Revertants Research Articles

Florfenicol/Chlortetracycline Effect on Pharmacodynamic Indices for Mutant Selection of Riemerella anatipestifer in Ducks.

Riemerella anatipestifer can cause septicemia and death in ducks and geese, leading to significant economic losses to animal farms. The emergence of resistance of R. anatipestifer to commonly used antibiotics increases the difficulty of treating R. anatipestifer infection. The aim of this study was to evaluate the utility of antibiotic combination to restrict mutant selection of multidrug-resistant (MDR) R. anatipestifer isolates. Pharmacokinetics of florfenicol and chlortetracycline in Pekin ducks were evaluated using both noncompartmental analysis and population pharmacokinetic models. The areas under the curve of florfenicol and chlortetracycline after single 20 and 10 mg/kg oral administration were 49.3 and 6.84 mg*h/L, respectively. Chlortetracycline exhibited high apparent clearance and low systemic exposure. Minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) values of the two antibiotics were determined in 10 and 2 MDR R. anatipestifer isolates, respectively, to derive fTMSW (the fraction of time over 24 hours wherein the free drug concentration was within the mutant selection window [MSW]) and fT>MPC (the fraction of time that the free drug concentration was above the MPC). Both fTMSW and fT>MPC were estimated from simulated concentration-time profiles relative to MIC and MPC. Florfenicol and chlortetracycline combination have additive activities against R. anatipestifer in majority of isolates and could significantly decrease monotherapy MPC of florfenicol and chlortetracycline, as well as optimize both fTMSW and fT>MPC parameters, provided that the bioavailability of chlortetracycline is improved. The application of pharmacokinetic/pharmacodynamic analyses to MPC concepts to restrict selection of mutant bacterial strains can help improve short- and long-term outcomes of antibiotic treatment in animal farms.

Synthesis and biological evaluation of anilide derivatives as epidermal growth factor receptor L858R/T790M and L858R/T790M/C797S inhibitors

AbstractDevelopment of a mutant epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor is important for the treatment of various cancers. Third‐generation EGFR tyrosine kinase inhibitors (TKIs) have been clinically used by targeting T790M specifically but are recently known to induce T790M/C797S mutation after the treatment. Therefore, there is an unmet need to develop novel kinase inhibitors targeting the mutant EGFRs. Here, we designed and synthesized 16 analogs by hybridizing EAI045 (1) and 3′,4′,5′‐trihydroxyflavone (2) and identified 9a, 9b, and 20b as EGFR L858R/T790M/C797S mutant inhibitors. In addition, we found that 10a and 10b can inhibit both L858R/T790M and L858R/T790M/C797S. Molecular docking study on a plausible binding mode of the compounds is also provided.

Abstract 3599: Helical stapled peptides derived from DIRAS3 block KRAS dimerization and downstream MEK/ERK signaling in pancreatic and ovarian carcinomas

Abstract The overall 5-year survival for pancreatic ductal adenocarcinoma (PDAC) has changed little over the past few decades, and PDAC is predicted to become the second leading cause of cancer-related mortality in the next decade in Western countries. Low grade serous ovarian cancer (LGSOC) is a slow-growing but generally chemo-resistant cancer with few effective treatments for recurrent disease. More than 90% of PDAC and up to 40% of LGSOC express mutationally activated KRAS that drives persistent cell division, anti-apoptosis, cell migration and metastasis. Preventing signaling from the KRAS oncoprotein has been challenging. While recent studies have shown promising results by targeting the binding pocket in Switch II for development of KRAS mutant-specific inhibitors for the G12C mutant and G12D mutant, targeting other mutations remains a work in progress. We have discovered that DIRAS3, a novel endogenous physiological RAS inhibitor, blocks KRAS activity by directly binding KRAS with high affinity, inhibiting KRAS dimerization/nanoclustering and blocking effector activation. In this study we have developed drug-like, helical 10-mer stapled peptides derived from DIRAS3 α5 domain. Preliminary studies show that these lead compounds associate with KRAS with low nanomolar affinity, are largely resistant to serum proteases, and rapidly cross the cellular membrane. Functionally, DIRAS3 peptides—but not control peptides—block KRAS homodimers in ReBiL split luciferase assays of KRAS dimerization and disrupt nanoclustering by TEM analysis of GFP-KRAS(G12V)-labeled gold nanoparticles on the inner leaflet of the plasma membrane of cells. Moreover, DIRAS3 peptides significantly inhibit phospho-ERK, decrease cell viability and induce apoptosis in pancreatic and low-grade ovarian cancer cells with KRAS mutations. Finally, daily treatment with stapled DIRAS3-derived peptides inhibited growth of ASPC-1 (KRAS G12D) PDAC xenografts and improved survival. Thus, our study suggests that development of DIRAS3 stapled peptides may provide a novel therapeutic approach to target mutant KRAS-driven cancers. Citation Format: Joshua P. Gray, Gamze E. Bildik, Margie N. Sutton, Yong Zhou, Steven Millward, John F. Hancock, Zhen Lu, Robert C. Bast. Helical stapled peptides derived from DIRAS3 block KRAS dimerization and downstream MEK/ERK signaling in pancreatic and ovarian carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3599.

