Clinical sequencing identifies potential actionable alterations in a high rate of urachal and primary bladder adenocarcinomas.

OBJECTIVE Administration of targeted therapies provides a promising treatment strategy for urachal adenocarcinoma (UrC) or primary bladder adenocarcinoma (PBAC); however, the selection of appropriate drugs remains difficult. Here, we aimed to establish a routine compatible methodological pipeline for the identification of the most important therapeutic targets and potentially effective drugs for UrC and PBAC. METHODS Next-generation sequencing, using a 161 cancer driver gene panel, was performed on 41 UrC and 13 PBAC samples. Clinically relevant alterations were filtered, and therapeutic interpretation was performed by in silico evaluation of drug-gene interactions. RESULTS After data processing, 45/54 samples passed the quality control. Sequencing analysis revealed 191 pathogenic mutations in 68 genes. The most frequent gain-of-function mutations in UrC were found in KRAS (33%), and MYC (15%), while in PBAC KRAS (25%), MYC (25%), FLT3 (17%) and TERT (17%) were recurrently affected. The most frequently affected pathways were the cell cycle regulation, and the DNA damage control pathway. Actionable mutations with at least one available approved drug were identified in 31/33 (94%) UrC and 8/12 (67%) PBAC patients. CONCLUSIONS In this study, we developed a data-processing pipeline for the detection and therapeutic interpretation of genetic alterations in two rare cancers. Our analyses revealed actionable mutations in a high rate of cases, suggesting that this approach is a potentially feasible strategy for both UrC and PBAC treatments

Abstracts from the 20th International Symposium on Signal Transduction at the Blood-Brain Barriers

https://deepblue.lib.umich.edu/bitstream/2027.42/138963/1/12987_2017_Article_71.pd

A Subdomain Interaction at the Base of the Lever Allosterically Tunes the Mechanochemical Mechanism of Myosin 5a

<div><p>The motor domain of myosin is the core element performing mechanochemical energy transduction. This domain contains the actin and ATP binding sites and the base of the force-transducing lever. Coordinated subdomain movements within the motor are essential in linking the ATPase chemical cycle to translocation along actin filaments. A dynamic subdomain interface located at the base of the lever was previously shown to exert an allosteric influence on ATP hydrolysis in the non-processive myosin 2 motor. By solution kinetic, spectroscopic and ensemble and single-molecule motility experiments, we determined the role of a class-specific adaptation of this interface in the mechanochemical mechanism of myosin 5a, a processive intracellular transporter. We found that the introduction of a myosin 2-specific repulsive interaction into myosin 5a via the I67K mutation perturbs the strong-binding interaction of myosin 5a with actin, influences the mechanism of ATP binding and facilitates ATP hydrolysis. At the same time, the mutation abolishes the actin-induced activation of ADP release and, in turn, slows down processive motility, especially when myosin experiences mechanical drag exerted by the action of multiple motor molecules bound to the same actin filament. The results highlight that subtle structural adaptations of the common structural scaffold of the myosin motor enable specific allosteric tuning of motor activity shaped by widely differing physiological demands.</p></div

ATP hydrolysis and P_i release remain rapid and non-rate-limiting, but actin-activation of ADP release is abolished in I67K-m5aS1.

