Loss of PERK Signaling Results in Impaired Granulopoiesis in Transgenic Mice Expressing Mutant Ela2.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 551-551
Author(s):  
Suparna Nanua ◽  
Jun Xia ◽  
Mark Murakami ◽  
Jill Woloszynek ◽  
Daniel C. Link
Keyword(s):  
Er Stress ◽  
Protein Misfolding ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Clinical Samples ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Granulocytic Differentiation ◽  
Disease Pathogenesis ◽  
Chemical Inducers

Abstract Abstract 551 Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis characterized by chronic neutropenia, a block in granulocytic differentiation at the promyelocyte/myelocyte stage, and a marked propensity to develop acute myeloid leukemia. Approximately 50% of cases of SCN are associated with germline heterozygous mutations of ELA2, encoding neutrophil elastase (NE). To date, 59 different, mostly missense, mutations of ELA2 have been reported. A unifying mechanism by which all of the different ELA2 mutants disrupt granulopoiesis is lacking. We and others previously proposed a model in which the ELA2 mutations result in NE protein misfolding, induction of endoplasmic reticulum (ER) stress, activation of the unfolded protein response (UPR), and ultimately apoptosis of granulocytic precursors. Testing this (and other) models has been limited by the rarity of SCN and difficulty in obtaining clinical samples for testing. We previously reported preliminary findings of a novel transgenic mouse expressing a truncation mutation of Ela2 (G193X) reproducing a similar mutation found in some patients with SCN (2008 ASH abstract #314). We showed that the G193X Ela2 allele produced the expected truncated protein that was rapidly degraded. Surprisingly, basal and stress granulopoiesis were normal. We hypothesized that reduced expression of Ela2 in murine compared with human granulocytic precursors resulted in less delivery of misfolded mutant NE protein to the ER, attenuating UPR activation and preserving granulopoiesis in G193X Ela2 mice. Consistent with this hypothesis, only modest evidence of UPR activation was observed in G193X Ela2 granulocytic precursors, and these cells displayed increased sensitivity to chemical inducers of ER stress compared with wildtype granulocytic precursors. The UPR model of disease pathogenesis predicts that inhibition of the cellular pathways that handle misfolded proteins may sensitize G193X Ela2 cells to ER stress and result in impaired granulocytic differentiation. To test this prediction, we crossed G193X Ela2 mice with mice lacking protein kinase RNA (PKR)-like ER kinase (PERK); PERK is one of three major ER-resident proteins that sense ER stress and activate the UPR. Of note, homozygous loss-of-function mutations of PERK (EIF2AK3) are responsible for Wolcott-Rallison syndrome, which is characterized by infantile diabetes and neutropenia in approximately 50% of cases. Since PERK deficiency is embryonic lethal, we transplanted fetal liver cells from PERK-/-, PERK-/- × G193X Ela2, and wild type embryos into irradiated recipients. Complete donor engraftment was observed in all cohorts. Basal granulopoiesis was normal in mice reconstituted with PERK-/- cells. However, in the PERK-/- × G193X Ela2 chimeras, though blood neutrophil counts were normal, a significant reduction in bone marrow neutrophils was observed [6.01 × 106/femur ± 0.92 (PERK-/-) versus 3.14 × 106 ± 0.88 (PERK-/- × G193X Ela2); p < 0.001]. These data show that loss of PERK signaling combined with G193X Ela2 expression results in impaired granulopoiesis, providing new evidence in support of the UPR model of disease pathogenesis. Disclosures: No relevant conflicts of interest to declare.

Rcor1 Deficiency Disrupts Myeloerythroid Lineage Differentiation and Causes Severe Myelodysplasia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3248-3248
Author(s):  
Devorah C Goldman ◽  
Huilan Yao ◽  
Guang Fan ◽  
Gail Mandel ◽  
William H. Fleming
Keyword(s):  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Ectopic Expression ◽  
Rapid Onset ◽  
Loss Of Function ◽  
Erythroid Progenitors ◽  
Monocytic Cells ◽  
Lineage Differentiation ◽  
Myeloid Neoplasia ◽  
Neutrophil Differentiation

