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Journal articles on the topic 'Chromosome de levure'
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1
Baby, Vincent, Fabien Labroussaa, Carole Lartigue, and Sébastien Rodrigue. "Chromosomes synthétiques." médecine/sciences 35, no. 10 (October 2019): 753–60. http://dx.doi.org/10.1051/medsci/2019153.
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Depuis les dix dernières années, les techniques de synthèse et d’assemblage d’ADN se sont grandement améliorées. La construction de molécules d’ADN synthétiques devient maintenant beaucoup plus simple et abordable de sorte qu’il est possible de reconstruire des chromosomes synthétiques complets. Nous assistons donc aux débuts de la génomique synthétique, qui vise la construction de génomes conçus sur mesure pour l’étude et l’utilisation de systèmes biologiques. De la synthèse des premiers génomes viraux jusqu’à la reconstruction des seize chromosomes de la levure, en passant par la première cellule bactérienne contrôlée par un génome entièrement synthétique, nous discutons des découvertes majeures, des aspects réglementaires et éthiques ainsi que du potentiel de cette nouvelle discipline pour le futur.
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ROGEL-GAILLARD, C. "Les banques de grands fragments d’ADN." INRAE Productions Animales 13, HS (December 22, 2000): 79–85. http://dx.doi.org/10.20870/productions-animales.2000.13.hs.3815.
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Les banques de grands fragments d’ADN constituent depuis une quinzaine d’années une avancée technologique remarquable pour l’étude des génomes complexes. En effet, chaque fragment cloné, jusqu’à 2 mégabases dans des chromosomes artificiels de levures (YAC) et 200 kilobases dans des chromosomes artificiels de bactéries (BAC), représente une petite région chromosomique qui contient un ou plusieurs gènes avec des éléments de régulation proximaux et distaux, ainsi que des marqueurs associés. Des collections de clones représentatives d’un génome entier ont été produites. Ces banques de grands fragments d’ADN sont intensivement utilisées pour la cartographie intégrée des génomes et sont les éléments fondateurs des programmes de grand séquençage, dont celui de l’Homme. Les clones de grands fragments d’ADN constituent, de plus, un matériel puissant pour élaborer les outils et concepts de la génomique fonctionnelle de demain.
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Nowell, Peter C., and Carlo M. Croce. "Philip Levine Award Lecture: Chromosome Translocations and Oncogenes in Human Lymphoid Tumors." American Journal of Clinical Pathology 94, no. 2 (August 1, 1990): 229–37. http://dx.doi.org/10.1093/ajcp/94.2.229.
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Labroussaa, Fabien, Vincent Baby, Sébastien Rodrigue, and Carole Lartigue. "La transplantation de génomes." médecine/sciences 35, no. 10 (October 2019): 761–70. http://dx.doi.org/10.1051/medsci/2019154.
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Le développement de la génomique synthétique (GS) a permis l’élaboration d’outils et de méthodes innovantes permettant la synthèse, l’assemblage et la modification génétique précise de chromosomes bactériens complets. La raison principale de ce succès, ayant abouti à la création de la première cellule synthétique quasi-minimale JCVI-syn3.0, est l’utilisation de la levure Saccharomyces cerevisiae comme hôte temporaire d’accueil et de modification de ces génomes. Cependant, une autre technique a joué un rôle considérable dans le succès retentissant de ces travaux : la transplantation de génomes bactériens (TG). Cette technique, encore mal comprise, permet d’installer des génomes complets naturels ou synthétiques dans un contexte cellulaire favorable à leur expression et donner la vie. Une meilleure compréhension du processus de TG permettrait d’élargir l’ensemble des techniques de GS, appliquées actuellement quasi exclusivement à l’étude des mycoplasmes, à de nombreuses autres bactéries d’intérêt, y compris des bactéries génétiquement non-modifiables à ce jour.
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Wu, Yingying, Xiujin Ye, Li Li, Jingjing Zhu, Wanzhuo Xie, Jie Zhang, Jingsong He, Zhen Cai, and He Huang. "Clinical Characterization and Prognostic Factors of 144 Adult Acute Monocytic Leukemia." Blood 120, no. 21 (November 16, 2012): 4798. http://dx.doi.org/10.1182/blood.v120.21.4798.4798.
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Abstract Abstract 4798 Background: Acute monocytic leukemia (M5) is one kind of acute leukemia with poor prognosis, which has the characterization of high number of white blood cells, high risk of extramedullary metastasis and abnormal chromosome karyotype. Objective: To analyze clinical characterization and prognostic factors of adult acute monocytic leukemia. Methods: Clinical data of 144 patients diagnosed between Jan 1st 2006 and Dec 31 2010 were collected. Clinical manifestation, cytogenetics, immunophenotyping and minimal residual disease (MRD) were analyzed respectively. The numeration data was tested by χ2 test, while the measurement data by Levene test to variance homogeneity and by T test to the mean difference. The data were analyzed by SPSS 17.0. Results: 81.9% of 144 patients reached complete remission. 27.5% patients were abnormal in cytogenetics, including +8, −5/del (5q), −7/del (7q). Patients were divided into three groups according to chromosome karyotype. Intermediate prognostic group has better OS and DFS than poor prognostic group (P<0.05). 26 patients treated with SCT.These patients have higher OS rate for one year and low relapse rate than those who did not receive SCT.CR rate for old patients(≥55) and young patients (<55) were 69.4% and 86.1% respectively. Old patients had shorter OS and DFS than young patients, but no singnificant difference in FAB type, number of white blood cell and immunophenotyping. Old patients occur chromosome abnormalities more frequently than young patients. Relapse rate is 20.7% in MRD+ group versus 11.5% in MRD- group. Multivariate COX analysis showed that chromosome karyotype and MRD had most significant correlation with relapse. Conclusion: Chromosome karyotype, age, therapy, MRD were all prognostic factors of M5. Chromosome karyotype and MRD had most important significance in prognosis. Disclosures: No relevant conflicts of interest to declare.
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Triche, Tim, Stephen Capone, Akil Merchant, Preet Chaudhary, and Giridharan Ramsingh. "DNA Methylation Changes In Aging Human CD34+ Cells Coincide With Hotspots Of Disordered Methylation In AML At Imprinted and Allelically Methylated Regions." Blood 122, no. 21 (November 15, 2013): 1193. http://dx.doi.org/10.1182/blood.v122.21.1193.1193.
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Abstract Aging of the hematopoietic system in humans is associated with increased incidence of myeloid malignancies. Epigenetic machinery such as DNA methylation is known to change with age, and is disproportionately impacted by recurrent genetic mutations in acute myeloid leukemia (AML). We hypothesized that epigenetic changes in CD34+ hematopoietic stem and progenitor cells (HSPCs) may precede recurrent genetic changes in AML, and might be detected in normal aging HSPCs with increasing frequency. We also hypothesized that areas with increased variability in methylation may be hot-spots for the emergence of epigenetically distinct HSPC clones. In order to characterize these changes, we performed methylome-wide profiles of human HSPCs from different age groups (20-25 years (10), 40-45 years (10) and >60 years (9)).Adult HSPCs were obtained from Leukocyte Reduction System cones from healthy platelet donors. CD34+ cells were then isolated by Fluorescence Assisted Cell Sorting. 200 ng of DNA extracted from the CD34+ cells was processed using the Infinium Methylation 450k Beadchip (Illumina). Differentially methylated regions (DMRs) were identified using the bump hunting procedure of Jaffe (2011) to pool information across CpG loci into regions of consistent change and to quantify statistical significance. 893 differentially methylated regions (DMRs) varied linearly with age in HSPCs; a set of 31 such regions yielded an accurate predictor of age in lineage-sorted cells (N=48, Reinius et al., 2012) and whole blood (N=656, Hannum et al., 2011), with a root-mean-squared error of 5.3 years. While age-related lymphopenia has previously been reported, DNA methylation marks for lineage commitment (Houseman et al., 2012) were nearly uniform within our subjects’ CD34+ cells, and exhibited no relationship with age. However, regional summaries of methylation provided more accurate age predictions than specific CpG loci. We reasoned that differential variability at individual loci might be the cause. We thus investigated regions where methylation variability increased with age. Known imprinted clusters and allelically methylated regions (AMRs) identified by Fang (2012, PNAS) were disproportionately represented among these; 27% of known imprinting regions and 33.3% of allelically methylated regions in the genome coincided with at least one such region, while comprising only 0.3% of the genome and 0.7% of loci assayed. Among these, the H19 imprinting control region has been shown to crucially regulate long-term HSPC homeostasis in mice via IGF2, and the allelically methylated WT1/WT1-AS region on chromosome 11p is a highly recurrent hotspot for disordered methylation in AML, as well as sequential epigenetic defects in Wilms’ tumor. The allelically methylated vault RNA VTRNA2-1 (recently shown to predict survival in AML) on chromosome 5q, and the monoallelically expressed TP73 and DIRAS3 genes on chromosome 1p, were also sites of greater methylation variability with age in normal HSPCs. Wu et al. (1997) showed that loss of imprinting at H19/IGF2 is common in AML, seemingly conferring a selective metabolic advantage, and global loss of imprinting in mice leads to widespread tumorigenesis (Holm et al., 2005). Recurrent methylation aberrations in induced pluripotent stem cells favor imprinted clusters (Nazor, 2012), and epigenetic polymorphisms arise in these regions over time in cultured cells (Tanay et al, 2012). However, to our knowledge, ours is the first report of this type of heterogeneity with age in normal human adult HSPCs. Clonal hematopoiesis has previously been documented in healthy elderly adults (Levine 2012), and the majority of patients in the Cancer Genome Atlas (TCGA) AML study exhibited mutations in one or more genes regulating epigenetic machinery. We propose that increased epigenetic heterogeneity in aging HSPCs, particularly at regions with allele-specific methylation (such as H19/IGF2), may precede malignant evolution in some leukemias, and warrants further investigation. Disclosures: No relevant conflicts of interest to declare.
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Malska, A. A., and O. B. Kuryliak. "Tetrallogy of Fallot and hypertrophic cardiomyopathy. Unusual association." UKRAINIAN JOURNAL OF PERINATOLOGY AND PEDIATRICS, no. 2(90) (June 30, 2022): 59–64. http://dx.doi.org/10.15574/pp.2022.90.59.
