Unmasking determinants of specificity in the human kinome.

Protein kinases control cellular responses to environmental cues by swift and accurate signal processing. Breakdowns in this high-fidelity capability are a driving force in cancer and other diseases. Thus, our limited understanding of which amino acids in the kinase domain encode substrate specificity, the so-called determinants of specificity (DoS), constitutes a major obstacle in cancer signaling. Here, we systematically discover several DoS and experimentally validate three of them, named the αC1, αC3, and APE-7 residues. We demonstrate that DoS form sparse networks of non-conserved residues spanning distant regions. Our results reveal a likely role for inter-residue allostery in specificity and an evolutionary decoupling of kinase activity and specificity, which appear loaded on independent groups of residues. Finally, we uncover similar properties driving SH2 domain specificity and demonstrate how the identification of DoS can be utilized to elucidate a greater understanding of the role of signaling networks in cancer (Creixell et al., 2015 [this issue of Cell]).

The Application of Nanotechnology for Quantification of Circulating Tumour DNA in Liquid Biopsies: A Systematic Review.

Technologies for quantifying circulating tumour DNA (ctDNA) in liquid biopsies could enable real-time measurements of cancer progression, profoundly impacting patient care. Sequencing methods can be too complex and time-consuming for regular point-of-care monitoring, but nanotechnology offers an alternative, harnessing the unique properties of objects tens to hundreds of nanometres in size. This systematic review was performed to identify all examples of nanotechnology-based ctDNA detection and assess their potential for clinical use. Google Scholar, PubMed, Web of Science, Google Patents, Espacenet and Embase/MEDLINE were searched up to 23rd March 2021. The review identified nanotechnology-based methods for ctDNA detection for which quantitative measures (e.g., limit of detection, LOD) were reported and biologically relevant samples were used. The pre-defined inclusion criteria were met by 66 records. LODs ranged from 10 zM to 50nM. 25 records presented an LOD of 10fM or below. Nanotechnology-based approaches could provide the basis for the next wave of advances in ctDNA diagnostics, enabling analysis at the point-of-care, but none are currently used clinically. Further work is needed in development and validation; trade-offs are expected between different performance measures e.g., number of sequences detected and time to result.

Quantitative Systems Pharmacology Modeling of Avadomide-Induced Neutropenia Enables Virtual Clinical Dose and Schedule Finding Studies.

Avadomide is a cereblon E3 ligase modulator and a potent antitumor and immunomodulatory agent. Avadomide trials are challenged by neutropenia as a major adverse event and a dose-limiting toxicity. Intermittent dosing schedules supported by preclinical data provide a strategy to reduce frequency and severity of neutropenia; however, the identification of optimal dosing schedules remains a clinical challenge. Quantitative systems pharmacology (QSP) modeling offers opportunities for virtual screening of efficacy and toxicity levels produced by alternative dose and schedule regimens, thereby supporting decision-making in translational drug development. We formulated a QSP model to capture the mechanism of avadomide-induced neutropenia, which involves cereblon-mediated degradation of transcription factor Ikaros, resulting in a maturation block of the neutrophil lineage. The neutropenia model was integrated with avadomide-specific pharmacokinetic and pharmacodynamic models to capture dose-dependent effects. Additionally, we generated a disease-specific virtual patient population to represent the variability in patient characteristics and response to treatment observed for a diffuse large B-cell lymphoma trial cohort. Model utility was demonstrated by simulating the avadomide effect in the virtual population for various dosing schedules and determining the incidence of high-grade neutropenia, its duration, and the probability of recovery to low-grade neutropenia.

Global view of the RAF-MEK-ERK module and its immediate downstream effectors.

Small molecule inhibitors of BRAF and MEK have proven effective at inhibiting tumor growth in melanoma patients, however this efficacy is limited due to the almost universal development of drug resistance. To provide advanced insight into the signaling responses that occur following kinase inhibition we have performed quantitative (phospho)-proteomics of human melanoma cells treated with either dabrafenib, a BRAF inhibitor; trametinib, a MEK inhibitor or SCH772984, an ERK inhibitor. Over nine experiments we identified 7827 class I phosphorylation sites on 4960 proteins. This included 54 phosphorylation sites that were significantly down-modulated after exposure to all three inhibitors, 34 of which have not been previously reported. Functional analysis of these novel ERK targets identified roles for them in GTPase activity and regulation, apoptosis and cell-cell adhesion. Comparison of the results presented here with previously reported phosphorylation sites downstream of ERK showed a limited degree of overlap suggesting that ERK signaling responses may be highly cell line and cue specific. In addition we identified 26 phosphorylation sites that were only responsive to dabrafenib. We provide further orthogonal experimental evidence for 3 of these sites in human embryonic kidney cells over-expressing BRAF as well as further computational insights using KinomeXplorer. The validated phosphorylation sites were found to be involved in actin regulation, which has been proposed as a novel mechanism for inhibiting resistance development. These results would suggest that the linearity of the BRAF-MEK-ERK module is at least context dependent.