Dual Mutant Research Articles

BCL2A1: A Novel Target in Refractory Acute Myeloid Leukemia with FLT3-ITD/D835 Dual Mutations

Internal tandem duplications in the juxtamembrane domain of FMS-like tyrosine kinase 3 gene (FLT3-ITD) and missense mutations in the gene's tyrosine-kinase domain (FLT3-TKD) play critical roles in the pathophysiology of acute myeloid leukemia (AML). Recent study revealed that AML cells resistant to quizartinib, a type II TKI, consist of heterogeneous clonal populations harboring wild-type FLT3 as well as FLT3-ITD, FLT3-TKD and FLT3-ITD/TKD mutations, and on- and off-target mechanisms may contribute to the resistance to TKIs. To overcome the heterogeneous resistance mechanisms of FLT3-ITD and TKD mutations, various combinatorial therapies have been investigated. For example, BCL-2 inhibition in the presence of TKIs increased survival a murine FLT3-ITD AML model, and a phase Ib/II clinical trial of a combination of quizartinib with venetoclax, a BCL2 inhibitor is ongoing in relapsed/refractory FLT3-mutant AML patients (NCT03735875). To dissect underlying mechanisms of drug-resistance and exploring new targets in refractory AML with FLT3-ITD and TKD mutations, we investigated alterations of transcriptome signatures by analyzing AML samples with FLT3-ITD/D835 dual mutations. Previously, we reported BCL2A1 transcriptomes were upregulated in primary AML cells with FLT3-ITD/D835 dual mutations compared to cells with FLT3-ITD mutations only. This was recapitulated in the MV4-11 cells harboring FLT3-ITD/D835 dual mutations after 6 month-exposure to quizartinib. The MV4-11 cells with the FLT3-ITD/D835 dual mutations became resistant to quizartinib, and the cells also became resistant to venetoclax, a BCL2 inhibitor (Yamatani et al. ASH 2019). In this study, we further investigated BCL2A1 as new target in refractory AML with FLT3-ITD/D835 dual mutations. First, we examined whether overexpression of BCL2A1 induces drug-resistance in MV4-11 and Molm13 cell lines with FLT3-ITD. While parental MV4-11 and Molm13 cells are sensitive to venetoclax and quizartinib, MV4-11 and Molm13 cells transfected with lentivirus carrying BCL2A1 became resistant to venetoclax (IC50: MV4-11 with BCL2A1 over-expression >1000 nM vs. mock vector 0.71 nM; Molm13 with over-expression >1000 nM vs. mock vector 0.38 nM, 72h). In contrast, the sensitivity to quizartinib was retained in the BCL2A1 overexpressing MV4-11 and Molm13 cells. These findings indicate that the overexpression of BCL2A1 could play a role in the acquired resistance to venetoclax, but not to quizartinib. Bromodomain-containing protein 4 (BRD4), a family member of bromodomain and extra-terminal motif (BET) is known to transcriptionally modulate BCL2A1 gene expression. Next, we examined effects of CPI-0610, a BET inhibitor, on MV4-11 cells with FLT3-ITD or the FLT3-ITD/D835 dual mutation. CPI-0610 inhibited cell growth of MV4-11 cells by inducing apoptosis irrespective of co-existing FLT3 mutations (IC50: FLT3-ITD/D835, 255 nM vs. FLT3-ITD, 191 nM, 72h). Immunoblotting analyses confirmed that BET inhibition by CPT-0610 decreased the expression of BCL2A1 in MV4-11 cells FLT3-ITD/D835. WIn conclusion, transcriptome analysis and molecular pharmacological approaches identified alterations in the anti-apoptotic BCL2 family proteins in double-mutant FLT3 leukemias. BCL2A1 upregulation might be involved in drug resistance of FLT3-ITD/D835 dual mutant AML cells, and could be a promising new target in refractory AML with FLT3-ITD/D835 dual mutations. Disclosures Konopleva: Cellectis: Research Funding; Eli Lilly: Research Funding; Calithera: Research Funding; Sanofi: Research Funding; Ascentage: Research Funding; AstraZeneca: Research Funding; Agios: Research Funding; Stemline Therapeutics: Consultancy, Research Funding; Kisoji: Consultancy; Genentech: Consultancy, Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Forty-Seven: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Rafael Pharmaceutical: Research Funding; Ablynx: Research Funding; Amgen: Consultancy. Carter:Syndax: Research Funding; AstraZeneca: Research Funding; Amgen: Research Funding; Ascentage: Research Funding. Andreeff:Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy.

Characteristics and Prognostic Effects of IDH Mutations across the Age Spectrum in AML: A Collaborative Analysis from COG, SWOG, and ECOG

Characteristics and Prognostic Effects of IDH Mutations across the Age Spectrum in AML: A Collaborative Analysis from COG, SWOG, and ECOG

Dual Oxidase Mutant Retards Mauthner-Cell Axon Regeneration at an Early Stage via Modulating Mitochondrial Dynamics in Zebrafish.