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Tetrallogy of Fallot (ToF) is the most widespread congenital heart defect characterized by a wide anatomic spectrum and clinical manifestations, which depend on the degree of stenosis of the pulmonary artery, and can be associated with chromosomal abnormalities. Hypertrophic cardiomyopathy (HCM) is a rare genetic disorder that often occurs in the autosomal-dominant type and has a high risk of cardiac death and is associated with abnormalities in certain gene loci. Clinical case. We present this rare association in a 9-month-old girl, without previous history of heart defect, who was admitted to intensive care unit with a clinical presentation of severe hypoxic spell. A rare case of the combination of ToF and HCM has been reported in a 9-month-old child admitted to the reanimation department of the Lviv Regional Children’s Clinical Hospital «OKHMATDYT» for malignant and cyanotic attacks. The girl was hospitalized for reanimation in a jaundice-cyanotic crisis with a saturation rate of 44%. On examination, a pronounced sciatica, pallor and cyanosis of the lips were detected. Auscultatively, the heart tones were rhythmic, muffled, the heart rate - 202 beats per second, systolic murmur was detected on the left side of the chest 5/6 on the Levine scale. Electrocardiogram showed signs of systolic hypertension and hypertrophy of the right ventricle. 2D-echocardiographic examination revealed hypertrophy of the left and right ventricular walls and interventricular septum as well as signs of ToF. On the magnetic resonance imaging the diagnosis was confirmed. After the stabilization of the general condition the child was transported to the Center of Pediatric Cardiology and Cardiac Surgery in Kyiv for surgical treatment, where it was recommended to continue the medical treatment with beta-blockers. After 2 months, the 1-year-old child was operated on routinely - radical correction of ToF, the post-operative state was good. Conclusions. Association of ToF and HCM is extremely rare. ToF is often associated with chromosomal aberration, while hypertrophic cardiomyopathy associates with certain gene loci. Surgical treatment of ToF associated with HCM differs greatly from surgical treatment of usual ToF and physiology of both conditions have to be considered prior to the surgery, as combination of ToF and HCM is associated with high postoperative mortality, as the LVOT progresses and increased risk of development of ventricular arrhythmias and heart failure develops. The association between ToF and HCM is extremely rare. ToF is associated with chromosomal abnormalities, whereas HCM is associated with abnormalities in certain gene loci. The prognosis for patients with TF and HCM is associated with high postoperative mortality due to progression of obstruction of the left ventricular tract, development of ventricular arrhythmias and cardiovascular failure during the postoperative period. The research was carried out in accordance with the principles of the Helsinki Declaration. The informed consent of the patient was obtained for conducting the studies. No conflict of interests was declared by the authors. Key words: Tetrallogy of Fallot, hypertrophic cardiomyopathy, cyanotic spell, echocardiography, children.
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Levine, Fayola, Emmanuel Asante-Asamani, Gargi Pal, Michael Liss, and Olorunseun Ogunwobi. "Abstract C060: Investigating the clinical relevance in prostate cancer of the serum biomarkers PVT1 exons 4A, 4B and 9 across risk levels and ethnicity/race." Cancer Epidemiology, Biomarkers & Prevention 32, no. 1_Supplement (January 1, 2023): C060. http://dx.doi.org/10.1158/1538-7755.disp22-c060.
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Abstract Chromosome 8q24 is of particular importance for cancer susceptibility. Located in this region is the Plasmacytoma Variant Translocation 1 (PVT1) gene, a long noncoding RNA that has been implicated in multiple cancers including prostate cancer. Amplification of the PVT1 gene locus is a common event in many malignant diseases and is associated with poor clinical outcomes. The pioneering role of PVT1, and its alternatively spliced transcripts, as a cancer biomarker is progressively becoming established. We have demonstrated that copy numbers of PVT1 exons 4A, 4B and 9 is quantifiable in cancer cells, tissue, and serum from cancer patients. In this study, we assessed clinically annotated serum samples from 40 prostate cancer patients to investigate the clinical relevance of PVT1 exons 4A, 4B, and 9 as a biomarker across cancer risk levels and ethnicity/race. Explorative data analysis for the development of composite score for prostate cancer was performed using Kruskal-Wallis Rank Sum Test. We observed significantly higher copy numbers of PVT1 exons 4B and 9 across all races (White, Black and Hispanic) and Blacks and Hispanics when compared to the control. Additionally, using a 3-level cancer risk rating assessment in which 0 = healthy, 1 = low risk and 2 = high risk, we observed that PVT1 exon 9 may distinguish between cancerous and noncancerous cases across all races, but may not help distinguish between indolent and aggressive cancer cases. Notably, PVT1 exon 4B may help distinguish between indolent and aggressive cancer cases for Blacks and Hispanics. The results of this study suggest that using PVT1 exon 4B or 9 may identify cancer regardless of ethnicity/race, and that utilization of serum PVT1 exon 4B copy number may help distinguish between indolent and aggressive prostate cancer in Blacks and Hispanics. Citation Format: Fayola Levine, Emmanuel Asante-Asamani, Gargi Pal, Michael Liss, Olorunseun Ogunwobi. Investigating the clinical relevance in prostate cancer of the serum biomarkers PVT1 exons 4A, 4B and 9 across risk levels and ethnicity/race [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C060.
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Kinnaman, Michael David, Julia Pena, Max Levine, Nancy Bouvier, Elli Papaemmanuil, Suzanne Forrest, Katherine Janeway, Paul Meyers, and Julia Glade Bender. "Abstract 2590: Analysis of homologous recombination deficiency (HRD) scores in osteosarcoma." Cancer Research 82, no. 12_Supplement (June 15, 2022): 2590. http://dx.doi.org/10.1158/1538-7445.am2022-2590.
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Abstract Objective: Outcomes for patients with metastatic or relapsed osteosarcoma (OS) remain poor. Many OS samples have demonstrated a high proportion of mutational signature 3 (ms3), suggestive of homologous recombination DNA repair deficiency (HRD) or a BRCAness molecular phenotype. The HRD score, which quantifies chromosomal structural rearrangements by combining loss of heterozygosity (LOH), large state transitions (LST), and telomere allelic imbalances (TAI), has been used to assess HRD status in patients with breast and ovarian cancer, with high scores found to be predictive of PARP inhibitor sensitivity. While high HRD scores have been described in OS, little is known about how HRD scores vary by stage, site, pre vs post-therapy, presence of homologous recombination pathway or TP53 alterations, and their correlation to ms3 contribution. Methods: To address this issue, we evaluated 48 OS samples with tumor purity ≥ 20% from 46 patients who had 80x whole genome sequencing (WGS) performed through our pediatric expanded genomics platform. HRD scores were then estimated using scarHRD, a computational tool, which quantifies LOH, LST, and TAI events from WGS data and has shown a good correlation to SNP-array based methods. Results: Of the 48 samples analyzed, 25 samples were from primary sites and 23 from metastatic sites of disease. The median HRD score was 45 with a minimum score of 6 and a maximum score of 79. The 25th, 75th, and 90th percentiles of values were 35.7, 57, and 66.3 respectively. There was a significant difference in HRD score in patients with localized disease (n=22) vs metastatic disease (n=26) at time of diagnosis with median HRD (mHRD) scores of 41 and 51.5 (p = 0.027). The presence of a TP53 structural variant or single nucleotide variant (mHRD=47) was associated with higher HRD scores than TP53 wild-type samples (mHRD=29.5, p=0.04). There was no statistically significant difference in HRD scores in pre vs post-treated samples or primary site vs metastatic site samples. Samples with homologous recombination pathway alterations (n=10) were not associated with increased HRD score. There was a positive correlation (Spearman’s rho=0.425, p=0.001) between the number of mutations attributed to ms3 and HRD score. Conclusions: The range and distribution of HRD scores in our OS cohort closely resemble BRCA1/2 deficient breast cancer. Patients with metastatic disease at diagnosis were found to have higher HRD scores, representing a higher degree of genomic instability and increasing potential to accumulate resistance mutations. Higher HRD scores in TP53 mutated samples may be due to chromosomal instability caused by mechanisms other than HRD. The BRCAness phenotype in OS is now being targeted in a phase II clinical trial, combining the PARP inhibitor olaparib with the ATR inhibitor ceralasertib (NCT0441706), which includes integrated correlative biology to prospectively assess biomarkers of response such as HRD score. Citation Format: Michael David Kinnaman, Julia Pena, Max Levine, Nancy Bouvier, Elli Papaemmanuil, Suzanne Forrest, Katherine Janeway, Paul Meyers, Julia Glade Bender. Analysis of homologous recombination deficiency (HRD) scores in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2590.
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Viny, Aaron D., Christopher J. Ott, Barbara Spitzer, Martin A. Rivas, Cem Meydan, Efthymia Papalexi, Dana Yelin, et al. "Dose-Dependent Role of the Cohesin Complex in Normal and Malignant Hematopoiesis." Blood 126, no. 23 (December 3, 2015): 435. http://dx.doi.org/10.1182/blood.v126.23.435.435.
