The Peptide-Drug Conjugate Melflufen Modulates the Unfolded Protein Response of Multiple Myeloma and Amyloidogenic Plasma Cells and Induces Cell Death.

Immunoglobulin light-chain (AL) amyloidosis is a rare disease caused by clonal plasma cell secretion of misfolded light chains that assemble as toxic amyloid fibrils, depositing in vital organs including the heart and kidneys, causing organ dysfunction. Plasma cell-directed therapeutics are expected to reduce production of toxic light chain by eliminating amyloidogenic cells in bone marrow, thereby diminishing amyloid fibril deposition and providing the potential for organ recovery. Melphalan flufenamide (melflufen) is a first-in-class peptide-drug conjugate that targets aminopeptidases and rapidly releases alkylating agents inside tumor cells. Melflufen is highly lipophilic, permitting rapid uptake by cells, where it is enzymatically hydrolyzed by aminopeptidases, resulting in intracellular accumulation of the alkylating agents, including melphalan. Previous data demonstrating sensitivity of myeloma cells to melflufen suggest that the drug might be useful in AL amyloidosis. We describe the effects of melflufen on amyloidogenic plasma cells in vitro and ex vivo, demonstrating enhanced cytotoxic effects in comparison to melphalan, as well as novel mechanisms of action through the unfolded protein response (UPR) pathway. These findings provide evidence that melflufen-mediated cytotoxicity extends to amyloidogenic plasma cells, and support the rationale for the evaluation of melflufen in patients with AL amyloidosis.

S100 Calcium Binding Protein Family Members Associate With Poor Patient Outcome and Response to Proteasome Inhibition in Multiple Myeloma.

Despite several new therapeutic options, multiple myeloma (MM) patients experience multiple relapses and inevitably become refractory to treatment. Insights into drug resistance mechanisms may lead to the development of novel treatment strategies. The S100 family is comprised of 21 calcium binding protein members with 17 S100 genes located in the 1q21 region, which is commonly amplified in MM. Dysregulated expression of S100 family members is associated with tumor initiation, progression and inflammation. However, the relationship between the S100 family and MM pathogenesis and drug response is unknown. In this study, the roles of S100 members were systematically studied at the copy number, transcriptional and protein level with patients' survival and drug response. Copy number analysis revealed a predominant pattern of gains occurring in S100 genes clustering in the 1q21 locus. In general, gains of genes encoding S100 family members associated with worse patient survival. However, S100 gene copy number and S100 gene expression did not necessarily correlate, and high expression of S100A4 associated with poor patient survival. Furthermore, integrated analysis of S100 gene expression and ex vivo drug sensitivity data showed significant negative correlation between expression of S100 family members (S100A8, S100A9, and S100A12) and sensitivity to some drugs used in current MM treatment, including proteasome inhibitors (bortezomib, carfilzomib, and ixazomib) and histone deacetylase inhibitor panobinostat. Combined proteomic and pharmacological data exhibited significant negative association of S100 members (S100A4, S100A8, and S100A9) with proteasome inhibitors and panobinostat. Clinically, the higher expression of S100A4 and S100A10 were significantly linked to shorter progression free survival in patients receiving carfilzomib-based therapy. The results indicate an association and highlight the potential functional importance of S100 members on chromosome 1q21 in the development of MM and resistance to established myeloma drugs, including proteasome inhibitors.

Aminopeptidase Expression in Multiple Myeloma Associates with Disease Progression and Sensitivity to Melflufen.

Multiple myeloma (MM) is characterized by extensive immunoglobulin production leading to an excessive load on protein homeostasis in tumor cells. Aminopeptidases contribute to proteolysis by catalyzing the hydrolysis of amino acids from proteins or peptides and function downstream of the ubiquitin-proteasome pathway. Notably, aminopeptidases can be utilized in the delivery of antibody and peptide-conjugated drugs, such as melflufen, currently in clinical trials. We analyzed the expression of 39 aminopeptidase genes in MM samples from 122 patients treated at Finnish cancer centers and 892 patients from the CoMMpass database. Based on ranked abundance, LAP3, ERAP2, METAP2, TTP2, and DPP7 were highly expressed in MM. ERAP2, XPNPEP1, DPP3, RNPEP, and CTSV were differentially expressed between relapsed/refractory and newly diagnosed MM samples (p < 0.05). Sensitivity to melflufen was detected ex vivo in 11/15 MM patient samples, and high sensitivity was observed, especially in relapsed/refractory samples. Survival analysis revealed that high expression of XPNPEP1, RNPEP, DPP3, and BLMH (p < 0.05) was associated with shorter overall survival. Hydrolysis analysis demonstrated that melflufen is a substrate for aminopeptidases LAP3, LTA4H, RNPEP, and ANPEP. The sensitivity of MM cell lines to melflufen was reduced by aminopeptidase inhibitors. These results indicate critical roles of aminopeptidases in disease progression and the activity of melflufen in MM.

