Hypomethylating Agents Do Not Alter Novel Splicing Events in Myeloid Neoplasms

Background: Aberrant mRNA splicing occurs in myeloid malignancies and affects genes involved in tumor suppression, heme biosynthesis and mitochondrial iron metabolism. Functional studies demonstrated impaired cellular differentiation upon targeting of aberrant splice variants. Hypomethylating agents (HMA) constitute the backbone of therapy of myeloid malignancies. Whether HMA treatment in myeloid malignancies alters the novel splicing transcriptional landscape and whether it correlates with responses remain largely unexplored. Methods: Total RNA sequencing was done on CD34+ cells from 79 patients bone marrow samples involved by acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), TF1 cell lines and CD34+ murine bone marrow cells. Novel alternatively spliced transcripts were detected using SplAdder and included the following splicing events: alterative 3' splice junction, alternative 5' splice junction, exon skipping, intron retention, multiple exon skipping and mutually exclusive exons. All alternatively splicing events were normalized to total transcript count in order to correct for total transcript levels. A false discovery rate of <0.1 was used to identify significant events. Results: A total of 79 myeloid disease patients (27.8% females, 72.1% males) with a median age of 70 years (range, 31-87 years) were included in this study. In aggregate analysis of all 79 myeloid malignancies (39.2% (n=31) pre-treatment and 60.8% (n=48) post-treatment), there were 160 versus 37 (4.3 folds), 112 versus 40 (2.8 folds), 292 versus 51 (5.7 folds), 172 versus 80 (2.1 folds) and 29 versus 9 (3.2 folds) and 2 versus 0 novel splicing events occurring in pre- versus post- HMA treatment, respectively, in alterative 3' splice junction, alternative 5' splice junction, exon skipping, intron retention, multiple exon skipping and mutually exclusive exons, respectively. This suggested that treatment with HMA led to downregulation of novel alternative splicing events after normalization to total transcripts. However, upon excluding AML patients from the analysis, there were no significant events associated with treatment suggesting that the findings could be due to random events. To further explore whether HMA therapy influenced novel splicing events, we examined the novel splicing pattern in 7 MDS patients with paired BM samples at pre- and post-HMA and found no significant differences in alternative splicing events before and after the treatment. We then examined TF1 (human erythroleukemia) cell lines at pre- and post- HMA time points, but did not identify notable differences in the novel alternative splicing events with respect to HMA treatment. To assess whether CD34+ bone marrow cells from mice treated with hypomethylating agents have differential novel alternatively spliced events, we conducted similar analysis and did not find any discernible differences pre- and post- HMA treatment. These findings suggest that HMA does not influence novel alternative splicing events. Conclusions: Aberrant splicing has been linked to myeloid neoplasms especially myelodysplastic syndrome with mutations in splice variant genes. Our findings suggest that HMA does not influence novel alternative splicing events in myeloid malignancies. Therefore, the alternative splicing in myeloid disease is inherent to the disease and not affected by treatment. Disclosures Garcia-Manero: H3 Biomedicine: Research Funding; Novartis: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Merck: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Onconova: Research Funding; Acceleron Pharmaceuticals: Consultancy, Honoraria; Amphivena Therapeutics: Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Helsinn Therapeutics: Consultancy, Honoraria, Research Funding; Jazz Pharmaceuticals: Consultancy; Astex Pharmaceuticals: Consultancy, Honoraria, Research Funding; AbbVie: Honoraria, Research Funding.

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Prediction of Response to Treatment with Lenalidomide in Patients with Non-Del 5q Myelodysplastic Syndrome by Gene Expression Profiling Remains Difficult.

Blood ◽

10.1182/blood.v114.22.2781.2781 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2781-2781

Author(s):

Wolf-Karsten Hofmann ◽

Florian Nolte ◽

Ouidad Benlasfer ◽

Eckhard Thiel ◽

Gerhard Ehninger ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Myelodysplastic Syndrome ◽