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Abstract Cohesin complex members have recently been identified as putative tumor suppressors in hematologic and epithelial malignancies. The cohesin complex guides chromosome segregation, however cohesin-mutant leukemias do not show genomic instability suggesting an alternate role in malignant transformation. We hypothesized reduced cohesin function alters chromatin structure and disrupts cis-regulatory architecture of hematopoietic stem/progenitor cells. We therefore investigated the impact of both complete loss and haploinsufficiency of Smc3, an obligate member of the cohesin complex, in normal hematopoiesis and in myeloid transformation by developing a conditional Smc3 knockout allele. Somatic loss of Smc3 in hematopoietic cells induced lethal bone marrow aplasia (median survival 11 days; p<0.001), with premature sister chromatid separation and abnormal nucleolar organization. Competitive transplant assays showed that Smc3 loss completely abrogated stem cell self-renewal in vivo. These data are consistent with an absolute requirement for the cohesin complex in hematopoietic stem/progenitor cells. By contrast, Smc3 haploinsufficiency increased self-renewal in vitro and in vivo, with increased serial replating, expanded hematopoietic stem/progenitor cells, and a self-renewal/engraftment advantage in competitive transplantation assays in vivo (Figure a). Smc3 haploinsufficiency altered coordinated transcriptional output, including reduced expression of master regulatory transcription factors governing lineage commitment. Consistent with these data, Smc3 loss resulted in expanded Cd150+ Cd48+ ST-HSC (p=0.008), reduction in Cd150+ Cd48- LT-HSC (p=0.001), and altered chromatin architecture with dysregulated expression of genes with specific chromatin architecture footprints. Smc3 haploinsufficiency cooperated with Flt3ITD to induce acute leukemia in vivo (Figure b), with dysregulated expression of hematopoietic master regulators and altered nucleolar topology similar to that observed in germline cohesinopathy syndromes and in AML patients with cohesin mutations (Figure c). To further explore the mechanism by which Smc3 loss cooperates with Flt3ITD to induce leukemia, we investigated chromatin cis-regulatory architecture with transposase hypersensitivity assays (ATAC-seq). We hypothesized that increased accessibility at cis-regulatory elements and the alterations in gene expression seen in cells with combined Smc3 haploinsufficiency and Flt3ITD may be in a large part driven by potentiated Stat signaling at chromatin. We analyzed 146 transcription factor recognition motifs within the THS differentially observed in Smc3Δ/+Flt3ITD and wild-type cells. Chromatin accessibility gained in Smc3Δ/+Flt3ITD cells are enriched in Stat family transcription factor binding sites, including Stat5. We also observed enrichment of the Stat5 gene expression signature in the Smc3Δ/+Flt3ITD cells compared to Smc3Δ/+, Flt3ITD and wild-type cells, suggesting the divergent mutations cooperate to potentiate oncogenic Stat5 signaling in HSPCs. Our results demonstrate a key dose-dependent role for the cohesin complex in hematopoiesis, and show that reduced cohesin functions to alter enhancer-mediated transcription and contribute to aberrant self-renewal and myeloid transformation. Figure 1. Figure 1. Disclosures Levine: Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy.
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Lu, Zhanping, Anna L. F. V. Assumpção, Aaron D. Viny, Ross L. Levine, and Xuan Pan. "YY1 Controls Hematopoietic Stem Cell Quiescence By Repressing Cohesin Expression." Blood 132, Supplement 1 (November 29, 2018): 3831. http://dx.doi.org/10.1182/blood-2018-99-118679.
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Abstract Hematopoietic stem cells (HSCs) are undifferentiated, self-renewing, pluripotent cells that have the capacity to differentiate into all mature lineage-specific cells in adult blood. Adult HSCs can remain in a quiescent state for a prolonged time, and quiescence is a fundamental characteristic of HSCs in adult bone marrow. Thus, the cell cycle must be precisely regulated. Yin Yang 1 (YY1) is a multifunctional transcription factor and Polycomb Group Protein (PcG) that is important for embryonic development, adult hematopoiesis, cell proliferation and maintaining higher-order chromosomal structure. We have generated YY1 conditional knockout mice (Yy1f/f Mx1-Cre) and showed that Yy1 deficient HSCs fail to self-renewal and had disrupted HSCs quiescence. Stem cell factor (SCF)/c-Kit signaling, a critical regulatory pathway in HSC development, is significantly downregulated in Yy1-/- HSCs. Interestingly, YY1 regulation of HSCs self-renewal and quiescence is independent on its PcG domain/function. Instead, YY1 occupied at Smc3 promoter area and repressed Smc3 expression. In Yy1-/-HSCs, Smc3 expression was upregulated. SMC3 is a core component of cohesin protein complex and plays critical roles in HSC self-renewal, myeloid differentiation and leukemogenesis. To further dissect the underlying mechanisms by which YY1 regulates SMC3 expression in HSCs, we have generated conditional knockout mice with YY1 homozygous deletion and SMC3 heterozygous deletion (Yy1f/f Smc3f/+Mx1-Cre). In Yy1-/- Smc3+/- bone marrow cells, SMC3 expression was normalized to the wild-type level. In adult bone marrow cells, YY1 physically interacted with cohesin complex proteins through its zinc finger domain. By analyzing the YY1, SMC1A and SMC3 ChIP-Seq database, our study showed that YY1 and cohesin co-occupied at promoter areas of genes that are critical for cell metabolism. Evidence from previous study showed that impaired metabolism, including increased reactive oxygen species (ROS) and decreased mitochondrial function, can cause defect in stem cell self-renewal and quiescence. In Yy1-/- Smc3+/-HSCs, cell quiescence was restored although HSC self-renewal was still impaired, indicates that YY1 and SMC3 may control HSC cell quiescence via regulating genes critical for cell metabolism. Our study identified YY1 as the first transcription factor that regulates expression of cohesin complex component SMC3. We are currently further dissecting underlying mechanisms and functional significances of metabolic pathways regulated by YY1-SMC3 axis in HSCs. Disclosures Levine: Imago: Equity Ownership; Isoplexis: Equity Ownership; Janssen: Consultancy, Honoraria; Gilead: Honoraria; Epizyme: Patents & Royalties; Loxo: Consultancy, Equity Ownership; C4 Therapeutics: Equity Ownership; Roche: Consultancy, Research Funding; Prelude: Research Funding; Qiagen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Novartis: Consultancy.
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Lavallee, Vincent-Philippe, Elham Azizi, Vaidotas Kiseliovas, Ignas Masilionis, Linas Mazutis, Ross L. Levine, and Dana Pe'er. "Comprehensive Single-Cell RNA-Sequencing Mapping of Primary Acute Myeloid Leukemias and Profiling of NPM1-Mutated Cells." Blood 132, Supplement 1 (November 29, 2018): 995. http://dx.doi.org/10.1182/blood-2018-99-111390.
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Abstract Introduction: Acute myeloid leukemia (AML) evolution is a multistep process in which cells evolve from hematopoietic stem and progenitor cells (HSPCs) that acquire genetic anomalies, such as chromosomal rearrangements and mutations, which define distinct subgroups. Mutations in Nucleophosmin 1 (NPM1), which occur in ~30% patients, are the most frequent subgroup-defining mutations in AML and appear to be a late driver event in this disease. Bulk RNA-sequencing studies have identified differentially expressed genes between AML subgroups, but they are uninformative of the composition of cell types populating each sample. Large scale Single-cell RNA sequencing (scRNA-seq) technologies now enable a detailed characterization of intra tumoral heterogeneity, and could help to better understand the stepwise evolution from normal to malignant cells. Methods: Twelve primary human AML specimens from MSKCC and Quebec Leukemia Cell Bank, including 8 with NPM1 mutations, were included in this cohort. Cells were subjected to scRNA-seq using 10X Genomics Chromium Single Cell 3' protocols and libraries were sequenced on Illumina HiSeq or NovaSeq platforms. FASTQ files were processed using SEQC pipeline (Azizi E et al, Cell 2018), resulting in a carefully filtered count matrix of > 100,000 single cells (4877 to 11532 cells per sample). Results: Using euclidean distance metrics and t-Distributed Stochastic Neighbor Embedding (t-SNE) visualization, we explored the phenotypic overlap between samples and showed that leukemia cells from different patients were mostly dissimilar, suggesting inter-sample heterogeneity. However, samples with similar morphology and similar NPM1 mutational status were phenotypically closer (Fig A), as anticipated from bulk RNA-sequencing data (TCGA, NEJM 2013). We partitioned cells into distinct clusters using Phenograph (Levine J et al, Cell 2015) (Fig B) and measured the diversity of samples per cluster using Shannon's entropy metric, revealing that mature cell types (B/plasma cells, T/NK and erythroid cells, Fig C), presumably excluded from the tumor bulk, are transcriptionally similar across samples. Most notably, the next most diverse cluster (C36), comprising 438 cells from 11/12 samples, contains cells with a HSPC-like phenotype, as suggested by i) highest correlation of the centroid of this cluster with HSC1 (lin-/CD133+/CD34dim) population from sorted bulk RNA-sequencing data (Novershtern N et al, Cell 2011), and ii) marked GSEA enrichment for stem cell signatures (top enrichment: Jaatinen_hematopoeitic_stem_cell_up, NES = 9.04, FDR q-val = 0). To study the extent to which NPM1 or other mutations drive heterogeneity in leukemia populations, we interrogated 3'-derived single-cell sequences for all recurrent mutations in AML and found that NPM1 gene has unique features (e.g. relatively high single-cell expression and 3' localization) that allow specific identification of mutations in 5 to 34% of cells per mutated sample. To control for the high frequency of false negatives caused by dropouts in scRNA-seq data, we normalized the abundance of mutated vs wild-type cells to provide an estimation of mutation frequency in different cell types (Fig D). As expected, NPM1 mutations were rare in B and T/NK lymphoid cells (also observed using RT-qPCR in sorted populations by Dvorakova D et al, Leuk Lymphoma 2013) and were found in the majority of leukemia and myeloid cells. Interestingly, these mutations were detected at various frequencies in erythroid cells, suggesting that NPM1 mutations are acquired in cells with different lineage commitment in different patients. Most notably, the HSPC-like cluster C36 also contained a subpopulation of cells that have acquired NPM1 mutations and are transcriptionally different from wild-type cells. Conclusion: This study presents a first comprehensive single-cell map of primary AML, and the first 3'-based interrogation of mutations in single cells. It led to the identification phenotypically distinct cells presenting a HSPC-like expression profile which were sub-clonally harboring NPM1 mutations, providing the means to identify deregulated genes in these important leukemia subpopulations. Figure Figure. Disclosures Levine: Epizyme: Patents & Royalties; Celgene: Consultancy, Research Funding; Janssen: Consultancy, Honoraria; Isoplexis: Equity Ownership; C4 Therapeutics: Equity Ownership; Prelude: Research Funding; Gilead: Honoraria; Imago: Equity Ownership; Novartis: Consultancy; Roche: Consultancy, Research Funding; Loxo: Consultancy, Equity Ownership; Qiagen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
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Tenedini, Elena, Isabella Bernardis, Valentina Artusi, Lucia Artuso, Enrica Roncaglia, Paola Guglielmelli, Lisa Pieri, et al. "Targeted Cancer Exome Sequencing Discovers Novel Recurrent Mutations In MPN." Blood 122, no. 21 (November 15, 2013): 4099. http://dx.doi.org/10.1182/blood.v122.21.4099.4099.