Abstract Previously, we showed that the co-repressor CoREST (Rcor1) is essential for the maturation of definitive erythroid cells in the mouse fetal liver. To elucidate Rcor1’s function in multilineage hematopoiesis in the adult, we conditionally deleted Rcor1 using Mx1-Cre. The loss of Rcor1 expression in hematopoietic cells led to the rapid onset of a severe anemia due to a complete block of erythropoiesis downstream of committed erythroid progenitors. By contrast, the production of megakaryocyte progenitors, megakaryocyte maturation and platelets were maintained. In the myelomonocytic lineages, although neutrophil differentiation was completely abrogated, the number of monocytic cells was significantly increased, resulting in a peripheral blood leukocytosis and monocytic infiltrations in the liver. Rcor1-deficient monocytes showed a 66% reduction in apoptosis and possessed ~100-fold more CFU-M activity than control cells. The CFU-M derived from Rcor1 deficient bone marrow could be serially replated up to 5 times; however, replating activity was entirely cytokine dependent. Defective myelomonocytic differentiation persisted following transplantation into wild type hosts for up to 12 months but did not progress to leukemia. To begin to understand at the molecular level how Rcor1 regulates monocyte expansion, we evaluated the expression levels of genes whose ectopic expression is associated with myeloid neoplasia. In Rcor1-deficient monocytes, Gata2, Meis1 and Hoxa9 were overexpressed by 8- to 200-fold. Taken together, these data demonstrate that Rcor1 is essential for both erythroid and myelomonocytic differentiation and that its loss of function causes significant myelodysplasia. Disclosures No relevant conflicts of interest to declare.

scholarly journals Increased hnRNP K Expression Impacts Myelopoiesis By Altering RUNX1 Splicing

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3317-3317
Author(s):  
Sean M Post ◽  
Marisa J Aitken ◽  
Prerna Malaney ◽  
Xiaorui Zhang ◽  
Todd Link ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Conflicts Of Interest ◽  
Rna Binding Proteins ◽  
Fetal Liver ◽  
Full Length ◽  
Clinical Samples ◽  
Differentiation Potential ◽  
Hnrnp K ◽  
Mutational Status

Abstract Mutations in RNA binding proteins have been identified as pathogenic drivers in many hematological malignancies. However, in addition to mutational status, expression changes in RNA binding proteins likely impact disease processes. Through our studies, we identified that overexpression of hnRNP K (heterogeneous ribonucleoprotein K) -a poly(C)-RNA binding protein that governs the expression of numerous genes and transcripts- plays a pivotal role in myeloid malignancies. Using clinical samples, we determined that hnRNP K overexpression is a recurrent abnormality, occurring in nearly 30% of AML cases. Importantly, elevated hnRNP K levels associate with decreased overall survival (24.3 months versus 48.7 months; HR 1.9; 95% CI 1.3-2.7). However, the role of hnRNP K overexpression in AML remains unclear. To evaluate its putative oncogenic potential, we overexpressed hnRNP K in murine fetal liver cells (FLCs). Using colony formation assays (CFAs), we demonstrated that hnRNP K-overexpressing FLCs have an altered differentiation potential (increased number of immature (c-kit +Sca-1 +) and decreased number of mature myeloid (Gr1 +CD11b +) cells) and an increase in self-renewal capacity (increased number of colonies) (p=0.008). Mice transplanted with hnRNP K overexpressing FLCs had markedly shortened survival compared to empty vector controls, despite similar engraftment (median survival 8.1 weeks versus median not reached (HR 3.0, 95% CI 1.2 - 7.3, p=0.02). Significantly, extramedullary hematopoiesis was observed in the spleens and the hepatic parenchyma of mice transplanted with FLCs that overexpress hnRNP K. This resulted in disrupted splenic architecture and the presence of immature hematopoietic cells and cells of myeloid origin (CD117, CD14, and myeloperoxidase). Furthermore, analyses of the bone marrow revealed an increase in myeloid cells in hnRNP K transplanted mice. We next used unbiased and biochemical approaches to discover a direct interaction between hnRNP K and the RUNX1 transcript-a critical transcriptional factor often dysregulated in leukemia. Molecular analyses revealed hnRNP K-dependent alternative splicing of RUNX1 (delExon6) , resulting in the generation of a functionally distinct isoform that is more stable than full-length RUNX1. RNA-Seq and reporter assays demonstrated that delExon6 has a unique transcriptional profile compared to full-length RUNX1, suggesting this spliced transcript may have a pathogenic role. To examine the functionality of delExon6, we performed CFAs. Here, we observed that delExon6 expression results in an increased proliferation potential that is mediated by hnRNP K's RNA binding activity. Together, these data establish hnRNP K as an oncogene in myeloid leukemia through its ability to directly bind the RUNX1 transcript, modify RUNX1 splicing, and subsequently alter its transcriptional activity. Disclosures No relevant conflicts of interest to declare.