Clinical Response ◽

Expression Profiling ◽

Research Funding ◽

Bone Marrow Cells ◽

Response To Treatment ◽

Specific Gene ◽

Prediction Of Response

Abstract Abstract 2781 Poster Board II-757 Lenalidomide belongs to a proprietary class of immunmodulatory drugs showing therapeutic activity in patients with myelodysplastic syndrome (MDS), in particular in those having the 5q-abnormality, but also in patients not showing this cytogenetical aberration. In 2008, Ebert et al. (PLos Med. 2, e35) could demonstrate that there is a specific gene expression profile in bone marrow cells collected from MDS-patients either with 5q- syndrome as well as MDS-patients having no 5q-abnormality which is strongly correlated with the clinical response to treatment with lenalidomide. Whereas this finding is not of clinical importance in patients with MDS del 5q (overall response 75 %) it may play a pivotal role for prediction of clinical response to lenalidomide in non-del 5q MDS-patients. Therefore, we have studied gene expression profile (HG-U133plus2.0, Affymetrix, Santa Clara, CA) of routinely isolated low-density mononuclear bone marrow cells from 8 patients with IPSS low/int-1 risk MDS having no deletion on chromosome 5 but were subsequently treated with lenalidomide 5 mg/day. All of the patients were transfusion dependent for red blood cells. The median duration of treatment with lenalidomide was 22 weeks. RNA was extracted by Trizol and quality controlled by using a Bioanalyzer 2100 system (Agilent, Waldborn, Germany) to exclude RNA degradation. Microarray hybridization was performed according to the standard Affymetrix protocol. Data were analyzed by Microarray Analysis Suites 5.0 (MAS 5.0; Affymetrix) and GeneSpring (Agilent Technologies, Santa Clara, CA). For clustering analysis we utilized the gene list of 68 discriminating genes as published by Ebert et al. the molecular analysis did clearly separate two groups of patients having specific gene expression profiles according to the responder/non-responder group as published previously. Furthermore, single sample prediction could discriminate three out of 8 patients to be possible responders to lenalidomide but this was not correlated to the clinical course of those patients while on treatment with lenalidomide. However, none of the MDS-patients receiving lenalidomide did show significant clinical response as defined by reduction of transfusion requirement by 50 % or transfusion independence. In conclusion, prediction of response to lenalidomide in non-del 5q patients by gene expression profiling so far remains critical. Prospective analysis of molecular changes including DNA analysis in larger clinical trials using lenalidomide in non-del 5q MDS-patients are required to establish reliable predictive markers in MDS. Disclosures: Hofmann: Celgene: Research Funding. Platzbecker:Celgene: Research Funding.

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Somatic Mutations in Schinzel-Giedion Syndrome Gene SETBP1 Determine Progression in Myeloid Malignancies

Blood ◽

10.1182/blood.v120.21.2.2 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2-2 ◽

Cited By ~ 1

Author(s):

Hideki Makishima ◽

Kenichi Yoshida ◽

Nhu Nguyen ◽

Masashi Sanada ◽

Yusuke Okuno ◽

...

Keyword(s):

Research Funding ◽

Somatic Mutations ◽

Bone Marrow Cells ◽

Germ Line ◽

Cancer Susceptibility ◽

Gain Of Function ◽

Myeloid Malignancies ◽

Runx1 Expression ◽

Myeloid Progenitors

Abstract Abstract 2 MDS and other chronic myeloid malignancies such as MDS/MPN are characterized by a frequent progression to secondary AML (sAML), a likely multistep process of acquisition of genetic abnormalities. Genes involved in congenital genetic cancer susceptibility syndromes are often targets of somatic mutations in various tumors. For instance, germ-line mutations of SETBP1 are associated with Schinzel-Giedion syndrome (SGS), which is characterized by skeletal malformations, mental retardation and frequent neuroepithelial tumors. While SETBP1 overexpression in myeloid malignancies links to poor prognosis, somatic mutations of SETBP1 were not previously identified in leukemias. When we performed whole exome sequencing of 20 cases with myeloid malignancies, in addition to detecting previously described lesions, such as TET2, CBL and ASXL1, we identified a somatic SETBP1 mutation (D868N) in 2 cases with RAEB. Analysis of DNA from CD3+ cells from these patients confirmed its somatic nature. Sanger sequencing was applied to all coding exons in an additional 48 cases, leading to detection of 2 additional somatic mutations (G870S and I871T) in 2 patients with CMML and sAML, respectively. These findings prompted us to further expand our screening cohort: targeted SETBP1 sequencing was performed in a total of 734 patients (283 with MDS, 106 with sAML, 167 with MDS/MPN, 138 MPN and 146 with primary AML): 52 mutations were detected in 52 patients (7.1%); D868N, G870S and I871T alterations were more frequently observed (N=27, N=16 and N=5, respectively), while D868Y, S869N, D880E and D880N were less prevalent. These mutations, of which 92% (48 out of 52) were identical to those in the SGS germ line, were detected in 15% with CMML (24/156), 15% with sAML (16/106) and 7% CML blast phase (2/28). Clinically, mutant cases were associated with higher age (p=.014), deletion of chromosome 7q (p=.0005) and shorter median survival (28 vs. 13 months, p<.0001). As shown in the analysis of 11 paired samples of progressing MDS patients, all SETBP1 mutations were acquired during leukemic evolution. In addition to mutations, SETBP1 overexpression can be found in 12% and 26% of cases of MDS and sAML, respectively, a finding linking higher activity of SETBP1 to leukemic progression. To directly test whether SETBP1 mutations represent gain-of-function, we performed retroviral transduction of murine Setbp1 engineered with two of the somatic mutations, D868N and I871T, and evaluated the ability of the mutants to immortalize normal murine myeloid progenitors. With a low viral titer of 1 x105 cfu, both Setbp1 mutants caused efficient immortalization of myeloid progenitors, similar to overexpressed WT Setbp1. In addition, cells immortalized with mutant Setbp1 proliferated faster than cells with WT Setbp1. These data suggest that mutations of SETBP1 in our study represent gain-of-function in leukemias. The in vitro immortalization effect of overexpressed WT Setbp1 was associated with and dependent on Hoxa9 and Hoxa10 overexpression. We performed quantitative RT-PCR and western blot experiments to evaluate expression of these genes in our mutant cases. Relative HOXA9 and HOXA10 mRNA expression values were higher in all mutant cases (N=7) than median of those in WT cases (N=4). Also, both HOXA9 and HOXA10 proteins were detected in all cases with SETBP1 mutations, suggesting that HOXA9 and HOXA10 induction is consistently associated with SETBP1 mutations similar to observations in forced expression of WT Setbp1. Moreover, in agreement with findings in primary cells showing that SETBP1 mutations or high SETBP1 expression share a common genetic association with RUNX1 mutations, Runx1 expression was reduced after in vitro immortalization of normal bone marrow cells by forced Setbp1 overexpression and two Runx1 promoter sequences were amplified after ChIP performed with antibody specific for exogenous Setbp1 protein. Moreover, Setbp1 shRNA knockdown resulted in enhanced Runx1 transcription consistent with the negative regulation of this gene by Setbp1. These results indicate that SETBP1 is associated with decreased activity of RUNX1 due to hypomorphic mutations or by direct down-modulation WT RUNX1 expression bypassing the need for mutations. In sum, somatic recurrent SETBP1 mutations are lead to gain of function and are associated with molecular pathogenesis of myeloid leukemic transformation of various primary myeloid subentities. Disclosures: Makishima: Scott Hamilton CARES Initiative: Research Funding. Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