Dual oxidase (duox)-derived reactive oxygen species (ROS) have been correlated with neuronal polarity, cerebellar development, and neuroplasticity. However, there have not been many comprehensive studies of the effect of individual duox isoforms on central-axon regeneration in vivo. Here, we explored this question in zebrafish, an excellent model organism for central-axon regeneration studies. In our research, mutation of the duox gene with CRISPR/Cas9 significantly retarded the single-axon regeneration of the zebrafish Mauthner cell in vivo. Using deep transcriptome sequencing, we found that the expression levels of related functional enzymes in mitochondria were down-regulated in duox mutant fish. In vivo imaging showed that duox mutants had significantly disrupted mitochondrial transport and redox state in single Mauthner-cell axon. Our research data provide insights into how duox is involved in central-axon regeneration by changing mitochondrial transport.

Abstract A21: Compound PIK3CA mutations support a mutational dose-response model for oncogene activation and response to PI3K inhibitor targeted therapy in breast cancer

Abstract PIK3CA mutations represent the most frequent oncogenic driver lesion found across all human cancers. Given that single PIK3CA mutations are a predictive biomarker for response to PI3K inhibition, and that these clinical benefits are small, we hypothesized that additional genomic factors may cooperate with single PIK3CA mutations in oncogenesis and response to PI3K inhibitors. We analyzed several large pan-cancer datasets and have discovered a phenomenon of dual PIK3CA mutations in 15% of all PIK3CA-mutant cancers, including breast cancer. Dual PIK3CA mutations are clonal, are found in both primary untreated and metastatic tumors, and are enriched at conserved amino acid positions. We have proved that the majority of dual PIK3CA mutations—including the most frequent dual PIK3CA mutant combinations in breast cancer—are in cis compound mutations (i.e., on the same allele, resulting in a protein molecule with two mutations) in patient samples and cell lines, as shown through multiple sequencing techniques including long-range single-molecule real-time sequencing (SMRT-seq). Compound PIK3CA mutations increase PI3K pathway signaling and growth proliferation in nontransformed cells and cancer cells in vitro and in vivo. Biochemical experiments using recombinant PI3K complexes reveal a mechanism where compound mutants increase PI3K complex thermal instability, resulting in increased kinase activity and liposome binding as compared to single mutants. Compound PIK3CA mutations render cells more sensitive to the PI3Kα inhibitors BYL719 and GDC-0077 than single PIK3CA mutations. These data translate to the clinic, where patients with dual PIK3CA-mutant metastatic breast cancer exhibit more durable responses to PI3K inhibition versus patients with single PIK3CA-mutant breast tumors. Our data support a novel mutational dose-response model for oncogene activation, where compound mutations amplify PI3K signaling and growth to a greater degree than single mutants, resulting in increased sensitivity to PI3Kα inhibitor targeted therapy. These findings also have therapeutic relevance, where compound PIK3CA mutations may be a superior predictive biomarker for response to PI3Kα inhibitors than single PIK3CA mutations, across PIK3CA-mutant cancers. Citation Format: Neil Vasan, Jared Johnson, Hong Shao, Pedram Razavi, Alexander Gorelick, Erik Ladewig, Alesia Antoine, Hardik Shah, Eneda Toska, Guotai Xu, Abiha Kazmi, Barry Taylor, Komal Jhaveri, Maura Dickler, Elisa de Stanchina, Eduard Reznik, Raul Rabadan, Melissa Smith, Robert Sebra, Lewis Cantley, Maurizio Scaltriti, Jose Baselga. Compound PIK3CA mutations support a mutational dose-response model for oncogene activation and response to PI3K inhibitor targeted therapy in breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A21.

Abstract 3429: Targeting gain-of-function of mutant p53 C-terminal and oligomerization domains to disrupt activated DNA replication in breast cancer