Abstract LB194: Discovery and characterization of a mutant selective PI3KαH1047Xinhibitor with a best-in-class profile

Abstract Oncogenic PIK3CA mutations are found in 14% of all cancers and most frequently occur at amino acids E542/E545 and H1047, involving the helical and kinase domains, respectively (Zhang 2017). Alpelisib is a PI3Kα isoform-selective, orthosteric kinase inhibitor, having equivalent activity on wild-type (wt) and mutant (mt) forms and was approved to treat PI3Kα mt, HR+/HER2- breast cancer in combination with fulvestrant, nearly doubling progression-free survival (Andre 2019). Hyperglycemia with insulin resistance is an on-target result of wt PI3Kα inhibition, leading to frequent alpelisib dose reductions, interruptions, and discontinuations. H1047X is the most common PIK3CA alteration, found in ~14% of all breast cancer patients (Martínez-Sáez 2020). We considered that selectively targeting this mutant could improve patient outcomes by sparing metabolic dysfunction related to wt inhibition. We have validated this therapeutic concept preclinically with the discovery of ST-814, an allosteric, CNS-penetrant, mutant selective PI3KαH1047X inhibitor, displaying excellent drug-like properties. ST-814 was 14-fold selective for PI3KαH1047R vs PI3Kαwt in biochemical assays (IC50 11 vs 146 nM) with exquisite isoform and kinome selectivity. Cell-based activity and selectivity were characterized in T47D (PI3KαH1047R) and SKBR3 (PI3KαWT) tumor cells grown in 10% FBS, with 8-fold mut-selectivity observed in target engagement (pAKT EC50 38 vs 304 nM) and 11-fold selectivity in viability (GI50 153 vs 1683 nM). ST-814 has robust anti-tumor activity in PI3KαH1047X in xenografts, with efficacy similar or superior to alpelisib dosed at 50 mg/kg (Table 1). To achieve this level of mouse efficacy, alpelisib exposure exceeded that in patients >200% (mouse vs human AUC0-24, 74,000 vs 33,224 ng*hr/mL). As such, repeat dosing of alpelisib at 50 mg/kg, caused profound elevations in serum insulin 1 hr post-dose (*p<0.01), whereas ST-814 showed no significant difference (Table 1). The metabolic consequences of repeated alpelisib (50 mg/kg) and ST-814 (100 mg/kg) dosing was further characterized in an insulin tolerance test. Insulin-mediated glucose clearance (AUC0-4) was significantly impaired by alpelisib (p<0.01, one-way ANOVA), but not by ST-814. These data support the potential of molecules like ST-814 to improve outcomes in patients harboring H1047X mut tumors. A clinical candidate from this program is currently in IND enabling studies. Table 1. CAL33 H1047R Oral Sq Cell Carcinoma Xenograft Alpelisib TGI% 50 mg/kg ST-814 TGI% 100 mg/kg Alpelisib ΔInsulin ng/mL ST-814 ΔInsulin ng/mL 79% 82% 4.42* 0.40 Citation Format: Leonard Buckbinder, David J. St. Jean, Trang Tieu, Weixue Wang, Gregory Kryukov, Philip Jonsson, Jacob Alltucker, Samantha Manimala, Brendon Ladd, Angel Guzman-Perez, Darrin Stuart. Discovery and characterization of a mutant selective PI3KαH1047Xinhibitor with a best-in-class profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB194.

Abstract 3647: Oncogenic Kras drives cancer-associated fibroblast heterogeneity and substate changes in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is currently the third-leading cause of cancer-related deaths in the United States with no effective or targeted therapies to improve outcomes. Two defining features of PDAC are the dense stroma composing the bulk of the tumor mass and mutations in KRAS, the primary oncogene in most PDAC patients. Advances in knowledge on both fronts have led to promising avenues for therapeutics: recently described heterogeneity within the cancer-associated fibroblast (CAF) population residing in the stroma offers insight into the lack of success with therapies only targeting CAFs; and mutant KRAS inhibitors directed at the G12C isoform have received accelerated FDA approval for treatment of locally advanced and metastatic non-small cell lung cancer. However, the nature of this CAF heterogeneity is not yet fully understood, and any effects of KRAS blockade on the stroma have not been evaluated with consideration of this heterogeneity in mind. To assess how loss of Kras affects CAF heterogeneity and plasticity, we employed a genetically engineered mouse model (GEMM) of PDAC harboring a reversible oncogenic KrasG12V allele conditionally expressed from the endogenous Kras locus. This FPC compound mouse (KrasFrt-LSL-G12V-Frt; p53LSL-R172H; PDX-CRE; Rosa26FlpOERT2) spontaneously develops pancreatic lesions and allows for irreversible excision of the mutant Kras allele upon the application of Tamoxifen. Using single-cell RNA sequencing (scRNAseq), fluorescence in situ hybridization, and functional in vitro and in vivo approaches, we found that oncogenic Kras ablation led to tumor regression and altered cancer cell gene programs, profoundly affecting the tumor microenvironment, specifically CAF heterogeneity. Mutant Kras loss leads to decreased Tgfb1 expression in the cancer cells, but surprisingly also to increased Il1a levels, correlating with a reduction in myofibroblastic CAFs (myCAFs) and an expansion in inflammatory CAFs (iCAFs), respectively. Our results suggest that mutant Kras drives paracrine signaling that shapes CAF heterogeneity and should be considered for the development of the most effective therapeutic strategies. Citation Format: Jennifer S. Thalappillil, Giuseppina Caligiuri, Sandeep Nadella, Brinda Alagesan, Georgi N. Yordanov, Mojdeh Shakiba, Benjamin Kaminow, Juliene Hinds, Pedro A. Perez-Mancera, Jonathan Preall, Alexander Dobin, Youngkyu Park, David A. Tuveson. Oncogenic Kras drives cancer-associated fibroblast heterogeneity and substate changes in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3647.