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Loss of Ezh2 Promotes the Development of Mutant-RUNX1 Induced MDS

Blood ◽

10.1182/blood.v120.21.402.402 ◽

2012 ◽

Vol 120 (21) ◽

pp. 402-402

Author(s):

Goro Sashida ◽

Satomi Tanaka ◽

Makiko Mochizuki-Kashio ◽

Atsunori Saraya ◽

Tomoya Muto ◽

...

Keyword(s):

Bone Marrow ◽

Tumor Suppressor ◽

Peripheral Blood ◽

Bone Marrow Cells ◽

The Self ◽

Post Transplantation ◽

Self Renewal ◽

Myeloid Malignancies ◽

Empty Vector ◽

Marrow Cells

Abstract Abstract 402 Polycomb group proteins are transcriptional repressors that epigenetically regulate transcription via histone modifications. There are two major polycomb-complexes, the Polycomb Repressive Complexes 1 and 2 (PRC1, PRC2). PRC2 contains SUZ12, EED, and EZH1/EZH2, and catalyzes the trimethylation of histone H3 at lysine 27 (H3K27me3), silencing target-genes. We have shown that the self-renewal of Ezh2-deficient HSCs is not compromised and H3K27me3 marks are not completely depleted in the absence of Ezh2, possibly as a result of Ezh1 complementation. EZH2 is generally thought to act as an oncogene in lymphoma and solid tumors by silencing tumor suppressor genes. Recently however, loss-of-function mutations of EZH2 have been found in myeloid malignancies such as AML, MDS and MPN, suggesting that EZH2 also functions as a tumor suppressor, although it remains unclear how EZH2 prevents the transformation of myeloid malignancies. RUNX1 is a critical transcription factor in the regulation of the self-renewal and differentiation of HSCs. RUNX1 mutations are frequently found in MDS, AML following MDS (MDS/AML) and de novo AML patients. One of the most frequent mutations, RUNX1S291fs, lacks the transactivation domain in C-terminus, but retains the RUNT DNA biding domain, resulting in a dominant negative phenotype. RUNX1S291fs-transduced bone marrow cells have been shown to generate MDS/AML in vivo. Given that RUNX1 and EZH2 mutations coexist in MDS and AML patients as reported recently, we generated a novel mouse model of MDS utilizing RUNX1S291fs retrovirus and Ezh2 conditional knockout mice in order to understand how EZH2 loss contributes to the pathogenesis of MDS upon genetic mutation of RUNX1. We first harvested CD34-Lin-Sca1+c-Kit+(LSK) HSCs from tamoxifen-inducible Cre-ERT;Ezh2wild/wild (EW) and Cre-ERT;Ezh2flox/flox (EF) mice (CD45.2) and transduced these cells with RUNX1S291fs retrovirus or an empty vector, which contains IRES-GFP. Then, we transplanted RUNX1S291fs-transduced Cre-ERT;Ezh2wild/wild (S291EW) or Cre-ERT;Ezh2flox/flox (S291EF) HSCs into lethally irradiated recipient mice (CD45.1) together with life saving dose 1×105 CD45.1 bone marrow cells. At 6 weeks post transplantation, we deleted Ezh2 via administration of tamoxifen, and observed disease progression until 12 months post transplantation. The empty vector transduced control mice with or without Ezh2 (EW and EF) did not develop myeloid malignancies. Two out of 16 S291EW mice died due to MDS progression, while 12 out of 16 and 1 out of 17 S291EF mice developed MDS and MDS/AML, respectively. S291EF mice showed significantly shorter median survival than S291EW mice (314 days versus undefined, p=0.037). In the peripheral blood, we observed significantly lower CD45.2+GFP+ chimerism in S291EF mice; however S291EF mice eventually showed macrocytic anemia and variable white blood cell counts accompanied with dysplastic features of MDS. Despite low CD45.2+GFP+ chimerism in peripheral blood, S291EF mice showed a higher chimerism of CD45.2+GFP+ cells in the bone marrow and had a significantly increased number of LSK and CD34-LSK cells compared to EW, EF, and S291EW mice, indicating that Ezh2 loss promoted HSCs/progenitors expansion, but impaired myeloid differentiation in the presence of RUNX1S291fs. We also saw enhanced apoptosis of CD71+Ter119+ erythroblasts in S291EF MDS mice, which may account for the anemia we observed. Since S291EF MDS bone marrow cells were transplantable in secondary experiments, we performed limiting-dilution assays to evaluate the frequency of MDS initiating cells and found that the frequency of MDS initiating cells was much higher in S291EF pre-MDS Lin-Mac1-Kit+ cells compared to S291EW pre-MDS Lin-Mac1-Kit+ cells. To understand this molecular mechanism, we performed gene expression analysis during MDS progression. S291EF MDS LSKs showed 1979 and 1875 dysregulated (>5-fold) genes, compared to EW LSK and S291EF pre-MDS LSK, respectively. We are now working to understand how these dysregulated genes are involved in the development of RUNX1S291fs-induced MDS after deletion of Ezh2. In summary, we have successfully recapitulated the clinical feature of MDS in mice reconstituted with Ezh2 null HSCs expressing a RUNX1 mutant, and demonstrated that Ezh2 functions as a tumor suppressor in this context. Disclosures: No relevant conflicts of interest to declare.

