Christopher Wilson

Manipulation of Hyaluronan Synthase Expression in Prostate Adenocarcinoma Cells Alters Pericellular Matrix Retention and Adhesion to Bone Marrow Endothelial Cells

Prostate cancer metastasis to bone marrow involves initial adhesion of tumor cells to the bone marrow endothelium, followed by transmigration and proliferation within the marrow. Rapid, specific adhesion of highly metastatic prostate adenocarcinoma cells (PC3M-LN4) to bone marrow endothelial cell (BMEC) lines requires a pericellular hyaluronan (HA) matrix and correlates with dramatically up-regulated HA synthase (HAS) expression. Non-metastatic prostate tumor cells (LNCaP) do not assemble a HA matrix, adhere poorly to BMECs, and express normal levels of HAS. Preferential bone metastasis of prostate carcinoma cells may therefore be facilitated by tumor cell HA biosynthesis. In this report, HAS gene expression was manipulated to investigate the direct impact of prostate tumor cell HA production on adhesion to BMECs. PC3M-LN4 cells stably transfected with antisense HAS2 and HAS3 failed to form pericellular matrices. Adhesion of these transfectants to BMECs was significantly diminished, comparable to the low level exhibited by LNCaP cells. Upon transfection with full-length HAS2 or HAS3, the non-adherent LNCaP cells retained pericellular HA and adhered to BMECs. The results of this study are consistent with a model in which HA matrix formation, BMEC adhesion, and metastatic potential are mediated by HAS expression.

Using ancient protein kinases to unravel a modern cancer drug’s mechanism

Evolution of dynamics affects function The drug Gleevac inhibits Abl kinases and is used to treat multiple cancers. The closely related Src kinases also play a role in cancer but are not inhibited effectively by Gleevac. Nevertheless, Gleevac-bound structures of Src and Abl are nearly identical. Based on this structural information and protein sequence data, Wilson et al. reconstructed the common ancestor of Src and Abl. Mutations that affected conformational dynamics caused Gleevac affinity to be gained on the evolutionary trajectory toward Abl and lost on the trajectory toward Src. Science, this issue p. 882 Characterization of ancestors of the kinases Src and Abl reveals why they respond differently to the cancer drug Gleevec. Macromolecular function is rooted in energy landscapes, where sequence determines not a single structure but an ensemble of conformations. Hence, evolution modifies a protein’s function by altering its energy landscape. Here, we recreate the evolutionary pathway between two modern human oncogenes, Src and Abl, by reconstructing their common ancestors. Our evolutionary reconstruction combined with x-ray structures of the common ancestor and pre–steady-state kinetics reveals a detailed atomistic mechanism for selectivity of the successful cancer drug Gleevec. Gleevec affinity is gained during the evolutionary trajectory toward Abl and lost toward Src, primarily by shifting an induced-fit equilibrium that is also disrupted in the clinical T315I resistance mutation. This work reveals the mechanism of Gleevec specificity while offering insights into how energy landscapes evolve.

Energetic dissection of Gleevec's selectivity toward human tyrosine kinases

Protein kinases are obvious drug targets against cancer, owing to their central role in cellular regulation. Since the discovery of Gleevec, a potent and specific inhibitor of Abl kinase, as a highly successful cancer therapeutic, the ability of this drug to distinguish between Abl and other tyrosine kinases such as Src has been intensely investigated but without much success. Using NMR and fast kinetics, we establish a new model that solves this longstanding question of how the two tyrosine kinases adopt almost identical structures when bound to Gleevec but have vastly different affinities. We show that, in contrast to all other proposed models, the origin of Abls high affinity lies predominantly in a conformational change after binding. An energy landscape providing tight affinity via an induced fit and binding plasticity via a conformational-selection mechanism is likely to be general for many inhibitors.