Abstract We recently documented that gain-of-function (GOF) mutant p53 (mtp53) R273H in triple negative breast cancer (TNBC) cells interacts with replicating DNA and PARP1 (Xiao et. al., Cancer Research 2019). The missense R273H GOF mtp53, without a functional central DNA binding domain, has the capacity to interact tightly with chromatin but it is unclear what regions of mtp53 are responsible for the chromatin-based DNA replication activities. GOF mtp53 proteins possess an intact C-terminal domain (CTD) and oligomerization domain (OD). In wild-type p53 the CTD and OD domain regulate non-specific DNA binding, post-translational modification, and protein tetramerization. We are targeting the CTD and OD regions of mtp53 to test if their disruption limits oncogenic GOF activity. We generated CRISPR/Cas9 edited R273H CTD and OD in the TNBC cell line MDA-MB-468, and also exogenously expressed such dual mutant proteins in p53 null cancers, to evaluate the roles played by oligomerization and non-specific DNA binding in mtp53 interactions with replicating DNA and PARP1. We determined the oligomerization state of R273H mtp53 using the glutaraldehyde crosslinking assay and observed that GOF mtp53 R273H mtp53 in breast cancer cells exists as a tetramer. Experiments are in progress to evaluate cell cycle progression, replication initiation, and mtp53 chromatin interactions in cells expressing missense mtp53 proteins with co-associated mutated CTDs or ODs. Our preliminary data suggests that disrupting the mtp53 CTD inhibits its interactions with chromatin and progression through the S-phase. Unpublished data on the roles played by the mtp53 CTD and OD in DNA replication will be presented at the 2020 AACR meeting. Acknowledgements: This work was funded by grants from the Breast Cancer Research Foundation BCRF-18-011 and BCRF-19-011 to Jill Bargonetti. Devon Lundine and Gu Xiao also received project support from TUFCCC/HC Regional Comprehensive Cancer Health Disparity Partnership, Award Number U54 CA221704(5) from the National Cancer Institute. Special thanks to Ella Freulich for help with CRISPR/Cas9 work and James J. Manfredi for mutant p53 plasmids to exogenously express protein. Citation Format: Devon Lundine, George Annor, Viola Ellison, Gu Xiao, Carol Prives, Jill Bargonetti. Targeting gain-of-function of mutant p53 C-terminal and oligomerization domains to disrupt activated DNA replication in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3429.

Allostery and Epistasis: Emergent Properties of Anisotropic Networks.

Understanding the underlying mechanisms behind protein allostery and non-additivity of substitution outcomes (i.e., epistasis) is critical when attempting to predict the functional impact of mutations, particularly at non-conserved sites. In an effort to model these two biological properties, we extend the framework of our metric to calculate dynamic coupling between residues, the Dynamic Coupling Index (DCI) to two new metrics: (i) EpiScore, which quantifies the difference between the residue fluctuation response of a functional site when two other positions are perturbed with random Brownian kicks simultaneously versus individually to capture the degree of cooperativity of these two other positions in modulating the dynamics of the functional site and (ii) DCIasym, which measures the degree of asymmetry between the residue fluctuation response of two sites when one or the other is perturbed with a random force. Applied to four independent systems, we successfully show that EpiScore and DCIasym can capture important biophysical properties in dual mutant substitution outcomes. We propose that allosteric regulation and the mechanisms underlying non-additive amino acid substitution outcomes (i.e., epistasis) can be understood as emergent properties of an anisotropic network of interactions where the inclusion of the full network of interactions is critical for accurate modeling. Consequently, mutations which drive towards a new function may require a fine balance between functional site asymmetry and strength of dynamic coupling with the functional sites. These two tools will provide mechanistic insight into both understanding and predicting the outcome of dual mutations.

N-glycosylation of \u03b11D-adrenergic receptor N-terminal domain is required for correct trafficking, function, and biogenesis

G protein-coupled receptor (GPCR) biogenesis, trafficking, and function are regulated by post-translational modifications, including N-glycosylation of asparagine residues. α1D-adrenergic receptors (α1D-ARs) – key regulators of central and autonomic nervous system function – contain two putative N-glycosylation sites within the large N-terminal domain at N65 and N82. However, determining the glycosylation state of this receptor has proven challenging. Towards understanding the role of these putative glycosylation sites, site-directed mutagenesis and lectin affinity purification identified N65 and N82 as bona fide acceptors for N-glycans. Surprisingly, we also report that simultaneously mutating N65 and N82 causes early termination of α1D-AR between transmembrane domain 2 and 3. Label-free dynamic mass redistribution and cell surface trafficking assays revealed that single and double glycosylation deficient mutants display limited function with impaired plasma membrane expression. Confocal microscopy imaging analysis and SNAP-tag sucrose density fractionation assays revealed the dual glycosylation mutant α1D-AR is widely distributed throughout the cytosol and nucleus. Based on these novel findings, we propose α1D-AR transmembrane domain 2 acts as an ER localization signal during active protein biogenesis, and that α1D-AR N-terminal glycosylation is required for complete translation of nascent, functional receptor.

Vorasidenib (AG-881): A First-in-Class, Brain-Penetrant Dual Inhibitor of Mutant IDH1 and 2 for Treatment of Glioma.

Inhibitors of mutant isocitrate dehydrogenase (mIDH) 1 and 2 cancer-associated enzymes prevent the accumulation of the oncometabolite d-2-hydroxyglutarate (2-HG) and are under clinical investigation for the treatment of several cancers harboring an IDH mutation. Herein, we describe the discovery of vorasidenib (AG-881), a potent, oral, brain-penetrant dual inhibitor of both mIDH1 and mIDH2. X-ray cocrystal structures allowed us to characterize the compound binding site, leading to an understanding of the dual mutant inhibition. Furthermore, vorasidenib penetrates the brain of several preclinical species and inhibits 2-HG production in glioma tissue by >97% in an orthotopic glioma mouse model. Vorasidenib represents a novel dual mIDH1/2 inhibitor and is currently in clinical development for the treatment of low-grade mIDH glioma.