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Use of the Immunoproteasome Subunit LMP7 Inhibitor to Ameliorate Gvhd in an MHC-Matched miHA-Disparate Murine Model

Blood ◽

10.1182/blood.v120.21.4106.4106 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4106-4106

Author(s):

Jenny Zilberberg ◽

Eugenia Dziopa ◽

Christopher J Kirk ◽

Robert Korngold

Keyword(s):

Bone Marrow ◽

Murine Model ◽

Research Funding ◽

Bone Marrow Cells ◽

Inflammatory Diseases ◽

Clinical Situation ◽

Unrelated Donor ◽

Mhc I ◽

Split Dose ◽

Ubiquitin Proteasome

Abstract Abstract 4106 Background: Therapeutic targeting of the ubiquitin-proteasome pathway is currently used for the treatment of multiple myeloma. The immunoproteasome however, is a distinct class of proteasome found predominantly in monocytes and lymphocytes, and it is known to regulate antigen presentation on class I major histocompatibility complexes (MHC-I). ONX 0914 is a LMP7-selective peptide-epoxyketone proteasome inhibitor that is currently in pre-clinical development for the treatment of inflammatory diseases. In the current study, we evaluated the feasibility of using ONX 0914 in the B10.BR→CBA MHC-matched (H2k), minor histocompatibility antigen (miHA)-disparate bone marrow transplantation (BMT) model to decrease the incidence of GVHD. This well established allogeneic BMT model is relevant to the MHC-matched sibling donor or completely matched unrelated donor clinical situation. Methods: To evaluate the effects of ONX 0914 on GVHD, we tested different compound regimens in the model. CBA recipient mice were lethally irradiated (11 Gy, split dose, 4 h apart) and transplanted with 2×106 anti-T cell depleted B10.BR bone marrow cells (ATBM) alone, or in combination with 1×107 enriched B10.BR T cells (GVHD control and ONX 0914 treated groups). ONX 0914 was administrated in full (s.c., once/day at 8 mg/kg) or as a split dose (half dose, twice/day approximately 6 hr apart). Results: Treatment with the ONX 0914 compound significantly improved the survival of mice compared to control GVHD recipients when dosing was performed once/day for three consecutive days (0,1,2 post-BMT) [p = 0.02]. Alternating dosage (days 0,2,4) or daily drug treatment (days 0–4) provided comparable amelioration of GVHD in this model. Conclusion: Our preclinical findings suggest that the immunoproteasome subunit LMP7 is potentially a novel therapeutic target in GVHD as demonstrated by the improved survival rate of recipient mice treated with ONX 0914 in the B10.BR→CBA BMT murine model. Disclosures: Zilberberg: Onyx Pharmaceuticals: Research Funding. Dziopa:Onyx Pharmaceuticals: Research Funding. Korngold:Onyx Pharmaceuticals: Research Funding.

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The Targeted Histone Deacetylase Inhibitor Tefinostat (CHR-2845) Shows Selective In Vitro Efficacy In Monocytoid-Lineage Acute Myeloid Leukaemia (AML)

Blood ◽

10.1182/blood.v122.21.1297.1297 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1297-1297 ◽

Cited By ~ 1

Author(s):

Joanna Zabkiewicz ◽

Marie Gilmour ◽

Robert K. Hills ◽

Elizabeth Bone ◽

Alan Davidson ◽

...