Evolutionary drivers of thermoadaptation in enzyme catalysis

With early life likely to have existed in a hot environment, enzymes had to cope with an inherent drop in catalytic speed caused by lowered temperature. Here we characterize the molecular mechanisms underlying thermoadaptation of enzyme catalysis in adenylate kinase using ancestral sequence reconstruction spanning 3 billion years of evolution. We show that evolution solved the enzyme’s key kinetic obstacle—how to maintain catalytic speed on a cooler Earth—by exploiting transition-state heat capacity. Tracing the evolution of enzyme activity and stability from the hot-start toward modern hyperthermophilic, mesophilic, and psychrophilic organisms illustrates active pressure versus passive drift in evolution on a molecular level, refutes the debated activity/stability trade-off, and suggests that the catalytic speed of adenylate kinase is an evolutionary driver for organismal fitness.

Effect of Changes in the Flexible Arm on tRNase Z Processing Kinetics

tRNAs are transcribed as precursors and processed in a series of reactions culminating in aminoacylation and translation. Central to tRNA maturation, the 3′ end trailer can be endonucleolytically removed by tRNase Z. A flexible arm (FA) extruded from the body of tRNase Z consists of a structured ααββ hand that binds the elbow of pre-tRNA. Deleting the FA hand causes an almost 100-fold increase in Km with little change in kcat, establishing its contribution to substrate recognition/binding. Remarkably, a 40-residue Ala scan through the FA hand reveals a conserved leucine at the ascending stalk/hand boundary that causes practically the same increase in Km as the hand deletion, thus nearly eliminating its ability to bind substrate. Km also increases with substitutions in the GP (α4–α5) loop and at other conserved residues in the FA hand predicted to contact substrate based on the co-crystal structure. Substitutions that reduce kcat are clustered in the β10–β11 loop.

Identification of mixed lineage leukemia 1(MLL1) protein as a coactivator of heat shock factor 1(HSF1) protein in response to heat shock protein 90 (HSP90) inhibition.

Background: The efficacy of HSP90 inhibitors may be limited by HSF1-mediated feedback mechanisms. Results: MLL1 regulates HSF1-target genes upon HSP90 inhibition, and MLL1 depletion shows a striking combination effect in human cancer. Conclusion: MLL1 functions as a coactivator of HSF1 upon HSP90 inhibition. Significance: This is the first report of MLL1 as a coactivator of HSF1 upon HSP90 inhibition. Heat shock protein 90 (HSP90) inhibition inhibits cancer cell proliferation through depleting client oncoproteins and shutting down multiple oncogenic pathways. Therefore, it is an attractive strategy for targeting human cancers. Several HSP90 inhibitors, including AUY922 and STA9090, show promising effects in clinical trials. However, the efficacy of HSP90 inhibitors may be limited by heat shock factor 1 (HSF1)-mediated feedback mechanisms. Here, we identify, through an siRNA screen, that the histone H3 lysine 4 methyltransferase MLL1 functions as a coactivator of HSF1 in response to HSP90 inhibition. MLL1 is recruited to the promoters of HSF1 target genes and regulates their expression in response to HSP90 inhibition. In addition, a striking combination effect is observed when MLL1 depletion is combined with HSP90 inhibition in various human cancer cell lines and tumor models. Thus, targeting MLL1 may block a HSF1-mediated feedback mechanism induced by HSP90 inhibition and provide a new avenue to enhance HSP90 inhibitor activity in human cancers.

Evolution and intelligent design in drug development

Sophisticated protein kinase networks, empowering complexity in higher organisms, are also drivers of devastating diseases such as cancer. Accordingly, these enzymes have become major drug targets of the twenty-first century. However, the holy grail of designing specific kinase inhibitors aimed at specific cancers has not been found. Can new approaches in cancer drug design help win the battle with this multi-faced and quickly evolving enemy? In this perspective we discuss new strategies and ideas that were born out of a recent breakthrough in understanding the molecular basis underlying the clinical success of the cancer drug Gleevec. An “old” method, stopped-flow kinetics, combined with old enzymes, the ancestors dating back up to about billion years, provides an unexpected outlook for future intelligent design of drugs.

Drug targets evolve, and so should the methods

ABSTRACT Design of specific kinase inhibitors is an appealing approach for developing new anticancer treatments. However, only a few success stories have been reported to date. Here we demonstrate how the combination of old-fashioned and new biophysical tools together with recent advances in genomics and molecular evolution can aid in overcoming existing limitations.