Abstract 6219: WM-S1, the novel small molecule inhibitor of mutant RTK/receptor tyrosine kinase, for the treatment of cancer

Abstract As the representative targeted anticancer drug for colon cancer patients, cetuximab is the EGFR targeted therapeutic antibody and used for treatment of KRAS wild type cancers. Even some patient with KRAS wt gene did not respond cetuximab. However, there is no treatment available for cetuximab-resistant patient group, which is almost 50% of KRAS WT gene holders. Recently, our team identified cetuximab primary resistant related proteins named as mtRTK (mutant receptor tyrosine kinase) by array analysis based cetuximab responder or non-responder colon cancer patient tissues. We investigated mtRTK’s oncogenic potential as a novel anti-cancer target. A large proportion of colon cancer patients (36.2% Caucasian, 56.9% Korean) expressed the mtRTK was identified, using the sequencing analysis of patient samples. Based on these results, our efforts have led to the discovery of WM-S1, mtRTK inhibitor, which is the first mtRTK inhibitor in clinical development. The potent enzyme inhibitor showed a high anticancer activity confirmed in Patient-Derived Cells (PDC) and Patient-Derived Xenograft (PDX) animal models expressing the mutation. In preclinical studies demonstrate that WM-S1 is well tolerated in rats and dogs. Furthermore, WM-S1 has potent anticancer activities for various solid tumor (NSCLC, cholangiocarcinoma, etc.) including activated mtKRAS colon cancer expressing the mtRTK. Currently we are investigating WM-S1 in a phase 1a trial in AUS, which is the first mtRTK inhibitor in clinical development. Meanwhile, the mtRTK inhibitor WM-S1 drives antitumor immunity (with anti-PD-L1) in NSCLC. Combinational approaches with immunotherapy showed that synergistic effect of WM-S1 and anti-PD1 monoclonal antibody, suppressing tumor growth by 75% in anti-PD1 resistance NSCLC-derived humanized mouse model. A phase 1b trial is expected to develop WM-S1 through not only indication expansion but also combination therapy with immuno-checkpoint inhibitors in the USA, AUS and KOR from Q2 2022. In conclusion, mtRTK is a potential oncogenic driver mutation in various solid tumor. A first-in-class anticancer agent WM-S1 targeting mtRTK can be promising therapeutic agents for cetuximab-resistant colon cancer patients regardless of KRAS mutation status and other cancers. Citation Format: Joseph Kim, Jai-Hee Moon, Kyung-Mi Lee, Hyun Ryu, Eun Hye Park, Sang Hee Kim, Jeong Seok Kim, Young Ok Ko, Yong Seok Kim, Hyo Jin Kim, Tae Young Kim Kim, Moon Seong Yoo, Soll Jin, Seongrak Kim, Yoon Sun Park, Min Ki Lee, Mi So Lee, Ji Hyun Go, Yu Geun Ji, Jun Hyung Lee, Haneul Lee, Min Hwa Kim, Eun Hee Ko, Yeo Jin Lee, Seung-Mi Kim, Joon-yee Jeong, Yeon-seoung Choi, Seung-geon Bae, Jinwoo Lee, Won Jun Lee, Min-Kyeong Kim, Ji min Shin, Dong-in Koh, Sun-Chul Hur, Chun-Ho Park, Hyun Ho Lee, Dong-Hoon Jin. WM-S1, the novel small molecule inhibitor of mutant RTK/receptor tyrosine kinase, for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6219.

