A saturation-mutagenesis analysis of the interplay between stability and activation in Ras.

Cancer mutations in Ras occur predominantly at three hotspots: Gly 12, Gly 13, and Gln 61. Previously, we reported that deep mutagenesis of H-Ras using a bacterial assay identified many other activating mutations (Bandaru et al., 2017). We now show that the results of saturation mutagenesis of H-Ras in mammalian Ba/F3 cells correlate well with the results of bacterial experiments in which H-Ras or K-Ras are co-expressed with a GTPase-activating protein (GAP). The prominent cancer hotspots are not dominant in the Ba/F3 data. We used the bacterial system to mutagenize Ras constructs of different stabilities and discovered a feature that distinguishes the cancer hotspots. While mutations at the cancer hotspots activate Ras regardless of construct stability, mutations at lower-frequency sites (e.g. at Val 14 or Asp 119) can be activating or deleterious, depending on the stability of the Ras construct. We characterized the dynamics of three non-hotspot activating Ras mutants by using NMR to monitor hydrogen-deuterium exchange (HDX). These mutations result in global increases in HDX rates, consistent with destabilization of Ras. An explanation for these observations is that mutations that destabilize Ras increase nucleotide dissociation rates, enabling activation by spontaneous nucleotide exchange. A further stability decrease can lead to insufficient levels of folded Ras - and subsequent loss of function. In contrast, the cancer hotspot mutations are mechanism-based activators of Ras that interfere directly with the action of GAPs. Our results demonstrate the importance of GAP surveillance and protein stability in determining the sensitivity of Ras to mutational activation.

Association of Radiation Therapy With Risk of Adverse Events in Patients Receiving Immunotherapy: A Pooled Analysis of Trials in the US Food and Drug Administration Database.

Importance: Immune checkpoint inhibitors (ICIs) and radiation therapy (RT) are widely used to treat various cancers, but little data are available to guide clinicians on ICI use sequentially with RT. Objective: To assess whether there is an increased risk of serious adverse events (AEs) associated with RT given within 90 days prior to an ICI. Design, Setting, and Participants: Individual patient data were pooled from 68 prospective trials of ICIs submitted in initial or supplemental licensing applications in the US Food and Drug Administration (FDA) databases through December 2019. Two cohorts were generated: (1) patients who received RT within the 90 days prior to beginning ICI therapy and (2) those who did not receive RT within the 90 days prior to beginning ICI therapy, and AE frequencies were determined. A 1:1 propensity score-matched analysis was performed. Interventions: All patients received an ICI (atezolizumab, avelumab, cemiplimab, durvalumab, ipilimumab, nivolumab, or pembrolizumab); 1733 received RT within the 90 days prior to starting ICI therapy, and 13 956 did not. Main Outcomes and Measures: The primary outcome was frequency and severity of AEs. Incidence of AEs was compared descriptively between participants who did vs did not receive RT in the propensity score-matched set. Because all analyses are exploratory (ie, not preplanned and no alpha allocated), assessment for statistical significance of the differences between groups was not considered appropriate. Results: A total of 25 469 patients were identified; 8634 were excluded because they lacked comparators who had received RT (n = 976), did not receive an ICI (n = 4949), received RT outside of the target window (n = 2338), or had missing data in 1 or more variables used in the propensity analysis (n = 371), leaving 16 835 patients included in the analysis. The majority were younger than 65 years (9447 [56.1%]), male (10 459 [62.1%]), and White (13 422 [79.7%]). Patients receiving RT had generally similar rates of AEs overall to those patients who did not receive RT. The average absolute difference in rates across the AEs was 1.2%, and the difference ranged from 0% for neurologic AEs to 8% for fatigue. No difference in grade 3 to 4 AEs was observed between the 2 groups (absolute difference ranged from 0.01% to 2%). These findings persisted after propensity score matching. Conclusions and Relevance: In this pooled analysis, administration of an ICI within 90 days following RT did not appear to be associated with an increased risk of serious AEs. Thus, it would appear to be safe to administer an ICI within 90 days of receiving RT. These findings should be confirmed in future prospective trials.

The Interdependent Activation of Son-of-Sevenless and Ras.

The guanine-nucleotide exchange factor (GEF) Son-of-Sevenless (SOS) plays a critical role in metazoan signaling by converting Ras•GDP (guanosine diphosphate) to Ras•GTP (guanosine triphosphate) in response to tyrosine kinase activation. Structural studies have shown that SOS differs from other Ras-specific GEFs in that SOS is itself activated by Ras•GTP binding to an allosteric site, distal to the site of nucleotide exchange. The activation of SOS involves membrane recruitment and conformational changes, triggered by lipid binding, that open the allosteric binding site for Ras•GTP. This is in contrast to other Ras-specific GEFs, which are activated by second messengers that more directly affect the active site. Allosteric Ras•GTP binding stabilizes SOS at the membrane, where it can turn over other Ras molecules processively, leading to an ultrasensitive response that is distinct from that of other Ras-specific GEFs.