<p><b><i>A,</i></b> Main panel: Single-turnover ATP hydrolysis profiles obtained on mixing 3.5 µM wt-m5aS1 (solid squares) or I67K-m5aS1 (open squares) with 2 µM γ-³²P-ATP in the quenched-flow apparatus. Double exponential fits to datasets shown yielded <i>k</i>_obs values of 1.1 s⁻¹ (37% fractional amplitude; limited by <i>K</i>₁<i>k</i>₂) and 0.026 s⁻¹ (limited by <i>k</i>₄) for wt-m5aS1, and 2.2 s⁻¹ (71% fractional amplitude) and 0.026 s⁻¹ for I67K-m5aS1. Data for wt-m5aS1 were taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062640#pone.0062640-Nagy1" target="_blank">[17]</a>. Inset: Multiple-turnover ATP hydrolysis profiles obtained on mixing 3 µM wt-m5aS1 (solid squares) or I67K-m5aS1 (open squares) with 30 µM γ-³²P-ATP in the quenched-flow apparatus. In the datasets shown, the rapid exponential burst had <i>k</i>_obs values (limited by <i>K</i>₁<i>k</i>₂) of 35 s⁻¹ and 39 s⁻¹ with amplitudes of 1.2 µM (0.40 mol P_i/mol m5aS1) and 1.7 µM (0.57 mol P_i/mol m5aS1) for wt-m5aS1 and I67K-m5aS1, respectively. The slope of the linear steady-state phase (limited by <i>k</i>₄) was 0.13 s⁻¹ and 0.050 s⁻¹ for wt-m5aS1 and I67K-m5aS1, respectively. <i>K</i>₃ equilibrium constants were calculated from amplitude data as described in the text. <b><i>B,</i></b> Kinetic traces of P_i release (monitored by MDCC-PBP fluorescence) recorded on mixing 0.5 µM wt-m5aS1 (black trace) or I67K-m5aS1 (gray trace) plus 10 µM actin with 100 µM ATP in the stopped-flow apparatus. The wt-m5aS1 trace shown contained an exponential rapid burst with a <i>k</i>_obs of 24 s⁻¹ and an amplitude of 0.44 mol P_i/mol m5aS1, and a linear steady-state phase with a slope of 5.8 s⁻¹. In the I67K-m5aS1 trace shown, the burst had a <i>k</i>_obs of 11 s⁻¹ and an amplitude of 0.74 mol P_i/mol m5aS1, and the steady-state slope was 1.7 s⁻¹. <b><i>C,</i></b> ATP concentration dependence of <i>k</i>_obs (main panel) and amplitudes (inset) of the rapid burst in experiments performed as in <b><i>B</i></b> (solid squares, wt-m5aS1; open squares, I67K-m5aS1). Hyperbolic fits to <i>k</i>_obs datasets yielded maximal rate constants (<i>k</i>_max ≤ <i>k</i>₄’) of 32 and 12 s⁻¹, with half-saturation at 13 and 9.1 µM ATP for wt-m5aS1 and I67K-m5aS1, respectively. Hyperbolic fits to the amplitude datasets yielded maximal amplitudes of 0.99 and 0.90 mol P_i/mol m5aS1 with half-saturation at 140 and 14 µM ATP for wt-m5aS1 and I67K-m5aS1, respectively. <b><i>D,</i></b> ADP release kinetics and ADP affinity of acto-m5aS1 monitored using PA fluorescence. Main panel, ADP concentration dependence of the fractional amplitudes of the slow phase of biphasic PA fluorescence transients recorded on rapidly mixing a premixture of 0.5 µM wt-m5aS1 (solid squares) or I67K-m5aS1 (open squares), 0.7 µM PA and the indicated ADP concentrations with 200 µM ATP in the stopped-flow apparatus (pre-mixing concentrations). In these conditions, the rapid and slow phases represented ATP-induced dissociation of the nucleotide-free and initially ADP-bound acto-m5aS1 fractions, respectively. In the case of the ADP-bound fraction, ADP release (<i>k</i>₅’) limited the <i>k</i>_obs of acto-m5aS1 dissociation. Hyperbolic fits to the datasets yielded <i>K</i>₅’ values of 3.7 and 1.0 µM for wt-m5aS1 and I67K-m5aS1, respectively. Inset: Representative transients obtained at a quasi-saturating ADP concentration (20 µM). The dominant slow phase had <i>k</i>_obs ( = <i>k</i>₅’) values of 14 s⁻¹ and 2.4 s⁻¹ in wt-m5aS1 (black trace) and I67K-m5aS1 (gray trace), respectively.</p

Functional properties of myosin 5a constructs^a.

a<p>Solution kinetic data are for m5aS1 and motility data are for m5aHMM. Nomenclature of kinetic constants refers to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062640#pone-0062640-g001" target="_blank"><b>Fig. 1B</b></a>. All parameters were measured at 25°C. Mean ± SD values for two to four independent experiments are shown.</p>b<p>At 1 mM ATP.</p>c<p>Half-saturating actin concentration (at 1 mM ATP).</p>d<p>Half-saturating ATP concentration (at 10 µM actin).</p>e<p>At 1 mM ATP.</p>f<p>Results of exponential analysis are shown (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062640#pone-0062640-g002" target="_blank"><b>Fig. 2</b></a>; rapid phase data for I67K-m5aS1). See also <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062640#pone.0062640.s001" target="_blank">Fig. S1</a></b> for global fitting results on I67K-m5aS1.</p>g<p>Lower bounds set by maximal rate constant of rapid pre-steady state burst at 30 µM ATP.</p>h<p>From steady-state slopes recorded at 100 µM ATP.</p>i<p>Lower bounds set by maximal rate constant of rapid pre-steady state burst at 10 µM actin.</p>j<p>Maximal amplitude of rapid pre-steady state burst at 10 µM actin.</p>k<p>Rate constants of the rapid (40±3% amplitude) and slow phases of biphasic mdADP release.</p>l<p>From ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062640#pone.0062640-Nagy1" target="_blank">[17]</a>.</p

I67K mutation influences ATP binding to m5aS1 both in the absence and presence of actin.