Keyword(s):

Bone Marrow ◽

Research Funding ◽

Bone Marrow Cells ◽

Therapeutic Window ◽

Normal Bone Marrow ◽

Inhibitory Effects ◽

Normal Bone ◽

Growth Inhibitory ◽

Deacetylase Inhibitor

Abstract Tefinostat (CHR-2845) is a novel monocyte/macrophage-targeted histone deacetylase inhibitor (HDACi) that is cleaved to an active acid, CHR-2847, by an intracellular esterase (human carboxylesterase-1, hCE-1), found only in cells of monocytoid lineage and hepatocytes. The clinical uptake of HDAC inhibition to date has been restricted by systemic toxicities including gastrointestinal disturbance, thrombocytopenia and fatigue. Accumulation of CHR-2847 in hCE-1-expressing cells results in a 20-100-fold increase in targeted anti-proliferative potency, considerably widening the potential therapeutic window in malignancies involving cells of monocytoid lineage (AML-M4, AML-M5 and CMML) by sparing the systemic toxicological effects associated with non-selective HDAC inhibition. The in vitro efficacy of tefinostat was assessed in primary AMLs using stored mononuclear cells obtained at diagnosis from 70 AML patients. Dose-dependent induction of apoptosis and significant growth inhibitory effects were seen in M4 /M5 AMLs (median IC50; 1.1µM+/-1.8) compared to non-M4/M5 FAB types (median IC50 5.1µM +/-4.7) (p=0.007). This potency and monocytoid specificity was not reproduced when using an alternative HDACi, tefinostat analogue CHR-8185 which is not cleaved by hCE-1. hCE-1 protein expression in patient samples was measured by both intracellular flow cytometry and immunoblotting, with highest levels seen in M4/M5 patients. This observation was validated by microarray analysis of hCE-1 mRNA in a further 130 AML samples with M4/M5 AMLs showing significant overexpression compared to normal bone marrow CD34+ cells (p=0.009). High levels of hCE-1 expression were found to drive a significant increase in tefinostat efficacy as measured by growth inhibition assays (p=0.001), and also strongly correlated with expression of the mature monocytoid marker CD14+. Sub-population analysis by flow cytometry revealed variable sensitivity to tefinostat within AML blasts, with CD14+ expressing cells showing maximum growth inhibition. This CD14+ response was accompanied by an induction of intracellular protein acetylation at nanomolar concentrations in tefinostat-responsive samples. Tefinostat-sensitive samples also showed strong induction of the cell cycle arrest and DNA damage sensor protein pH2AX, which is a potential biomarker of patient responsiveness. Importantly, no growth inhibitory effects were seen in normal bone marrow cells (n=5) exposed to AML-toxic doses of tefinostat while, in comparison, equivalent concentrations of the non-hCE-1-dependent analogue CHR-8185 caused considerable cytotoxicity, again emphasising the potential for expansion of the clinical therapeutic window using an hCE-1-dependent agent. In vitro synergy was demonstrated in combination experiments with tefinostat and cytarabine (median Combination Index value=0.68) which is likely to be a logical combination for future clinical evaluation. In summary, monocytoid targeting of HDACi activity was achieved using tefinostat in primary AML samples of monocytoid lineage, with minimal toxicity to normal bone marrow cells at equimolar concentrations. Given the absence of significant toxicity seen in a recently-published phase 1 study of tefinostat in patients with advanced haematological malignancies, further larger scale clinical evaluation of this compound is warranted in haematological malignancies involving cells of monocytoid lineage. Disclosures: Zabkiewicz: Chroma Therapeutics: Research Funding. Gilmour:Chroma Therapeutics: Research Funding. Hills:Chroma Therapeutics: Research Funding. Bone:Chroma Therapeutics: Employment. Davidson:Chroma Therapeutics: Employment. Burnett:Chroma Therapeutics: Research Funding. Knapper:Chroma Therapeutics: Research Funding.

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Leukemia-Associated Mutations Of DNMT3A Inhibit Differentiation Of Hematopoietic Stem Cells and Promote Leukemic Transformation Through Abberant Recruitment Of Bmi1

Blood ◽

10.1182/blood.v122.21.469.469 ◽

2013 ◽

Vol 122 (21) ◽

pp. 469-469

Author(s):

Junji Koya ◽

Keisuke Kataoka ◽

Takako Tsuruta-Kishino ◽

Hiroshi Kobayashi ◽

Kensuke Narukawa ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Research Funding ◽