Somatic Bzip Mutations of CEBPA Are Associated with Favorable Outcome Regardless of Presence As Single Vs. Double Mutation

Somatic mutations of CEBPA in AML are present at similar rates in all age groups and have been shown to be associated with favorable outcome. The most common cooperating variant in CEBPA mutations is a second CEBPA mutation, where the cooperation of TAD and bZip mutations lead to a highly penetrant variant which is sufficient for malignant transformation. Data suggested that only double CEBPA mutations (CEBPA-dm) were associated with favorable outcome. Currently, WHO classification of AML includes CEBPA-dm as a diagnostic entity in AML. We previously reported that in children and young adults, somatic single CEBPA mutations, which almost exclusively occurred in the bZip domain, had similar outcomes to CEBPA-dm patients. We have also shown that CSF3R and GATA2 mutations occur in childhood AML and are highly associated with CEBPA mutations. In this large analysis, we sought to define the prognostic impact of CEBPA-dm vs single bZip mutations and evaluate the impact of common co-occurring mutations. This study included a total of 2,958 patients (age 0-29.94 years) treated on the 4 large Children's Oncology Group trials CCG2961 (n=562), AAML03P1 (n=266), AAML0531 (n=917), and AAML1031(n=1233). DNA binding domain of CEBPA (bZip domain) was analyzed by fragment length analysis and 160 patients with mutations were identified and verified with sanger sequencing (n=23 2961; n=15 03P1; n=51 0531; n=71 1031). In all cases where bZip mutations were detected, N terminal region was sequenced for identification of TAD mutations. Of the 160 cases with bZip mutation, 132 patients had a secondary TAD mutation (CEBPA-dm; 82.5%) and in the remaining 28 cases (17.5%) no TAD mutation was detected (CEBPA-sm). Comprehensive NGS data was available in a cohort of patients (n=106) and demonstrated dm vs. sm prevalence of 81% and 19% respectively, similar to the cohort overall. We compared clinical and biologic characteristics among CEBPA-dm vs. CEBPA-sm patients. The two groups showed no significant differences across karyotype, complete remission rates, FLT3/ITD or NPM1 status, cyto-molecular risk group, age, or diagnostic WBC. CEBPA mutations were more prevalent in Asian patients (10.5%) compared to other races (p=0.019). Evaluation of outcomes for those with CEBPA-dm vs. sm in the entire cohort demonstrated that those with CEBPA bZip mutations regardless of dm vs. sm status had identical event free survival (EFS) of 64% (Fig 1A; p=0.777). Similarly, the overall survival (OS) for CEBPA-sm was 89% vs. 81% for CEBPA-dm (p=0.259; Figure 1A). Evaluation by study cohort demonstrated that presence of bZip mutations whether as sm or dm had similar favorable clinical impact (Figure 1B-D; p=NS in each study for EFS and OS). Analysis of CEBPA+ patients with comprehensive NGS data identified somatic mutations in GATA2 (n=20; 19%) and CSF3R (n=12; 11%)(Figure 1E). Co-occurrence of a GATA2 mutation did not impact the favorable outcome conferred by CEBPA+ on EFS or OS (p=0.356 and p=0.749 respectively; Figure 1F). However, presence of CSF3R mutation significantly modulated the clinical implications of CEBPA mutations. We previously showed an overlap with CEBPA and CSF3R mutations, which occur as SNVs in the extracellular domain or truncating mutations in the cytoplasmic domain. CSF3R mutations result in aberrant CSF3R activation and expression and have been shown to be amenable to targeted therapy with either JAK or SRC family inhibitors. Given the large number of CEBPA+ patients available for analysis, we evaluated the outcomes of patients with CEBPA+/CSF3R+ (n=12) and without CSF3R mutations (n=94). Dual mutant CEBPA+/CSF3R+ patients had a significantly inferior EFS of 17% vs. 63% (p<0.001; Figure 1F) due to high rate of relapse. Importantly, despite high relapse rates these dual mutant patients were salvageable after relapse with an OS of 73% vs. 83% for CEBPA+ patients without CSF3R (p=0.563). In a large study of CEBPA mutations in pediatric AML, we show that patients with a bZip mutation, regardless of sm vs. dm status have a favorable prognosis. Further, we confirm the significant overlap of CEBPA and CSF3R mutations previously reported, and demonstrate that CEBPA+/CSF3R+ patients are at high risk for relapse and thus should not be considered a low risk cohort. Given the poor outcomes with current regimens, patients with CEBPA+/CSF3R+ mutations could be considered for addition of tyrosine kinase inhibitors with upfront therapy. Disclosures No relevant conflicts of interest to declare.

