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Hyer et al . generate a potent and specific small-molecule inhibitor of the E1 ubiquitin-activating enzyme UBE1 that has antitumor activity in mice against a wide variety of tumor types. The ubiquitin–proteasome system (UPS) comprises a network of enzymes that is responsible for maintaining cellular protein homeostasis. The therapeutic potential of this pathway has been validated by the clinical successes of a number of UPS modulators, including proteasome inhibitors and immunomodulatory imide drugs (IMiDs). Here we identified TAK-243 (formerly known as MLN7243) as a potent, mechanism-based small-molecule inhibitor of the ubiquitin activating enzyme (UAE), the primary mammalian E1 enzyme that regulates the ubiquitin conjugation cascade. TAK-243 treatment caused depletion of cellular ubiquitin conjugates, resulting in disruption of signaling events, induction of proteotoxic stress, and impairment of cell cycle progression and DNA damage repair pathways. TAK-243 treatment caused death of cancer cells and, in primary human xenograft studies, demonstrated antitumor activity at tolerated doses. Due to its specificity and potency, TAK-243 allows for interrogation of ubiquitin biology and for assessment of UAE inhibition as a new approach for cancer treatment.

Background MicroRNAs (miRNA) are varied in length, under 25 nucleotides, single-stranded noncoding RNA that regulate post-transcriptional gene expression via translational repression or mRNA degradation. Elevated levels of miRNAs can be detected in systemic circulation after tissue injury, suggesting that miRNAs are released following cellular damage. Because of their remarkable stability, ease of detection in biofluids, and tissue specific expression patterns, miRNAs have the potential to be specific biomarkers of organ injury. The identification of miRNA biomarkers requires a systematic approach: 1) determine the miRNA tissue expression profiles within a mammalian species via next generation sequencing; 2) identify enriched and/or specific miRNA expression within organs of toxicologic interest, and 3) in vivo validation with tissue-specific toxicants. While miRNA tissue expression has been reported in rodents and humans, little data exists on miRNA tissue expression in the dog, a relevant toxicology species. The generation and evaluation of the first dog miRNA tissue atlas is described here. Results Analysis of 16 tissues from five male beagle dogs identified 106 tissue enriched miRNAs, 60 of which were highly enriched in a single organ, and thus may serve as biomarkers of organ injury. A proof of concept study in dogs dosed with hepatotoxicants evaluated a qPCR panel of 15 tissue enriched miRNAs specific to liver, heart, skeletal muscle, pancreas, testes, and brain. Dogs with elevated serum levels of miR-122 and miR-885 had a correlative increase of alanine aminotransferase, and microscopic analysis confirmed liver damage. Other non-liver enriched miRNAs included in the screening panel were unaffected. Eli Lilly authors created a complimentary Sprague Dawely rat miRNA tissue atlas and demonstrated increased pancreas enriched miRNA levels in circulation, following caerulein administration in rat and dog. Conclusion The dog miRNA tissue atlas provides a resource for biomarker discovery and can be further mined with refinement of dog genome annotation. The 60 highly enriched tissue miRNAs identified within the dog miRNA tissue atlas could serve as diagnostic biomarkers and will require further validation by in vivo correlation to histopathology. Once validated, these tissue enriched miRNAs could be combined into a powerful qPCR screening panel to identify organ toxicity during early drug development.

In non-clinical studies, the proteasome inhibitor ixazomib inhibits cell growth in a broad panel of solid tumor cell lines in vitro. In contrast, antitumor activity in xenograft tumors is model-dependent, with some solid tumors showing no response to ixazomib. In this study we examined factors responsible for ixazomib sensitivity or resistance using mouse xenograft models. A survey of 14 non-small cell lung cancer (NSCLC) and 6 colon xenografts showed a striking relationship between ixazomib activity and KRAS genotype; tumors with wild-type (WT) KRAS were more sensitive to ixazomib than tumors harboring KRAS activating mutations. To confirm the association between KRAS genotype and ixazomib sensitivity, we used SW48 isogenic colon cancer cell lines. Either KRAS-G13D or KRAS-G12V mutations were introduced into KRAS-WT SW48 cells to generate cells that stably express activated KRAS. SW48 KRAS WT tumors, but neither SW48-KRAS-G13D tumors nor SW48-KRAS-G12V tumors, were sensitive to ixazomib in vivo. Since activated KRAS is known to be associated with metabolic reprogramming, we compared metabolite profiling of SW48-WT and SW48-KRAS-G13D tumors treated with or without ixazomib. Prior to treatment there were significant metabolic differences between SW48 WT and SW48-KRAS-G13D tumors, reflecting higher oxidative stress and glucose utilization in the KRAS-G13D tumors. Ixazomib treatment resulted in significant metabolic regulation, and some of these changes were specific to KRAS WT tumors. Depletion of free amino acid pools and activation of GCN2-eIF2α-pathways were observed both in tumor types. However, changes in lipid beta oxidation were observed in only the KRAS WT tumors. The non-clinical data presented here show a correlation between KRAS genotype and ixazomib sensitivity in NSCLC and colon xenografts and provide new evidence of regulation of key metabolic pathways by proteasome inhibition.