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Abstract The discovery of the JAK2V617F mutation in 2005 [Kralovics R, N Engl J Med 2005] represented a major breakthrough in the understanding of the molecular pathogenesis of Philadelphia chromosome negative chronic myeloproliferative neoplasms (MPN). Nevertheless several observations suggest that the JAK2V617F mutation may not be the disease funding mutation, at least in most instances. Therefore, a great deal of effort is ongoing with the aim to identifying novel genetic lesions contributing to the disease pathogenesis. The two major theoretical and technical drawbacks to the identification of new somatic mutations are represented, respectively, by the huge number of genes potentially involved in tumorigenesis of MPN and by the availability of a “pure” germline control DNA. Buccal swabs and saliva have been generally considered as readily available sources of DNA of non-hematopoietic origin, but detection of the JAK2V617F mutation in at least some of these samples indeed suggested the presence of myeloid cell contamination [Levine RL, Cancer Cell 2005]. So, in order to discover novel mutations in MPN using upfront technologies based on next-generation sequencing (NGS) we designed a “cancer exome” capture panel of 2000 unique genes and microRNAs. This panel was used to capture libraries generated from genomic DNA extracted from granulocytes and in vitro expanded CD3+ T-lymphocytes as germline control, in a cohort of 20 MPN patients. These captured libraries were than massively sequenced using the Roche 454 FLX platform. DNA samples had been collected at the diagnosis of PV in 9 subjects and PMF in 6 subjects, while the remaining 5 DNA samples were from 5 of the 9 PV patients at the time they evolved to post-PV myelofibrosis. After extensive bioinformatics analysis and multiple control adjustments, we finally produced a list of 171 novel “true” somatic mutations occurring in genes and microRNAs coding regions of those MPN samples; some of these mutations have been already described in MPN, whereas novel variants represent the vast majority. Despite patients harbored different numbers of somatic mutations, spanning from four to twenty-one variants, only 22 genes appear recurrently mutated. It is worth of notice the acquisition of additional mutations and/or the occurrence of loss of some mutations at the time of disease evolution from PV to a post-PV Myelofibrosis in the five patients for whom samples were available at both disease phases. Some of them, either acquired (NTRK1, PRDM2, BRCA2 and BARD1) or lost (APC, CARS, MLL3 and FAT2) had been found also in another PV or PMF sample. To test the recurrence of these mutations, we screened a different cohort of 189 patients composed by PMF (91 samples), PV (50 patients) and post-PV Myelofibrosis (48 samples) by Ion AmpliSeq technology on an Ion Torrent PGM platform. Deep amplicon sequencing of granulocytes DNA achieved a sample median of 1000-fold coverage. Excluding JAK2, MPL, IDH2, ASXL1 known variants, for 7 genes (SCRIB, MIR662, BARD1, TCF12, FAT4, DAP3, NRAS) we demonstrated in MPN a global mutation frequency greater than the 3%. Whereas some new variants need functional validation to prove causal mechanisms, some other mutations have a well-known pathogenic role in solid cancers but here are described for the first time in MPN. Disclosures: No relevant conflicts of interest to declare.
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Zhang, Kejie, Yuhan Chen, Yongli Zhang, Qin Yao, and Huiqin Zhuo. "Novel CRM1 Inhibitor Prevent the Nuclear Export of Beclin-1 Blocks Autophagy in Mantle Cell Lymphoma." Blood 124, no. 21 (December 6, 2014): 3596. http://dx.doi.org/10.1182/blood.v124.21.3596.3596.
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Abstract Mantle cell lymphoma (MCL) is an aggressive histotype of B-cell non-Hodgkin’s lymphoma (NHL-B) with poor response to conventional chemotherapy and short survival which prompt the urgent need for novel therapeutic agents. Chromosomal Region Maintenance 1 (CRM1, Exportin 1, Xpo 1), the major mammalian export protein, plays a critical role in nuclear-cytosolic transport of multiple tumor suppressors which lose their normal function once they have exited the nucleus. We had found that CRM1 expression was higher in MCL cell lines and primary MCL cells than in normal B lymphocytes. Inhibiting CRM1 with small interfering RNA inhibited MCL cell growth. Our findings suggest that CRM1 plays a key role in the pathophysiology of MCL cells and that targeting CRM1 in MCL could have therapeutic value. Small molecule selective inhibitors of nuclear export (SINE) block XPO1-mediated nuclear export, inducing cancer cell death and sensitizing cancer cells to other cytotoxic drugs. Although the cytotoxic effects of SINE on MCL cells have now been established, the mechanism of cell death is still not fully understood. The objective of our study was to elucidate the mechanism of CRM1 inhibitor-mediated cytotoxic effects on MCL cells. We used 8 established MCL cell lines and primary cells from 4 patients with relapsed/refractory MCL. Flow cytometry analysis with fluorescence-labeled Annexin V and propidium iodide showed that KPT-185,a KPT-SINE compound, induced MCL cells apoptosis in both time- and dose-dependent manners. However, specific knockdown of CRM1 has minimal effect on apoptosis induction. We therefore hypothesized that KPT-185 may induce other types of programmed cell death, autophagy than apoptosis. Autophagic features were determined by detecting acidic vesicular organelles (AVO) by MDC staining under fluorescence microscope after flow cytometry analysis. The autophagy related protein expressions of Beclin-1, a well-known key regulator of autophagy, and LC3-II were assessed by Western blotting. The results showed that after KPT-185 treatment autophagy declined in MCL cells and the declination was most obvious at 2 h and at 50nM. the combination of KPT-185 with chloroquine, an autophagy inhibitor that block lysosome acidification and consequent autophagosome fusion, resulted in synergistic cell death (CI 0.2-0.6) characterized by enhanced induction of both apoptosis and autophagy in MCL cells. There is no LC3-II expression being detected by Western blotting in MCL cells with low basal autophagy. As shown in Western blot and confocal microscopy, inhibiting CRM1 activity with KPT185 in MCL cells up-regulated the protein expression of Beclin-1 and trapping Beclin-1 in the nucleus where fail to promote autophagy. The same results were reported by Beth Levine that the nuclear export mutant of Beclin-1 fails to promote nutrient deprivation-induced autophagy in MCF7 cells. In summary, our studies are the first to suggest that autophagy is a survival pathway used by MCL cells. Targeting autophagy has therapeutic potential in MCL.The CRM1 nuclear export pathway may be important in the functional regulation of autophagic growth control that is enhanced in response to combined treatment with chloroquine. Our data suggest that KPT185 combination with chloroquine provides a potential therapy for patients with MCL. Disclosures No relevant conflicts of interest to declare.
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Knight, Thomas G., Myra Robinson, Jing Ai, Brittany K. Ragon, Rhonda Davis, Cindy Shiflett, Erica Ruston, et al. "Patient Reported Financial Toxicity in Myeloproliferative Neoplasms." Blood 134, Supplement_1 (November 13, 2019): 2099. http://dx.doi.org/10.1182/blood-2019-128858.
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Background: Financial Toxicity (FT) is increasingly recognized as a major contributor to morbidity and mortality in a variety of cancers. Previous research has demonstrated patients with myeloproliferative neoplasms (MPNs) exhibit a substantial comorbidity burden and have an increased risk of mortality. The purpose of this study was to define rates of FT and the implications on morbidity and mortality in this population using patient reported data. Methods: All patients seen at the Levine Cancer Institute, a tertiary hospital-based specialty practice, were surveyed prior to their visit over a six-month period. All patients were aged ≥18 years and diagnosed with Philadelphia chromosome−negative classical MPNs including myelofibrosis (MF), polycythemia vera (PV), and essential thrombocythemia (ET). The survey consisted of the PROMIS Global-10 measure and two questions from the COST measure. FT was defined as scoring 4 or less (maximum: 10) in agreement with the COST questions: "I know that I have enough money in savings, retirement, or assets to cover the costs of my treatment" and "I am satisfied with my current financial situation." Patient disease and treatment characteristics were summarized with frequencies and proportions for categorical variables and medians and ranges for continuous variables. Correlation of numerical FT scores with PROMIS scores was assessed with Pearson correlation coefficients and ANOVA regression. Additionally, model selection was carried out using logistic regression to identify factors impacting the incidence of financial toxicity (where FT score <=4). Kaplan Meier methods were used to estimate overall survival distributions and log rank tests were used to compare between groups. Results: A total of 51 patients were surveyed. Disease type consisted of 45.1% MF, 31.4% PV, and 23.5% ET. Median age was 62 years. Most patients were high risk by disease specific scoring systems (58.8%), Caucasian (82.4%), and had commercial insurance (56.9%). Median distance from the clinic was 21 miles and median time from diagnosis was 2.2 years. Of the 51 patients surveyed, 20 (39.2%) met the predefined definition of exhibiting severe FT. Lower FT scores (indicating a higher degree of FT) were associated with lower global physical (p < .001) and mental (p < .002) scores by the PROMIS measures (Figure 1). There was no statistically significant difference in survival between patients with FT scores >4 compared to patients with FT scores <=4; however, there was a trend toward decreased survival in those with lower FT scores. The rate of mortality in those with FT score ≤4 was 15.0% compared to 3.2% in those with FT score >4 (p =.287). There also appeared to be a separation of the survival curves when looking at both time from diagnosis and time from survey administration (Figures 2 and 3). Age, race, gender, insurance type, distance from the hospital, disease type, disease specific risk classification, medications utilized, and history of blood/marrow transplant were not found to be significantly different in the two groups. Conclusions: Patients with myeloproliferative neoplasms represent an extremely vulnerable population for financial toxicity with quantifiably increased distress related to this toxicity increasing morbidity and potentially mortality. These findings should be validated in a larger patient cohort and interventions devised to reduce financial distress. Disclosures Knight: Foundation for Financial Planning: Research Funding. Ai:InCyte: Speakers Bureau; Amgen: Speakers Bureau. Trivedi:Incyte: Speakers Bureau. Avalos:Best Practice-Br Med J: Patents & Royalties: receives royalties from a coauthored article on evaluation of neutropenia; Juno: Membership on an entity's Board of Directors or advisory committees. Symanowski:Boston Biomedical: Membership on an entity's Board of Directors or advisory committees; Carsgen Therapeutics: Membership on an entity's Board of Directors or advisory committees; Eli Lilly: Membership on an entity's Board of Directors or advisory committees; Immatics: Membership on an entity's Board of Directors or advisory committees. Grunwald:Amgen: Consultancy; Novartis: Research Funding; Genentech/Roche: Research Funding; Pfizer: Consultancy; Daiichi Sankyo: Consultancy; Trovagene: Consultancy; Agios: Consultancy; Incyte: Consultancy, Research Funding; Cardinal Health: Consultancy; Forma Therapeutics: Research Funding; Abbvie: Consultancy; Celgene: Consultancy; Merck: Consultancy; Medtronic: Equity Ownership; Janssen: Research Funding.