Dual Driver Mutant BCR-ABL1-Negative Myeloid Neoplasms: Multicenter Clinicopathologic and Genomic Analysis

Background: Mutational profiling using next-generation sequencing (NGS) has enhanced our biological understanding of and improved prognostic abilities for patients with BCR-ABL1-negative myeloproliferative neoplasms (MPN). Mutations in JAK2, CALR, and MPL, all of which activate the JAK-STAT pathway, account for the majority of MPN driver mutations, and are thought to be mutually exclusive. However, recent data has demonstrated that multiple JAK-STAT activating mutations may coexist in the same patient. Whether such patients demonstrate unique disease biology or clinical manifestations of disease has not been elucidated. Furthermore, characterization of the genomic architecture and histopathologic details of such cases has largely not been described. Methods: We queried genomically-annotated clinical databases at Mount Sinai Hospital and Memorial Sloan Kettering Cancer center to identify patients with myeloid malignancies with dual driver mutations in either JAK2, CALR, and MPL, with absence of BCR-ABL1. We performed chart review to record clinical parameters. Sequencing was carried out using a commercial CLIA-certified myeloid malignancy NGS panel (Genoptix, N=2) or a CLIA-certified myeloid/lymphoid malignancy NGS panel at Memorial Sloan Kettering (N=10). Only variants deemed to be oncogenic or likely oncogenic based on published reports or publicly-available annotated databases were included. We assigned cytogenetic risk for primary myelofibrosis (PMF) based on DIPSS. Histopathologic analysis was performed on bone marrow biopsy and aspirate samples simultaneous with mutational profiling. Results: We identified 11 MPN patients and 1 de novo AML patient out of a total of 680 sequenced patients, with two JAK-STAT activating mutations (Table 1). No cases were identified with more than two activating JAK-STAT mutations. We identified canonical mutations in JAK2, MPL, and CALR in most cases, with some cases harboring mutations reported at a lower frequency in these genes. Co-occurring mutations in genes frequently identified in myeloid malignancies such as TET2 and splicing factors were also identified. Clonal architecture was inferred from mutant-allele fraction (MAF) ratios in these cases. We identified two predominant patterns of clonal architecture across these cases. First, cases in which the MAFs of the two JAK-STAT driver mutations approximate each other (Figure 1A) and second, cases in which one of the JAK-STAT mutations represents the dominant clone (Figure 1B). The greatest discordance in driver mutation MAF occurred in patients with ET, whereas several cases of PMF demonstrated a relative concordance of driver mutation MAF. Karyotype was normal in 4 patients and not performed in 3 patients; the remaining patients had: gain of 9, t(7;12), del(16q), and +der(9)(p10),del(9)(p12p21). Additionally, aCGH+SNP uncovered cryptic CNLOH of 7q22.37q31.2 in one patient. Histopathologic analysis of these cases demonstrated findings consistent with classical description of MPNs (Figures 2A, 2B). These histologic sections from a patient with concurrent MPL and JAK2 mutations showed features suggestive of ET. However, megakaryocytes showed variable morphology ranging from bulbous forms to slightly hyperlobulated forms. Overall, the megakaryocytes were not overly enlarged. Conclusions: Consistent with prior observations, in a proportion of cases with dual driver mutants, the MAF between the two genes is highly discordant. However, in a substantial proportion of cases we have identified, the MAFs of co-occurring driver mutations were relatively concordant. These findings suggest the possibility of clonal interference, as has been described in RAS-mutant core-binding factor AML (Itzykson R, et al. Blood 132.2 (2018): 187-196). Co-existing clonal hematopoiesis could also explain these results. These findings may have clinical implications for the natural history of the disease. Of greater importance, this may have implications for response to therapeutic modalities such as interferon, as differences in response rates to interferon have been described based on the type of JAK-STAT driver mutation present. Thus, clonal selection may occur. Clinical and preclinical data pertaining to the impact of interferon and JAK inhibitors on clonal architecture in dual JAK-STAT driver mutation cases will be presented at the meeting. Disclosures Arcila: Invivoscribe, Inc.: Consultancy, Honoraria. Mascarenhas:Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Roche: Consultancy, Research Funding; Merck: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CTI Biopharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Research Funding; Promedior: Research Funding; Merus: Research Funding; Pharmaessentia: Consultancy, Membership on an entity's Board of Directors or advisory committees. Hoffman:Merus: Research Funding. Rampal:Celgene: Consultancy; Jazz: Consultancy; Blueprint: Consultancy; Constellation: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Incyte: Research Funding.

Cage Transcriptome Analysis Reveals BCL2A1 Upregulation in FLT3-ITD/D835 Dual Mutated AML Cells Harboring Complex Co-Mutations

Cage Transcriptome Analysis Reveals BCL2A1 Upregulation in FLT3-ITD/D835 Dual Mutated AML Cells Harboring Complex Co-Mutations

A steroid-resistant nephrotic syndrome in an infant resulting from a consanguineous marriage with COQ2 and ARSB gene mutations: a case report

