Discovery and Preclinical Pharmacology of NX-2127, an Orally Bioavailable Degrader of Bruton's Tyrosine Kinase with Immunomodulatory Activity for the Treatment of Patients with B Cell Malignancies.

Bruton's tyrosine kinase (BTK), a member of the TEC family of kinases, is an essential effector of B-cell receptor (BCR) signaling. Chronic activation of BTK-mediated BCR signaling is a hallmark of many hematological malignancies, which makes it an attractive therapeutic target. Pharmacological inhibition of BTK enzymatic function is now a well-proven strategy for the treatment of patients with these malignancies. We report the discovery and characterization of NX-2127, a BTK degrader with concomitant immunomodulatory activity. By design, NX-2127 mediates the degradation of transcription factors IKZF1 and IKZF3 through molecular glue interactions with the cereblon E3 ubiquitin ligase complex. NX-2127 degrades common BTK resistance mutants, including BTKC481S. NX-2127 is orally bioavailable, exhibits in vivo degradation across species, and demonstrates efficacy in preclinical oncology models. NX-2127 has advanced into first-in-human clinical trials and achieves deep and sustained degradation of BTK following daily oral dosing at 100 mg.

Improvement in plan quality after Implementation of clinical goals in a large network of cancer centers.

Clinical Goals (CG) is a tool available in the Varian Eclipse planning system to objectively and visually evaluate the quality of treatment plans based upon user-defined dose-volume parameters. We defined a set of CG for Stereotactic Radiosurgery (SRS) and Intensity-Modulated Radiotherapy (IMRT) based on published data and guidelines and implemented this in a network of cancer centers in India (American Institute of Oncology). A dosimetric study was performed to compare brain SRS and breast IMRT plan quality before and after CG implementation.The CG defined for SRS plans were target V100% ≥ 98%, dose gradient measure (GM) ≤ 0.5 cm, conformity index (CI) 1.0 to 1.2. For breast IMRT plans, CG defined target V100% ≥ 97%, V95% ≥ 95%, V107% ≤ 2%, V105% ≤ 10%, and Dmax ≤ 2.4 Gy. Dose limits to organs-at-risk (OAR) were summarize in supplemental materials. Twenty brain SRS and 10 breast IMRT treatment plans that were previously delivered on patients were selected and re-planned using CG. The pre and postoptimized plan parameters were compared using student t-tests.For brain SRS plans, the V100, GM, and CI for the pre- and post-Clinical-Goals plans were 93.22% ± 7.2% vs 97.96% ± 0.29% (p = 0.009), 0.63 ± 0.16 vs 0.42 ± 0.05 (p < 0.001) and 1.07 ± 0.18 vs 1.06 ± 0.06 (p = 0.79), respectively. There were no differences in max dose to OARs. In breast IMRT plans, the target V107% for pre and postimplemented plans were 16.50% ± 10.98% vs 0.32% ± 0.32%, respectively (p = 0.001). The average target V105% were 44.00% ± 15.72% and 8.69% ± 4.53%, respectively (p < 0.001). No differences were found in the average target V100% (p = 0.128) and V95% (p = 0.205). The average target Dmax were 112.28% ± 1.59% and 109.14% ± 0.73%, respectively (p < 0.001). There were only minor differences in doses to OARs.The implementation of CG in Varian Eclipse significantly improved SRS and IMRT plan quality with enhanced coverage, dose GM, and CI without increased dose to OARs.

Involvement of glutamine synthetase 2 (GS2) amplification and overexpression in Amaranthus palmeri resistance to glufosinate.