Bone Marrow Cells ◽

Cpg Island ◽

Hematopoietic Stem ◽

Cell Accumulation ◽

Exogenous Expression ◽

Marrow Cells

Whole genome sequencing has revealed DNMT3A mutation is present in over 20% of cytogenetically normal acute myeloid leukemia (CN-AML) and R882 is the most frequent and recurrent mutated site. Cumulating clinical data have emphasized the importance of the mutation as a poor prognostic factor of AML. Since the functional role of DNMT3A mutation in leukemogenesis remains largely unknown, we aimed to elucidate the impact of DNMT3A mutation on the development and maintenance of AML. To investigate the effect of exogenous expression of DNMT3A R882 mutant (Mut) in hematopoiesis, we transplanted 5-FU primed mouse bone marrow cells transduced with empty vector (EV), DNMT3A wild type (WT), or DNMT3A Mut to lethally irradiated mice. Recipients transplanted with DNMT3A Mut-transduced cells exhibited hematopoietic stem cell (CD150+CD48-Lin-Sca1+c-Kit+) accumulation and enhanced repopulating capacity compared with EV and DNMT3A WT recipients. To identify the downstream target genes of DNMT3A Mut that evoked hematopoietic stem cell accumulation, we sorted vector-transduced LSK cells from transplanted mice and conducted quantitative PCR (Q-PCR) of various hematopoiesis-related genes. Q-PCR revealed that Hoxb cluster expression was up-regulated and differentiation-associated genes, such as PU.1 and C/ebpa, were down-regulated in DNMT3A Mut-transduced LSK cells. Targeted bisulfite sequencing showed hypomethylation of the Hoxb2 promoter-associated CpG island in DNMT3A Mut-transduced cells compared with EV-transduced cells, which suggests dominant-negative effect of DNMT3A R882 mutation. DNMT3A Mut caused no change in methylation status of PU.1 promoter-associated CpG island, indicating that DNA methylation-independent mechanism underlies PU.1 downregulation. Given that DNMT3A interacts with several histone modifiers to regulate target gene transcription, we performed co-immunoprecipitation to investigate whether these interactions are altered by DNMT3A mutation. We found that DNMT3A Mut has the emhanced capacity to interact with polycomb repressive complex 1 (PRC1), which is thought to be a potential mechanism of the DNMT3A Mut-induced differentiation defect. Co-immunoprecipitation experiments showed that DNMT3A R882H and R882C mutant exhibited augmented interaction with BMI1 and MEL18, respectively. In addition, RING1B, an essential component of PRC1, co-localized with DNMT3A Mut more frequently than WT, irrespective of the type of amino acid substitution. Furthermore, heterozygosity of Bmi1 restored the PU.1 mRNA to the normal level and canceled the effect of stem cell accumulation in mice transplanted with DNMT3A Mut bone marrow cells. Chromatin immunoprecipitation in AML cell lines showed that BMI1 and RING1B were more efficiently recruited to the upstream regulatory element of PU.1 upon expression of DNMT3A Mut than WT, while the amount of DNMT3A recruited were comparable between DNMT3A WT and Mut. In the murine transplantation model, we found that exogenous PU.1 expression impaired repopulating capacity in both EV and R882H-transduced cells to the similar level. Exogenous expression of DNMT3A WT inhibited proliferation and induced terminal myeloid differentiation, whereas DNMT3A Mut-transduced cells remained immature in AML cell lines. DNMT3A Mut-transduced cells were resistant to ATRA-induced differentiation compared to EV-transduced cells. Furthermore, R882 mutation promoted blastic transformation of murine c-Kit+ bone marrow cells in vitro in combination with HOXA9 which is highly expressed in clinical cases harboring DNMT3A mutation. Morphological and surface marker analysis revealed these cells were F4/80+ monocytic blasts, consistent with clinical observation that DNMT3A mutation is found frequently in FAB M4/M5 leukemia. These results indicate a distinct role for DNMT3A Mut as well as a potential collaboration between DNMT3A Mut and HOXA9 in malignant transformation of hematopoietic cells. Interestingly, Bmi1 heterozygosity impaired this monoblastic transformation of R882H and HOXA9 co-transduced progenitors. Taken together, our results highlight the functional role of DNMT3A mutation in differentiation block of hematopoietic stem cells and in promoting leukemic transformation via aberrant recruitment of Bmi1 and other PRC1 components. Disclosures: Kurokawa: Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding.

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Dynamic Changes in Clonal Clearance with Decitabine Therapy in AML and MDS Patients

Blood ◽

10.1182/blood.v126.23.689.689 ◽

2015 ◽

Vol 126 (23) ◽

pp. 689-689

Author(s):

John S. Welch ◽

Allegra Petti ◽

Christopher A. Miller ◽

Daniel C. Link ◽

Matthew J. Walter ◽

...

Keyword(s):