BackgroundTreatment of steroid-resistant nephrotic syndrome (SRNS) remains a challenge for paediatricians. SRNS accounts for 10~20% of childhood cases of nephrotic syndrome (NS). Individuals with SRNS overwhelmingly progress to chronic kidney disease (CKD) and end-stage kidney disease (ESRD). Genetic research is of great significance for diagnosis and treatment. More than 39 recessive or dominant genes have been found to cause human SRNS, including COQ2. COQ2 gene mutations not only cause primary coenzyme Q10 deficiency but also cause SRNS without extrarenal manifestations. The concept of COQ2 nephropathy has been proposed for a long time. Mutations in the COQ2 gene have rarely been reported. Worldwide, only 5 cases involving 4 families have been reported.Case presentationWe present the case of a 6-month-old girl with steroid-resistant glomerulopathy due to a COQ2 defect with no additional systemic symptoms. The patient was identified as a homozygote for the c.832 T > C (p. Cys278Arg) missense mutation and a single base homozygous mutation in ARSB gene in c.1213 + 1G > A. The father and mother were heterozygous mutation carriers in both COQ2 and ARSB, and her healthy sister was only a heterozygous mutation carrier in COQ2. In this case, hormone therapy was ineffective, and progressive deterioration of renal function occurred within 1 week after onset, leading to acute renal failure and eventual death.ConclusionsWe reported a consanguinity married family which had COQ2 and ARSB dual mutant. Kidney diseases caused by COQ2 gene mutations can manifest as SRNS, with poor prognosis. The C. 832 T > c (p.csc 278arg) is a new mutation site. Genetic assessment for children with steroid-resistant nephrotic syndrome, especially in infancy, is very important. Families with a clear family history should receive genetic counselling and prenatal examinations, and children without a family phenotype should also receive genetic screening as early as possible.

AML with Mutations in IDH1 and DNMT3A Exhibits a Distinct Epigenetic Signature with Poorer Overall Survival

Acute Myeloid Leukemia (AML) is a biologically diverse disease with subtypes that can be identified through integrated cytogenetic, mutational, and epigenetic characterization. Mutations in epigenetic regulators such as IDH1, IDH2, and DNMT3A are among the most common gene mutations found in AML (Cancer Genome Atlas Research et al. 2013; Papaemmanuil et al. 2016). Targeted inhibitors of IDH1 and IDH2 have recently been approved by the FDA for the treatment of AML (DiNardo et al. 2018; Stein et al. 2015). In addition, IDH/DNMT3A dual mutant AMLs exhibit a distinct epigenetic signature and are characterized by increased in vitro sensitivity to MEK inhibition (Glass et al. 2017).Here we explore the clinical course of AML patients with IDH1, IDH2, and DNMT3A mutations treated at Memorial Sloan Kettering Cancer Center (MSKCC) between 2012 and 2018. Treatment regimens included standard chemotherapy, as well as investigational therapies such as IDH1, IDH2, and pan-IDH inhibitors (Table 1). Using overall survival as our endpoint, we assessed contributions of WHO risk, age, treatment regimen, and gene mutations using multivariate Cox Proportional Hazards modeling. Among intermediate risk patients, we found that the presence of concurrent IDH1/DNMT3A mutations (n=16) conferred the highest mortality risk compared to DNMT3A mutation alone (HR=2.31; p=0.046). In contrast, IDH2/DNMT3A mutated AML conferred similar mortality risk compared to DNMT3A mutation alone (HR=1.07; p=0.85). When compared to intermediate risk IDH1/DNMT3A mutated AML, intermediate risk IDH2/DNMT3A mutated AML had a lower risk of mortality (HR=0.345; p=0.038), (Fig. 1). This adverse risk of IDH1/DNMT3A mutations persists after adjusting for IDH inhibitor therapy (HR=2.42; p=0.05). In multivariate analysis, IDH1 mutation and use of low intensity therapeutic regimens conferred an increased risk of mortality after adjusting for WHO risk (HR=2.25; p=0.016 and HR=1.71; p=0.036, respectively). Induction chemotherapy provided an overall survival benefit after adjusting for WHO Risk and the presence of IDH1, IDH2, DNMT3A, or dual IDH/DNMT3A mutations (HR=0.50; p<0.01). The increased risk associated with IDH1/DNMT3A dual mutations approached statistical significance after adjustment for WHO Risk and therapeutic modality (HR=2.00; p=0.08).Overall, our findings suggest that AML with dual IDH1/DNMT3A mutations constitutes a higher risk disease entity. Further study of targeted treatment modalities such as dual IDH1/MEK inhibition or combined chemotherapy/IDH1 inhibition may be helpful in the treatment of these higher risk AML patients. [Display omitted] DisclosuresLevine:Isoplexis: Equity Ownership; Qiagen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Imago: Equity Ownership; Gilead: Honoraria; Janssen: Consultancy, Honoraria; C4 Therapeutics: Equity Ownership; Novartis: Consultancy; Epizyme: Patents & Royalties; Loxo: Consultancy, Equity Ownership; Prelude: Research Funding; Roche: Consultancy, Research Funding; Celgene: Consultancy, Research Funding. Tallman:AbbVie: Research Funding; Cellerant: Research Funding; Orsenix: Other: Advisory board; BioSight: Other: Advisory board; AROG: Research Funding; Daiichi-Sankyo: Other: Advisory board; ADC Therapeutics: Research Funding. Goldberg:Abbvie: Research Funding; AROG: Research Funding; Pfizer: Research Funding; Celgene: Research Funding. Stein:Pfizer: Consultancy; Celgene: Consultancy; Bayer: Consultancy; Daiichi Sankyo: Consultancy; Agios: Consultancy; Novartis: Consultancy.