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Mantha, Simon, Ross L. Levine, and Raajit K. Rampal. "MLL Exons 4-8 Duplication in a Patient with Calr-Mutated Essential Thrombocythemia." Blood 124, no. 21 (December 6, 2014): 5583. http://dx.doi.org/10.1182/blood.v124.21.5583.5583.
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Background: About 25% of individuals with essential thrombocythemia (ET) harbor a somatic mutation of the cal reticulin (CALR) gene which is thought to be the primary driving factor for the myeloid clone in those cases. Different secondary mutations have been described in association with CALR defects, however to our knowledge mixed-lineage leukemia (MLL) gene alterations have not been described before in this setting before progression to MDS or AML. Case Report: A 68 year old woman with a past medical history significant for thyroid cancer in remission following treatment with radioactive iodine was initially noted to have a platelet count of 629 K/mcL at the occasion of a routine CBC (normal=160-400 K/mcL). Total white blood cell count, differential and hemoglobin levels were normal. Platelet count had been normal at 377 K/mcL about one year prior to presentation. Testing for mutations of the JAK2 and MPL genes came back negative. There was no splenomegaly; initial bone marrow biopsy showed mild hypercellularity with maturing trilineage hematopoiesis and atypical megakaryocytosis without an increase in blasts. A very small population of clonal B-cells was detected (<2% of total). Cytogenetic analysis did not reveal any chromosomal rearrangement. The patient was started on hydroxyurea (HU) 500 mg PO daily one month after initial presentation. There was evidence of mild iron deficiency, which was corrected with administration of IV iron sucrose at a total dose of 400 mg. Four months after starting HU the platelet count had decreased to 543 K/mcL. Repeat bone marrow biopsy around that time showed mild hypocellularity (10-20%) along with persisting normal maturing trilineage hematopoiesis and atypical megakaryocytosis. There was no increase in reticulin fibers and blast count was normal. Stainable iron was present but no ring sideroblasts were noted. Karyotype was still normal and FISH revealed no evidence of deletion 5q, monosomy 5, deletion 7q, monosomy 7, trisomy 8, 11q23 translocation or 20q deletion. Given the absence of clonal defect and the lack of symptoms, HU was withheld following which the patient was observed closely. The platelet count then increased progressively, reaching a maximum of level of 977 K/mcL about 2 months after discontinuing cytotoxic treatment. The drug was then resumed and testing with the FoundationOne HemeTM panel was obtained, looking for a select list of base substitutions, insertions, deletions, copy number alterations and other DNA rearrangements for more than 400 genes known to be somatically altered in hematologic malignancies. This assay revealed the presence of an acquired anomaly of the MLL gene, consisting of a duplication of exons 4-8. Subsequent testing specific for the CALR gene also revealed an exon 9 insertion/deletion. HU was progressively increased, up to a dose of 1000 mg alternating with 1500 mg daily. With this treatment, the platelet count has decreased to 396 K/mcL one year after presentation. Total white blood cell count, differential and hemoglobin have remained normal. The patient is also taking aspirin 81 mg daily and remains clinically stable with no B symptoms, bleeding or thrombotic manifestations. Discussion: Since mutations of the CALR gene were found to be associated with ET and myelofibrosis, several secondary genetic defects were demonstrated. Those are thought to represent clonal evolution from a primary subset of cells carrying either the JAK2 V617F mutation or a CALR exon 9 alteration. To our knowledge, MLL gene mutations have not been described as early “hits” in myeloproliferative neoplasms. However they have been abundantly documented in myelodysplastic syndrome and acute myeloid leukemia (AML), where they are often a marker of poor prognosis. Conclusion: This is the first report of MLL exons 4-8 duplication in an individual with CALR-mutated ET. Interestingly, the patient presented here did not exhibit any neutrophilia, dysplasia or increase in bone marrow blasts. It remains unclear if in this setting such a genomic alteration confers an increased risk of progression to MDS or AML. Disclosures Levine: Foundation Medicine: Consultancy. Rampal:Foundation Medicine: Consultancy.
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Quek, Lynn, Muriel David, Alison Kennedy, Marlen Metzner, Michael Amatangelo, Alan H. Shih, Bilyana Stoilova, et al. "Clonal Heterogeneity in Differentiation Response and Resistance to the IDH2 Inhibitor Enasidenib in Acute Myeloid Leukemia." Blood 130, Suppl_1 (December 7, 2017): 724. http://dx.doi.org/10.1182/blood.v130.suppl_1.724.724.
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Abstract Background Mutations in Isocitrate Dehydrogenase 2 (IDH2) occur in many cancers including Acute Myeloid Leukemia (AML). In preclinical models mutant IDH2 (mIDH2) causes partial hemopoietic differentiation block1. Recently, we showed that single agent enasidenib, a first-in-class, selective mIDH2 inhibitor, produces a 40% response in relapsed/refractory AML patients by promoting differentiation2. Here, we studied response and acquired resistance to enasidenib, in sequential samples treated in the Phase 1 study of Enasidenib in relapsed/ refractory AML patients. Results We studied a cytogenetically and genetically representative subset of 25 patients enriched for enasidenib responders, genotyped by whole exome sequencing (WES) or cancer gene panel targeted re-sequencing. Pre-enasidenib, differentiation arrest in these AML patients resulted in abnormally expanded leukaemic progenitors or precursors and diminished mature haematopoietic populations. Complete remission (CR) post-enasidenib was associated with in increased mature populations, near-normalisation of haematopoietic progenitor profiles, and restoration of in vitro progenitor function. In most patients, mature blood cells (of erythroid and granulocyte-monocyte lineages) post-enasidenib are IDH2 mutant, consistent with enasidenib inducing differentiation of IDH2 mutant leukaemic progenitors/ precursors. Each mIDH2 patient studied had on average 13 somatic, non-synonymous exonic or splice site mutations in addition to IDH2 . We used single cell genotyping (SCG) to reveal linear or branching clonal structures in mIDH2 AML. We combined clonal structure data and immunophenotyping of haematopoietic progenitor, precursor and mature populations to track functional behaviour of mIDH2 clones before, and during enasidenib treatment. We demonstrate, for the first time, that mIDH2 subclones within the same patient are functionally heterogeneous: both in their ability to differentiate pre-enasidenib, and in their sensitivity to Enasidenib-induced differentiation. This suggests that different combinations of co-operating mutations result in functional heterogeneity of mIDH2 clones. When we studied the contribution of mIDH2 clones to functional haematopoiesis at CR, we found that this was supported by either ancestral or leukaemic terminal mIDH2 clones. Despite a median survival of 18-21 months in patients who respond to enasidenib, most patients eventually relapse3. In contrast to targeted therapies such as tyrosine kinase inhibitors, in all 12 relapse samples studied, none had second site mutations in IDH2 . Furthermore, 2-hydroxyglurate (2HG) levels remain suppressed in most patients suggesting enasidenib remains effective in inhibiting mIDH2 enzyme. Instead, mIDH2 clones, which had persisted at CR or partial remission (PR) acquired additional mutations or aneuploidy, highlighting bypass pathways which re-impose differentiation arrest. We found 4 patterns: i) acquisition of IDH1 codon R132 mutations which resulted in a rise in 2HG (n=2), ii) deletion of chromosome 7q (n=4), iii) gain of function mutations in genes implicated in cell proliferation (FLT3, CSF3R) (n=3) and iv) mutation in hematopoietic transcription factors (GATA2, RUNX1) (n=2). We also found mutations in 4 genes (DHX15 and DEAF1 (n=1) ; NFKB1 (n=1) and MTUS1 (n=1)) not previously implicated in haematopoietic differentiation arrest which were selected for, or evolved in mIDH2 subclones at relapse. Conclusion This study provides a paradigm of how deep clonal single cell analysis in purified hemopoietic compartments in sequential samples through therapy reveals clonal complexity and the impact of the selective pressure of therapy on clonal architecture. Furthermore, we gain insights into the functional heterogeneity of mIDH2 subclones in their ability to differentiate pre-and post-Enasidenib. Further analysis of this kind in a larger cohort of IDH2 -inhibitor-treated patients would also provide insight to improve efficacy of this novel class of therapeutics, and design of combination therapies in AML and other cancers. Finally, this provides a platform for further study of the pathways mediating enasidenib resistance. References 1. Kats, L.M. , et al. Cell Stem Cell14, 329-341 (2014). 2. Amatangelo, M.D. , et al. Blood (2017). 3. Stein, E.M. , et al. Blood (2017). Disclosures Quek: Celgene Corporation: Research Funding. Amatangelo: Celgene Corporation: Employment. Agresta: Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Yen: Agios: Employment, Equity Ownership. Stein: Pfizer: Consultancy, Other: Travel expenses; Agios Pharmaceuticals, Inc.: Consultancy, Research Funding; Constellation Pharma: Research Funding; Novartis: Consultancy, Research Funding; GSK: Other: Advisory Board, Research Funding; Celgene Corporation: Consultancy, Other: Travel expenses, Research Funding; Seattle Genetics: Research Funding. De Botton: Agios: Honoraria, Research Funding; Celgene: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Servier: Honoraria. Thakurta: Celgene Corporation: Employment, Equity Ownership. Levine: Qiagen: Equity Ownership; Qiagen: Equity Ownership; Celgene: Research Funding; Roche: Research Funding; Celgene: Research Funding; Roche: Research Funding. Vyas: Jazz Pharmaceuticals: Speakers Bureau; Celgene Corporation: Speakers Bureau.