Summary Purpose Ixazomib is an investigational proteasome inhibitor with demonstrated antitumor activity in xenograft models of multiple myeloma (MM), lymphoma, and solid tumors. This open-label, phase 1 study investigated intravenous (IV) ixazomib, in adult patients with advanced non-hematologic malignancies. Methods Patients received IV ixazomib twice-weekly for up to twelve 21-day cycles. The 0.125 mg/m 2 starting dose was doubled (one patient/dose) until 1.0 mg/m 2 based on dose-limiting toxicities (DLTs) in cycle 1. This was followed by 3 + 3 dose-escalation and expansion at the maximum tolerated dose (MTD). Primary objectives included safety and MTD assessment. Secondary objectives included assessment of pharmacokinetics, pharmacodynamics, and disease response. Results Ixazomib was escalated from 0.125 to 2.34 mg/m 2 to determine the MTD ( n =  23); patients were then enrolled to MTD expansion ( n  = 73) and pharmacodynamic ( n  = 20) cohorts. Five patients experienced DLTs (1.0 and 1.76 mg/m 2 : grade 3 pruritic rash; 2.34 mg/m 2 : grade 3 and 4 thrombocytopenia, and grade 3 acute renal failure); thus, the MTD was 1.76 mg/m 2 . Drug-related grade ≥3 adverse events (AEs) included thrombocytopenia (23 %), skin and subcutaneous (SC) tissue disorders (16 %), and fatigue (9 %). Among 92 evaluable patients, one (head and neck cancer) had a partial response and 30 had stable disease. Ixazomib terminal half-life was 3.8–7.2 days; plasma exposures increased dose-proportionally and drug was distributed to tumors. Inhibition of whole-blood 20S proteasome activity and upregulation of ATF-3 in tumor biopsies demonstrated target engagement. Conclusions In patients with solid tumors, ixazomib was associated with a manageable safety profile, limited antitumor activity, and evidence of downstream proteasome inhibition effects.

An anti-idiotypic serum prepared against the combining site (idiotype) of specific anti-diphtheria toxoid antibodies was characterized with respect to its interaction with highly diphtheria toxin-sensitive Vero cells. Although the anti-idiotypic serum protected Vero cells against the cytotoxic action of diphtheria toxin, it did not prevent the binding of 125I-labeled diphtheria toxin to the cells but did inhibit the internalization and degradation of 125I-labeled toxin. This anti-idiotypic serum immunoprecipitated a cell-surface protein from radiolabeled Vero cells with an apparent Mr of ≈ 15,000. These results are consistent with the hypothesis that the antiidiotypic serum contains antibodies that carry an internal image of an internalization site on the toxin and that a cell-surface protein involved in toxin internalization possesses a complementary site recognized by both the toxin and the anti-idiotypic antibodies.

Immunization of Guinea pigs with diphtheria toxoid generated antibodies of the IgG class that were capable of neutralizing native toxin in vivo. Sera from these animals were used to affinity purify idiotypic antibodies (AB1). AB1 vaccines derived from the IgG1 class and from F(ab$\\sp\\prime)\\sb2$ of IgG1 + IgG2 (IgG1/2) classes were effective in inducing a syngeneic anti-idiotype (AB2) response. Animals immunized with AB1 consisting of both IgG1/2 did not elicit a detectable AB2 response. These findings are consistent with previous reports that the IgG Fc domain can influence the immune response to the idiotype. Binding of homologous $\\sp{125}$I-F(ab$\\sp\\prime)\\sb2$ (AB1) to the antiidiotype was inhibited 90% in the presence of DT. F(ab$\\sp\\prime)\\sb2$ derived from preimmune serum or had no inhibitory effects on the idiotype-antiidiotype interaction. Two groups of outbred guinea pigs were vaccinated with alum absorbed F(ab$\\sp\\prime)\\sb2$ of anti-idiotype IgG1/2 (AB2). Of the ten animals inoculated with AB2, three tested positive by RIA against $\\sp{125}$I-DT. Two of the RIA positive sera contained antibodies that neutralized diphtheria toxin in a rabbit intracutaneous assay. Purification of guinea pig IgG by protein A-Sepharose affinity chromatography resulted in the separation of three distinct IgG populations. As previously described, the first two peaks eluting at pH 4.9 and 4.3 correspond to the IgG1 and IgG2 subclasses respectively. Further elution at pH 2.3 revealed a third peak of IgG that appears to be a unique population of antibody differing in protein A binding affinity, electrophoretic mobility and amino acid content of the Fc domain. This third immunoglobulin peak had an immunoelectrophoresis pattern similar to the IgG2 class. However, a line of partial identity was formed between the IgG1 class and this third immunoglobulin. Conversely, amino acid analysis of Fc crystals prepared from each of the protein A-Sepharose purified immunoglobulin fractions indicated that the amino acid composition of the third immunoglobulin was more similar to the IgG1 class. On the basis of these diverse biochemical differences this third immunoglobulin isolated from protein A-Sepharose was termed IgG3.

