Cellulose synthase encoding polynucleotides and uses thereof

The invention relates to isolated polynucleotides encoding functional cellulose synthases and UDP-glucose binding domain thereof, transgenic plants and plant cells transformed with the polynucleotides. The invention further relates to methods of transforming plants and plant cells with cellulose synthase or UDP-encoding polynucleotides.https://digitalcommons.mtu.edu/patents/1113/thumbnail.jp

Cellulose synthase promoter and method for modifiying cellulose and lignin biosynthesis in plants

This invention relates to an isolated cellulose synthase promoter, methods for genetically altering cellulose and lignin biosynthesis, and to methods for improving strength properties of juvenile wood and fiber in trees. The invention further relates to methods for identifying regulatory elements in a cellulose synthase promoter and to methods for augmenting expression of polynucleotides operably linked to a cellulose synthase promoter.https://digitalcommons.mtu.edu/patents/1037/thumbnail.jp

The role of the tissue microenvironment in the regulation of cancer cell motility and invasion

During malignant neoplastic progression the cells undergo genetic and epigenetic cancer-specific alterations that finally lead to a loss of tissue homeostasis and restructuring of the microenvironment. The invasion of cancer cells through connective tissue is a crucial prerequisite for metastasis formation. Although cell invasion is foremost a mechanical process, cancer research has focused largely on gene regulation and signaling that underlie uncontrolled cell growth. More recently, the genes and signals involved in the invasion and transendothelial migration of cancer cells, such as the role of adhesion molecules and matrix degrading enzymes, have become the focus of research. In this review we discuss how the structural and biomechanical properties of extracellular matrix and surrounding cells such as endothelial cells influence cancer cell motility and invasion. We conclude that the microenvironment is a critical determinant of the migration strategy and the efficiency of cancer cell invasion

Coniferyl aldehyde 5-hydroxylation and methylation direct syringyl lignin biosynthesis in angiosperms

A central question in lignin biosynthesis is how guaiacyl intermediates are hydroxylated and methylated to the syringyl monolignol in angiosperms. To address this question, we cloned cDNAs encoding a cytochrome P450 monooxygenase (LsM88) and a caffeate O-methyltransferase (COMT) from sweetgum (Liquidambar styraciflua) xylem. Mass spectrometry-based functional analysis of LsM88 in yeast identified it as coniferyl aldehyde 5-hydroxylase (CAld5H). COMT expressed in Escherichia coli methylated 5-hydroxyconiferyl aldehyde to sinapyl aldehyde. Together, CAld5H and COMT converted coniferyl aldehyde to sinapyl aldehyde, suggesting a CAld5H/COMT-mediated pathway from guaiacyl to syringyl monolignol biosynthesis via coniferyl aldehyde that contrasts with the generally accepted route to sinapate via ferulate. Although the CAld5H/COMT enzyme system can mediate the biosynthesis of syringyl monolignol intermediates through either route, k(cat)/K(m) of CAld5H for coniferyl aldehyde was ≈140 times greater than that for ferulate. More significantly, when coniferyl aldehyde and ferulate were present together, coniferyl aldehyde was a noncompetitive inhibitor (K(i) = 0.59 μM) of ferulate 5-hydroxylation, thereby eliminating the entire reaction sequence from ferulate to sinapate. In contrast, ferulate had no effect on coniferyl aldehyde 5-hydroxylation. 5-Hydroxylation also could not be detected for feruloyl-CoA or coniferyl alcohol. Therefore, in the presence of coniferyl aldehyde, ferulate 5-hydroxylation does not occur, and the syringyl monolignol can be synthesized only from coniferyl aldehyde. Endogenous coniferyl, 5-hydroxyconiferyl, and sinapyl aldehydes were detected, consistent with in vivo operation of the CAld5H/COMT pathway from coniferyl to sinapyl aldehydes via 5-hydroxyconiferyl aldehyde for syringyl monolignol biosynthesis

Genetic and Epigenetic Defects in the Autophagy Machinery in Myelodysplastic Syndromes