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Getta, Bartlomiej M., Emily C. Zabor, Sean Devlin, Kristina Marie Knapp, Minal Patel, Abhinita Mohanty, Marcel van den Brink, et al. "RAS Pathway Mutations Are Associated with Proliferative Features and Frequently Co-Occur with TET2 mutationsin Philadelphia Negative MPN Subtypes." Blood 128, no. 22 (December 2, 2016): 4269. http://dx.doi.org/10.1182/blood.v128.22.4269.4269.
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Abstract Introduction: The Philadelphia-chromosome negative myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell disorders, which include polycythemia vera, essential thrombocythemia and myelofibrosis. These disorders are characterized by activation of the JAK-STAT pathway via somatic mutations in JAK2, MPL, and CALR. However, a number of recurrent somatic mutations outside the JAK-STAT pathway have been described. Many of these mutations, such as ASXL1 and EZH2, are know to confer risk for disease progression. Other mutations, such as TET2 mutations, appear to alter the biology of disease in preclinical and clinical studies. Thus, further investigation of co-occurring mutation events is important to further the understanding of disease biology. Recurrent activating mutations in the RAS signaling pathway have been described in patients with MPNs. We have sought to determine the clinical impact of RAS mutations in patients with MPN, and to assess the pattern of co-mutational events in patients with RAS mutations. Methods : A targeted 28-gene, amplicon based next-generation sequencing assay was used to sequenced bone marrow aspirate or peripheral blood samples from 211 pts with Philadelphia negative MPN including: ET=61, PV=35, PMF=54, Post ET MF=16, Post PV MF=17, Post MPN AML=20, MPN unclassified=5 and systemic mastocytosis=3. Pts with MDS/MPN overlap syndromes were excluded. NRAS and KRAS mutations were grouped together. FisherÕs exact test and Wilcoxon rank-sum test were used to compare categorical and continuous variables, respectively. Results: N/KRAS mutations occurred at a frequency of 6 % in this cohort (Fig 1a). The median variant allele frequency (VAF) of RAS mutations was 15.6%. This was lower than the VAF of JAK2 V617F (44.6%) (p<0.001), TET2 (43.0%) (p=0.011) and ASXL1 (29.5%) (p=0.029) suggesting that RAS mutations are sub-clonal relative to other MPN associated mutations. By contrast, mutant RAS VAF did not significantly differ from VAFs of genes frequently enriched at the time of leukemic transformation, such as IDH (33.7%) (p=0.079) and TP53 (18.2%) (p=0.582) (Fig 1b). The presence of concurrent JAK-STAT activating mutations with RAS mutations was associated with distinct clinical features. RAS mutations occurred more frequently in patients with fibrotic MPN and post MPN AML and were less frequent in patients with ET and PV (table 1). RAS mutant patients had clinical features of more proliferative disease with higher total WBC, absolute monocytes, percentage circulating blasts, higher frequency of splenomegaly, and were associated with higher DIPSS score in pts with myelofibrosis (Table 1). RAS mutations were associated with mutations in TET2 with 4/32 (13%) with mut-TET2 vs 6/152 (4%) wild type TET2 having RAS mutations, though this result did not reach statistical significance (p=0.074) (Fig 1d). No other gene mutation was positively associated with mutations in RAS. 3/6 pts with TET2/RAS co-mutation had post MPN AML, while the remaining 3 had PMF or post ET MF (Fig 1c). The VAF of mut-RAS was always lower than mut-TET2 in patients where these were co-mutated, a pattern not seen for RAS VAF relative to JAK2 V617F VAF. This suggests that RAS mutations were acquired after TET2 in all cases assessed. Patients with RAS/TET2 co-mutation had the highest incidence of post-MPN AML. In those with wild type TET2 and mutant RAS proliferative disease features were noted including high WBC, monocyte count and splenomegaly. Finally, patients with myelofibrosis and RAS mutations were significantly more likely to have high DIPSS scores. Conclusions: RAS mutations occur in 6% of pts with Philadelphia negative MPN. Patients with co-occurring JAK-STAT pathway activating mutations and RAS mutations had more proliferative disease and a higher incidence of post MPN AML. RAS mutations were more frequent in patients with mutant TET2 and in this context they were associated with post MPN AML. The RAS VAF was always lower relative to TET2 and other MPN driver mutations suggesting they are acquired later in disease evolution. These data suggest the presence of RAS mutations may alter the disease biology of MPNs, and raise the question of the possible clinical efficacy of utilizing therapeutic agents, such as MEK inhibitors, in MPN patients with RAS mutations. Further preclinical and clinical evaluation of the role of this pathway in MPN pathobiology is warranted. Disclosures Levine: Qiagen: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.
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Hatlen, Megan A., Kanika Arora, Vladimir Vacic, Ewa A. Grabowska, Willey Liao, Bridget Riley-Gillis, Dayna M. Oschwald, et al. "Integrative Analysis of the Mutational Landscape of Mouse and Human AML Identifies Functionally Relevant Leukemia Disease Alleles." Blood 126, no. 23 (December 3, 2015): 1247. http://dx.doi.org/10.1182/blood.v126.23.1247.1247.
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Abstract t(8;21) is the most frequent chromosomal abnormality in acute myeloid leukemia (AML), occurring in 4-12% of adult and 12-30% of pediatric patients. This translocation fuses the N-terminus of AML1 to nearly the entire coding region of ETO, resulting in expression of the fusion protein AML1-ETO. Observations that mice expressing AML1-ETO develop AML only if treated with mutagenic agents have suggested that AML1-ETO requires cooperating disease alleles for leukemogenesis. Consistent with this, t(8;21)+ AML patients harbor multiple genetic abnormalities. Recent exome/genome sequencing studies have expanded the number of known mutations in t(8;21)+ AML patients; however, efforts to distinguish driver from passenger mutations have yielded few cooperative events and the requirements for AML1-ETO leukemogenesis remain largely unknown. To better define the genetic landscape in AML and distinguish driver from passenger mutations, we compared the mutational profiles of two specific AML1-ETO driven mouse models of leukemia to the mutational profiles of human AML patients. We found that the mouse models of AML1-ETO driven AML were phenotypically similar in terms of their extensive latency, myeloid progenitor immunophenotype, and the acquired secondary disease alleles. The first model relies upon the expression of AML1-ETO in transplanted p21 null cells, while the second model relies upon the expression of AML1-ETO9a, a splice variant of AML1-ETO, in transplanted wild type cells. p21 is neither disrupted, nor methylated in t(8;21)+ AML. Because loss of p21 prevents the repair of damaged DNA, leukemogenesis may occur in this model once a cooperating disease allele has been naturally acquired in an AML1-ETO positive hematopoietic progenitor. AML1-ETO9a itself deregulates the expression of several DNA repair genes, suggesting that AML1-ETO9a could similarly facilitate the acquisition of a cooperating disease allele. When we compared the mutational landscape of these murine leukemias to AML patients, we found that the murine leukemias enrich for disease alleles present in human AML (hypergeometric p ≤ 4.26x10-20) and that there is a significant tendency for disease alleles mutated in both species to possess mutations in the same protein domain (hypergeometric p ≤ 4.23x10-3). Furthermore, domains mutated in both species were affected by recurrent mutations (Spearman correlation of domain p-values r = 0.53, p ≤ 2.73x10-8). While the frequency with which various protein classes were affected by mutations was significantly different in MLL-AF9 and AML1-ETO/AML1-ETO9a positive murine AML compared to MLL-fusion and t(8;21)+ positive human AML (p = 0.049), the protein classes targeted in AML1-ETO/AML1-ETO9a murine AML vs. human t(8;21)+ AML were not significantly different (p = 0.327). To identify disease alleles capable of cooperating with AML1-ETO, we determined that of the 424 genes mutated in both species, 38 of those genes were significantly mutated in human AML (Genome MuSiC SMG FDR ≤ 30%). These 38 genes represented 45 mouse orthologues, 38 of which were significantly mutated in AML1-ETO driven murine leukemias (FDR ≤ 10%). These 38 orthologues corresponded to 32 human orthologues, 3 of which were annotated in COSMIC as cancer-related genes: TET2, PTPN11, and THRAP3. Using retroviral transduction and transplantation experiments, we demonstrated that the expression of AML1-ETO in transplanted Tet2 null cells or PTPN11 D61Y cells was sufficient for leukemogenesis. At euthanasia, mice exhibited leukocytosis, anemia, thrombocytopenia, splenomegaly, and an expansion in the myeloid progenitor compartment. Our identification of Tet2 loss as a cooperating allele implicates mutations in epigenetic regulators as potential driving events in t(8;21)+ AML, while the discovery of PTPN11 D61Y solidifies the role of constitutive MAPK signaling in t(8;21)+ AML. This integrative genetic profiling approach allowed us to accurately predict cooperating events in t(8;21)+ AML in a robust and unbiased manner, while also revealing functional convergence in mouse and human AML. Collectively, these findings illustrate the power of integrating murine and human genomic profiling to identify functionally relevant disease alleles in AML. Disclosures Levine: CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy.
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Przychodzen, Bartlomiej, Xiaorong Gu, Dewen You, Cassandra M. Hirsch, Michael J. Clemente, Aaron D. Viny, Ross L. Levine, et al. "PHF6 - Somatic Mutations and Their Role in Pathophysiology of MDS and AML." Blood 126, no. 23 (December 3, 2015): 1259. http://dx.doi.org/10.1182/blood.v126.23.1259.1259.
