Life after transplant: are we becoming high maintenance in AML?

Allogeneic hematopoietic cell transplantation (allo-HCT) for patients with AML is increasingly able to impact the historically poor outcomes in this disease. Nonetheless, even with transplant, the rates of post-HCT relapse are unacceptably high, and remain a great challenge in the treatment of patients with AML. Maintenance therapies after allo-HCT, given to patients at high risk of relapse or with evidence of minimal residual disease (MRD), may provide a way to reduce relapse rates and improve survival. New therapies may offer acceptable toxicity profiles in the post-HCT setting, and investigations are ongoing using hypomethylating agents, histone deacetylase inhibitors, immunomodulatory drugs, targeted tyrosine kinase inhibitors, drug-antibody conjugates and cellular therapies. Future directions in the field of post-HCT therapies may include better risk stratification with MRD, as well as the exploitation of novel mechanisms such as immune checkpoint inhibition and modified chimeric antigen receptor (CAR) T cells. In this mini review, we discuss the current landscape of clinical research in post-HCT maintenance therapies, as well as future therapeutic strategies of interest. Although there is great potential for post-HCT agents to improve AML outcomes, these will need to be evaluated prospectively through well-designed randomized clinical trials.

High dietary fat and the development of osteoarthritis in a rabbit model.

OBJECTIVE: Osteoarthritis (OA) is associated with obesity, although this relationship remains unclear. Proposed etiologies of OA in obesity include mechanical loading of malaligned joints and possible toxicity of dietary fat. The hypothesis tested in the present study was that increased dietary fat worsens OA in both malaligned and normal joints, detected by biochemical and histological cartilage markers. METHOD: 83 New Zealand white rabbits were divided among two conditions related to OA: bowing of the knee and a 14%kcal vs 47.8%kcal fat diet. Rabbit weights and knee angles were compared throughout the experiment. At 28 and 38 weeks, intra-articular forces were measured, animals sacrificed, and knee cartilage examined for histological changes, glycosaminoglycan content, 35S uptake, and aggrecanase-1 expression. RESULTS: There were no differences in animal weights or intra-articular forces between the two diets. Despite increased fat content in their diet, animals on the 47.8%kcal fat diet did not gain excess weight. Representative histology showed atypical shearing of articular cartilage among animals on the high fat diet. Animals on the 47.8%kcal fat diet had suppression of protein synthesis compared to the 14%kcal fat diet: lower glycosaminoglycan content and aggrecanase-1 expression in all knee compartments at both times, and lower 35S uptake at 38 weeks. CONCLUSION: These results suggest dietary fat, independent of animal weight, results in altered chondrocyte function. Increased dietary fat was associated with changes in rabbit cartilage in vivo and appears to be a risk factor for the development of OA.

Alcohol-inducible gene expression in transgenic Populus.

We tested the efficiency and optimized the conditions for controlled alcohol-inducible transgene expression in Populus using gus as a reporter gene. Specificity of induction, efficiency in different organs, effect of three chemical inducers, and induction methods were tested using up to 10 independent transgenic events generated in two different Populus genotypes. The optimal inducer concentration and the duration of induction period were determined in dose-response and in time-course experiments. Under in vitro conditions, beta-glucuronidase (GUS) induction was efficient both in the aerial parts and in the roots of regenerated plantlets. Among the chemical inducers tested, ethanol was the most effective activator with no apparent phytotoxicity when concentrations were at or below 2%. After 5 days of treatment, fluorometrically-determined the GUS activity could be detected when inducing with ethanol at concentrations as low as 0.5%. Prolonged induction by ethanol vapors significantly increased the GUS activity in leaves from both the tissue culture plants and greenhouse-grown plants.

A DEFICIENS homolog from the dioecious tree black cottonwood is expressed in female and male floral meristems of the two-whorled, unisexual flowers.

We isolated PTD, a member of the DEFICIENS (DEF) family of MADS box transcription factors, from the dioecious tree, black cottonwood (Populus trichocarpa). In females, in situ hybridization experiments showed that PTD mRNA was first detectable in cells on the flanks of the inflorescence meristem, before differentiation of individual flowers was visually detectable. In males, the onset of PTD expression was delayed until after individual flower differentiation had begun and floral meristems were developing. Although PTD was initially expressed throughout the inner whorl meristem in female and male flowers, its spatial expression pattern became sex-specific as reproductive primordia began to form. PTD expression was maintained in stamen primordia, but excluded from carpel primordia, as well as vegetative tissues. Although PTD is phylogenetically most closely related to the largely uncharacterized TM6 subfamily of the DEF/APETELA3(AP3)/TM6 group, its spatio-temporal expression patterns are more similar to that of DEF and AP3 than to other members of the TM6 subfamily.