Abstract 2666: Anti-tumor effects of the novel KIT mutant inhibitor M4205 in patient-derived gastrointestinal stromal tumor (GIST) xenograft models

Abstract Background: The majority of GISTs are driven by constitutively activated KIT/PDGFRA kinases and susceptible to treatment with tyrosine kinase inhibitors such as imatinib, sunitinib and regorafenib. During treatment most tumors will develop heterogeneous secondary mutations in KIT or PDGFRA inducing drug resistance, so there is an unmet need for novel therapies. We tested the efficacy of M4205, a novel specific KIT inhibitor with high activity towards the most relevant KIT mutations, in patient-derived GIST xenograft models. Methods: NMRI nu/nu mice (n=146) were transplanted with patient-derived GIST xenograft models UZLX-GIST9 (KIT:p.P577del;W557LfsX5;D820G) known to be resistant to both imatinib and sunitinib, with the dose-dependent imatinib-sensitive and sunitinib-sensitive models UZLX-GIST2B (KIT:p.A502_Y503dup), UZLX-GIST25 (KIT: p.K642E) and the cell-line derived model GIST882 (KIT: p.K642E)*. Mice were treated daily with vehicle (control), imatinib (100mg/kg), avapritinib (5mg/kg), sunitinib (20mg/kg), or M4205 (10mg/kg, 25mg/kg). Efficacy was assessed by tumor volume evolution, histopathology [hematoxilin & eosine staining (H&E)] and immunohistochemistry [Ki-67, phospho-Histone H3 (pHH3), cleaved PARP]. Histologic response (HR) was graded as previously described°. Mann Whitney U and Wilcoxon Matched Pairs tests were used for statistical analysis, with p<0.05 considered as significant. Results: M4205 (25mg/kg) caused tumor volume shrinkage in UZLX-GIST2B, -GIST25 and GIST882 with relative decrease to 45.6%, 35.1% and 57.3% on the last day as compared to baseline. In UZLX-GIST9 tumor growth to 132.4% was observed in M4205 (25mg/kg)-treated tumors as compared to baseline. In all models we observed significant smaller tumor volumes in M4205 (25mg/kg)-treated tumors compared to control, and this antitumor activity was superior to imatinib in UZLX-GIST9, -GIST2B and GIST882, and to sunitinib in -GIST25. Compared to controls, M4205 (25mg/kg) induced a significant decrease in mitosis (H&E) in all models, confirmed on pHH3 and Ki-67 immunostainings in three models. In -GIST25 and GIST882 grade 2-4 HR with myxoid degeneration was observed in all tumors. All treatments were well tolerated. Conclusion: M4205 has significant antitumor activity in patient- and cell line-derived GIST xenograft models. The novel kinase inhibitor induces volumetric responses, decreases mitotic activity, has antiproliferative effects and in models with KIT exon 13 mutation leads to characteristic myxoid degeneration. * Cornillie et al. Mol Cancer Ther 2019; 18(6):1168-1178 ° Agaram et al. Clin Cancer Res 2007; 13:170-81 Citation Format: Luna De Sutter, Agnieszka Wozniak, Jasper Verreet, Ulla Vanleeuw, Lore De Cock, Nina Linde, Christine Drechsler, Christina Esdar, Raf Sciot, Patrick Schöffski. Anti-tumor effects of the novel KIT mutant inhibitor M4205 in patient-derived gastrointestinal stromal tumor (GIST) xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2666.

Adaptation of Listeria monocytogenes to perturbation of c-di-AMP metabolism underpins its role in osmoadaptation and identifies a fosfomycin uptake system.

The human pathogen Listeria monocytogenes synthesizes and degrades c-di-AMP using the diadenylate cyclase CdaA and the phosphodiesterases PdeA and PgpH respectively. c-di-AMP is essential because it prevents the uncontrolled uptake of osmolytes. Here, we studied the phenotypes of cdaA, pdeA, pgpH and pdeA pgpH mutants with defects in c-di-AMP metabolism and characterized suppressor mutants restoring their growth defects. The characterization of the pdeA pgpH mutant revealed that the bacteria show growth defects in defined medium, a phenotype that is invariably suppressed by mutations in cdaA. The previously reported growth defect of the cdaA mutant in rich medium is suppressed by mutations that osmotically stabilize the c-di-AMP-free strain. We also found that the cdaA mutant has an increased sensitivity against isoleucine. The isoleucine-dependent growth inhibition of the cdaA mutant is suppressed by codY mutations that likely reduce the DNA-binding activity of encoded CodY variants. Moreover, the characterization of the cdaA suppressor mutants revealed that the Opp oligopeptide transport system is involved in the uptake of the antibiotic fosfomycin. In conclusion, the suppressor analysis corroborates a key function of c-di-AMP in controlling osmolyte homeostasis in L. monocytogenes.

Genetic suppressors of Δgrx3 Δgrx4, lacking redundant multidomain monothiol yeast glutaredoxins, rescue growth and iron homeostasis.

