The inflammasome is a multiprotein complex that triggers the activation of proinflammatory cytokines. The adapter ASC and its isoform ASCb mediate inflammasome assembly via self-association and ...oligomerization with other inflammasome proteins by homotypic interactions of their two identical Death Domains, PYD and CARD, connected by a linker of different length: 23 (ASC) and 4 (ASCb) amino acids long. However, ASC is a more potent inflammasome activator compared to ASCb. Thus, adapter isoforms might be involved in the regulation of the inflammatory response. As previously reported, ASC’s faster and less polydisperse self-association compared to ASCb points to interdomain flexibility resulting from the linker length as a key factor in inflammasome regulation. To test the influence of linker length in self-association, we have engineered the isoform ASC3X with identical PYD and CARD connected by a 69 amino acid-long linker (i.e., three-times longer than ASC’s linker). Real-time NMR and dynamic light scattering data indicate that ASC3X polymerization is less effective and more polydisperse compared to ASC or ASCb. However, transmission electron micrographs show that ASC3X can polymerize into filaments. Comparative interdomain dynamics of the three isoforms obtained from NMR relaxation data reveal that ASCb tumbles as a rod, whereas the PYD and CARD of ASC and ASC3X tumble independently with marginally higher interdomain flexibility in ASC3X. Altogether, our data suggest that ASC’s linker length is optimized for self-association by allowing enough flexibility to favor intermolecular homotypic interactions but simultaneously keeping both domains sufficiently close for essential participation in filament formation.
The purpose of this analysis is to examine how the progressive discipline approach has evolved with the modern practice of human capital management and employee engagement. A review of relevant ...literature informed the exploration of current discipline approaches, historical foundations, and applied examples from organizations. Examples are analyzed outlining current strategies for implementing discipline approaches. It is well understood that the current composition of the workforce brings many new and non-traditional beliefs, attitudes, and values. The issue of traditional punitive approaches to discipline represents a significant concern for modern managers. This paper seeks to outline why a new collaborative approach to performance management and employee discipline is needed, in order to maintain high levels of employee engagement and honor organizational claims that human capital is indeed the most valued asset. A model will be presented with an emphasis on healthcare organizations.
Abstract Oncogenic RAS binds to multiple effector proteins to activate downstream signaling pathways, including the MAPK and PI3K axes. While RAS-mediated MAPK activation has a well-validated role in ...driving cancer progression, the contribution of PI3K activation has been more difficult to assess. Understanding the degree to which different KRAS-mutant genotypes stimulate PI3K activity may inform biomarker and combination strategies for RAS inhibitors currently undergoing clinical evaluation.RMC-7977 is a RASMULTI(ON) inhibitor that targets GTP-bound RAS(ON) and blocks effector protein binding. RMC-7977 exhibits similar potency for wild-type and oncogenic mutant RAS and is a powerful preclinical tool, representative of the investigational RASMULTI(ON) inhibitor RMC-6236, for comparing how RAS-mutant genotypes differ with respect to signal activation. MAPK signaling proceeds through a protein phosphorylation cascade that is readily detectable using standard cell biology methods, but PI3K is a lipid kinase whose activity is not amenable to protein characterization techniques. An assay of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) was developed to directly measure the activity of PI3K