Diverse effects of overexpression of LEAFY and PTLF, a poplar (Populus) homolog of LEAFY/FLORICAULA, in transgenic poplar and Arabidopsis.

PTLF, the Populus trichocarpa homolog of LEAFY (LFY) and FLORICAULA, was cloned to assess its function in a dioecious tree species. In situ hybridization studies showed that the gene was expressed most strongly in developing inflorescences. Expression was also seen in leaf primordia and very young leaves, most notably in apical vegetative buds near inflorescences, but also in seedlings. Although ectopic expression of the PTLF cDNA in Arabidopsis accelerated flowering, only one of the many tested transgenic lines of Populus flowered precociously. The majority of trees within a population of 3-year-old transgenic hybrid Populus lines with PTLF constitutively expressed showed few differences when compared to controls. However, phenotypic effects on growth rate and crown development, but not flowering, were seen in some trees with strong PTLF expression and became manifest only as the trees aged. Competence to respond to overexpression of LFY varied widely among Populus genotypes, giving consistent early flowering in only a single male P. tremula x P. tremuloides hybrid and causing gender change in another hybrid genotype. PTLF activity appears to be subject to regulation that does not affect heterologously expressed LFY, and is dependent upon tree maturation. Both genes provide tools for probing the mechanisms of delayed competence to flower in woody plants.

Structure and expression of duplicate AGAMOUS orthologues in poplar.

To investigate the homeotic systems underlying floral development in a dioecious tree, and to provide tools for the manipulation of floral development, we have isolated two Populus trichocarpa genes, PTAG1 and PTAG2, homologous to the Arabidopsis floral homeotic gene AGAMOUS (AG). PTAG1 and PTAG2 are located on separate linkage groups, but their non-coding regions are highly similar, consistent with a phylogenetically recent duplication. Intron/exon structure is conserved in relation to AG and the Antirrhinum AG orthologue, PLENA (PLE), and low-stringency Southern analysis demonstrated the absence of additional genes in the poplar genome with significant PTAG1/2 homology. PTAG1 and PTAG2 exhibit an AG-like floral expression pattern, and phylogenetic analysis of the AG subfamily strongly supports evolutionary orthology to C-class organ identity genes. The high degree of similarity shared by PTAG1 and PTAG2 in both sequence (89% amino acid identity) and expression indicates that they are unlikely to be functionally associated with specification of tree gender. Unexpectedly, PTAG transcripts were consistently detected in vegetative tissues.

Site-directed mutagenesis of glycosylation sites in the transforming growth factor-beta 1 (TGF beta 1) and TGF beta 2 (414) precursors and of cysteine residues within mature TGF beta 1: effects on secretion and bioactivity.

The transforming growth factor-beta 1 (TGF beta 1) and -beta 2 (414) precursors both contain three predicted sites of N-linked glycosylation within their pro regions. These are located at amino acid residues 72, 140, and 241 for the TGF beta 2 (414) precursor and at residues 82, 136, and 176 for the TGF beta 1 precursor; both proteins contain mannose-6-phosphate (M-6-P) residues. The major sites of M-6-P addition are at Asn (82) and Asn (136), the first two sites of glycosylation, for the TGF beta 1 precursor. We now show that the major site of M-6-P addition within the TGF beta 2 (414) precursor is at Asn241, the third glycosylation site. To determine the importance of N-linked glycosylation to the secretion of TGF beta 1 and -beta 2, site-directed mutagenesis was used to change the Asn residues to Ser residues; the resulting DNAs were transfected into COS cells, and their supernatants were assayed for TGF beta activity. Substitution of Asn (241) of the TGF beta 2 (414) precursor resulted in an 82% decrease in secreted TGF beta 2 bioactivity. Mutation at Asn72 resulted in a 44% decrease, while mutation at Asn140 was without effect. Elimination of all three glycosylation sites resulted in undetectable levels of TGF beta 2. These results were compared with similar mutations made in the cDNA encoding the TGF beta 1 precursor. Mutagenesis of the two M-6-P-containing sites (Asn82 and Asn136) resulted in an 83% decrease in secreted TGF beta 1; replacement of Asn82 and Asn136 with Ser individually resulted in 85% and 42% decreases in activity, respectively. Substitution of Asn176 with Ser was without effect, while substitution of all three sites of glycosylation resulted in undetectable levels of TGF beta 1 activity, similar to the results obtained with TGF beta 2. The nine Cys residues within the mature region of TGF beta 1 were mutated to serine, and their effects on TGF beta 1 secretion were evaluated. Mutation of most Cys residues resulted in undetectable levels of TGF beta 1 protein or activity in conditioned medium. Mutation of Cys (355) led to the secretion of inactive TGF beta 1 monomers, suggesting that this residue is either directly involved in dimer formation or required for correct interchain disulfide bond formation.