Saccharomyces cerevisiae Grx3 and Grx4 are multidomain monothiol glutaredoxins that are redundant with each other. They can be efficiently complemented by heterologous expression of their mammalian ortholog, PICOT, which has been linked to tumor development and embryogenesis. PICOT is now believed to act as a chaperone distributing Fe-S clusters, although the first link to iron metabolism was observed with its yeast counterparts. Like PICOT, yeast Grx3 and Grx4 reside in the cytosol and nucleus where they form unusual Fe-S clusters coordinated by two glutaredoxins with CGFS motifs and two molecules of glutathione. Depletion or deletion of Grx3/Grx4 leads to functional impairment of virtually all cellular iron-dependent processes and loss of cell viability, thus making these genes the most upstream components of the iron utilization system. Nevertheless, the Δgrx3/4 double mutant in the BY4741 genetic background is viable and exhibits slow but stable growth under hypoxic conditions. Upon exposure to air, growth of the double deletion strain ceases, and suppressor mutants appear. Adopting a high copy-number library screen approach, we discovered novel genetic interactions: overexpression of ESL1, ESL2, SOK1, SFP1 or BDF2 partially rescues growth and iron utilization defects of Δgrx3/4. This genetic escape from the requirement for Grx3/Grx4 has not been previously described. Our study shows that even a far-upstream component of the iron regulatory machinery (Grx3/4) can be bypassed, and cellular networks involving RIM101 pH sensing, cAMP signaling, mTOR nutritional signaling, or bromodomain acetylation, may confer the bypassing activities.

Modulation of autoimmune diabetes by N-ethyl-N-nitrosourea- induced mutations in non-obese diabetic mice.

ABSTRACTGenetic association studies of type 1 diabetes (T1D) in humans, and in congenic non-obese diabetic (NOD) mice harboring DNA segments from T1D-resistant mice, face the challenge of assigning causation to specific gene variants among many within loci that affect disease risk. Here, we created random germline mutations in NOD/NckH mice and used automated meiotic mapping to identify mutations modifying T1D incidence and age of onset. In contrast with association studies in humans or congenic NOD mice, we analyzed a relatively small number of genetic changes in each pedigree, permitting implication of specific mutations as causative. Among 844 mice from 14 pedigrees bearing 594 coding/splicing changes, we identified seven mutations that accelerated T1D development, and five that delayed or suppressed T1D. Eleven mutations affected genes not previously known to influence T1D (Xpnpep1, Herc1, Srrm2, Rapgef1, Ppl, Zfp583, Aldh1l1, Col6a1, Ccdc13, Cd200r1, Atrnl1). A suppressor mutation in Coro1a validated the screen. Mutagenesis coupled with automated meiotic mapping can detect genes in which allelic variation influences T1D susceptibility in NOD mice. Variation of some of the orthologous/paralogous genes may influence T1D susceptibility in humans.

Multicenter phase II study of abemaciclib and ramucirumab in metastatic esophageal/gastroesophageal junction carcinoma.

TPS4169 Background: Cyclin dependent kinases (CDKs) are serine/threonine kinases that are responsible for phosphorylating the intracellular proteins that coordinate cell-cycle progression. The Cancer Genome Atlas (TCGA) molecular analysis of esophageal/gastroesophageal junction (E/GEJ) carcinomas has highlighted significant dysregulation of CDKN2A, the gene coding for the tumor suppressor p16, through deletion, mutation or epigenetic silencing in up to 76% of cases and the associated significant upregulation of the CDK4/6-Cyclin D-axis (The Cancer Genome Atlas Research Network. Integrated genomic characterization of oesophageal carcinoma. Nature 2017). Preclinical experiments demonstrated that single agent abemaciclib has potent antitumor efficacy both in vitro and in vivo in esophageal adenocarcinoma with direct pathway inhibition (Kosovec J et al. CDK4/6 dual inhibitor abemaciclib demonstrates compelling preclinical activity against esophageal adenocarcinoma: a novel therapeutic options for a deadly disease. Oncotarget 2017 Nov 1;8[59]). Significant activity was seen in the OE33 (p=0.048) and FLO1 (p=0.043) esophageal cancer cell lines while western blot revealed downregulation of Cyclin D, phospho-pRb, E2F1, and Cyclin A2 (Kosovec J et al, 2017). In vivo experiments , utilizing the Levrat model of end-to-side esophagojejunostomy performed on Sprague-Dawley rats demonstrated that 78.9% of animals given abemaciclib demonstrated >20% tumor volume decrease (placebo 0%). Post treatment comparison between placebo and abemaciclib of qRT-PCR gene expression demonstrated downregulation of pathway correlates, including CDK4 (p=0.023), CDK6 (p=0.068), E2F1 (p=0.005), Rb1 (p=0.067), Cyclin D (p=0.031), and Cyclin A (p=0.039) (Kosovec J et al, 2017). The JPBJ phase 1b study has previously demonstrated the safety of combining abemaciclib with ramucirumab in NSCLC. Methods: Subjects with previously treated metastatic esophageal or gastroesophageal junction adenocarcinomas are eligible for this study which is currently open and enrolling patients. Squamous cell and mixed histology with neuroendocrine features are excluded. A total of 30 patients will be enrolled across 3 sites at Baylor University Medical Center in Dallas, The Allegheny Health Network in Pittsburgh and at Johns Hopkins Hospital in Baltimore. The primary objective is to describe the safety profile of abemaciclib (150mg po bid) and ramucirumab (8mg/kg iv every 2 weeks) in this patient population as assessed according to Common Terminology Criteria for Adverse Events version 4.0. Secondary objectives are to assess response rate, progression free and overall survival. In addition, we will determine the rate of stable disease at 3 months post targeted therapy. Correlative studies investigating changes in the expression of selected serum/tissue genomic markers of interest will be performed. Clinical trial information: NCT04921904.