Prognostic significance of esterase gene expression in multiple myeloma.

BACKGROUND: Esterase enzymes differ in substrate specificity and biological function and may display dysregulated expression in cancer. This study evaluated the biological significance of esterase expression in multiple myeloma (MM). METHODS: For gene expression profiling and evaluation of genomic variants in the Institute for Molecular Medicine Finland (FIMM) cohort, bone marrow aspirates were obtained from patients with newly diagnosed MM (NDMM) or relapsed/refractory MM (RRMM). CD138+ plasma cells were enriched and used for RNA sequencing and analysis, and to evaluate genomic variation. The Multiple Myeloma Research Foundation (MMRF) Relating Clinical Outcomes in MM to Personal Assessment of Genetic Profile (CoMMpass) dataset was used for validation of the findings from FIMM. RESULTS: MM patients (NDMM, n = 56; RRMM, n = 78) provided 171 bone marrow aspirates (NDMM, n = 56; RRMM, n = 115). Specific esterases exhibited relatively high or low expression in MM, and expression of specific esterases (UCHL5, SIAE, ESD, PAFAH1B3, PNPLA4 and PON1) was significantly altered on progression from NDMM to RRMM. High expression of OVCA2, PAFAH1B3, SIAE and USP4, and low expression of PCED1B, were identified as poor prognostic markers (P < 0.05). The MMRF CoMMpass dataset provided validation that higher expression of PAFAH1B3 and SIAE, and lower expression of PCED1B, were associated with poor prognosis. CONCLUSIONS: Esterase gene expression levels change as patients progress from NDMM to RRMM. High expression of OVCA2, PAFAH1B3, USP4 and SIAE, and low expression of PCED1B, are poor prognostic markers in MM, suggesting a role for these esterases in myeloma biology.

Opposite Expression Patterns of Spry3 and p75NTR in Cerebellar Vermis Suggest a Male-Specific Mechanism of Autism Pathogenesis.

Autism is a genetically complex neurobehavioral disorder with a population prevalence of more than 1%. Cerebellar abnormalities, including Purkinje cell deficits in the vermis, are consistently reported, and rodent models of cerebellar dysfunction exhibit features analogous to human autism. We previously analyzed the regulation and expression of the pseudoautosomal region 2 gene SPRY3, which is adjacent to X chromosome-linked TMLHE, a known autism susceptibility gene. SPRY3 is a regulator of branching morphogenesis and is strongly expressed in Purkinje cells. We previously showed that mouse Spry3 is not expressed in cerebellar vermis lobules VI-VII and X, regions which exhibit significant Purkinje cell loss or abnormalities in autism. However, these lobules have relatively high expression of p75NTR, which encodes a neurotrophin receptor implicated in autism. We propose a mechanism whereby inappropriate SPRY3 expression in these lobules could interact with TrkB and p75NTR signaling pathways resulting in Purkinje cell pathology. We report preliminary characterization of X and Y chromosome-linked regulatory sequences upstream of SPRY3, which are polymorphic in the general population. We suggest that an OREG-annotated region on chromosome Yq12 ∼60 kb from SPRY3 acts as a silencer of Y-linked SPRY3 expression. Deletion of a ß-satellite repeat, or alterations in chromatin structure in this region due to trans-acting factors, could affect the proposed silencing function, leading to reactivation and inappropriate expression of Y-linked SPRY3. This proposed male-specific mechanism could contribute to the male bias in autism prevalence.

Computational and mutational analysis of TatD DNase of Bacillus anthracis.

The role of TatD DNases as DNA repair enzymes or cell death (apoptotic) nucleases is well established in prokaryotes as well as eukaryotes. The current study aims to characterize the TatD nuclease from Bacillus anthracis (Ba TatD) and to explore its key histidine catalytic residues. Ba TatD was found to be a metal-dependent, nonspecific endonuclease which could efficiently cleave double-stranded DNA substrates. Moreover, Ba TatD nuclease was observed to be thermostable up to 55°C and act in a wide pH range indicating its industrial applicability. Diethyl pyrocarbonate-based histidine-selective alkylation of the Ba TatD resulted in a loss of its nuclease activity suggesting a crucial role of the histidine residues in its activity. The key residues of Ba TatD were predicted using sequence analysis and structure-based approaches, and then the predicted residues were further tested by mutational analysis. Upon mutational analysis, H128 and H153 have been found to be crucial for Ba TatD activity, though H153 seems to bear an important but a dispensable role for the Ba TatD nuclease. Ba TatD had a uniform expression in the cytosol of B. anthracis, which indicates a significant role of the protein in the pathogen's life cycle. This is the first study to identify and characterize the TatD DNase from B. anthracis and will be helpful in gaining more insights on the role of TatD proteins in Gram-positive bacteria where it remains unexplored.