Background. Diarrhea remains a common complaint among US patients who seek medical attention. Methods. We performed a prospective study to determine the etiology of diarrheal illness among patients and control subjects of all ages presenting to the emergency departments and outpatient clinics of 2 large academic hospitals in Baltimore, Maryland, and New Haven, Connecticut. We used molecular methods to detect the presence of diarrheagenic Escherichia coli pathotypes, including enteroaggregative E. coli (EAEC), as well as Shiga toxin–producing, cytodetaching, enterotoxigenic and enteropathogenic E. coli. Results. Of the pathotypes sought, only EAEC was found in an appreciable proportion (4.5%) of case patients, and it was found more frequently among case patients than control subjects (P < .02). Surprisingly, EAEC was the most common bacterial cause of diarrhea in our population. EAEC was common in all age strata and was not associated with foreign travel or immunodeficiency. EAEC infection is frequently accompanied by fever and abdominal pain, though this did not happen more frequently in patients with EAEC infection than in patients with diarrhea due to other causes. Conclusions. Our data suggest that EAEC infection should be considered among persons with diarrhea that does not yield another known etiologic agent.

BackgroundAntibody-dependent cell-mediated cytotoxicity (ADCC) is an effective tool where antibody-coated cells are targeted and killed by effector immune cells. The application of ADCC therapies has been expanded for both solid tumors as well as hematologic malignancies. However, the immunosuppressive mechanisms present in the immune tumor microenvironment (TME) pose a formidable challenge to immune cell efficacy in addition to hinderance of immune cell infiltration by tumor stromal elements. Hence, it is important to develop clinically relevant platforms to assess the efficacy of antibodies for ADCC. Here we utilized our 3D-EX platform using tumoroids of fresh patient tumor samples to assess ADCC-mediated tumor cell killing.MethodsAll human tumor samples were obtained with proper patient consent and IRB approval. Fresh patient tumor tissue of various histologic types including non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) was processed to generate uniform sized live 3D tumoroids measuring 150 µm in size. Treatment groups included cetuximab alone or in combination with nivolumab and/or ipilimumab. Culture supernatants were collected for multiplex analysis of cytokine release in media. Multiplex flow cytometry was used to assess the activation profile of tumor resident immune cells in combination with high-content confocal imaging to determine extent of ADCC-mediated tumor cell death in the intact tumor extracellular matrix.ResultsUsing fresh patient-derived tumor organoids, we observed ADCC-dependent death of EGFR expressing tumor cells. Flow cytometric analysis of immune cell populations demonstrated treatment mediated activation of resident immune cells, which coincided with cytokine profiles determined by Luminex multiplex cytokine analysis. Additionally, tumor cell killing observed through high-content confocal imaging and quantitative image analysis showed tumor cell death with the 3D tumoroids.ConclusionsIn this comprehensive study we demonstrate that the 3D-EX ex vivo model is a robust system to assess the efficacy of ADCC and to develop novel therapeutic combinations with other immuno-oncology therapies. Furthermore, implementation of this platform in clinical studies may also allow for determination of the most effective combinatorial immuno-oncology therapy strategies for specialized individual patient care.Ethics ApprovalThe study was approved by Chesapeake IRB Pro00014313.

Voters are starting to feel the full scope of Trump 2.0 as wehit day 100 of the new Congress. And Democrats see an opening with anewly energized base. President Trump's domestic policies rest inlarge part on whether the conservative supermajority in the SupremeCourt will go along with them as he tests the boundaries of his powersand federal law. Trump's team closes ranks while his tariff whiplashrattles Americans. After a volatile week where he sent marketsspiraling with his whiplash tariff policies, there are growingquestions about the president's strategy and fresh signs the Americanpublic is losing confidence in his handling of the economy. PresidentTrump putting Democratic governors through the wringer as they startmaking moves ahead of 2028. GUESTS: Marianna Sotomayor, Dasha Burns, Jonah Goldberg

Trump speaks ahead of signing Laken Riley Act; Trump says Guantanamo to be used for holding migrants; Laken Riley Act to be signed by U.S. President Trump; Laken Riley Act being signed by U.S. President Trump; White House rescinds federal aid freeze; Trump demands federal workers return to office; Senators grill RFK Jr. on vaccines, abortion; Senate confirmation hearing for health secretary pick RFK Jr. GUESTS: Lynn Sweet