Profiling oncogenic KRAS mutant drugs with a cell-based Lumit p-ERK immunoassay.

KRAS is one of the most heavily mutated oncogenes in cancer and targeting mutant KRAS with drugs has proven difficult. However, recent FDA approval of the KRAS G12C selective inhibitor sotorasib (AMG-510), has breathed new life into the drive to develop mutant KRAS inhibitors. In an effort to study RAS inhibitors in cells and identify new compounds that inhibit Ras signaling, western blotting and ELISA assays are commonly used. These traditional immunoassays are tedious, require multiple washing steps, and are not easily adaptable to a high throughput screening (HTS) format. To overcome these limitations, we applied Lumit immunoassay technology to analyze RAS signaling pathway activation and inhibition through the detection of phosphorylated ERK. The assay we developed was used to rank order potencies of allele specific inhibitors within cell lines harboring various activating KRAS mutations. An inhibition profile was obtained indicating various potencies and selectivity of the inhibitors, including MRTX-1133, which was shown to be highly potent against KRAS G12D signaling. MRTX-1133 had approximately 40 and 400 times less inhibitory potency against G12C and G12V mutant KRAS, respectively, while no inhibition of WT KRAS was observed. The potency of PROTAC compound LC-2 targeting selective degradation of KRAS G12C was also tested using the Lumit pERK immunoassay, and a maximal decrease in RAS signaling was achieved. Lumit immunoassays provide a rapid, homogeneous platform for detecting signaling pathway activation and inhibition. Our results demonstrate that this bioluminescent technology can streamline the analysis of signaling pathways of interest, such as RAS-dependent pathways, and be used to identify much needed inhibitors. The results further imply that similar assay designs could be applied to other signaling pathway nodes.

Comparison of Experimental Approaches Used to Determine the Structure and Function of the Class D G Protein-Coupled Yeast α-Factor Receptor.

The Saccharomyces cerevisiae α-factor mating pheromone receptor (Ste2p) has been studied as a model for the large medically important family of G protein-coupled receptors. Diverse yeast genetic screens and high-throughput mutagenesis of STE2 identified a large number of loss-of-function, constitutively-active, dominant-negative, and intragenic second-site suppressor mutants as well as mutations that specifically affect pheromone binding. Facile genetic manipulation of Ste2p also aided in targeted biochemical approaches, such as probing the aqueous accessibility of substituted cysteine residues in order to identify the boundaries of the seven transmembrane segments, and the use of cysteine disulfide crosslinking to identify sites of intramolecular contacts in the transmembrane helix bundle of Ste2p and sites of contacts between the monomers in a Ste2p dimer. Recent publication of a series of high-resolution cryo-EM structures of Ste2p in ligand-free, agonist-bound and antagonist-bound states now makes it possible to evaluate the results of these genetic and biochemical strategies, in comparison to three-dimensional structures showing activation-related conformational changes. The results indicate that the genetic and biochemical strategies were generally effective, and provide guidance as to how best to apply these experimental strategies to other proteins. These strategies continue to be useful in defining mechanisms of signal transduction in the context of the available structures and suggest aspects of receptor function beyond what can be discerned from the available structures.

Vitamin B6 Deficiency Promotes Loss of Heterozygosity (LOH) at the Drosophila warts (wts) Locus

The active form of vitamin B6, pyridoxal 5′-phosphate (PLP), is a cofactor for more than 200 enzymes involved in many metabolic pathways. Moreover, PLP has antioxidant properties and quenches the reactive oxygen species (ROS). Accordingly, PLP deficiency causes chromosome aberrations in Drosophila, yeast, and human cells. In this work, we investigated whether PLP depletion can also cause loss of heterozygosity (LOH) of the tumor suppressor warts (wts) in Drosophila. LOH is usually initiated by DNA breakage in heterozygous cells for a tumor suppressor mutation and can contribute to oncogenesis inducing the loss of the wild-type allele. LOH at the wts locus results in epithelial wts homozygous tumors easily detectable on adult fly cuticle. Here, we found that PLP depletion, induced by two PLP inhibitors, promotes LOH of wts locus producing significant frequencies of wts tumors (~7% vs. 2.3%). In addition, we identified the mitotic recombination as a possible mechanism through which PLP deficiency induces LOH. Moreover, LOH of wts locus, induced by PLP inhibitors, was rescued by PLP supplementation. These data further confirm the role of PLP in genome integrity maintenance and indicate that vitamin B6 deficiency may impact on cancer also by promoting LOH.