PausePred and Rfeet: webtools for inferring ribosome pauses and visualizing footprint density from ribosome profiling data.

The process of translation is characterized by irregularities in the local decoding rates of specific mRNA codons. This includes the occurrences of long pauses that can take place when ribosomes decode certain peptide sequences, encounter strong RNA secondary structures, or decode "hungry" codons. Examples are known where such pausing or stalling is used for regulating protein synthesis. This can be achieved at the level of translation via direct alteration of ribosome progression through mRNA or by altering mRNA stability via NoGo decay. Ribosome pausing has also been implicated in the cotranslational folding of proteins. Ribosome profiling data often are used for inferring the locations of ribosome pauses. However, no dedicated online software is available for this purpose. Here we present PausePred (https://pausepred.ucc.ie/), which can be used to infer ribosome pauses from ribosome profiling (Ribo-seq) data. Peaks of ribosome footprint density are scored based on their magnitude relative to the background density within the surrounding area. The scoring allows the comparison of peaks across the transcriptome or genome. In addition to the score, PausePred reports the coordinates of the pause, the footprint density at the pause site, and the surrounding nucleotide sequence. The pauses can be visualized in the context of Ribo-seq and RNA-seq density plots generated for specific transcripts or genomic regions with the Rfeet tool. PausePred does not require input on the location of protein coding ORFs (although gene annotations can be optionally supplied). As a result, it can be used universally and its output does not depend on ever evolving annotations.

Stimulation of reverse transcriptase generated cDNAs with specific indels by template RNA structure: retrotransposon, dNTP balance, RT-reagent usage.

RNA dependent DNA-polymerases, reverse transcriptases, are key enzymes for retroviruses and retroelements. Their fidelity, including indel generation, is significant for their use as reagents including for deep sequencing. Here, we report that certain RNA template structures and G-rich sequences, ahead of diverse reverse transcriptases can be strong stimulators for slippage at slippage-prone template motif sequence 3' of such 'slippage-stimulatory' structures. Where slippage is stimulated, the resulting products have one or more additional base(s) compared to the corresponding template motif. Such structures also inhibit slippage-mediated base omission which can be more frequent in the absence of a relevant stem-loop. Slippage directionality, base insertion and omission, is sensitive to the relative concentration ratio of dNTPs specified by the RNA template slippage-prone sequence and its 5' adjacent base. The retrotransposon-derived enzyme TGIRT exhibits more slippage in vitro than the retroviral enzymes tested including that from HIV. Structure-mediated slippage may be exhibited by other polymerases and enrich gene expression. A cassette from Drosophila retrotransposon Dme1_chrX_2630566, a candidate for utilizing slippage for its GagPol synthesis, exhibits strong slippage in vitro. Given the widespread occurrence and importance of retrotransposons, systematic studies to reveal the extent of their functional utilization of RT slippage are merited.

Specific reverse transcriptase slippage at the HIV ribosomal frameshift sequence: potential implications for modulation of GagPol synthesis.

Synthesis of HIV GagPol involves a proportion of ribosomes translating a U6A shift site at the distal end of the gag gene performing a programmed -1 ribosomal frameshift event to enter the overlapping pol gene. In vitro studies here show that at the same shift motif HIV reverse transcriptase generates -1 and +1 indels with their ratio being sensitive to the relative concentration ratio of dNTPs specified by the RNA template slippage-prone sequence and its 5' adjacent base. The GGG sequence 3' adjacent to the U6A shift/slippage site, which is important for ribosomal frameshifting, is shown here to limit reverse transcriptase base substitution and indel 'errors' in the run of A's in the product. The indels characterized here have either 1 more or less A, than the corresponding number of template U's. cDNA with 5 A's may yield novel Gag product(s), while cDNA with an extra base, 7 A's, may only be a minor contributor to GagPol polyprotein. Synthesis of a proportion of non-ribosomal frameshift derived GagPol would be relevant in efforts to identify therapeutically useful compounds that perturb the ratio of GagPol to Gag, and pertinent to the extent in which specific polymerase slippage is utilized in gene expression.