MAIN CONCLUSION: Amplification and overexpression of the target site glutamine synthetase, specifically the plastid-located isoform, confers resistance to glufosinate in Amaranthus palmeri. This mechanism is novel among glufosinate-resistant weeds. Amaranthus palmeri has recently evolved resistance to glufosinate herbicide. Several A. palmeri populations from Missouri and Mississippi, U.S.A. had survivors when sprayed with glufosinate-ammonium (GFA, 657 g ha-1). One population, MO#2 (fourfold resistant) and its progeny (sixfold resistant), were used to study the resistance mechanism, focusing on the herbicide target glutamine synthetase (GS). We identified four GS genes in A. palmeri; three were transcribed: one coding for the plastidic protein (GS2) and two coding for cytoplasmic isoforms (GS1.1 and GS1.2). These isoforms did not contain mutations associated with resistance. The 17 glufosinate survivors studied showed up to 21-fold increase in GS2 copies. GS2 was expressed up to 190-fold among glufosinate survivors. GS1.1 was overexpressed > twofold in only 3 of 17, and GS1.2 in 2 of 17 survivors. GS inhibition by GFA causes ammonia accumulation in susceptible plants. Ammonia level was analyzed in 12 F1 plants. GS2 expression was negatively correlated with ammonia level (r = - 0.712); therefore, plants with higher GS2 expression are less sensitive to GFA. The operating efficiency of photosystem II (ϕPSII) of Nicotiana benthamiana overexpressing GS2 was four times less inhibited by GFA compared to control plants. Therefore, increased copy and overexpression of GS2 confer resistance to GFA in A. palmeri (or other plants). We present novel understanding of the role of GS2 in resistance evolution to glufosinate.

Genetic architecture underlying HPPD-inhibitor resistance in a Nebraska Amaranthus tuberculatus population.

BACKGROUND: Amaranthus tuberculatus is a problematic weed species in Midwest USA agricultural systems. Inhibitors of 4-hydroxyphenylpyruvate dioxygenase (HPPD) are an important chemistry for weed management in numerous cropping systems. Here, we characterize the genetic architecture underlying the HPPD-inhibitor resistance trait in an A. tuberculatus population (NEB). RESULTS: Dose-response studies of an F1 generation identified HPPD-inhibitor resistance as a dominant trait with a resistance factor of 15.0-21.1 based on dose required for 50% growth reduction. Segregation analysis in a pseudo-F2 generation determined the trait is moderately heritable (H2  = 0.556) and complex. Bulk segregant analysis and validation with molecular markers identified two quantitative trait loci (QTL), one on each of Scaffold 4 and 12. CONCLUSIONS: Resistance to HPPD inhibitors is a complex, largely dominant trait within the NEB population. Two large-effect QTL were identified controlling HPPD-inhibitor resistance in A. tuberculatus. This is the first QTL mapping study to characterize herbicide resistance in a weedy species.

Evolution of Glyphosate-Resistant Weeds.

Widespread adoption of glyphosate-resistant crops and concomitant reliance on glyphosate for weed control set an unprecedented stage for the evolution of herbicide-resistant weeds. There are now 48 weed species that have evolved glyphosate resistance. Diverse glyphosate-resistance mechanisms have evolved, including single, double, and triple amino acid substitutions in the target-site gene, duplication of the gene encoding the target site, and others that are rare or nonexistent for evolved resistance to other herbicides. This review summarizes these resistance mechanisms, discusses what is known about their evolution, and concludes with some of the impacts glyphosate-resistant weeds have had on weed management.

Draft Genomes of Amaranthus tuberculatus, Amaranthus hybridus, and Amaranthus palmeri.

Amaranthus tuberculatus, Amaranthus hybridus, and Amaranthus palmeri are agronomically important weed species. Here, we present the most contiguous draft assemblies of these three species to date. We utilized a combination of Pacific Biosciences long-read sequencing and chromatin contact mapping information to assemble and order sequences of A. palmeri to near-chromosome-level resolution, with scaffold N50 of 20.1 Mb. To resolve the issues of heterozygosity and coassembly of alleles in diploid species, we adapted the trio binning approach to produce haplotype assemblies of A. tuberculatus and A. hybridus. This approach resulted in an improved assembly of A. tuberculatus, and the first genome assembly for A. hybridus, with contig N50s of 2.58 and 2.26 Mb, respectively. Species-specific transcriptomes and information from related species were used to predict transcripts within each assembly. Syntenic comparisons of these species and Amaranthus hypochondriacus identified sites of genomic rearrangement, including duplication and translocation, whereas genetic map construction within A. tuberculatus highlighted the need for further ordering of the A. hybridus and A. tuberculatus contigs. These multiple reference genomes will accelerate genomic studies in these species to further our understanding of weedy evolution within Amaranthus.