Deconstruction of the Ras switching cycle through saturation mutagenesis.

Ras proteins are highly conserved signaling molecules that exhibit regulated, nucleotide-dependent switching between active and inactive states. The high conservation of Ras requires mechanistic explanation, especially given the general mutational tolerance of proteins. Here, we use deep mutational scanning, biochemical analysis and molecular simulations to understand constraints on Ras sequence. Ras exhibits global sensitivity to mutation when regulated by a GTPase activating protein and a nucleotide exchange factor. Removing the regulators shifts the distribution of mutational effects to be largely neutral, and reveals hotspots of activating mutations in residues that restrain Ras dynamics and promote the inactive state. Evolutionary analysis, combined with structural and mutational data, argue that Ras has co-evolved with its regulators in the vertebrate lineage. Overall, our results show that sequence conservation in Ras depends strongly on the biochemical network in which it operates, providing a framework for understanding the origin of global selection pressures on proteins.

Identification of mRNAs bound and regulated by human LIN28 proteins and molecular requirements for RNA recognition.

Human LIN28A and LIN28B are RNA-binding proteins (RBPs) conserved in animals with important roles during development and stem cell reprogramming. We used Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) in HEK293 cells and identified a largely overlapping set of ∼3000 mRNAs at ∼9500 sites located in the 3' UTR and CDS. In vitro and in vivo, LIN28 preferentially bound single-stranded RNA containing a uridine-rich element and one or more flanking guanosines and appeared to be able to disrupt base-pairing to access these elements when embedded in predicted secondary structure. In HEK293 cells, LIN28 protein binding mildly stabilized target mRNAs and increased protein abundance. The top targets were its own mRNAs and those of other RBPs and cell cycle regulators. Alteration of LIN28 protein levels also negatively regulated the abundance of some but not all let-7 miRNA family members, indicating sequence-specific binding of let-7 precursors to LIN28 proteins and competition with cytoplasmic miRNA biogenesis factors.

FMRP targets distinct mRNA sequence elements to regulate protein expression.

Fragile X syndrome (FXS) is a multi-organ disease that leads to mental retardation, macro-orchidism in males and premature ovarian insufficiency in female carriers. FXS is also a prominent monogenic disease associated with autism spectrum disorders (ASDs). FXS is typically caused by the loss of fragile X mental retardation 1 (FMR1) expression, which codes for the RNA-binding protein FMRP. Here we report the discovery of distinct RNA-recognition elements that correspond to the two independent RNA-binding domains of FMRP, in addition to the binding sites within the messenger RNA targets for wild-type and I304N mutant FMRP isoforms and the FMRP paralogues FXR1P and FXR2P (also known as FXR1 and FXR2). RNA-recognition-element frequency, ratio and distribution determine target mRNA association with FMRP. Among highly enriched targets, we identify many genes involved in ASD and show that FMRP affects their protein levels in human cell culture, mouse ovaries and human brain. Notably, we discovered that these targets are also dysregulated in Fmr1(-/-) mouse ovaries showing signs of premature follicular overdevelopment. These results indicate that FMRP targets share signalling pathways across different cellular contexts. As the importance of signalling pathways in both FXS and ASD is becoming increasingly apparent, our results provide a ranked list of genes as basis for the pursuit of new therapeutic targets for these neurological disorders.

Mass conservation and inference of metabolic networks from high-throughput mass spectrometry data.

We present a step towards the metabolome-wide computational inference of cellular metabolic reaction networks from metabolic profiling data, such as mass spectrometry. The reconstruction is based on identification of irreducible statistical interactions among the metabolite activities using the ARACNE reverse-engineering algorithm and on constraining possible metabolic transformations to satisfy the conservation of mass. The resulting algorithms are validated on synthetic data from an abridged computational model of Escherichia coli metabolism. Precision rates upwards of 50% are routinely observed for identification of full metabolic reactions, and recalls upwards of 20% are also seen.

A human B-cell interactome identifies MYB and FOXM1 as master regulators of proliferation in germinal centers.

Assembly of a transcriptional and post-translational molecular interaction network in B cells, the human B-cell interactome (HBCI), reveals a hierarchical, transcriptional control module, where MYB and FOXM1 act as synergistic master regulators of proliferation in the germinal center (GC). Eighty percent of genes jointly regulated by these transcription factors are activated in the GC, including those encoding proteins in a complex regulating DNA pre-replication, replication, and mitosis. These results indicate that the HBCI analysis can be used for the identification of determinants of major human cell phenotypes and provides a paradigm of general applicability to normal and pathologic tissues.