Mechanically induced localisation of SECONDARY WALL INTERACTING bZIP is associated with thigmomorphogenic and secondary cell wall gene expression.

Plant growth requires the integration of internal and external cues, perceived and transduced into a developmental programme of cell division, elongation and wall thickening. Mechanical forces contribute to this regulation, and thigmomorphogenesis typically includes reducing stem height, increasing stem diameter, and a canonical transcriptomic response. We present data on a bZIP transcription factor involved in this process in grasses. Brachypodium distachyon SECONDARY WALL INTERACTING bZIP (SWIZ) protein translocated into the nucleus following mechanostimulation. Classical touch-responsive genes were upregulated in B. distachyon roots following touch, including significant induction of the glycoside hydrolase 17 family, which may be unique to grass thigmomorphogenesis. SWIZ protein binding to an E-box variant in exons and introns was associated with immediate activation followed by repression of gene expression. SWIZ overexpression resulted in plants with reduced stem and root elongation. These data further define plant touch-responsive transcriptomics and physiology, offering insights into grass mechanotranduction dynamics.

Preclinical discovery and initial clinical data of WVT078, a BCMA × CD3 bispecific antibody.

B-cell maturation antigen (BCMA) is an ideal target in multiple myeloma (MM) due to highly specific expression in malignant plasma cells. BCMA-directed therapies including antibody drug conjugates, chimeric antigen receptor-T cells and bispecific antibodies (BsAbs) have shown high response rates in MM. WVT078 is an anti-BCMA× anti-CD3 BsAb that binds to BCMA with subnanomolar-affinity. It was selected based on potent T cell activation and anti-MM activity in preclinical models with favorable tolerability in cynomolgus monkey. In the ongoing first-in-human phase I dose-escalation study (NCT04123418), 33 patients received intravenous WVT078 once weekly at escalated dosing. At the active doses of 48-250 µg/kg tested to date (n = 26), the overall response rate (ORR) was 38.5% (90% CI: 22.6-56.4%) and the complete response rate (CRR, stringent complete response + complete response) was 11.5%, (90% CI: 3.2-27.2%). At the highest dose level tested, the ORR was 75% (3 of 4 patients). 26 (78.8%) patients reported at least one Grade ≥3 AE and 16 of these AEs were suspected to be drug related. 20 patients (60.6%) experienced cytokine release syndrome. WVT078 has an acceptable safety profile and shows preliminary evidence of clinical activity at doses tested to date.

CRISPR screening identifies T cell-intrinsic regulators of CD3-bispecific antibody responses.

CD3-engaging bispecific antibodies (BsAbs) enable the formation of an immune synapse between T cells and tumor cells, resulting in robust target cell killing not dependent on a preexisting tumor specific T cell receptor. While recent studies have shed light on tumor cell-specific factors that modulate BsAb sensitivity, the T cell-intrinsic determinants of BsAb efficacy and response durability are poorly understood. To better clarify the genes that shape BsAb-induced T cell responses, we conducted targeted analyses and a large-scale unbiased in vitro CRISPR/Cas9-based screen to identify negative regulators of BsAb-induced T cell proliferation. These analyses revealed that CD8+ T cells are dependent on CD4+ T cell-derived signaling factors in order to achieve sustained killing in vitro. Moreover, the mammalian target of rapamycin (mTOR) pathway and several other candidate genes were identified as intrinsic regulators of BsAb-induced T cell proliferation and/or activation, highlighting promising approaches to enhancing the utility of these potent therapeutics.

Inhibition of MDM2 Promotes Antitumor Responses in p53 Wild-Type Cancer Cells through Their Interaction with the Immune and Stromal Microenvironment.