Safety and immunogenicity of an attenuated Chinese pseudorabies variant by dual deletion of TK&gE genes

BackgroundSince the outbreak of a new emerging virulent pseudorabies virus mutant in Chinese pig herds, intensive research has been focused on the construction of novel gene deletion vaccine based on the variant virulent viruses. An ideal vaccine candidate is expected to have a balanced safety and immunogenicity.ResultsFrom the infectious clone of PRV AH02LA strain, a TK deletion mutant was generated through two-step Red mutagenesis. After homologous recombination with a transfer vector, a TK&gE dual deficient mutant PRV (PRVΔTK&gE-AH02) was generated, and its structure verified by PCR, RFLP and sequencing. Growth kinetics test showed that PRVΔTK&gE-AH02 reached a titer of 107.5 TCID50 /mL on ST cells.The PRVΔTK&gE-AH02 at a dose of 106.0 TCID50 /animal was not virulent in mice or 1-day-old piglets with maternal PRV antibodies. No clinical signs or virus shedding were detected in 28~ 35-day-old piglets without maternal PRV antibodies after nasal or intramuscular administration with a dose of 106.0 TCID50, although it caused one death of four 1-day-old piglets without maternal PRV antibodies. In the efficiency test of PRVΔTK&gE-AH02, all four 28~ 35-day-old piglets without PRV antibody in the challenge control showed typical clinical symptoms and virus shedding, and two died at 4~ 5 days post challenge. All piglets in 105.0, 104.0 and 103.0 TCID50/dose PRVΔTK&gE-AH02 groups provided complete protection against challenge at only 7 days post intramuscular vaccination. More importantly, PRVΔTK&gE-AH02 stopped virus shedding in these piglets. In contrast, all four piglets in PRV Bartha K61 vaccine group developed high body temperature (≥40.5 °C) and viral shedding, despite they had mild or even no clinical symptoms.ConclusionsThe constructed TK&gE dual deletion mutant PRVΔTK&gE-AH02 can reach high titers on ST cells. The live vaccine of PRVΔTK&gE-AH02 is highly safe, and can not only provide clinical protection but also stops virus shedding. This study suggests that PRVΔTK&gE-AH02 might work as a promising vaccine candidate to combat the PRV variant emerging in Chinese herds since 2011.

Enhanced production of L-threonine by an α,ε-diamopimilic acid and thiamine-HCl dual auxotrophic mutant Corynebacterium glutamicum X1870

Enhanced production of L-threonine by an α,ε-diamopimilic acid and thiamine-HCl dual auxotrophic mutant Corynebacterium glutamicum X1870

Improved catalytic properties of a serine hydroxymethyl transferase from Idiomarina loihiensis by site directed mutagenesis

A novel glyA gene was screened from a marine bacterium, Idiomarina loihiensis encoding a thermo-stable serine hydroxymethyl transferase (SHMT; 418 AA; 45.4 kDa). The activities of wild type (WT) and mutants were analyzed against d-phenylserine using pyrodoxal-5-phosphate (PLP) as cofactor under optimized conditions. Based on homology modelling and molecular docking, several residues were found that may be able to improve the activity of WT-SHMT. Site directed mutagenesis was conducted. The activity and thermostability of the wild type SHMT was improved by two variants H61G and G132P, which showed a noteworthy change in the thermo-stability and activity as compared to WT. To investigate the mechanism of activity of mutants, we combined two residues into one mutant DUAL. WT showed the optimum activity at 50 °C, whereas H61G, G132P and DUAL had the temperature optima of 55, 60 and 60 °C, respectively. These mutants G132P, H61G and DUAL were quite stable at 45 and 55 °C as compared to WT. Dual mutant was relatively more stable at all tested pH(s) while WT loses its activity in alkaline pH(s). Kinetics studies indicated the 1.52, 2.42 and 4.54 folds increase in the kcat value of H61G, G132P and Dual mutants as compared to WT respectively. The molecular docking indicated that hydrophobic interactions are more prominent than hydrogen-bonding and had more influence on ligand binding and active site cavity. The molecular dynamics showed the changed RMSD values for ligand and formation of new hydrogen bonds, hydrophobic interaction which considerably increased the activity and thermo-stability of the mutant proteins as compared to WT. Thus, increased stabilities at higher temperatures and activities can be attributed to new hydrogen bonding, altered active site geometry and increased ligand interactions.