Abstract Autophagy is a degradation process for the turnover of damaged organelles and long-lived proteins that also plays an important role during erythropoiesis. Accordingly, knockout of the essential autophagy gene Atg7 in mice leads to clinico-morphologic features of MDS. To date, no study has determined the prevalence and impact of defects (mutations, aberrant expression) in the autophagy machinery in MDS. We interrogated the occurrence of alterations in 180 autophagy genes by analyzing WES of patients with MDS (N=120). For comparison, we analyzed results from other hematologic neoplasms (N=103) and TCGA (N=202). We detected somatic mutations in autophagy genes in 40/425 patients (9%). Mutations were enriched in MDS (12%;14/120) and prevalent in higher risk MDS patients (30%;12/40). Mutations were found in: ATG2A, ATG4C, ATG14, ATG16L1, BCL2, CDKN2AIPNL, COG8, DNM1L, DNM2, GYS1, HIF1A, KIF1B, LAMP2, MLST8, MTOR, NOD2, PIK3CB, PIK3C2A/B, PIK3C2G, PPP2R2A/B, PPP2R3A, PRKAA1/2, PRKACB, PRKAG1/G2, PTPN2, RICTOR, RPTOR, SEC22B, SMURF1, SQSTM1, STAT3, SUPT20H, TAB2, TNFSF10/13B, ULK4, USP10, VPS11/33B, VTI1A, WDFY3/4, WAC. Twenty-five mutations had a cut-off >20% VAF. Most patients (31/40;78%) had a sole mutation, 4 patients carried 2 mutations each (ULK4, WDFY3), (KIF1B, SEC22B), (VIT1A, TAB2), (DNM2, WAC). PRKACB mutations were found in 3 patients. NOD2, PTPN2, PRKAG1, SEC22B, ULK4, VPS11 and WAC mutations were found in 2 patients. inces autophagy genes has been Loss of function mutations were observed in ULK4, NOD2 and WAC. Four genes mapped to commonly deleted regions e.g., 5q (CDKN2AIPNL, SQSTM1) or 7q (SMURF1, PRKAG2) and coincided with haploinsufficient expression, while 3 genes had hemizygous configuration (SMURF1, PPP2R3A, PIK3C2G). Reactome analysis clustered the mutations in effectors/inhibitors and early stage. The analysis of 263,973 germline variants detected that PIK3C2G, NOD2 and HIF1A were also associated with exonic germline variants predicted to be significantly deleterious. Mutations or aberrant expression of core components of the autophagy network have been associated with poor outcomes in multiple diseases. In our cohort, 21 patients died. Most (57%;21/37) of patients had abnormal karyotype with 4 patients having complex karyotype including -17 and -7. Among the cases with abnormal karyotype, 4 cases had del(5q). Mutant patients had worse survival trending toward significance compared to WT patients (MUTvs. WT=20 vs. 90; median 14 vs. 20 months; LogR=.09). Among disease groups, autophagy mutations were associated with significantly inferior survival in MDS (MUT vs. WT=13 vs. 61; 17 vs. 35 months; LogR=.018) and MDS/MPN (MUT vs. WT=4 vs. 31; 12 vs. 30 months; LogR=.037). Seven mutant patients who received hypomethylating agents had no response. Comparative analyses (Sanger, TruSeq, WES, TCGA) identified that autophagy gene mutations were significantly associated with TET2 (28%;11/40; P=.02) among other mutations [RUNX1, STAG2 (20%;8/40), SRSF2 (18%;7/40), DNMT3A, ASXL1 (15%;6/40)]. Clonal hierarchy showed that autophagy gene mutations were mainly secondary events, were ancestral events in 7 (ATG2A, DNML1, PRKACB, PRKAG1, PTPN2, SEC22B, STAT3) and co-dominant in 2 patients (NOD2, MLST8). When autophagy genes mutations were secondary, the most represented ancestral mutationswere in splicing factors (N=9; SRSF2, PRPF8, U2AF1) and DNA methylation (N=4; TET2, DNMT3A). RNA sequencing determined that changes in autophagy gene expression are overrepresented in specific MDS subtypes with distinct mutational profiles. The expression levels of 2 ULK family members commonly elevated during erythroid maturation were found in SF3B1K700E compared to SF3B1WT MDS patients (N=6; ULK1, FC=2; ULK3, FC=4; P=.05). Erythroid cells of these SF3B1K700E patients showed increased autophagosomes compared to SF3B1WT cells. In vivo administration of the mTOR inhibitor, temsirolimus (10 mg/kg i.p. 5d/week for 2 wk) improved the erythropoiesis of a transgenic Sf3b1 mouse model by increasing CD71+ cells (10-20% vs. 5%) and ameliorating anemia (Hgb: 7.9 vs. 6.6 g/dL; P=.07; MCV: 43.5 vs. 42.4 fL; P=.08). In sum, defects in autophagy genes are present in MDS, co-occur with other mutations and impact survival. Changes in expression levels of autophagy genes may be associated with MDS phenotypes and modulated by autophagy inducing drugs as evidenced in models of SF3B1 mutations. Disclosures Kelly: Novartis: Consultancy, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Consultancy, Speakers Bureau. Maciejewski:Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Speakers Bureau; Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Speakers Bureau

