects Of Dead/H-Box Helicases In Myeloid Neoplasms

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 655-655
Author(s):  
Chantana Polprasert ◽  
Mikkael A. Sekeres ◽  
Hideki Makishima ◽  
Bartlomiej P Przychodzen ◽  
Naoko Hosono ◽  
...  
Keyword(s):  
Cell Lines ◽  
Research Funding ◽  
Snp Array ◽  
Rna Helicase ◽  
Differential Sensitivity ◽  
Mrna Levels ◽  
Rna Helicases ◽  
Early Presentation ◽  
Myeloid Neoplasms ◽  
Lower Expression

Abstract Familial MDS is rare and usually associated with early presentation in childhood. We encountered identical twins presenting with concordant MDS (RCMD-subtype) and a strong family history of leukemia. Their diseases demonstrated several unifying features, including a clinical response to lenalidomide despite neither twin expressing the del(5q) abnormality, resulting in transfusion-independence and restoration of normal counts. Whole exome sequencing (WES) for confirmatory deep targeted sequencing identified the same somatic heterozygous missense mutation of DDX41, c.G1574A (p.R525H) in both cases. In addition, we found diversifying accessory somatic mutations; in case 1 the clonal architecture consisted of DDX41, PHF6 (c.59delG, p.C20fs) and DNMT3A (c.T1180C, p.C394R) in 45%, 14%, and 15% reads, respectively, while in the 2nd case the clone consisted of 37% DDX41 and 50% JAK2 (c.G1849T, p.V617F) mutant reads. We also found a rare non-synonymous alteration of DDX41, c.T1187C (p.I396T) present in the germline DNA in both twins (the prevalence of this germline minor allele in the general population is <.05%). DDX41 is a member of the DEAD-box helicase family, the largest of the helicase families. RNA helicases are involved in RNA metabolism, spliceosomal function, ribosome biogenesis, pre-mRNA splicing and translation initiation. To assess the clinical importance of DDX41 and other RNA helicase mutations, we screened a cohort of 763 patients (pts) with myeloid neoplasms and also analyzed the TCGA AML cohort. We found 1.04% (N=10/960) of DDX41 mutations among MDS/AML pts and other RNA helicases mutations including DDX23 as follows : 0.47% (N=2/419), DDX4 : 0.24% (N=1/419), DDX54 : 0.1% (N=1/960) and DHX33 : 0.48%(N=2/419) were demonstrated. We also found 2 rare germline events of DHX29: c.G1627A, p.V543M and c.G1561A, p.E521K. We further focused on the most prevalent DDX41 lesions located on the long arm of chromosome 5. SNP-array identified deletions of 5q involving the DDX41 locus (5q35.3) in 16% of cases. Expression analysis found that DDX41 mRNA levels were significantly lower in cases with del(5q) affecting the DDX41 locus than in those without deletions (P=.0004). The majority of cases with the DDX41 defect showed advanced disease (higher-risk MDS and sAML). Cases with lower expression of DDX41 showed a significantly worse OS than those with higher expression (HR=1.6, 95%CI=1.04-2.19). As both twins showed a remarkable responsiveness to lenalidomide despite lack of del(5q), we analyzed an additional 94 pts (MDS,MPN,MDS/MPN) who received lenalidomide for the presence of DDX41. In lower-risk group pts, those who demonstrated DDX41 locus deletions/DDX41 mutations showed a significantly higher sensitivity to lenalidomide than those who did not (P=.024). When we compared expression levels of DDX41 in responders (n=10) and refractory patients (n=9), lenalidomide responders showed significantly lower expression of DDX41 (P=.048), indicating a relationship between DDX41 and lenalidomide sensitivity. To study the biological consequences of DDX41 low expression, we knocked down DDX41 expression in leukemic cell lines (HL-60 and K562 cell lines) via lentiviral transduced shRNA. Significantly higher proliferation rates were observed in both DDX41 knockdown cell lines compared to control. Based on a synthetic lethal approach, we used the DDX41 knockdown cells to test the effects of helicase inhibitors (C14H15N3O): DDX41 low expressing cells showed differential sensitivity to helicase inhibitors as compared with control cells. In sum, DDX41 mutant or haploinsuffcient cases are associated with MDS (as a secondary driver) and may signal lenalidomide responsiveness. DDX41 and other helicases represent a novel class of genes found to carry mutations in MDS. DDX41 and possibly other helicases may be targeted by RNA helicase inhibitors. Disclosures: Polprasert: MDS foundation: Research Funding. Makishima:Scott Hamilton CARES grant: Research Funding; AA & MDS international foundation: Research Funding. Maciejewski:NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding.

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