in the context of RAS inhibition.In isogenic LIM1215 cell lines expressing different KRASG12X mutants (KRASG12D, KRASG12V, KRASG12C and KRASG12R), RAS inhibition by RMC-7977 completely suppressed PI3K activity in cells with wild-type KRAS and KRASG12D, had a moderate inhibitory effect in KRASG12C and KRASG12V, and had no effect in KRASG12R. To confirm that this inhibition is dependent on a direct interaction between RAS(ON) and PI3K, CRISPR engineering was used to introduce mutations in PIK3CA to perturb the RAS-binding domain. In cells with disrupted RAS-PI3K interactions, the basal levels of PIP3 are significantly reduced and no longer change in response to treatment with RMC-7977.Based on the isogenic cell line results, KRASG12D and KRASG12R-mutant pancreatic cancer cell lines were profiled for their response to RAS inhibition. RMC-7977 decreased the levels of MAPK signaling in both KRASG12D and KRASG12R genotypes, but PI3K activity was only inhibited in KRASG12D lines. MEK inhibitors only inhibited MAPK signaling and had no effect on PI3K activity in either genotype. The simultaneous inhibition of MAPK and PI3K signaling by RMC-7977 led to deeper suppression of cyclin D levels and a more pronounced G1 cell cycle arrest as compared with MAPK inhibitors.We demonstrate that the coupling between RAS activation and PI3K signaling varies between oncogenic mutants, with KRASG12D showing the greatest degree of PI3K stimulation. In KRASG12D-mutant pancreatic cancer cell lines, a RASMULTI(ON) inhibitor suppresses both PI3K and MAPK signaling. These preclinical results may be used to inform potential, rational, mechanism-based combination treatment strategies for RAS(ON) inhibitors. Citation Format: Priyanka S. Bapat, Kyle Seamon, Andrea Gould, David Wildes. The RASMULTI(ON) inhibitor RMC-7977 blocks downstream MAPK and PI3K pathway activation in KRASG12X_mutant cancers abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4709.
The discovery of small-molecule inhibitors requires suitable binding pockets on protein surfaces. Proteins that lack this feature are considered undruggable and require innovative strategies for ...therapeutic targeting.
KRAS
is the most frequently activated oncogene in cancer, and the active state of mutant KRAS is such a recalcitrant target. We designed a natural product–inspired small molecule that remodels the surface of cyclophilin A (CYPA) to create a neomorphic interface with high affinity and selectivity for the active state of KRAS
G12C
(in which glycine-12 is mutated to cysteine). The resulting CYPA:drug:KRAS
G12C
tricomplex inactivated oncogenic signaling and led to tumor regressions in multiple human cancer models. This inhibitory strategy can be used to target additional KRAS mutants and other undruggable cancer drivers. Tricomplex inhibitors that selectively target active KRAS
G12C
or multiple RAS mutants are in clinical trials now (NCT05462717 and NCT05379985).
Editor’s summary
KRAS is one of the most common oncogenes, but unfortunately it is also commonly thought of as “undruggable” because it lacks a suitable binding pocket for small-molecule drug candidates. To get around this limitation, Schulze
et al
. built on observations from natural product–derived drugs to go after oncogenic KRAS indirectly (see the Perspective by Liu). The authors identified a naturally occurring compound that binds cyclophilin A, a type of cellular chaperone, and then modified this compound to also bind oncogenic mutant KRAS in a three-way complex. The authors used this approach to design multiple small molecules that effectively bound mutant KRAS in complex with cyclophilin A. These molecules were very effective at inhibiting the downstream pathways involved in cell proliferation and at suppressing tumor growth in multiple models. —Yevgeniya Nusinovich
Small molecules recruit cyclophilin A to the active state of mutant KRAS to disrupt oncogenic signaling and tumor growth.