scholarly journals Second-Generation Pharmacological Chaperones: Beyond Inhibitors

Molecules ◽  
2020 ◽  
Vol 25 (14) ◽  
pp. 3145 ◽  
Author(s):  
My Lan Tran ◽  
Yves Génisson ◽  
Stéphanie Ballereau ◽  
Cécile Dehoux
Keyword(s):  
Protein Misfolding ◽  
First Generation ◽  
Second Generation ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Pharmacological Chaperone ◽  
Conformational Diseases ◽  
Pharmacological Chaperones ◽  
Drug Candidates ◽  
Binding Pockets

Protein misfolding induced by missense mutations is the source of hundreds of conformational diseases. The cell quality control may eliminate nascent misfolded proteins, such as enzymes, and a pathological loss-of-function may result from their early degradation. Since the proof of concept in the 2000s, the bioinspired pharmacological chaperone therapy became a relevant low-molecular-weight compound strategy against conformational diseases. The first-generation pharmacological chaperones were competitive inhibitors of mutant enzymes. Counterintuitively, in binding to the active site, these inhibitors stabilize the proper folding of the mutated protein and partially rescue its cellular function. The main limitation of the first-generation pharmacological chaperones lies in the balance between enzyme activity enhancement and inhibition. Recent research efforts were directed towards the development of promising second-generation pharmacological chaperones. These non-inhibitory ligands, targeting previously unknown binding pockets, limit the risk of adverse enzymatic inhibition. Their pharmacophore identification is however challenging and likely requires a massive screening-based approach. This review focuses on second-generation chaperones designed to restore the cellular activity of misfolded enzymes. It intends to highlight, for a selected set of rare inherited metabolic disorders, the strategies implemented to identify and develop these pharmacologically relevant small organic molecules as potential drug candidates.

Induction of the Unfolded Protein Response but Normal Basal Granulopoiesis in Mice Expressing G192X ELA2

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 314-314
Author(s):  
Mark Murakami ◽  
Jill Woloszynek ◽  
Jun Xia ◽  
Fulu Liu ◽  
Daniel Link
Keyword(s):  
Unfolded Protein Response ◽  
Protein Misfolding ◽  
Embryonic Stem ◽  
Clinical Samples ◽  
Developmental Defect ◽  
Granulocytic Differentiation ◽  
Transgenic Mouse Line ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

Abstract Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis characterized by chronic neutropenia, a block in granulocytic differentiation at the promyelocyte/myelocyte stage, and a marked propensity to develop acute myeloid leukemia. Most cases of SCN are associated with germline heterozygous mutations of ELA2, encoding neutrophil elastase (NE). To date, 59 different, mostly missense, mutations of ELA2 have been reported. A unifying mechanism by which all of the different ELA2 mutants disrupt granulopoiesis is lacking. We and others previously proposed a model in which the ELA2 mutations result in NE protein misfolding, induction of the unfolded protein response (UPR), and ultimately apoptosis of granulocytic precursors. Testing this (and other) models has been limited by the rarity of SCN and difficulty in obtaining clinical samples for testing. Herein, we report the preliminary description of a novel transgenic mouse line that expresses G192X Ela2, reproducing the G193X ELA2 mutation found in some patients with SCN. The G192X mutation was introduced into the murine Ela2 locus by homologous recombination in embryonic stem cells. Heterozygous or homozygous G192 Ela2 “knock-in” mice were healthy with no apparent developmental defect. While expression of Ela2 mRNA was normal, no mature NE protein was detected in the neutrophils of homozygous G192X Ela2 mice. However, in granulocytic precursors (mainly promyelocytes/myelocytes) a small amount of heavily glycosylated mutant NE protein was detected. Together, these observations suggest that G192X NE protein is retained in the endoplasmic reticulum (ER) and rapidly degraded. Consistent with ER stress and induction of the UPR, a significant increase in BiP/GRP78 and ATF6 mRNA expression in mutant granulocytic precursors were observed. Surprisingly, G192X Ela2 mice have normal basal granulopoiesis. The number of circulating neutrophils, granulocytic differentiation in the bone marrow, and number and cytokine responsiveness of myeloid progenitors were comparable to wild type mice. In summary, the G192X Ela2 mice appear to reproduce the NE protein misfolding and UPR activation observed in human SCN granulocytic precursors. However, expression of G192X Ela2 is not sufficient to disrupt basal granulopoiesis in mice. Studies of stress granulopoiesis are underway.