Targeting Autophagy in Myelodysplastic Syndromes

Abstract Autophagy is a conserved mechanism of protein degradation that plays a physiologic role in iron homeostasis. The RARS subtype of MDS is characterized by inefficient erythropoiesis and the presence of erythroblasts with iron-laden mitochondria (ring sideroblasts, RS). Patients with RARS exhibit an accumulation of iron due to the inability of their erythroid precursors to properly process iron intended for hemoglobin synthesis. RARS erythroid cells display increased autophagy compared to cells from healthy subjects and mice lacking the essential autophagy genes Atg1 and Atg7 develop progressive anemia due to the inefficient removal of defective mitochondria. We previously showed that the presence of SF3B1 mutations, which are frequent in RARS patients, are associated with the presence of increased mitochondrial iron content and low levels of apoptosis. This finding suggests that cell death-independent mechanisms may temper excess iron by triggering the clearance of mitochondria during erythroid maturation. RNA sequencing analyses have shown that the autophagy pathway is upregulated in SF3B1MUT cells. We investigated the role of mitochondrial autophagy in the alleviation of iron-related damage in RARS erythroid cells and the potential therapeutic benefit of autophagy-stimulating agents for the selective improvement of erythropoiesis and iron homeostasis in SF3B1MUT RARS cells. Transmission electron microscopy (TEM) demonstrated an accumulation of iron in the mitochondria of SF3B1MUT (K700E, n=4) compared to SF3B1WT RARS erythroblasts. Flow cytometry confirmed increased mitochondrial iron in SF3B1MUT (n=10) compared to SF3B1WT (n=10) RARS cells (82% ± 10 vs. 24% ± 5; P = 0.004). Autophagic vacuolization of the cytoplasm and an increased number of autophagosomes were found in SF3B1MUT (K700E) compared to SF3B1WT erythroblasts (n=2). The mRNA expression levels of numerous autophagy genes were elevated in SF3B1MUT vs. SF3B1WT RARS cells: ATG complexes (ATG2A/B, FC=2; ATG4A, FC=2; ATG9A, FC=5; ATG4C, P=0.05; ATG18 (FC=4.8; P= 0.02), autophagy-initiating kinases (ULK1, FC=2; ULK3, FC=3.9; P=0.05), and cathepsins involved in late stage autophagy (CTSL1, FC=20; CTSD, FC=5.8; P=0.05; CTSB, FC=2.1; CTSE, FC=5.9; CTSD, FC=2; P=0.01). qRT-PCR confirmed that SF3B1MUT cells expressed elevated mRNA levels of selected genes. We next administered 3 FDA-approved drugs with established autophagy-stimulating properties [temsirolimus, metformin, arsenic trioxide (ATO)] to a transgenic SF3B1 mouse model (SF3B1+/-) that exhibits anemia, dysplasia, and RS (Visconte, J Hematol Oncol 2014) to evaluate drug-mediated improvement of erythropoiesis and autophagic clearance of excess iron. Eight-month-old SF3B1+/- mice (n = 10 per group) were treated with vehicle control and the following: temsirolimus (10 mg/kg i.p. 5d/week for 2 wk), metformin (250 mg/kg/d, gavage for 2 wk), and ATO (10 mg/kg i.p. 5 d/wk for 2 wk). All agents were well tolerated and triggered morphologic features of autophagy including increased autophagosome-like structures by TEM. No effects on BM cellularity and/or dysplasia were noted, although changes in the morphology of myeloid cells (numerous swollen nuclei) were detected by Wright stain. Mitochondria that were engulfed in autophagosomes were frequently seen. After 2 weeks, temsirolimus-treated SF3B1+/- mice showed an incremental increase in hemoglobin by 1.2 g/dL (7.9 vs. 6.6 g/dL; P=0.07) and in mean corpuscular volume (43.5 vs. 42.4 fL; P=0.08). Erythropoiesis was improved as shown by increased levels of CD71-positive cells by immunohistochemistry in cells post-temsirolimus treatment compared to pre-treatment (10-20% vs. 5%). Increased dividing erythroid cells with binucleation and budding were also observed in cells following temsirolimus treatment. These hematologic changes were not detected with ATO or metformin. In sum, our data support a role for activated autophagy in the pathogenesis of RARS and indicate that stimulating autophagy with approved existing drugs or novel investigational drugs may yield therapeutic benefit in SF3B1MUT RARS patients. Disclosures Kelly: Pharmacyclics: Consultancy, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Speakers Bureau. Advani:Pfizer: Consultancy, Research Funding. Carraway:Novartis: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Baxalta: Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Research Funding, Speakers Bureau