<p><b><i>A,</i></b> Normalized Trp fluorescence transients recorded on mixing 0.5 µM wt-m5aS1 with 7.5 µM ATP (black trace) or 0.5 µM I67K-m5aS1 with 10 µM ATP (gray trace) in the stopped-flow apparatus. Single exponential fit to the wt-m5aS1 trace shown yielded a <i>k</i>_obs of 7.1 s⁻¹. I67K-m5aS1 traces were biphasic, with <i>k</i>_obs values of 15 s⁻¹ (70% fractional amplitude) and 2.5 s⁻¹ in the example shown. <b><i>B,</i></b> ATP concentration dependence of <i>k</i>_obs values obtained as in <b><i>A</i></b> (solid squares, wt-m5aS1; open squares and circles, I67K-m5aS1 rapid and slow phases, respectively). Hyperbolic fits to wt-m5aS1 and rapid-phase I67K-m5aS1 datasets shown in the main panel yielded maximal rate constants (<i>k</i>₃+ <i>k</i>_–3) of 320 s⁻¹ and 820 s⁻¹, respectively, with an initial slope (<i>K</i>₁<i>k</i>₂) of 1.5 µM⁻¹s⁻¹ in both cases. <b><i>C,</i></b> ATP concentration dependence of rapid-phase (open squares), slow-phase (open circles) and total (solid circles) amplitudes of I67K-m5aS1 transients obtained as in <b><i>A</i></b>. <b><i>D,</i></b> Normalized PA fluorescence transients recorded on mixing 0.35 µM PA plus 0.25 µM wt-m5aS1 (black trace) or I67K-m5aS1 (gray trace) with 50 µM ATP in the stopped-flow apparatus. Single exponential fit to the wt-m5aS1 trace shown yielded a <i>k</i>_obs of 37 s⁻¹. I67K-m5aS1 traces were biphasic, with <i>k</i>_obs values of 71 s⁻¹ (62% fractional amplitude) and 2.3 s⁻¹ in the example shown. <b><i>E,</i></b> ATP concentration dependence of <i>k</i>_obs of transients obtained as in <b><i>D</i></b> (solid squares, wt-m5aS1; open squares and circles, I67K-m5aS1 rapid and slow phases, respectively). Linear fits to wt-m5aS1 and rapid-phase I67K-m5aS1 datasets shown in the main panel yielded slopes (<i>K</i>₁’<i>k</i>₂’) of 0.55 and 0.73 µM⁻¹s⁻¹, respectively. The <i>y</i> intercept was not significantly different from zero in wt-m5aS1, whereas it was 18 s⁻¹ in I67K-m5aS1 in the example shown. <b><i>F,</i></b> ATP concentration dependence of rapid-phase (open squares), slow-phase (open circles) and total (solid circles) amplitudes of I67K-m5aS1 transients obtained as in <b><i>D</i></b>. AU, arbitrary units.</p

I67K mutant exhibits slowed actin-activated ATPase activity and high steady-state actin attachment.

<p><b><i>A,</i></b> Actin (main panel) and ATP (inset) concentration dependence of wt-m5aS1 (solid squares) and I67K-m5aS1 (open squares) steady-state ATPase activity (concentrations used: 100 nM m5aS1 (both panels), 1 mM ATP (main panel), 10 µM actin (inset)). Hyperbolic fits to the datasets shown yielded maximal activities (<i>k</i>_cat) of 8.7 and 2.4 s⁻¹ with half-saturation (<i>K</i>_actin) at 5.6 and 1.8 µM actin (main panel); and 7.1 and 1.3 s⁻¹ with half-saturation (<i>K</i>_ATP) at 12 and 2.8 µM ATP (inset) for wt-m5aS1 and I67K-m5aS1, respectively. <b><i>B,</i></b> Main panel, wt-m5aS1 (solid symbols) and I67K-m5aS1 (open symbols) concentration dependence of fluorescence intensities of 150 nM PA in the absence of nucleotide (rigor, squares) and in 1 mM ATP (circles). Quadratic fits to the rigor datasets (based on an equation described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062640#pone.0062640-Geeves2" target="_blank">[40]</a>) and hyperbolic fits to the ATP datasets shown yielded apparent m5aS1 binding <i>K</i>_d values and m5aS1-saturated PA fluorescence levels (normalized to that in the absence of m5aS1) of less than 0.5 µM and 0.19 (for both wt-m5aS1 and I67K-m5aS1 in rigor); 1.7 µM and 0.23 (wt-m5aS1 in ATP); and 0.88 µM and 0.14 (I67K-m5aS1 in ATP). Inset, actin concentration dependence of the fractional actin attachment of 1 µM wt-m5aS1 (solid squares) or I67K-m5aS1 (open squares) in the presence of 15 mM ATP, determined in acto-m5aS1 cosedimentation experiments. Hyperbolic fits to the datasets indicated half-saturation at 5.5 and less than 2 µM actin for wt-m5aS1 and I67K-m5aS1, respectively.</p