A Mutant Era GTPase Suppresses Phenotypes Caused by Loss of Highly Conserved YbeY Protein in Escherichia coli.

Ribosome assembly is a complex fundamental cellular process that involves assembling multiple ribosomal proteins and several ribosomal RNA species in a highly coordinated yet flexible and resilient manner. The highly conserved YbeY protein is a single-strand specific endoribonuclease, important for ribosome assembly, 16S rRNA processing, and ribosome quality control. In Escherichia coli, ybeY deletion results in pleiotropic phenotypes including slow growth, temperature sensitivity, accumulation of precursors of 16S rRNA, and impaired formation of fully assembled 70S subunits. Era, an essential highly conserved GTPase protein, interacts with many ribosomal proteins, and its depletion results in ribosome assembly defects. YbeY has been shown to interact with Era together with ribosomal protein S11. In this study, we have analyzed a suppressor mutation, era(T99I), that can partially suppress a subset of the multiple phenotypes of ybeY deletion. The era(T99I) allele was able to improve 16S rRNA processing and ribosome assembly at 37°C. However, it failed to suppress the temperature sensitivity and did not improve 16S rRNA stability. The era(T99I) allele was also unable to improve the 16S rRNA processing defects caused by the loss of ribosome maturation factors. We also show that era(T99I) increases the GroEL levels in the 30S ribosome fractions independent of YbeY. We propose that the mechanism of suppression is that the changes in Era’s structure caused by the era(T99I) mutation affect its GTP/GDP cycle in a way that increases the half-life of RNA binding to Era, thereby facilitating alternative processing of the 16S RNA precursor. Taken together, this study offers insights into the role of Era and YbeY in ribosome assembly and 16S rRNA processing events.

Genetic analysis suggests a surface of PAT-4 (ILK) that interacts with UNC-112 (kindlin).

Integrin plays a crucial role in the attachment of cells to the extracellular matrix. Integrin recruits many proteins intracellularly, including a 4-protein complex (kindlin, ILK, PINCH, and parvin). Caenorhabditis elegans muscle provides an excellent model to study integrin adhesion complexes. In Caenorhabditis elegans, UNC-112 (kindlin) binds to the cytoplasmic tail of PAT-3 (β-integrin) and to PAT-4 (ILK). We previously reported that PAT-4 binding to UNC-112 is essential for the binding of UNC-112 to PAT-3. Although there are crystal structures for ILK and a kindlin, there is no co-crystal structure available. To understand the molecular interaction between PAT-4 and UNC-112, we took a genetic approach. First, using a yeast 2-hybrid method, we isolated mutant PAT-4 proteins that cannot bind to UNC-112 and then isolated suppressor mutant UNC-112 proteins that restore interaction with mutant PAT-4 proteins. Second, we demonstrated that these mutant PAT-4 proteins cannot localize to attachment structures in nematode muscle, but upon co-expression of an UNC-112 suppressor mutant protein, mutant PAT-4 proteins could localize to attachment structures. Third, overexpression of a PAT-4 mutant results in the disorganization of adhesion plaques at muscle cell boundaries and co-expression of the UNC-112 suppressor mutant protein alleviates this defect. Thus, we demonstrate that UNC-112 binding to PAT-4 is required for the localization and function of PAT-4 in integrin adhesion complexes in vivo. The missense mutations were mapped onto homology models of PAT-4 and UNC-112, and taking into account previously isolated mutations, we suggest a surface of PAT-4 that binds to UNC-112.

Virtual Screening Based on Machine Learning Explores Mangrove Natural Products as KRASG12C Inhibitors

Mangrove secondary metabolites have many unique biological activities. We identified lead compounds among them that might target KRASG12C. KRAS is considered to be closely related to various cancers. A variety of novel small molecules that directly target KRAS are being developed, including covalent allosteric inhibitors for KRASG12C mutant, protein–protein interaction inhibitors that bind in the switch I/II pocket or the A59 site, and GTP-competitive inhibitors targeting the nucleotide-binding site. To identify a candidate pool of mangrove secondary metabolic natural products, we tested various machine learning algorithms and selected random forest as a model for predicting the targeting activity of compounds. Lead compounds were then subjected to virtual screening and covalent docking, integrated absorption, distribution, metabolism and excretion (ADME) testing, and structure-based pharmacophore model validation to select the most suitable compounds. Finally, we performed molecular dynamics simulations to verify the binding mode of the lead compound to KRASG12C. The lazypredict function package was initially used, and the Accuracy score and F1 score of the random forest algorithm exceeded 60%, which can be considered to carry a strong ability to distinguish the data. Four marine natural products were obtained through machine learning identification and covalent docking screening. Compound 44 and compound 14 were selected for further validation after ADME and toxicity studies, and pharmacophore analysis indicated that they had a favorable pharmacodynamic profile. Comparison with the positive control showed that they stabilized switch I and switch II, and like MRTX849, retained a novel binding mechanism at the molecular level. Molecular dynamics analysis showed that they maintained a stable conformation with the target protein, so compound 44 and compound 14 may be effective inhibitors of the G12C mutant. These findings reveal that the mangrove-derived secondary metabolite compound 44 and compound 14 might be potential therapeutic agents for KRASG12C.