Activation of the Unfolded Protein Response with the First-in-Class P97 Inhibitor CB-5083 Induces Stable Disease Regression and Overcomes Ara-C Resistance in AML

Abstract Acute myeloid leukemia (AML) therapy has remained relatively unchanged for more than 40 years with the majority of patients not achieving long-term remission when treated with currently available agents. Novel strategies are urgently needed to improve outcomes. The constitutive dysregulation of protein synthesis/turnover contributes to disease progression and drug resistance in many forms of cancer including AML. p97 (VCP) is a master regulator of protein turnover that has been implicated in oncogenesis and malignant pathogenesis. CB-5083 is a first-in-class selective and potent orally available inhibitor of p97 that in currently being evaluated in phase I clinical trials in patients with multiple myeloma and advanced solid tumors. To assess the potential benefit of p97 inhibition as a novel approach for AML therapy, we investigated the efficacy, pharmacodynamics (PD), and pharmacokinetics (PK) of CB-5083 in a panel of human AML cell lines with diverse genetic backgrounds, primary AML specimens from both newly diagnosed and relapsed/refractory patients, and xenograft mouse models of AML. In vitro treatment with CB-5083 potently diminished the viability of AML cell lines (n = 7) and primary CD34+ blasts obtained from patients (n = 10) with IC50s significantly below 1 µM (range 200 - 700 nM) in all lines and specimens evaluated to date. Diminished viability was associated with reduced clonogenic survival and increased apoptosis in AML cell lines and primary blasts. In contrast to many conventional and experimental drugs that are less active against primary AML cells than established AML cell lines, primary cells exhibited sensitivity to CB-5083 that was similar to cell lines. Additionally, CB-5083 was highly active in 3 different cell line models of cytarabine resistance and primary cells from refractory AML patients. This suggests that CB-5083 may be effective for patients who are relapsed/refractory to conventional therapy. In vitro PD analyses demonstrated that CB-5083 rapidly triggered the accumulation of ubiquitin-conjugated proteins, activated the unfolded protein response (UPR), disrupted STAT5 signaling, reduced levels of key STAT5 targets including BCL-xL and PIM-2, and induced apoptosis. The pro-apoptotic effects of CB-5083 were associated with activation of the endoplasmic reticulum (ER) resident initiator caspase-4 and induction of the BH3-only protein NOXA, which has been previously demonstrated to be an important mediator of cell death induced by other agents that disrupt protein homeostasis. RNA sequencing (RNASeq) gene ontology (GO) analyses of MV4-11 and MOLM-13 AML cells following treatment with CB-5083 demonstrated that short-term treatment (6h) caused significant increases in multiple regulators of the unfolded protein response, protein biosynthesis, and other ubiquitin-related pathways (p<0.001). Results were confirmed by qRT-PCR. The in vivo anti-leukemic activity of CB-5083 was investigated in two different xenograft mouse models of AML: the FLT3-ITD+ MV4-11 cell line and APML HL-60 cells. Oral administration of CB-5083 (once daily, 4 days on, 3 days off) was well tolerated and induced disease regression in both xenograft models (p<0.01). In vivo PD studies demonstrated that administration of CB-5083 led to reduced AML cell proliferation (PCNA), to the induction of apoptosis (active caspase-3), and pathway inhibition as evidenced by poly-ubiquitin accumulation and elevated expression of CHOP, GRP78, and NOXA. PK-PD analyses demonstrated a correlation between the kinetics of the in vivo PD effects and drug exposure. Our collective preclinical data demonstrate that p97 inhibition is a very effective novel anti-leukemic strategy and support clinical investigation of CB-5083 in patients with relapsed/refractory AML. Disclosures LE Moigne: Cleave Biosciences: Employment. Rolfe:Cleave Biosciences: Employment. Djakovic:Cleave Biosciences: Employment. Anderson:Cleave Biosciences: Employment. Wustrow:Cleave Biosciences: Employment. Zhou:Cleave Biosciences: Employment. Wong:Cleave Biosciences: Employment. Sekeres:TetraLogic: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Carew:Boehringer Ingelheim: Research Funding