p53 is a transcription factor that plays a central role in guarding the genomic stability of cells through cell-cycle arrest or induction of apoptosis. However, the effects of p53 in antitumor immunity are poorly understood. To investigate the role of p53 in controlling tumor-immune cell cross-talk, we studied murine syngeneic models treated with HDM201, a potent and selective second-generation MDM2 inhibitor. In response to HDM201 treatment, the percentage of dendritic cells increased, including the CD103+ antigen cross-presenting subset. Furthermore, HDM201 increased the percentage of Tbet+Eomes+ CD8+ T cells and the CD8+/Treg ratio within the tumor. These immunophenotypic changes were eliminated with the knockout of p53 in tumor cells. Enhanced expression of CD80 on tumor cells was observed in vitro and in vivo, which coincided with T-cell-mediated tumor cell killing. Combining HDM201 with PD-1 or PD-L1 blockade increased the number of complete tumor regressions. Responding mice developed durable, antigen-specific memory T cells and rejected subsequent tumor implantation. Importantly, antitumor activity of HDM201 in combination with PD-1/PD-L1 blockade was abrogated in p53-mutated and knockout syngeneic tumor models, indicating the effect of HDM201 on the tumor is required for triggering antitumor immunity. Taken together, these results demonstrate that MDM2 inhibition triggers adaptive immunity, which is further enhanced by blockade of PD-1/PD-L1 pathway, thereby providing a rationale for combining MDM2 inhibitors and checkpoint blocking antibodies in patients with wild-type p53 tumors. SIGNIFICANCE: This study provides a mechanistic rationale for combining checkpoint blockade immunotherapy with MDM2 inhibitors in patients with wild-type p53 tumors.

Secondary Wall Regulating NACs Differentially Bind at the Promoter at a CELLULOSE SYNTHASE A4 Cis-eQTL.

Arabidopsis thaliana CELLULOSE SYNTHASE A4/7/8 (CESA4/7/8) are three non-redundant subunits of the secondary cell wall cellulose synthase complex. Transcript abundance of these genes can vary among genotypes and expression quantitative trait loci (eQTL) were identified in a recombinant population of the accessions Bay-0 and Shahdara. Genetic mapping and analysis of the transcript levels of CESAs between two distinct near isogenic lines (NILs) confirmed a change in CESA4 expression that segregates within that interval. We sequenced the promoters and identified 16 polymorphisms differentiating CESA4Sha and CESA4Bay . In order to determine which of these SNPs could be responsible for this eQTL, we screened for transcription factor protein affinity with promoter fragments of CESA4Bay, CESA4Sha , and the reference genome CESA4Col . The wall thickening activator proteins NAC SECONDARY WALL THICKENING PROMOTING FACTOR2 (NST2) and NST3 exhibited a decrease in binding with the CESA4Sha promoter with a tracheary element-regulating cis-element (TERE) polymorphism. While NILs harboring the TERE polymorphisms exhibited significantly different CESA4 expression, cellulose crystallinity and cell wall thickness were indistinguishable. These results suggest that the TERE polymorphism resulted in differential transcription factor binding and CESA4 expression; yet A. thaliana is able to tolerate this transcriptional variability without compromising the structural elements of the plant, providing insight into the elasticity of gene regulation as it pertains to cell wall biosynthesis and regulation. We also explored available DNA affinity purification sequencing data to resolve a core binding site, C(G/T)TNNNNNNNA(A/C)G, for secondary wall NACs referred to as the VNS element.

Functional characterization of cinnamyl alcohol dehydrogenase and caffeic acid O-methyltransferase in Brachypodium distachyon.

BACKGROUND: Lignin is a significant barrier in the conversion of plant biomass to bioethanol. Cinnamyl alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the pathway of lignin monomer biosynthesis. Brown midrib mutants in Zea mays and Sorghum bicolor with impaired CAD or COMT activity have attracted considerable agronomic interest for their altered lignin composition and improved digestibility. Here, we identified and functionally characterized candidate genes encoding CAD and COMT enzymes in the grass model species Brachypodium distachyon with the aim of improving crops for efficient biofuel production. RESULTS: We developed transgenic plants overexpressing artificial microRNA designed to silence BdCAD1 or BdCOMT4. Both transgenes caused altered flowering time and increased stem count and weight. Downregulation of BdCAD1 caused a leaf brown midrib phenotype, the first time this phenotype has been observed in a C3 plant. While acetyl bromide soluble lignin measurements were equivalent in BdCAD1 downregulated and control plants, histochemical staining and thioacidolysis indicated a decrease in lignin syringyl units and reduced syringyl/guaiacyl ratio in the transgenic plants. BdCOMT4 downregulated plants exhibited a reduction in total lignin content and decreased Maule staining of syringyl units in stem. Ethanol yield by microbial fermentation was enhanced in amiR-cad1-8 plants. CONCLUSION: These results have elucidated two key genes in the lignin biosynthetic pathway in B. distachyon that, when perturbed, may result in greater stem biomass yield and bioconversion efficiency.