Caulobacter lipid A is conditionally dispensable in the absence of fur and in the presence of anionic sphingolipids.

SUMMARYLipid A, the membrane-anchored portion of lipopolysaccharide (LPS), is an essential component of the outer membrane (OM) of nearly all Gram-negative bacteria. Here we identify regulatory and structural factors that together render lipid A nonessential in Caulobacter crescentus. Mutations in the ferric uptake regulator fur allow Caulobacter to survive in the absence of either LpxC, which catalyzes an early step of lipid A synthesis, or CtpA, a tyrosine phosphatase homolog we find is needed for wild-type lipid A structure and abundance. Alterations in Fur-regulated processes, rather than iron status per se, underlie the ability to survive when lipid A synthesis is blocked. Fitness of lipid A-deficient Caulobacter requires an anionic sphingolipid, ceramide phosphoglycerate (CPG), which also mediates sensitivity to the antibiotic colistin. Our results demonstrate that, in an altered regulatory landscape, anionic sphingolipids can support the integrity of a lipid A-deficient OM.

Systematic discovery of mutation-directed neo-protein-protein interactions in cancer

Comprehensive sequencing of patient tumors reveals genomic mutations across tumor types that enable tumorigenesis and progression. A subset of oncogenic driver mutations results in neomorphic activity where the mutant protein mediates functions not engaged by the parental molecule. Here, we identify prevalent variant-enabled neomorph-protein-protein interactions (neoPPI) with a quantitative high-throughput differential screening (qHT-dS) platform. The coupling of highly sensitive BRET biosensors with miniaturized coexpression in an ultra-HTS format allows large-scale monitoring of the interactions of wild-type and mutant variant counterparts with a library of cancer-associated proteins in live cells. The screening of 17,792 interactions with 2,172,864 data points revealed a landscape of gain of interactions encompassing both oncogenic and tumor suppressor mutations. For example, the recurrent BRAF V600E lesion mediates KEAP1 neoPPI, rewiring a BRAFV600E/KEAP1 signaling axis and creating collateral vulnerability to NQO1 substrates, offering a combination therapeutic strategy. Thus, cancer genomic alterations can create neo-interactions, informing variant-directed therapeutic approaches for precision medicine.

Pharmacokinetics and pharmacokinetic/pharmacodynamic-based dosing regimens of long-acting oxytetracycline in Nile tilapia (Oreochromis niloticus) broodstock to minimize selection of drug resistance

This study aimed to determine the pharmacokinetics of long-acting oxytetracycline (OTC-LA) after intraperitoneal (IP) administration in Nile tilapia broodstock. Pharmacokinetic/pharmacodynamic (PK/PD) relationships were evaluated to determine the optimal dosing regimens of OTC-LA against Streptococcus agalactiae. A total of 120 healthy male tilapia (450 ± 37.47 g) were arbitrarily divided into two groups (60 fish/group) that individually received OTC-LA single IP injection at dosages of 50 or 100 mg/kg body weight. Blood samples were collected at various times posttreatment, and plasma oxytetracycline (OTC) was analyzed using high-performance liquid chromatography. For pharmacodynamic study, 56 S. agalactiae isolates were subjected to agar dilution to determine the minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC). Results showed that the Cmax and Tmax of OTC-LA were both 110.70 ± 5.61 μg/mL at 2 h for the dosage of 50 mg/kg and 287.85 ± 8.03 μg/mL at 4 h for the dosage of 100 mg/kg. Plasma OTC levels remained at 3.99 ± 0.48 and 23.00 ± 2.51 μg/mL at 168 h (7 day) after OTC-LA administration at 50 and 100 mg/kg, respectively. The MIC50 and MIC90 of S. agalactiae were 0.5 and 1 μg/mL, respectively, and its MPC50 and MPC90 were 32 and 128 μg/mL, respectively. The mutant selection window of MIC50 and MPC50 was 0.5–32 μg/mL, and that of MIC90 and MPC90 was 1–128 μg/mL. OTC-LA at 50 and 100 mg/kg provided plasma OTC levels above MIC for at least 7 days. However, only OTC-LA at 100 mg/kg can provide plasma OTC to prevent the growth of resistant mutant subpopulation. Our study suggested that a single IP administration of OTC-LA at 100 mg/kg body weight is the optimal dosing regimen to attain therapeutic efficacy against S. agalactiae infection in tilapia and prevent the emergence of resistant mutant subpopulation.