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Abstract Recently, rare somatic nonsense PHF6 mutations and deletions have been reported in patients with T-ALL, AML and blast crisis CML. Germ line PHF6 mutations have been described in Borjeson−Forssman−Lehmann syndrome (BFLS), a hereditary X-linked disorder characterized by mental retardation and somatic deformities. Patients with BFLS have been also reported to develop leukemia, suggesting PHF6 mutations may predispose to cancer. PHF6 is a highly conserved 41kDa protein showing ubiquitous expression in a variety of tissues, including bone marrow, CD34+ cells and leukocytes. The function and molecular pathogenesis in hematological disorders is unknown. PHF6 has been suggested to be a tumor suppressor gene (TSG) involved in the control of rRNA synthesis. Recent CHIPseq experiments showed that PHF6 binds upstream of the regulatory sequence of RUNX1. In an index case of a young adult female patient with proliferative CMML with dysmorphic features, we have identified remarkable GL mosaicism for PHF6 mutation (p.K44fs), confirmed by deep sequencing of bone marrow, CD3+ cells, spleen and skin tissue. Subsequently, we screened patients with myeloid neoplasms by targeted multi-amplicon sequencing to determine the prevalence and distribution of PHF6 gene alterations. Sequencing results from 1122 cases were analyzed (778 by targeted deep sequencing and 344 by whole exome sequencing). In total, we identified 45 cases with PHF6 mutations, 32 of which were frameshift or nonsense mutations. Previously, PHF6 have been included in screening panels by Haferlach et al., (Leukemia 2014) and Papaemmanuil et al., (Blood 2013) and somatic mutations were found in 24/944 and 21/738 cases of MDS, respectively. The somatic nature of these defects was confirmed by analysis of non-clonal CD3+ lymphocytes, Thus the incidence of PHF6 mutations ranges from 4.3% in current study to 2.8% and 2.5% reported by others and are most frequently observed among patients with secondary AML (33%), suggesting that PHF6 mutations are not uncommon driver events in myeloid neoplasia. Gender distribution showed a strong male predominance (76%), indicating that retention of a single copy of PHF6 may be protective. There was no significant sex difference in the transcriptional expression of PHF6 itself. The most frequent chromosomal aberration observed in conjunction with PHF6 mutations was trisomy-8 (p=.08). The most commonly associated somatic mutations were in RUNX1 (p=.001) and IDH2 (p=.008). Concomitant PHF6 and RUNX1 mutations are associated with a poor prognosis in AML, and occur predominantly in males. There was no association observed between low expressors of PHF6 and RUNX1 mutations or RUNX1 expression levels. Conversely, RUNX1 mutant cases without somatic PHF6 mutations were not observed to have low transcriptional PHF6 levels. Subsequent analysis of clonal architecture using variant allelic frequency calculations and serial sampling suggested that mutated PHF6 may function as a founder driver gene in proportion of cases, while RUNX1 mutations are acquired as secondary events. Recent studies proposed that PHF6 deficiency leads to impaired cell proliferation, cell cycle arrest at the G2/M phase and an increase in DNA damage. To delineate a possible pathophysiological pathway involving PHF6 we compared transcriptional expression profiles of low expressors to those with normal levels of PHF6. The most notably deregulated group of genes were clustered to a functionally related group of ribosomal RNA proteins (p<.00001). To better understand functional properties of PHF6 we conducted PHF6 specific immuno-precipitation followed by mass spectrometric fingerprinting on K562 cells to identify protein partners. We have identified a novel association of PHF6 in RNA degradation/stability and ribosomal proteins, including MOV and PABPC families. In conclusion, our results indicate that PHF6 mutations are generally present in more aggressive types of myeloid neoplasms and arefrequently associated with RUNX1/IDH2 mutations. Our functional in vitro studies, along with recently published reports, suggest an association of PHF6 deficiency with transcriptional regulation and thereby provide a basis for a transcriptional repressor phenotype conveyed by ancestral lesions, consistent with a role for PHF6 as a TSG. Disclosures Levine: Foundation Medicine: Consultancy; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees. Sekeres:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees.
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Getta, Bartlomiej, Franck Rapaport, Sean Devlin, Chen Zhao, Kristina Marie Knapp, Minal Patel, Abhinita Mohanty, et al. "Targeted Sequencing Reveals a Relationship Between Mutational Burden and Clinical Phenotype in MPNs." Blood 126, no. 23 (December 3, 2015): 4061. http://dx.doi.org/10.1182/blood.v126.23.4061.4061.
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Abstract The Philadelphia-chromosome negative myeloproliferative neoplasms (MPNs) Essential Thrombocytosis (ET), Polycythemia Vera (PV), and Myelofibrosis (MF) are characterized by mutations, which drive JAK-STAT pathway activation. Several studies have demonstrated the presence of recurrent somatic mutations outside of the JAK-STAT pathway, which accumulate over time, and may impact disease phenotype and outcome. We sought to determine the influence of somatic mutations on clinical phenotype and prognosis. We sequenced a total of 30 genes recurrently mutated in myeloid malignancies in a cohort of 162 MPN patients (pts) using a next generation sequencing platform. The cohort included 49 pts with ET, 26 PV, 38 Primary Myelofibrosis (MF), 11 Post ET MF, 14 Post PV MF, 12 with leukemic transformations of MPN (LT), 7 with MPN-unclassified (MPN-U) and 5 others. Median age was 59 years and 79 were men. A Total of 288 gene mutations were identified with the most commonly mutated genes being JAK2 (n=121, 74%), TET2 (n=31, 19%), DNMT3A (n=18, 11%), ASXL1 (n=16, 10%), IDH2 (n=10, 6%), RAS (n=12, 7%), TYK2 (n=8, 5%) and TP53 (n=7, 4%). We did not find any mutations in NPM1, CBL, SRSF2 and no FLT3 -ITD. CALR was not assessed in 20 pts and these were excluded from mutation number analysis. Importantly, we identified a relationship between the absolute number of mutations found per pt, disease phenotype, and age (table 1). Pts with/without prior chemotherapy or radiotherapy exposure did not have a difference in mutation number (1.5 vs. 1.9). Cases of ET or PV with fibrotic transformation had more mutations in ASXL1, RAS, EZH2, PHF6 and MPL than pre fibrotic ET or PV suggesting these may be relevant in disease progression and development of fibrosis. Mutations in TET2, RAS and PHF6 were more frequent in cases with LT compared to those with chronic phase MPN. Pts over 40 were more likely to have mutations in TET2 (p=0.026) and JAK2 (p=0.019) and ASXL1 mutations were more common in pts with abnormal cytogenetics than in those with normal cytogenetics (p=0.003). Thrombotic events, which are an important cause of morbidity in MPN patients, negatively correlated with mutations in ASXL1 (p=0.044). Prognosis as measured by DIPPS and DIPSS-Plus scores appeared to correlate with the average number of mutations found in MF patients (table 2). We examined several cases for which serial samples were available, and noted the acquisition of new mutational events despite ongoing therapy. We noted that the most commonly acquired mutations occurred in epigenetic modifying (DNMT3A, TET, IDH, ASXL1) and in growth signaling pathway (RAS, CBL) genes. These occurred despite active therapy and often without an overt change in clinical phenotype. Further details of these serial samples will be presented. We conclude that the number and spectrum of somatic mutations correlate with disease phenotype of MPN. Younger pts have fewer mutations, as do pts with normal cytogenetics. JAK2 and TET2 mutations were more common in older pts. We show that a subset of pts acquire mutations in epigenetic modifiers and in genes involved in growth signaling pathways during disease course, and that mutations in TET2, RAS and PHF6 were enriched at the time of leukemic transformation. Taken together, these results indicate that mutations outside the JAK-STAT pathway influence disease phenotype, and that the acquisition of mutations over time may predict for disease progression. Serial evaluation of mutational burden over time therefore warrants exploration in the clinical setting. Table 1. Average number of mutations appeared to correlate with disease phenotype, age and abnormal cytogenetics. Average Number of Mutations N Mean (SD) P-value Age < 40 years 13 1.4 (0.9) 0.026 Age > 40 years 12 2 (1) No Thrombosis 113 2 (1) 0.712 Thrombosis 28 1.9 (1) Normal Cytogenetics 64 1.8 (0.9) 0.016 Abnormal Cytogenetics 40 2.3 (1.2) ET/PV/PMF 99 1.8 (0.8) 0.029 LT 10 3 (1.5) ET/PV 66 1.6 (0.7) 0.01 Post ET/PV MF 22 2.3 (1.1) ET 44 1.6 (0.7) < 0.001 PV 22 1.5 (0.9) PMF 33 2.2 (0.9) Post ET/PV MF 22 2.3 (1.1) LT 10 3 (1.5) Table 2. Disease prognostic scores in MF appear to correlate with the average number of mutations found per patient. Risk category N Average number mutations DIPSS Low 8 1.5 Intermediate-1 19 2.4 Intermediate-2 9 1.8 High 1 5 DIPSS-Plus Low 6 1.5 Intermediate-1 12 2 Intermediate-2 14 2.2 High 1 5 Figure 1. Comutation map of genomic alterations. Each hash mark on x-axis represents an individual patient. Figure 1. Comutation map of genomic alterations. Each hash mark on x-axis represents an individual patient. Disclosures Levine: CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy.
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Chen, Bo-Rui, Anagha Deshpande, Maria Kleppe, Narayana Yeddula, Sunnie Yoh, Scott A. Armstrong, Ross L. Levine, Sumit Chanda, and Aniruddha J. Deshpande. "Genomic and Proteomic Profiling of AF10-Fusion Oncoproteins Reveal Mechanisms of Leukemogenesis and Actionable Targets." Blood 132, Supplement 1 (November 29, 2018): 544. http://dx.doi.org/10.1182/blood-2018-99-119210.