Modeling MLL-PTD Related Myelodysplastic Syndromes in Mouse.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2811-2811
Author(s):  
Xiaomei Yan ◽  
Yue Zhang ◽  
Goro Sashida ◽  
Aili Chen ◽  
Xinghui Zhao ◽  
...  
Keyword(s):  
De Novo ◽  
Conflicts Of Interest ◽  
Gene Dosage ◽  
Fetal Liver ◽  
Loss Of Function ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
De Novo Aml

Abstract Abstract 2811 MLL partial tandem duplication (MLL-PTD) is found in 5–8% of human MDS, secondary acute myeloid leukemia (s-AML) and de novo AML. The molecular and clinical features of MLL-PTD+ AML are different from MLL-fusion+ AML, although they share similar worse outcomes. Mouse knock-in model of Mll-PTD has been generated to understand its underlining mechanism (Dorrance et al. JCI. 2006). Using this model, we've recently reported hematopoietic stem/progenitor cell (HSPC) phenotypes of MllPTD/WT mice. Their HSPCs showed increased apoptosis and reduced cell number, but they have a proliferative advantage over wild-type HSPCs. Furthermore, the MllPTD/WT–derived phenotypic ST-HSCs/MPPs and even GMPs have self-renewal capabilities. However, MllPTD/WT HSPCs never develop MDS or s-AML in primary or transplanted recipient mice, suggesting that additional genetic and/or epigenetic defects are necessary for transformation (Zhang et al. Blood. 2012). Recently, high frequent co-existences of both MLL-PTD and RUNX1 mutations have been reported in several MDS, s-AML and de novo AML clinical cohorts, which strongly suggest a potential cooperation for transformation between these two mutations. Our previous study has shown that MLL interacts with and stabilizes RUNX1 (Huang et al. Blood. 2011). Thus, we hypothesize that reducing RUNX1 dosage may facilitate the MLL-PTD mediated transformation toward MDS and/or s-AML. We first generated the mice containing one allele of Mll-PTD in a Runx1+/− background and assessed HSPCs of MllPTD/wt/Runx1+/− double heterozygous (DH) mice. The DH newborns are runty; they frequently die in early postnatal stage and barely survive to adulthood, compared to the normal life span of wild type (WT) or single heterozygous (Mllwt/wt/Runx1+/− and MllPTD/wt/Runx1+/+) mice. We studied DH embryos fetal liver hematopoiesis and found reduced LSK and LSK/SLAM+ cells, partly because of increased apoptosis. Enhanced proliferation was found in DH fetal liver cells (FLCs) in vitro CFU replating assays over WT and MllPTD/wt/Runx1+/+ controls. DH FLCs also showed dominant expansion in both serial competitive and serial non-competitive BMT assays compared to WT controls. The DH derived phenotypic ST-HSCs/MPPs and GMPs also have enhanced self-renewal capabilities, rescuing hematopoiesis by giving rise to long-term repopulating cells in recipient mice better than cells derived from MllPTD/wt/Runx1+/+ mice. However, DH HSPCs didn't develop MDS or s-AML in primary or in serial BMT recipient mice. We further generated MllPTD/wt/Runx1Δ/Δ mice using Mx1-Cre mediated deletion. These mice showed thrombocytopenia 1 month after pI-pC injection, and developed pancytopenia 2–4 months later. All these MllPTD/wt/Runx1Δ/Δ mice died of MDS induced complications within 7–8 months, and tri-lineages dysplasias (TLD) were found in bone marrow aspirate. However, there are no spontaneous s-AML found in MllPTD/wt/Runx1Δ/Δ mice, which suggests that RUNX1 mutants found in MLL-PTD+ patients may not be simply loss-of-function mutations and present gain-of-function activities which cooperate with MLL-PTD in human diseases onsets. In conclusion, our study demonstrates that: 1) RUNX1 gene dosage reverse-correlates with HSPCs self-renewal activity; 2) Runx1 complete deletion causes MDS in Mll-PTD background. Future studies are needed to fully understand the collaboration between MLL-PTD and RUNX1 mutations for MDS development and leukemic transformation, which should facilitate improved therapies and patient outcomes. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A new murine model of Barth Syndrome neutropenia links TAFAZZIN deficiency to increased ER stress induced apoptosis