Target-Site Mutations Conferring Herbicide Resistance.

Mutations conferring evolved herbicide resistance in weeds are known in nine different herbicide sites of action. This review summarizes recently reported resistance-conferring mutations for each of these nine target sites. One emerging trend is an increase in reports of multiple mutations, including multiple amino acid changes at the glyphosate target site, as well as mutations involving two nucleotide changes at a single amino acid codon. Standard reference sequences are suggested for target sites for which standards do not already exist. We also discuss experimental approaches for investigating cross-resistance patterns and for investigating fitness costs of specific target-site mutations.

Cancer stem cell populations in lymphoma in dogs and impact of cytotoxic chemotherapy.

Cancer relapse following chemotherapy has been attributed in part to the presence of cancer stem cells (CSC), which drive tumour growth and metastasis and are highly resistant to the effects of cytotoxic chemotherapy. As a result, treatment with cytotoxic chemotherapy selects for drug-resistant CSC populations that eventually drive tumour recurrence. Little is known currently regarding the role of CSC in dogs with lymphoma, nor the impact of chemotherapy on CSC populations. Therefore, we prospectively quantitated CSC populations in dogs with B-cell (BCL) and T-cell lymphoma (TCL), using tumour aspirates and flow cytometric analysis with a panel of CSC markers. In addition, in vitro studies were carried out to determine the impact of chemotherapy resistance on the stem cell phenotype and stem cell properties of lymphoma cells. We found that the percentages of tumour cells expressing CSC markers were significantly increased in dogs with BCL, compared with B cells from normal lymph nodes. Similar findings were observed in dogs with TCL. In vitro studies revealed that lymphoma cells selected for resistance to CHOP chemotherapy had significantly upregulated expression of CSC markers, formed spheroids in culture more readily, and expressed significantly greater aldehyde dehydrogenase activity compared with chemotherapy-sensitive tumour cells. Similar results were observed in tumour samples dogs with relapsed BCL. These findings suggest that cytotoxic chemotherapy can lead to a relative enrichment of tumour cells with CSC properties, which may be associated with lymphoma recurrence.

A quantitative assay for Amaranthus palmeri identification.

BACKGROUND: Amaranthus palmeri recently has been brought into the Midwestern USA as a contaminant in Conservation Reserve Program seeding mixes. Rapid species screening is required to mitigate the risk of continued species movement. RESULTS: Markers were developed for A. palmeri-specific nucleotide polymorphisms in the internal transcribed spacer of the ribosomal coding region. A quantitative polymerase chain reaction (qPCR) assay successfully identified A. palmeri from single-plant samples, simulated mixed-plant samples and seed mixtures. CONCLUSION: A qPCR assay for distinguishing A. palmeri from 12 other Amaranthus spp. was developed and validated. The assay can consistently detect a single A. palmeri seed when present in a pool of 100 total Amaranthus spp. seeds. © 2017 Society of Chemical Industry.

The SLC2A14 gene: genomic locus, tissue expression, splice variants, and subcellular localization of the protein.

The SLC2A14 gene encodes for GLUT14, an orphan member of the facilitated membrane glucose transporter family, which was originally described to be exclusively expressed in human testis. However, genetic variations in SLC2A14 are associated with chronic diseases such as Alzheimer's disease and Inflammatory Bowel Disease, which cannot be explained by a strictly testicular expression. Therefore we analyzed available information on the SLC2A14 gene to update knowledge of the locus and its encoded products. This report presents an expanded SLC2A14 gene locus and a more diverse tissue expression, concurring with the existing evidence for disease associations. The exon utilization is tissue specific, with major expression in testis. When the 2 major testicular protein isoforms were expressed in mammalian cells, they located to the plasmalemma membrane, providing early evidence that GLUT14 could function as a membrane transporter.

Ammonia as a preferred additive in chiral and achiral applications of supercritical fluid chromatography for small, drug-like molecules.