Two adjacent phenylalanines in the NMDA receptor GluN2A subunit M3 domain interactively regulate alcohol sensitivity and ion channel gating

The N-methyl-d-aspartate (NMDA) receptor is a key target of ethanol action in the central nervous system. Alcohol inhibition of NMDA receptor function involves small clusters of residues in the third and fourth membrane-associated (M) domains. Previous results from this laboratory have shown that two adjacent positions in the M3 domain, F636 and F637, can powerfully regulate alcohol sensitivity and ion channel gating. In this study, we report that these positions interact with one another in the regulation of both NMDA receptor gating and alcohol action. Using dual mutant cycle analysis, we detected interactions among various substitution mutants at these positions with respect to regulation of glutamate EC50, steady-state to peak current ratios (Iss:Ip), mean open time, and ethanol IC50. This interaction apparently involves a balancing of forces on the M3 helix, such that the disruption of function due to a substitution at one position can be reversed by a similar substitution at the other position. For example, tryptophan substitution at F636 or F637 increased or decreased channel mean open time, respectively, but tryptophan substitution at both positions did not alter open time. Interestingly, the effects of a number of mutations on receptor kinetics and ethanol sensitivity appeared to depend upon subtle structural differences, such as those between the isomeric amino acids leucine and isoleucine, as they could not be explained on the basis of sidechain molecular volume or hydrophilicity.

Resistance to Mutant Group 2 Influenza Virus Neuraminidases of an Oseltamivir-Zanamivir Hybrid Inhibitor.

Oseltamivir and zanamivir are the two major antiviral drugs available for the treatment of influenza virus infections. However, multidrug-resistant viruses have emerged in clinical cases, which pose a challenge for the development of new drugs. N1 and N2 subtypes exist in the viruses which cause seasonal epidemics and past pandemics. Although N1 drug resistance is well characterized, the molecular mechanisms underlying N2 drug resistance are unknown. A previous report showed that an N2 E119V/I222L dual mutant conferred drug resistance to seasonal influenza virus. Here, we confirm that these substitutions result in multidrug resistance and dramatically reduced sensitivity to NAI. We further elucidate the molecular mechanism underlying N2 drug resistance by solving crystal structures of the N2 E119V and I222L mutants and the dual mutant. Most importantly, we found that a novel oseltamivir-zanamivir hybrid inhibitor, MS-257, remains more effective against drug-resistant N2 and is a promising candidate as a next-generation anti-influenza virus drug.

Dynamic Bcl-xL (S49) and (S62) Phosphorylation/Dephosphorylation during Mitosis Prevents Chromosome Instability and Aneuploidy in Normal Human Diploid Fibroblasts.

Bcl-xL proteins undergo dynamic phosphorylation/dephosphorylation on Ser49 and Ser62 residues during mitosis. The expression of Bcl-xL(S49A), (S62A) and dual (S49/62A) phosphorylation mutants in tumor cells lead to severe mitotic defects associated with multipolar spindle, chromosome lagging and bridging, and micro-, bi- and multi-nucleated cells. Because the above observations were made in tumor cells which already display genomic instability, we now address the question: will similar effects occur in normal human diploid cells? We studied normal human diploid BJ foreskin fibroblast cells expressing Bcl-xL (wild type), (S49A), (S49D), (S62A), (S62D) and the dual-site (S49/62A) and (S49/62D) mutants. Cells expressing S49 and/or S62 phosphorylation mutants showed reduced kinetics of cell population doubling. These effects on cell population doubling kinetics correlated with early outbreak of senescence with no impact on the cell death rate. Senescent cells displayed typical senescence-associated phenotypes including high-level of senescence-associated β-galactosidase activity, interleukin-6 (IL-6) secretion, tumor suppressor p53 and cyclin-dependent kinase inhibitor p21Waf1/Cip1 activation as well as γH2A.X-associated nuclear chromatin foci. Fluorescence in situ hybridization analysis and Giemsa-banded karyotypes revealed that the expression of Bcl-xL phosphorylation mutants in normal diploid BJ cells provoked chromosome instability and aneuploidy. These findings suggest that dynamic Bcl-xL(S49) and (S62) phosphorylation/dephosphorylation cycles are important in the maintenance of chromosome integrity during mitosis in normal cells. They could impact future strategies aiming to develop and identify compounds that could target not only the anti-apoptotic domain of Bcl-xL protein, but also its mitotic domain for cancer therapy.

Conditional Budding Yeast Mutants with Temperature-Sensitive and Auxin-Inducible Degrons for Screening of Suppressor Genes.

The conditional control of protein expression is useful to characterize the function of proteins, especially of those that are essential for cell viability. Two degron-based systems, temperature-sensitive and auxin-inducible degrons, can be used to generate conditional mutants of budding yeast, simply by transforming appropriate cells with PCR-amplified DNA. We describe a protocol for the generation of temperature-sensitive and auxin-inducible degron mutants. We also show that a conditional mutant with few spontaneous revertants was generated by combining two degron systems for the Inn1 protein. Finally, we describe a suppressor screening method that uses the dual degron-Inn1 mutant to identify mutant proteins that suppress Inn1-K31A, which has a defect in cytokinesis.