2022 ◽  
Author(s):  
Jihee Sohn ◽  
Jelena Milosevic ◽  
Thomas Brouse ◽  
Najihah Aziz ◽  
Jenna Elkhoury ◽  
...  
Keyword(s):  
Er Stress ◽  
Murine Model ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Model System ◽  
Barth Syndrome ◽  
Loss Of Function ◽  
Inner Mitochondrial Membrane ◽  
Hematopoietic Stem ◽  
Increased Sensitivity

Barth syndrome is an inherited X-linked disorder that leads to cardiomyopathy, skeletal myopathy and neutropenia. These symptoms result from the loss of function of the enzyme TAFAZZIN, a transacylase located in the inner mitochondrial membrane that is responsible for the final steps of cardiolipin production. The link between defective cardiolipin maturation and neutropenia remains unclear. To address potential mechanisms of neutropenia, we examined myeloid progenitor development within the fetal liver of TAFAZZIN knock-out animals as well as within the adult bone marrow of wild-type recipients transplanted with TAFAZZIN KO hematopoietic stem cells. We also used the ER Hoxb8 system of conditional immortalization to establish a new murine model system for the ex vivo study of TAFAZZIN-deficient neutrophils. The TAFAZZIN KO cells demonstrated the expected dramatic differences in cardiolipin maturation that result from a lack of TAFAZZIN enzyme activity. Contrary to our hypothesis, we did not identify any significant differences in neutrophil development or neutrophil function across a variety of assays including phagocytosis, and the production of cytokines or reactive oxygen species. However, transcriptomic analysis of the TAFAZZIN-deficient neutrophil progenitors demonstrated an upregulation of markers of endoplasmic reticulum stress and confirmatory testing demonstrated that the TAFAZZIN-deficient cells had increased sensitivity to certain ER stress mediated and non ER stress mediated triggers of apoptosis. While the link between increased sensitivity to apoptosis and the variably penetrant neutropenia phenotype seen in some Barth syndrome patients remains to be clarified, our studies and new model system set a foundation for further investigation.

scholarly journals Activation of the unfolded protein response is associated with impaired granulopoiesis in transgenic mice expressing mutant Elane

Blood ◽  
2011 ◽  
Vol 117 (13) ◽  
pp. 3539-3547 ◽  
Author(s):  
Suparna Nanua ◽  
Mark Murakami ◽  
Jun Xia ◽  
David S. Grenda ◽  
Jill Woloszynek ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Er Stress ◽  
Unfolded Protein Response ◽  
Protein Misfolding ◽  
Strong Support ◽  
Chemical Induction ◽  
Granulocytic Differentiation ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

Abstract Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis that in many cases is caused by mutations of the ELANE gene, which encodes neutrophil elastase (NE). Recent data suggest a model in which ELANE mutations result in NE protein misfolding, induction of endoplasmic reticulum (ER) stress, activation of the unfolded protein response (UPR), and ultimately a block in granulocytic differentiation. To test this model, we generated transgenic mice carrying a targeted mutation of Elane (G193X) reproducing a mutation found in SCN. The G193X Elane allele produces a truncated NE protein that is rapidly degraded. Granulocytic precursors from G193X Elane mice, though without significant basal UPR activation, are sensitive to chemical induction of ER stress. Basal and stress granulopoiesis after myeloablative therapy are normal in these mice. Moreover, inaction of protein kinase RNA-like ER kinase (Perk), one of the major sensors of ER stress, either alone or in combination with G193X Elane, had no effect on basal granulopoiesis. However, inhibition of the ER-associated degradation (ERAD) pathway using a proteosome inhibitor resulted in marked neutropenia in G193X Elane. The selective sensitivity of G913X Elane granulocytic cells to ER stress provides new and strong support for the UPR model of disease patho-genesis in SCN.