Supercritical fluid chromatography is routinely utilized by analytical separations groups in the pharmaceutical industry to efficiently handle separations for discovery medicinal chemistry purposes. Purifications are performed on samples ranging from a few milligrams up to hundreds of grams. Basic additives dissolved into the liquid component of the SFC mobile phase are commonly used to improve peak shape and efficiency in achiral and chiral separations. While for purposes of analysis there is minimal consequence to additive introduction in the mobile phase, for preparative separations one needs to consider the potential effect of an additive's presence when concentrated with the desired compound. Following an SFC purification using an additive-containing modifier, the resulting fractions will contain an easily evaporated modifier, and after its evaporation perhaps still significant levels of the less volatile additive. Depending on the aqueous solubility and basicity of the final product, the process of removing basic amine additives can be time-consuming and can result in reduced yields. NMR analysis following preparative isolation and evaporation often reveals the fact of insufficient removal of the chromatographic additive even after aqueous work up steps. In this study, ammonia is evaluated as an alternative additive to strong bases such as diethylamine (DEA) in SFC purification and analysis and to the authors' knowledge no previous publication has been written describing the application of methanolic ammonia as an additive for SFC separations. Dimethylethylamine (DMEA), a more volatile additive than DEA, is also evaluated relative to ammonia for its potential to simplify the isolation process after purification and in terms of chromatographic performance. The loss in concentration of ammonia in methanol modifier over time due to evaporation and effects of that loss are also described. Furthermore, for ammonia the analytical benefit is shown to extend to on-line mass spectrometric detection relative to other basic additives.

One-bead-one-compound library of end-capped dipeptides and deconvolution by microflow NMR.

As part of our program to identify novel small molecules with interesting biological activity, we have designed and synthesized a library of end-capped dipeptides with an emphasis on compound diversity, complexity, and membrane permeability. An approximately 1500-member library was synthesized manually on large polystyrene beads using the mix-and-split method. The final compounds were cleaved into 384-well plates to generate individual stock solutions for input into high-throughput biological screens. Individual compounds were decoded using a combination of mass spectrometry and microflow NMR spectroscopy. In principle, this approach to deconvolution obviates the need for complicated binary encoding-decoding strategies for one-bead-one-compound libraries.

Cymetra.

Soft tissue replacement using a filler as a temporary scaffold to encourage revascularization and tissue in growth is an exciting concept. Sheets of acellular human dermal matrix, called Alloderm (Lifecell Corp, Branchburg, NJ), have been shown to do just that. When implanted into a patient, tissue growth and revascularization have both been observed. The company manufactures a micronized or injectable form called Cymetra. This article reviews the manufacturing, tissue interaction, clinical applications, and anticipated clinical results.

Gamma knife subthalamotomy for Parkinson disease: the subthalamic nucleus as a new radiosurgical target. Case report.

The authors present the neuroimaging, treatment planning, and radiosurgical technique for the first reported case of unilateral radiosurgical subthalamotomy, which was performed to control motor symptoms associated with advanced Parkinson disease (PD) in a patient who had undergone previous contralateral radiofrequency (RF) pallidotomy. A 73-year-old woman with end-stage PD had undergone RF pallidotomy of the right globus pallidus with resolution of symptoms. Two years following this procedure, due to the natural progression of her disease, she suffered recurrent motor fluctuations, dyskinesia, and worsening bradykinesia of the right side. Her Parkinson's Disease Disability Rating (PDDR) score was 28. Computerized tomography and magnetic resonance (MR) imaging were used to localize the left subthalamic nucleus (STN). The patient underwent gamma knife radiosurgery-a single shot of 120 Gy was administered using the 4-mm collimator helmet. The patient was evaluated up to 42 months after the procedure. The dyskinesia became minimal. Right-sided motor control improved as did her balance. At 3 months after treatment MR imaging demonstrated the radiosurgical lesion in the left STN. At 3.5 years postradiosurgery, she experienced minimal focal (oral) dyskinesia, no bradykinesia or rigidity, and her PDDR score was 11. Radiosurgery of the STN in this case was safe and effective. The STN is a readily localized anatomical target with neuroimaging. Radiosurgery avoids the risks of open procedures.