Cancer treatment decisions are increasingly guided by which specific genes are mutated within each patient’s tumor. For example, agents inhibiting the epidermal growth factor receptor (EGFR) benefit many colorectal cancer (CRC) patients, with the general exception of those whose tumor includes a KRAS mutation. However, among the various KRAS mutations, that which encodes the G13D mutant protein (KRASG13D) behaves differently; for unknown reasons, KRASG13D CRC patients benefit from the EGFR-blocking antibody cetuximab. Controversy surrounds this observation, because it contradicts the well-established mechanisms of EGFR signaling with regard to RAS mutations. Here, we identified a systems-level, mechanistic explanation for why KRASG13D cancers respond to EGFR inhibition. A computational model of RAS signaling revealed that the biophysical differences between the three most common KRAS mutants were sufficient to generate different sensitivities to EGFR inhibition. Integrated computation with experimentation then revealed a nonintuitive, mutant-specific dependency of wild-type RAS activation by EGFR that is determined by the interaction strength between KRAS and the tumor suppressor neurofibromin (NF1). KRAS mutants that strongly interacted with and competitively inhibited NF1 drove wild-type RAS activation in an EGFR-independent manner, whereas KRASG13D weakly interacted with and could not competitively inhibit NF1 and, thus, KRASG13D cells remained dependent on EGFR for wild-type RAS activity. Overall, our work demonstrates how systems approaches enable mechanism-based inference in genomic medicine and can help identify patients for selective therapeutic strategies.

The structural flexibility found in human immunodeficiency virus (HIV) envelope glycoproteins creates a complex relationship between antigenicity and sensitivity to antiviral antibodies. The study of this issue in the context of viral particles is particularly problematic as conventional virus capture approaches can perturb antigenicity profiles. Here, we employed a unique analytical system based on fluorescence correlation spectroscopy (FCS), which measures antibody-virion binding with all reactants continuously in solution. Panels of nine anti-envelope monoclonal antibodies (MAbs) and five virus types were used to connect antibody binding profiles with neutralizing activities. Anti-gp120 MAbs against the 2G12 or b12 epitope, which marks functional envelope structures, neutralized viruses expressing CCR5-tropic envelopes and exhibited efficient virion binding in solution. MAbs against CD4-induced (CD4i) epitopes considered hidden on functional envelope structures poorly bound these viruses and were not neutralizing. Anti-gp41 MAb 2F5 was neutralizing despite limited virion binding. Similar antigenicity patterns occurred on CXCR4-tropic viruses, except that anti-CD4i MAbs 17b and 19e were neutralizing despite little or no virion binding. Notably, anti-gp120 MAb PG9 and anti-gp41 MAb F240 bound to both CCR5-tropic and CXCR4-tropic viruses without exerting neutralizing activity. Differences in the virus production system altered the binding efficiencies of some antibodies but did not enhance antigenicity of aberrant gp120 structures. Of all viruses tested, only JRFL pseudoviruses showed a direct relationship between MAb binding efficiency and neutralizing potency. Collectively, these data indicate that the antigenic profiles of free HIV particles generally favor the exposure of functional over aberrant gp120 structures. However, the efficiency of virion-antibody interactions in solution inconsistently predicts neutralizing activity in vitro.

The HIV-1 envelope glycoprotein, gp120, undergoes multiple molecular interactions and structural rearrangements during the course of host cell attachment and viral entry, which are being increasingly defined at the atomic level using isolated proteins. In comparison, antigenic markers of these dynamic changes are essentially unknown for single HIV-1 particles bound to target cells. Such markers should indicate how neutralizing and/or non-neutralizing antibodies might interdict infection by either blocking infection or sensitizing host cells for elimination by Fc-mediated effector function. Here we address this deficit by imaging fluorescently labeled CCR5-tropic HIV-1 pseudoviruses using confocal and superresolution microscopy to track the exposure of neutralizing and non-neutralizing epitopes as they appear on single HIV-1 particles bound to target cells. Epitope exposure was followed under conditions permissive or non-permissive for viral entry to delimit changes associated with virion binding from those associated with post-attachment events. We find that a previously unexpected array of gp120 epitopes is exposed rapidly upon target cell binding. This array comprises both neutralizing and non-neutralizing epitopes, the latter being hidden on free virions yet capable of serving as potent targets for Fc-mediated effector function. Under non-permissive conditions for viral entry, both neutralizing and non-neutralizing epitope exposures were relatively static over time for the majority of bound virions. Under entry-permissive conditions, epitope exposure patterns changed over time on subsets of virions that exhibited concurrent variations in virion contents. These studies reveal that bound virions are distinguished by a broad array of both neutralizing and non-neutralizing gp120 epitopes that potentially sensitize a freshly engaged target cell for destruction by Fc-mediated effector function and/or for direct neutralization at a post-binding step. The elucidation of these epitope exposure patterns during viral entry will help clarify antibody-mediated inhibition of HIV-1 as it is measured in vitro and in vivo.

Fluid shear stress (FSS) exerted on endothelial cell surfaces induces actin cytoskeleton remodeling through mechanotransduction. This study was designed to determine whether FSS activates Jun N-terminal kinase (JNK), to examine the spatial and temporal distribution of active JNK relative to the actin cytoskeleton in endothelial cells exposed to different FSS conditions, and to evaluate the effects of active JNK on actin realignment. Exposure to 15 and 20 dyn/cm2 FSS induced higher activity levels of JNK than the lower 2 and 4 dyn/cm2 flow conditions. At the higher FSS treatments, JNK activity increased with increasing exposure time, peaking 30 minutes after flow onset with an 8-fold activity increase compared to cells in static culture. FSS-induced phospho-JNK co-localized with actin filaments at cell peripheries, as well as with stress fibers. Pharmacologically blocking JNK activity altered FSS-induced actin structure and distribution as a response to FSS. Our results indicate that FSS-induced actin remodeling occurs in three phases, and that JNK plays a role in at least one, suggesting that this kinase activity is involved in mechanotransduction from the apical surface to the actin cytoskeleton in endothelial cells.

Necroptosis, an inflammatory form of cell death, is initiated by the activation of receptor-interacting protein kinase 3 (RIPK3), which depends on its interaction with RIPK1. Although catalytically inactive, the RIPK3 mutant D161N still stimulates RIPK1-dependent apoptosis and embryonic lethality in RIPK3 D161N homozygous mice. Whereas the absence of RIPK1 rescues RIPK3 D161N homozygous mice, we report that the absence of RIPK1 leads to embryonic lethality in RIPK3 D161N heterozygous mice. This suggested that the kinase domain of RIPK3 had a noncatalytic function that was enhanced by a conformation induced by the D161N mutation. We found that the RIPK3 kinase domain homodimerized through a surface that is structurally similar to that of the RAF family members. Mutation of residues at the dimer interface impaired dimerization and necroptosis. Kinase domain dimerization stimulated the activation of RIPK3 through cis-autophosphorylation. This noncatalytic, allosteric activity was enhanced by certain kinase-deficient mutants of RIPK3, including D161N. Furthermore, apoptosis induced by certain RIPK3 inhibitors was also dependent on the kinase dimerization interface. Our studies reveal that the RIPK3 kinase domain exhibits catalytically independent function that is important for both RIPK3-dependent necroptosis and apoptosis.