Site-directed mutagenesis of cysteine residues in the pro region of the transforming growth factor beta 1 precursor. Expression and characterization of mutant proteins.

Three cysteine residues are located in the pro region of the transforming growth factor beta 1 (TGF-beta 1) precursor at amino acid positions 33, 223, and 225. Previous studies (Gentry, L. E., Lioubin, M. N., Purchio, A. F., and Marquardt, H. (1988) Mol. Cell. Biol. 8, 4162-4168) with purified recombinant TGF-beta 1 (rTGF-beta 1) precursor produced by Chinese hamster ovary (CHO) cells revealed that Cys-33 can form a disulfide bond with at least 1 cysteine residue in mature TGF-beta 1, contributing to the formation of a 90-110-kDa protein. We now show that Cys-223 and Cys-225 form interchain disulfide bonds. Site-directed mutagenesis was used to change these Cys codons to Ser codons, and mutant constructs were transfected into COS cells. Analysis of recombinant proteins by immunoblotting showed that by substituting Cys-33 the 90-110-kDa protein is not formed, and thus, more mature dimer (24 kDa) is obtained, corresponding to a 3- to 5-fold increase in biological activity. Substitution of Cys-223 and/or Cys-225 resulted in near wild-type levels of mature TGF-beta 1. Furthermore, cells transfected with plasmid coding for Ser at positions 223 and 225 expressed only monomeric precursor proteins and released bioactive TGF-beta 1 that did not require acid activation, suggesting that dimerization of the precursor pro region may be necessary for latency.

Transforming growth factor beta 1: importance of glycosylation and acidic proteases for processing and secretion.

The role of glycosylation of the transforming growth factor-beta 1 (TGF-beta 1) precursor was investigated by treating a transfected Chinese hamster ovary (CHO) cell line expressing high levels of recombinant TGF-beta 1 (TGF-beta 3-2000 cells) with a series of glycosylational inhibitors. Tunicamycin, a nucleoside antibiotic which prevents the formation of the dolichol intermediate necessary for oligosaccharide addition of the nascent polypeptide chain, appeared to block secretory exit and led to an increase in the cellular associated, nonglycosylated pro-TGF-beta 1 form. 1-Deoxymannojirimycin and swainsonine, inhibitors of the mannosidases I and II, respectively, blocked complete glycoprotein processing of the TGF-beta 1 precursor as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by sensitivity to glycosidases. However, the abnormal TGF-beta 1 polypeptides containing the altered carbohydrate side chains were secreted readily by the CHO cells. In contrast, inhibitors of the glucosidases at the first step in glycoprotein remodeling, 1-deoxynojirimycin and castanospermine, markedly inhibited secretion of the TGF-beta 1 polypeptides from transfected CHO cells. In all cases, these inhibitors did not appear to affect proteolytic processing of the TGF-beta 1 polypeptides. Furthermore, inhibitor treatment did not affect mannose-6-phosphorylation of the TGF-beta 1 polypeptides. These results suggest that glycosylation and early stage remodeling of oligosaccharide side chains are necessary for secretion of TGF-beta 1. Treatment of the transfected CHO cells with weak bases (NH4Cl and chloroquine), or a monovalent ionophore (monensin), prevented proteolytic processing of the TGF-beta 1 precursor indicating that cleavage occurs by proteases in an acidic cellular compartment.

Identification of mannose 6-phosphate in two asparagine-linked sugar chains of recombinant transforming growth factor-beta 1 precursor.