Bone Marrow ◽

Complete Remission ◽

Clinical Response ◽

Exome Sequencing ◽

Board Of Directors ◽

Research Funding ◽

Bone Marrow Cells ◽

Advisory Committees ◽

Clonal Hematopoiesis ◽

Clinical Responses

Abstract To determine how AML subclonal architecture changes during decitabine treatment, and whether specific mutations might correlate with sensitivity vs. resistance to decitabine, we performed exome sequencing at multiple time points during single agent decitabine therapy. We enrolled 69 patients with either AML (age ≥ 60, or with relapsed/refractory disease, N = 45) or MDS (N = 24) on a phase I clinical trial. All subjects were treated with decitabine 20 mg/m2 on days 1-10 of 28 day cycles. With a median follow-up of 13.7 months, the intention to treat clinical response (complete remission with or without complete count recovery: CR/CRi) is 40%, with survival correlating with response (median survival - CR/CRi: 583 days; partial response/stable disease (PR/SD): 260 days; progressive disease (PD) or failure to complete cycle 1: 36 days, p < 0.0001). We performed exome sequencing on unfractionated bone marrow cells at diagnosis (day 0), cycle 1 day 10, cycle 1 day 28, cycle 2 day 28, and, when possible, during remission and at clinical relapse/progression. We have completed sequencing analysis for the first 34 cases (outcomes: 5 CR, 15 CRi, 3 PR, 8 SD, and 3 PD). Several important themes have emerged, as follows: 1) We correlated mutation status at diagnosis with clinical response. All six patients with TP53 mutations obtained clinical CR or CRi, and exome analysis demonstrated near complete elimination of the TP53- associated founding clones by the end of cycle 2 (p < 0.03). Long-term outcomes were similar in these patients compared with other patients who achieved CR/CRi: four patients relapsed after 8, 9, 10, or 17 cycles; 1 patient is doing well post-transplant; and one patient died of an infectious complication after cycle 2. No other mutations were significantly associated with clinical response or with consistent mutation clearance. 2) We observed a reduction in blast counts, which preceded mutation elimination in fourteen cases with CR, CRi or PR. This suggests that decitabine may induce morphological blast differentiation in vivo prior to mutation elimination. 3) In eight of nine cases with a clinical response followed by relapse, clinical progression was associated with expansion of a pre-existing subclone. We have not yet observed any recurrent mutations that reliably predict whether a subclone will contribute to relapse. Intriguingly, in two of these cases, the relapse-associated subclone was detectable at diagnosis and was eliminated more slowly than the founding clone mutations, suggesting that this subclone harbored intrinsic decitabine-resistance. 4) Complete remission can occur with concomitant non-malignant, clonal hematopoiesis. In three cases with a CR, a new clonal population was selected for during the remission. In two of these cases, there were no shared mutations between the founding clone and the emergent, non-malignant, clonal hematopoiesis, suggesting that these clones were unrelated. 5) Mutational architecture is generally stable, but differential chemo-sensitivity can be detected even between subclones in the same patient. In ten cases with PR or SD, we observed minimal shifts within the mutational burden over the course of eight weeks, suggesting that "clonal drift" is a relatively slow process. However, in four cases with SD, what appeared clinically to be simple persistent disease was in fact a dynamic elimination of one subclone, and its replacement by a different subclone. Similarly, in three cases with CRi, we observed rapid clearance of a subclone with slower clearance of the founding clone, again suggesting differential chemo-sensitivity among subclones. 6) Finally, we correlated pharmacologic markers with clinical outcomes. We observed no correlation between steady-state plasma decitabine levels and clinical responses. Using Illumina 450k methylation arrays, we observed a correlation between response and the extent of decitabine-induced hypomethylation in total bone marrow cells that persisted on cycle 1 day 28 (p < 0.01), but not on cycle 1 day 10 (p < 0.1). In summary, these data reveal that response to decitabine is associated with morphologic blast clearance before mutations are eliminated, that relapse is associated with subclonal outgrowth that may be identified early in the treatment course, and that TP53 mutations may be predictive of rapid clinical responses, although, like most responses to decitabine, these are not necessarily durable. Disclosures Off Label Use: Decitabine treatment of AML.. Uy:Novartis: Research Funding. Oh:CTI Biopharma: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Abboud:Novartis: Research Funding; Gerson Lehman Group: Consultancy; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding; Merck: Research Funding; Teva Pharmaceuticals: Research Funding. Cashen:Celgene: Speakers Bureau. Schroeder:Celgene: Other: Azacitidine provided for this trial by Celgene; Incyte: Consultancy. Jacoby:Sunesis: Research Funding; Novo Nordisk: Consultancy.

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Targeted RNA-Seq profiling of splicing pattern in the DMD gene: exons are mostly constitutively spliced in human skeletal muscle

Scientific Reports ◽

10.1038/srep39094 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 13

Author(s):

Anne-Laure Bougé ◽

Eva Murauer ◽

Emmanuelle Beyne ◽

Julie Miro ◽

Jessica Varilh ◽

...

Keyword(s):

Skeletal Muscle ◽

Alternative Splicing ◽

Cdna Sequence ◽

454 Sequencing ◽

Exon Skipping ◽

Rna Seq ◽

Splicing Pattern ◽

Alternative Splicing Events ◽

Diagnostic Research ◽

Dmd Gene

Abstract We have analysed the splicing pattern of the human Duchenne Muscular Dystrophy (DMD) transcript in normal skeletal muscle. To achieve depth of coverage required for the analysis of this lowly expressed gene in muscle, we designed a targeted RNA-Seq procedure that combines amplification of the full-length 11.3 kb DMD cDNA sequence and 454 sequencing technology. A high and uniform coverage of the cDNA sequence was obtained that allowed to draw up a reliable inventory of the physiological alternative splicing events in the muscular DMD transcript. In contrast to previous assumptions, we evidenced that most of the 79 DMD exons are constitutively spliced in skeletal muscle. Only a limited number of 12 alternative splicing events were identified, all present at a very low level. These include previously known exon skipping events but also newly described pseudoexon inclusions and alternative 3′ splice sites, of which one is the first functional NAGNAG splice site reported in the DMD gene. This study provides the first RNA-Seq-based reference of DMD splicing pattern in skeletal muscle and reports on an experimental procedure well suited to detect condition-specific differences in this low abundance transcript that may prove useful for diagnostic, research or RNA-based therapeutic applications.

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Prognostic Significance of Bone Marrow Cellularity in the Outcome of Patients with Myelodysplastic Syndromes Treated with Azacyitidine: A Retrospective Analysis from the Hellenic MDS Study Group

Blood ◽

10.1182/blood-2019-131003 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5417-5417

Author(s):

Elena E. Solomou ◽

Alexandra Kourakli ◽

Anna Vardi ◽

Ioannis Kotsianidis ◽

Panagiotis Zikos ◽

...