Abstract RASG12D mutant cancers represent a significant unmet medical need with 55,000 new diagnoses annually in the US. The RASG12D mutation increases the abundance of the active, GTP-bound state of ...RAS (RASG12D(ON)) and occurs commonly in multiple tumor histotypes, including about 20%, 29% and 17% of RAS mutant colorectal, pancreatic, and non-small cell lung cancers, respectively. RMC-6236, an investigational RASMULTI(ON) inhibitor currently in clinical testing, selectively targets the active, GTP-bound state of both mutant and wild-type RAS variants, including RASG12D(ON) and has shown clinical anti-tumor activity against tumors harboring KRASG12D. Efforts to find a mutant-selective RASG12D(ON) inhibitor led to the discovery of RMC-9805.The investigational agent RMC-9805 is a first-in-class, orally bioavailable, mutant selective covalent inhibitor of RASG12D(ON) that forms a tri-complex between the abundant intracellular chaperone cyclophilin A (CypA) and the “ON” state of RASG12D, enabling selective covalent engagement of Asp-12 and disrupting downstream RAS signaling by steric occlusion of effector binding. In contrast to other examples in the literature of covalent inhibitors of KRASG12D that rely on highly reactive, low stability warheads to overcome the low intrinsic reactivity of aspartic acid, RMC-9805 employs a warhead with low intrinsic reactivity and high stability under biological conditions. Rapid, selective, covalent modification of RASG12D by RMC-9805 is enabled by our tri-complex technology. We used structure-guided design to position the warhead in the CypA-RAS interface in an optimal orientation that enhanced crosslinking of Asp-12 allowing us to use a warhead that showed no intrinsic reactivity to a model aspartic acid system and was stable to enable oral dosing. RMC-9805 drove selective and persistent covalent modification of KRASG12D in human cancer cell lines in vitro, leading to deep and durable suppression of RAS pathway activity, inhibition of cell proliferation, and apoptosis. RMC-9805 monotherapy induced tumor regressions at well-tolerated doses in a majority of preclinical PDAC and NSCLC models harboring KRASG12D. Though a more heterogeneous response was observed in KRASG12D CRC models, combinations of RMC-9805 with either RMC-6236 (RASMULTI(ON) inhibitor) or an anti-EGFR antibody improved the depth of response and delayed the onset of resistance in vivo. In addition, RMC-9805 promoted cancer-associated neoantigen recognition and synergized with immunotherapy in preclinical models. RMC-9805 is also CNS penetrant and drove regressions in intracranial xenograft models of human KRASG12D PDAC. RMC-9805 is a first-in-class orally bioavailable, mutant selective and covalent RASG12D inhibitor currently in Phase 1 clinical trial (NCT06040541). Citation Format: John E. Knox, G. Leslie Burnett, Caroline Weller, Lingyan Jiang, Dongyu Zhang, Nicole Vita, Abby Marquez, Kyle J. Seamon, Andrea Gould, Marie Menard, Elsa Quintana, Zhe Chen, Zhican Wang, Zhengping Wang, Elena S. Koltun, Malika Singh, Jingjing Jiang, David Wildes, Jacqueline A.M. Smith, Adrian L. Gill. Discovery of RMC-9805, an oral, covalent tri-complex KRASG12D(ON) inhibitor abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr ND03.
Adaptive intelligence - the ability to sense like a GPS that constantly recalculates and revises for optimum positioning - can be cultivated with specific skill development, a leading priority in ...today's workplace.
Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2006.
Title from electronic title page (viewed Oct. 10, 2007). "... in partial fulfillment of the requirements for the degree of Doctor ...of Philosophy in the Department of Psychology." Discipline: Psychology; Department/School: Psychology.
Dynamic Image Networks for Action Recognition Bilen, Hakan; Fernando, Basura; Gavves, Efstratios ...
2016 IEEE Conference on Computer Vision and Pattern Recognition (CVPR),
06/2016
Conference Proceeding
Odprti dostop
We introduce the concept of dynamic image, a novel compact representation of videos useful for video analysis especially when convolutional neural networks (CNNs) are used. The dynamic image is based ...on the rank pooling concept and is obtained through the parameters of a ranking machine that encodes the temporal evolution of the frames of the video. Dynamic images are obtained by directly applying rank pooling on the raw image pixels of a video producing a single RGB image per video. This idea is simple but powerful as it enables the use of existing CNN models directly on video data with fine-tuning. We present an efficient and effective approximate rank pooling operator, speeding it up orders of magnitude compared to rank pooling. Our new approximate rank pooling CNN layer allows us to generalize dynamic images to dynamic feature maps and we demonstrate the power of our new representations on standard benchmarks in action recognition achieving state-of-the-art performance.
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