Recombinant transforming growth factor-beta 1 (TGF-beta 1) precursor produced and secreted by a clone of Chinese hamster ovary cells was found to be glycosylated and phosphorylated. Treatment of 32P-labeled precursor protein with N-glycanase indicated that phosphate was incorporated into asparagine-linked complex carbohydrate moieties. Fractionation of 32P-labeled glycopeptides followed by amino acid sequence analysis indicated that greater than 95% of the label was incorporated into two out of three glycosylation sites at Asn-82 and Asn-136 of the TGF-beta 1 precursor. Two-dimensional electrophoretic analysis of acid hydrolyzed precursor protein and precursor protein-derived glycopeptides indicated that 32P was incorporated as mannose 6-phosphate. Binding studies with the purified receptor for mannose 6-phosphate indicated that the TGF-beta 1 precursor could bind to this receptor and the binding was specifically inhibited with mannose 6-phosphate.

Recombinant type 1 transforming growth factor beta precursor produced in Chinese hamster ovary cells is glycosylated and phosphorylated.

Analyses of cDNA clones coding for simian type 1 transforming growth factor beta (TGF-beta 1) suggest that there are three potential sites for N-linked glycosylation located in the amino terminus of the precursor region. Analysis of [3H]glucosamine-labeled serum-free supernatants from a line of Chinese hamster ovary cells which secrete high levels of recombinant TGF-beta 1 indicate that the TGF-beta 1 precursor, but not the mature form, is glycosylated. Digestion with neuraminidase resulted in a shift in migration of the two TGF-beta 1 precursor bands, which suggests that they contain sialic acid residues. Endoglycosidase H had no noticeable effect. Treatment with N-glycanase produced two faster-migrating sharp bands, the largest of which had a molecular weight of 39 kilodaltons. TGF-beta 1-specific transcripts produced by SP6 polymerase programmed the synthesis of a 42-kilodalton polypeptide which, we suggest, is the unmodified protein backbone of the precursor. Labeling with 32Pi showed that the TGF-beta 1 precursor was phosphorylated in the amino portion of the molecule.

Type 1 transforming growth factor beta: amplified expression and secretion of mature and precursor polypeptides in Chinese hamster ovary cells.

Recombinant type 1 transforming growth factor beta (TGF-beta) was expressed to high levels in CHO cells by using dihydrofolate reductase (dhfr) gene amplification. The expression plasmid was derived from the pSV2 vectors and contained, in tandem, the simian TGF-beta and mouse dhfr cDNAs. Transcription of both cDNAs was controlled by the simian virus 40 early promoter. Stepwise selection of transfected CHO cells in increasing concentrations of methotrexate yielded cell lines that expressed amplified TGF-beta nucleic acid sequences. The expression plasmid DNA was amplified greater than 35-fold in one of the methotrexate-selected transfectants. The major proteins secreted by these cells consisted of latent TGF-beta and TGF-beta precursor polypeptides, as judged by immunoblots by using site-specific anti-peptide antibodies derived from various regions of the TGF-beta precursor. Levels of recombinant TGF-beta protein secreted by these cells approached 30 micrograms/24 h per 10(7) cells and required prior acidification for optimal activity; nonacidified supernatants were approximately 1% as active as acidified material. Antibodies directed toward sequences present in the mature growth factor readily identified a proteolytically processed recombinant TGF-beta which, on sodium dodecyl sulfate-polyacrylamide gels, comigrated with highly purified natural TGF-beta. In addition to mature recombinant TGF-beta, site-specific antibodies demonstrated the existence of larger TGF-beta precursor polypeptides. The availability of biologically active recombinant type 1 TGF-beta and precursor forms should provide a means to examine the structure, function, and potential in vivo therapeutic use of this growth factor.

Dual evolutionary modes in the bovine globin locus.

Five bovine globin pseudogenes were subjected to sequence analysis. These genes include the three pseudogenes in the beta-type globin gene cluster as well as two allelic forms. Comparison of the sequences with those of the adult and fetal bovine globin genes shows that together they form a multigene family that was created by large-scale duplication. The structures are explained by invoking sequence exchange mediated by gene conversion. After their creation these genes evolved in a concerted fashion, exchanging sequence freely by intrachromosomal gene conversion. Subsequently, one by one, the genes were uncoupled from this exchange. This was accomplished by the creation of nonhomologies that formed barriers to gene conversion. These nonhomologies were several hundred bases in length and were formed by either deletion or by insertion of short repetitive sequences within the gene structures. In this way the genes made the transition from a rapid, coupled mode to a slow, solitary mode of evolution. Allelic gene polymorphisms were distributed inhomogeneously in the bovine globin family. It is proposed that this was due to interruption of interchromosomal gene conversion by a recent pseudogene duplication in the fetal globin gene cluster.