scholarly journals Pathogenic Mechanisms Underlying Stargardt Macular Degeneration Linked to Mutations in the Transmembrane Domains of ABCA4

2020 ◽  
Author(s):  
Fabian A. Garces ◽  
Jessica F. Scortecci ◽  
Robert S. Molday
Keyword(s):  
Atpase Activity ◽  
Protein Misfolding ◽  
Substrate Binding ◽  
Transmembrane Domains ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Stargardt Disease ◽  
Photoreceptor Outer Segments ◽  
Novel Treatments ◽  
Stargardt Macular Degeneration

AbstractABCA4 is an ATP-binding cassette (ABC) transporter predominantly expressed in photoreceptors where it transports the substrate N-retinylidene-phosphatidylethanolamine across disc membranes thereby facilitating the clearance of retinal compounds from photoreceptor outer segments. Loss of function mutations in ABCA4 cause the accumulation of bisretinoids leading to Stargardt disease (STGD1) and other retinopathies. In this study, we examined the expression and functional properties of ABCA4 harboring disease-causing missense mutations in the two transmembrane domains (TMDs) of ABCA4. Our results indicate that these mutations lead to protein misfolding, loss in substrate binding, decreased ATPase activity or a combination of these properties. Additionally, we identified an arginine (R653) in transmembrane segment 2 of ABCA4 as a residue essential for substrate binding and substrate-stimulated ATPase activity. The expression and functional activity of the TMD variants correlate well with the severity of STGD1. Our studies provide a basis for developing and evaluating novel treatments for STGD1.

scholarly journals DNMT3A Mutations in Acute Myeloid Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. SCI-31-SCI-31
Author(s):  
Timothy J. Ley ◽  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Gene Expression Signature ◽  
Adverse Outcomes ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Hematopoietic Stem

Abstract Abstract SCI-31 DNMT3A is one of the two human de novo methyltransferases; both are essential for methylating specific cytosine residues, a process that is critical for regulating gene expression during early development. In 2010, recurring mutations in DNMT3A were discovered in patients with AML (1, 2); mutations were not found in the other de novo methyltransferase (DNMT3B) or in the binding partner for both (DNMT3L). After discovering the first mutation by whole genome sequencing, we sequenced all of the exons of DNMT3A in 281 additional AML patients and found a total of 62 mutations (22% of all cases) in the gene that were predicted to affect translation. Eighteen different missense mutations were found, the most common of which was at amino acid R882 (37 patients). We identified six frameshift, six nonsense, and three splice site mutations, and also a 1.5 MB deletion that included the entire DNMT3A gene. DNMT3A mutations are highly enriched in patients with intermediate risk cytogenetics (56 of 166 patients, 33.7%) and were absent in 79 patients with favorable risk cytogenetics (p < 0.001 for both). In our series, DNMT3A mutations were independently associated with poor outcomes in multivariate analyses. Since the initial reports, DNMT3A mutations have been identified in ∼5%–10% of patients with myelodysplastic syndromes (3, 4), in 10%–20% of patients with myeloproliferative neoplasms and secondary AML (5), and in pediatric AML cases, but at much lower frequencies than adult AML (0%–1%) (6, 7). The adverse clinical outcomes associated with DNMT3A mutations have been confirmed by Yan et al (8) and by Thol et al (9). DNMT3A mutations are therefore among the most common in AML and other myeloid malignancies, and may be important for identifying patients with adverse outcomes. However, the mechanisms by which DNMT3A mutations influence AML pathogenesis are not yet clear. In our original study, we did not identify a clear gene expression signature associated with DNMT3A mutations, nor were we able to identify global or focal alterations in DNA methylation patterns that are clearly caused by the mutations. Yan et al (8) reported that DNMT3A mutations were associated with increased expression and hypomethylation of several HOXB genes, but this could not be validated with our data. Many important questions about DNMT3A mutations remain to be answered, including: 1) What are the relationships between DNMT3A mutations and other mutations that affect outcomes?, 2) Do DNMT3A mutations predict responses to hypomethylating agents or other regimens?, 3) Are the recurring mutations at R882 associated with a gain-of-function activity or do they act as dominant negatives?, and 4) Do the missense mutations alter methylase activity, DNA binding and/or methylation specificity, protein/protein interactions, or other (as yet unknown) functions of DNMT3A? Although the mechanism of action of DNMT3A mutations is currently unknown, Challen et al (10) noted that loss of Dnmt3a in the hematopoietic cells of mice caused a dramatic expansion of stem cells upon serial transplantation. These results strongly suggest that loss-of-function mutations in DNMT3A may affect the self-renewal properties of hematopoietic stem cells, which may be relevant for AML pathogenesis. Disclosures: No relevant conflicts of interest to declare.