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Abstract The AF10/MLLT10 gene is recurrently involved in chromosomal rearrangements in human leukemia. AF10 rearrangements are linked to a poor prognosis in AML and T-ALL, underscoring the need to identify targeted therapies for AF10-fusion positive leukemia. Defining the molecular mechanisms of oncogenesis mediated by AF10-fusion proteins (AF10-FPs) may unravel novel actionable targets in leukemias with AF10-gene rearrangements. Towards this end, we established tetracycline (Tet)-inducible models of MLL-AF10 and CALM-AF10 AML and performed RNA-seq in AML cells treated with doxycycline (Dox) compared to vehicle treated counterparts. Since Dox treatment completely abrogates AF10-fusion gene expression from the Tet-regulated promoter, these models can be used to characterize the transcriptional landscape of potential AF10-FP target genes. We observed that among transcripts significantly downregulated upon Dox treatment, 168 genes were common in both the MLL-AF10 Tet-Off or CALM-AF10 Tet-Off conditions, indicating a high overlap between potential transcriptional targets of these distinct AF10-FPs. Expectedly, this list included genes previously implicated in leukemogenesis including Hoxa cluster genes, Meis1, Flt3, Mecom, Cd34, Gfi1b, Eya1 and Nkx2-3. Importantly, in addition to these well-characterized genes, we identified a number of novel pathways that were downregulated in the AF10-FP Tet-Off state. The most striking molecular signature of potential AF10-FP-regulated genes emerging from these analyses were factors involved in innate immunity and pro-inflammatory cytokine signaling. Prominent drivers of these molecular signatures included genes of the Jak/Stat and NFkB signaling pathways as well as Interferon response genes. We confirmed that AF10-FPs strongly activated Jak-Stat and NFkB signaling by performing Western blotting for key factors involved in these pathways. Since pro-inflammatory cytokines have been shown to play a role in AML cell survival, we tested the impact of cytokine depletion on murine AF10-FPs-driven AML cells. Proliferation assays demonstrated that AF10-FP-transformed cells could survive significantly better in cytokine-free medium compared to those transformed with other oncogenes such as MLL-AF9, which were completely dependent on cytokines for survival and proliferation in vitro. These results suggest that activation of cytokine signaling may contribute to increased survival of AF10-FP-driven AML cells. Next, we performed proteomic studies in which affinity-purified epitope-tagged AF10-FPs were evaluated for interacting proteins using Mass Spectrometry (MS). While studies on MLL-AF10 fusion are ongoing, our studies revealed that the strongest interactor of the CALM-AF10 fusion protein was the Janus kinase protein Jak1. We confirmed this finding by immunoprecipitation experiments in CALM-AF10 AML cells using a Jak1-specific antibody. Given the role of JAK1 in cytokine-mediated pro-inflammatory signaling, our findings indicate that CALM-AF10 may activate this pathway through direct recruitment of the Jak1 kinase. We sought to directly test the role of JAK1 in AF10-FP-mediated leukemogenesis. For this, we transformed bone marrow stem and progenitor cells from Jak1 floxed mice with the CALM-AF10 fusion. Deletion of Jak1 using Cre-recombinase in CALM-AF10 AML significantly reduced their proliferation in vitro. Furthermore, Jak1 deletion led to a highly significant reduction in the number of colony forming units (CFUs) from CALM-AF10 AML cells, with a particularly striking decrease in the number of blast-like colonies. We also observed a significant increase in differentiation of CALM-AF10 AML cells following Jak1 deletion, demonstrating that Jak1 activity is important for maintaining the CALM-AF10 leukemia cells in an undifferentiated state. Importantly, these results were recapitulated with two different small-molecule JAK1 inhibitors itacitinib and filgotinib that are being tested in clinical trials for a variety of human diseases. Treatment of CALM-AF10 AML cells with these selective JAK1 inhibitors led to a significant, dose-dependent decrease in proliferation accompanied by growth arrest and apoptosis. Taken together, our studies demonstrate that AF10 fusions activate pro-inflammatory signaling by co-opting the Jak-Stat pathway, presenting a potential therapeutic target in AF10-fusion-driven AML. Disclosures Levine: Janssen: Consultancy, Honoraria; Celgene: Consultancy, Research Funding; Qiagen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Prelude: Research Funding; Loxo: Consultancy, Equity Ownership; Imago: Equity Ownership; C4 Therapeutics: Equity Ownership; Novartis: Consultancy; Gilead: Honoraria; Isoplexis: Equity Ownership; Epizyme: Patents & Royalties; Roche: Consultancy, Research Funding. Deshpande:A2A Pharma: Membership on an entity's Board of Directors or advisory committees; Salgomed Therapeutics: Membership on an entity's Board of Directors or advisory committees.
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Papaemmanuil, Elli. "Somatic Mutations in Myelodysplastic Syndrome." Blood 124, no. 21 (December 6, 2014): SCI—22—SCI—22. http://dx.doi.org/10.1182/blood.v124.21.sci-22.sci-22.
Full textAbstract:
Abstract Myelodysplastic syndromes (MDS) are clonal stem cell neoplasms affecting patients usually over 60 years old that typically present into the clinic with common symptoms including cytopenias, recurrent infections, bleeding and bruising. Approximately 20-30% of MDS patients progress to acute myeloid leukemia (AML) and are associated with inferior survival1. Diagnosis of MDS relies on findings from peripheral blood counts, examination of bone marrow morphology and evaluation of cytogenetic profiles for chromosomal aberrations. Using the WHO 2008 criteria, the proportion of blasts in the bone marrow, the number of cell lineages affected and the presence of del(5q) are collectively evaluated to classify patients into one of the five MDS categories [refractory anemia, refractory anemia with ring sideroblasts, refractory cytopenia multilineage dysplasia, refractory anemia with excess blasts, MDS with del(5q)]. The International Prognostication Scoring System (IPSS & IPSS-R) is the most widely used prognostic system in MDS. IPSS utilizes morphological variables to assign patients into low, intermediate or high-risk groups2. Accurate classification into one of these prognostic categories is critical as it determines selection of therapy regimes. Recent systematic profiling screens of MDS genomes have unraveled a complex network of cellular pathways that are causally implicated in MDS pathogenesis. Mutations have now been characterized in a number of key components of the spliceosome machinery (SF3B1, SRSF2, U2AF1, U2AF2, ZRSR2), regulators of DNA methylation (DNMT3A, IDH1, IDH2, TET2), chromatin modification (ASXL1, EZH2), transcription (EVI1, RUNX1, GATA2), signal transduction (NRAS, JAK2, KRAS, CBL) and cell cycle control (TP53)3-9. Collectively, more than 40 genes are significantly mutated in MDS; these mutations account for nearly 90% of MDS patients. The majority of patients present with two or more oncogenic mutations at diagnosis, and significant patterns of gene-gene interactions and mutual exclusivity have been reported10,11. Systematic integration of mutation data with large and well-annotated clinical datasets offers an unprecedented opportunity to decipher both the diagnostic as well as prognostic potential of these mutations as clinical biomarkers. However, the underlying genetic heterogeneity imposes significant challenges and important considerations that need to be accounted for when interpreting observed correlations between genotype, morphology and patient outcome. To unravel the interlocking genetic heterogeneity in MDS, Bejar et al., Papaemmanuil et al., and Haferlach et al. have studied the prevalence of acquired gene mutations in MDS and closely related chronic myeloid neoplasms in ~ 2100 MDS patients with well-annotated diagnostic and clinical outcome variables10-12. Univariate analysis has identified more than 10 genes to be significantly correlated with clinical outcome, including SF3B1, SRSF2, ASXL1, RUNX1, TP53, BCOR, RUNX1, EZH2, IDH2, ZRSR2, U2AF1 and CUX1. The total number of oncogenic mutations identified in each patient is selected as one of the most significant genetic predictors of outcome. Mutations in gene components of the spliceosome machinery are observed in approximately 50% of MDS patients, identifying pre-mRNA splicing as the most frequently altered biological process in MDS. Additionally, clonal relationship analysis of these mutations identifies that mutations in splicing genes occur early, followed by mutations in preferred partner genes, and mutations in different genes of the spliceosome machinery are associated with distinct morphological classification groups. The present talk will provide an overview of our current understanding of the underlying molecular mechanisms that underpin MDS biology. It will also evaluate how the genetic architecture of MDS can be incorporated in developing reliable and informative patient classification as well as outcome prediction models that can support clinical decision making in the future. References: 1. Tefferi A, Vardiman JW. Myelodysplastic syndromes. N Engl J Med. 2009;361(19):1872-1885. 2. Greenberg PL, Tuechler H, Schanz J, et al. Revised International Prognostic Scoring System (IPSS-R) for myelodysplastic syndromes. Blood. 2012. 3. Yoshida K, Sanada M, Shiraishi Y, et al. Frequent pathway mutations of splicing machinery in myelodysplasia. Nature. 2011;478(7367):64-69. 4. Graubert TA, Shen D, Ding L, et al. Recurrent mutations in the U2AF1 splicing factor in myelodysplastic syndromes. Nat Genet. 2012;44(1):53-57. 5. Ernst T, Chase AJ, Score J, et al. Inactivating mutations of the histone methyltransferase gene EZH2 in myeloid disorders. Nat Genet. 2010;42(8):722-726. 6. Ley TJ, Ding L, Walter MJ, et al. DNMT3A mutations in acute myeloid leukemia. N Engl J Med. 2010;363(25):2424-2433. 7. Mardis ER, Ding L, Dooling DJ, et al. Recurring mutations found by sequencing an acute myeloid leukemia genome. N Engl J Med. 2009;361(11):1058-1066. 8. Gelsi-Boyer V, Trouplin V, Adelaide J, et al. Mutations of polycomb-associated gene ASXL1 in myelodysplastic syndromes and chronic myelomonocytic leukaemia. Br J Haematol. 2009;145(6):788-800. 9. Shih AH, Levine RL. Molecular biology of myelodysplastic syndromes. Semin Oncol. 2011;38(5):613-620. 10. Haferlach T, Nagata Y, Grossmann V, et al. Landscape of genetic lesions in 944 patients with myelodysplastic syndromes. Leukemia. 2014;28(2):241-247. 11. Papaemmanuil E, Gerstung M, Malcovati L, et al. Clinical and biological implications of driver mutations in myelodysplastic syndromes. Blood. 2013;122(22):3616-3627; quiz 3699. 12. Bejar R, Stevenson K, Abdel-Wahab O, et al. Clinical effect of point mutations in myelodysplastic syndromes. N Engl J Med. 2011;364(26):2496-2506. Disclosures No relevant conflicts of interest to declare.