TORC1 and TORC2 work together to regulate ribosomal protein S6 phosphorylation in Saccharomyces cerevisiae.

Nutrient-sensitive phosphorylation of the S6 protein of the 40S subunit of the eukaryote ribosome is highly conserved. However, despite four decades of research, the functional consequences of this modification remain unknown. Revisiting this enigma in Saccharomyces cerevisiae, we found that the regulation of Rps6 phosphorylation on Ser-232 and Ser-233 is mediated by both TOR complex 1 (TORC1) and TORC2. TORC1 regulates phosphorylation of both sites via the poorly characterized AGC-family kinase Ypk3 and the PP1 phosphatase Glc7, whereas TORC2 regulates phosphorylation of only the N-terminal phosphosite via Ypk1. Cells expressing a nonphosphorylatable variant of Rps6 display a reduced growth rate and a 40S biogenesis defect, but these phenotypes are not observed in cells in which Rps6 kinase activity is compromised. Furthermore, using polysome profiling and ribosome profiling, we failed to uncover a role of Rps6 phosphorylation in either global translation or translation of individual mRNAs. Taking the results together, this work depicts the signaling cascades orchestrating Rps6 phosphorylation in budding yeast, challenges the notion that Rps6 phosphorylation plays a role in translation, and demonstrates that observations made with Rps6 knock-ins must be interpreted cautiously.

Genomic survey, gene expression analysis and structural modeling suggest diverse roles of DNA methyltransferases in legumes.

DNA methylation plays a crucial role in development through inheritable gene silencing. Plants possess three types of DNA methyltransferases (MTases), namely Methyltransferase (MET), Chromomethylase (CMT) and Domains Rearranged Methyltransferase (DRM), which maintain methylation at CG, CHG and CHH sites. DNA MTases have not been studied in legumes so far. Here, we report the identification and analysis of putative DNA MTases in five legumes, including chickpea, soybean, pigeonpea, Medicago and Lotus. MTases in legumes could be classified in known MET, CMT, DRM and DNA nucleotide methyltransferases (DNMT2) subfamilies based on their domain organization. First three MTases represent DNA MTases, whereas DNMT2 represents a transfer RNA (tRNA) MTase. Structural comparison of all the MTases in plants with known MTases in mammalian and plant systems have been reported to assign structural features in context of biological functions of these proteins. The structure analysis clearly specified regions crucial for protein-protein interactions and regions important for nucleosome binding in various domains of CMT and MET proteins. In addition, structural model of DRM suggested that circular permutation of motifs does not have any effect on overall structure of DNA methyltransferase domain. These results provide valuable insights into role of various domains in molecular recognition and should facilitate mechanistic understanding of their function in mediating specific methylation patterns. Further, the comprehensive gene expression analyses of MTases in legumes provided evidence of their role in various developmental processes throughout the plant life cycle and response to various abiotic stresses. Overall, our study will be very helpful in establishing the specific functions of DNA MTases in legumes.

Genomewide discovery of DNA polymorphisms in rice cultivars with contrasting drought and salinity stress response and their functional relevance.

Next-generation sequencing technologies provide opportunities to understand the genetic basis of phenotypic differences, such as abiotic stress response, even in the closely related cultivars via identification of large number of DNA polymorphisms. We performed whole-genome resequencing of three rice cultivars with contrasting responses to drought and salinity stress (sensitive IR64, drought-tolerant Nagina 22 and salinity-tolerant Pokkali). More than 356 million 90-bp paired-end reads were generated, which provided about 85% coverage of the rice genome. Applying stringent parameters, we identified a total of 1 784 583 nonredundant single-nucleotide polymorphisms (SNPs) and 154 275 InDels between reference (Nipponbare) and the three resequenced cultivars. We detected 401 683 and 662 509 SNPs between IR64 and Pokkali, and IR64 and N22 cultivars, respectively. The distribution of DNA polymorphisms was found to be uneven across and within the rice chromosomes. One-fourth of the SNPs and InDels were detected in genic regions, and about 3.5% of the total SNPs resulted in nonsynonymous changes. Large-effect SNPs and InDels, which affect the integrity of the encoded protein, were also identified. Further, we identified DNA polymorphisms present in the differentially expressed genes within the known quantitative trait loci. Among these, a total of 548 SNPs in 232 genes, located in the conserved functional domains, were identified. The data presented in this study provide functional markers and promising target genes for salinity and drought tolerance and present a valuable resource for high-throughput genotyping and molecular breeding for abiotic stress traits in rice.