SF3B1 Mutations Frequently Occur With Both ATM Mutations and TP53 Mutations In CLL Patients

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2868-2868
Author(s):  
Veronika Navrkalova ◽  
Jitka Malcikova ◽  
Barbara Kantorova ◽  
Ludmila Sebejova ◽  
Karla Plevova ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Hot Spot ◽  
Statistical Significance ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Protein Loss ◽  
Tp53 Mutations ◽  
Active Involvement ◽  
Mutation Profile ◽  
Complete Protein

Abstract Abnormalities of TP53 and ATM genes are well established adverse prognostic markers in CLL. Mutations in splicing factor SF3B1 have recently been described as recurrent and predominantly subclonal aberration. Previous studies inconsistently suggested mutual exclusivity or partial overlap with TP53 mutations. Concerning ATM defects, the association between SF3B1 mutations and del(11q) was reported, but relation to ATM mutations remains unclear. The aims were: (a) to assess association between SF3B1 mutations and the most adverse classic genetic lesions represented by TP53 mutations and del(11q); (b) to investigate association between SF3B1 mutations and ATM mutations in a subset of ATM-characterized patients, and (c) to delineate SF3B1 mutation profile and proportion. We used Sanger sequencing of SF3B1 hot-spot exons 14-16, FASAY analysis of TP53 exons 4-10 and resequencing microarray for ATM mutation detection (all 62 coding exons). We analyzed unfavorable cohort of 338 patients characterized by prevalence of unmutated IGHV (72%). At the time of analysis, 82.5% of the patients were previously untreated. We observed SF3B1 mutation in 17.5% (59/338), TP53 mutation in 20% (68/338), and del(11q) in 27.5% (93/338) of cases. All these genetic defects were significantly more frequent in treated patients (SF3B1: P=0.008, TP53: P<0.0001, del(11q): P=0.0295). Interestingly, we observed quite frequent co-occurrence of SF3B1 and TP53 mutations; 28% (19/68) of p53-affected patients in comparison with 15% (40/270) of p53-wt patients harbored SF3B1 mutation (P=0.019). This co-occurrence was apparent (albeit without statistical significance, P=0.166) also in patients investigated at diagnosis, when 20.6% (7/34) of p53-affected patients but only 11.5% (19/165) of p53-wt patients exhibited SF3B1 mutation. The previously reported increased SF3B1 mutation frequency in patients with del(11q) was also apparent but not significant (P=0.078) in our cohort since mutation frequencies were 24% (22/93) and 15% (37/244) in groups with and without del(11q). To analyze the relation of SF3B1 and ATM mutations we used samples with characterized ATM mutation status (n=112). The p53-defective samples and samples with sole del(11q) were omitted since these more frequently harbored SF3B1 mutation. In the remaining 37 patients we observed that SF3B1 and ATM mutations frequently co-occur: 8/21 ATM-mutated patients (38%) but only 2/16 ATM-TP53-wt patients (12.5%) exhibited SF3B1 mutation (P=0.137). This association should be, however, verified on larger cohort. Concerning the SF3B1 mutation profile, we observed previously reported hot-spot missense mutations with the most abundant mutation K700E (21/59, 36%). In addition, we found 2 short in-frame deletions. Altogether it shows that rather than SF3B1 loss-of-function only partial impairment or gain-of-function is possible. The vast majority of samples had mutation in heterozygous state that correlates with presumably preserved second allele. Interestingly, we found 4 mutations in a proportion around 90% that could be explained by loss of heterozygosity in the locus 2q33.1 by any cause. Our study indicates that SF3B1 mutations frequently overlap with mutations in the DNA damage response pathway genes at least in prognostically unfavorable cohort. It seems that SF3B1 mutations do not lead to complete protein loss indicating rather active involvement of mutated SF3B1 in splicing processes in CLL cells. The work was supported by grants NT13519-4, NT11218-6, MSM0021622430, MUNI/A/0723/2012. Disclosures: No relevant conflicts of interest to declare.

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