Keyword(s):

Bone Marrow ◽

Overall Survival ◽

Board Of Directors ◽

Research Funding ◽

Hypomethylating Agents ◽

Advisory Committees ◽

Female Ratio ◽

Bone Marrow Cellularity ◽

Marrow Cellularity ◽

Male To Female

Introduction: Clinical trials in patients with high risk myelodysplastic syndromes (MDS) have shown that these patients benefit from the available hypomethylating agents 5-azacytidine and decitabine. The majority of these patients display hypercellular bone marrow, but a small proportion despite the excess of blasts, exhibit marrow hypocellularity (<30% cellularity). Data are limited for the efficacy and safety of treatment with hypomethylating agents in this patient subgroup. In the present study we examined the effect of bone marrow cellularity in the overall survival in patients with MDS treated with azacitidine. Patients & Methods: This is a retrospective multicenter study from the Hellenic National MDS Registry (EAKMYS) on behalf of the Hellenic MDS Study Group. Between 1.1.2009 and 31.12.2018 a total of 1161 MDS patients who have received treatment with azacytidine have been registered. Complete patient information and follow-up were available for 989 patients, and all these have been included in the final analysis. Statistical analysis was performed and overall survival (OS) was evaluated, using Kaplan-Meier estimates (GraphPad Prism software, CA). A p value less than 0.05 was considered statistically significant. Results: Forty nine patients had a hypocellular bone marrow (hMDS), representing the 4.95% of the whole patient population. Of these patients 39 were men (5.3% of all men included in the study) and 10 were women representing the 2.98% of all women enrolled (male to female ratio 3.9). In the non-hypoplastic group, 750 were men and 358 were women (male to female ratio 2.09). The median age at diagnosis for the hMDS group was 70.8 years, compared to 72.8 years in the non-hypoplastic group. The IPSS-R prognostic risk categorization included 15 hMDS patients in the low group, 9 in the intermediate, 14 in the high and 11 in the very high risk group. Twenty-six patients (53%) of the hMDS group had bone marrow blasts between 10 and 20%, and the remaining 23 (47%) had 5-10% blasts. The patients with hMDS received an average of 10 cycles of azacytidine treatment during the follow-up period (range 2-29 cycles). The outcomes tested were overall survival and progression to AML. The median overall survival of patients with hMDS, following azacytidine treatment start, was not significantly different from the median survival of patients with non-hypoplastic MDS [20 months versus 16 months in the non-hypoplastic group (95% CI of ratio: 0.839 to 1.863). The survival curves were not significantly different between the hMDS and non-hypoplastic MDS group (p=0.32, Figure 1). Progression to AML was also evaluated. Eleven (22.4 %) hMDS patients showed disease progression to AML. Patients with hMDS had significantly prolonged estimated median time to AML transformation, compared to the non-hypoplastic MDS population (31.7 versus 22 months respectively, p<0.001). There were not any major safety issues among patients with hMDS, despite the increased RBC and Platelet transfusion needs. The infectious episodes and the hospitalization courses did not differ significantly between the hMDS and the non-hypoplastic group. Discussion and Conclusive remarks: In this retrospective study, in which a large number of MDS patients was analyzed, we showed that bone marrow cellularity does not affect the outcome in patients treated with azacyitidine. Patients with hMDS show statistically significant slower AML progression compared to non-hypoplastic MDS. Bone marrow cellularity should not be a contraindication for using hypomethylating agents as a therapeutic option, and this type of treatment can be used safely, when indicated, also in patients with hMDS. Disclosures Pappa: Amgen: Research Funding; Gilead: Honoraria, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene / GenesisPharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Vassilakopoulos:Merck: Honoraria; Takeda Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genesis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria; Winmedica: Honoraria; Servier: Membership on an entity's Board of Directors or advisory committees. Symeonidis:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; MSD: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tekeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Genome-wide transcriptome analysis identifies alternative splicing regulatory network and key splicing factors in mouse and human psoriasis

10.1101/259291 ◽

2018 ◽

Author(s):

Jin Li ◽

Peng Yu

Keyword(s):

Alternative Splicing ◽

Computational Analysis ◽

Potential Role ◽

Exon Skipping ◽

Chronic Inflammatory Disease ◽

Splicing Factors ◽

The Novel ◽

Rna Seq ◽

Genome Wide

AbstractPsoriasis is a chronic inflammatory disease that affects the skin, nails, and joints. For understanding the mechanism of psoriasis, though, alternative splicing analysis has received relatively little attention in the field. Here, we developed and applied several computational analysis methods to study psoriasis. Using psoriasis mouse and human datasets, our differential alternative splicing analyses detected hundreds of differential alternative splicing changes. Our analysis of conservation revealed many exon-skipping events conserved between mice and humans. In addition, our splicing signature comparison analysis using the psoriasis datasets and our curated splicing factor perturbation RNA-Seq database, SFMetaDB, identified nine candidate splicing factors that may be important in regulating splicing in the psoriasis mouse model dataset. Three of the nine splicing factors were confirmed upon analyzing the human data. Our computational methods have generated predictions for the potential role of splicing in psoriasis. Future experiments on the novel candidates predicted by our computational analysis are expected to provide a better understanding of the molecular mechanism of psoriasis and to pave the way for new therapeutic treatments.

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