Mark E. Burkard

Predicting oligonucleotide affinity to nucleic acid targets

A computer program, OligoWalk, is reported that predicts the equilibrium affinity of complementary DNA or RNA oligonucleotides to an RNA target. This program considers the predicted stability of the oligonucleotide-target helix and the competition with predicted secondary structure of both the target and the oligonucleotide. Both unimolecular and bimolecular oligonucleotide self structure are considered with a user-defined concentration. The application of OligoWalk is illustrated with three comparisons to experimental results drawn from the literature.

Chemical genetics reveals the requirement for Polo-like kinase 1 activity in positioning RhoA and triggering cytokinesis in human cells.

Polo-like kinases (Plks) play crucial roles in mitosis and cell division. Whereas lower eukaryotes typically contain a single Plk, mammalian cells express several closely related but functionally distinct Plks. We describe here a chemical genetic system in which a single Plk family member, Plk1, can be inactivated with high selectivity and temporal resolution by using an allele-specific, small-molecule inhibitor, as well as the application of this system to dissect Plk1s role in cytokinesis. To do this, we disrupted both copies of the PLK1 locus in human cells through homologous recombination and then reconstituted Plk1 activity by using either the wild-type kinase (Plk1wt) or a mutant version whose catalytic pocket has been enlarged to accommodate bulky purine analogs (Plk1as). When cultured in the presence of these analogs, Plk1as cells accumulate in prometaphase with defects that parallel those found in PLK1Δ/Δ cells. In addition, acute treatment of Plk1as cells during anaphase prevents recruitment of both Plk1 itself and the Rho guanine nucleotide exchange factor (RhoGEF) Ect2 to the central spindle, abolishes RhoA GTPase localization to the equatorial cortex, and suppresses cleavage furrow formation and cell division. Our studies define and illuminate a late mitotic function of Plk1 that, although difficult or impossible to detect in Plk1-depleted cells, is readily revealed with chemical genetics.

Plk1 self-organization and priming phosphorylation of HsCYK-4 at the spindle midzone regulate the onset of division in human cells

Self-regulated movement of Polo-like kinase 1 to the midzone of the mitotic spindle initiates a local signaling cascade that activates the cell division machinery at the cells equator.

Cytotoxicity of Paclitaxel in Breast Cancer Is due to Chromosome Missegregation on Multipolar Spindles

The chemotherapy drug paclitaxel causes tumor regression and cell death by inducing high rates of chromosome missegregation on multipolar spindles. The Secret Life of Paclitaxel The classic chemotherapy drug paclitaxel is a standard part of treatment for breast cancer and other malignancies. Although it is commonly understood to act as a microtubule poison and lead to mitotic arrest, this knowledge is largely based on studies of cells in culture, with drug concentrations that may not be realistic. Now, Zasadil and coauthors measured the concentration of paclitaxel in real patients undergoing treatment with the drug, and then investigated the response of cancer cells to paclitaxel at these lower and more realistic concentrations. Unexpectedly, the cells treated under these conditions did not undergo mitotic arrest, but instead proceeded through mitosis with abnormal spindles, resulting in chromosome missegregation, which leads to tumor cell death. This intriguing discovery demonstrates that we may not know as much as we thought about the effects of one of our most common chemotherapy drugs. In addition, the findings from this study may lead to clinical applications both in optimizing the selection of chemotherapy drug combinations and in determining which patients are likely to respond to paclitaxel treatment. The blockbuster chemotherapy drug paclitaxel is widely presumed to cause cell death in tumors as a consequence of mitotic arrest, as it does at concentrations routinely used in cell culture. However, we determine here that paclitaxel levels in primary breast tumors are well below those required to elicit sustained mitotic arrest. Instead, cells in these lower concentrations of drug proceed through mitosis without substantial delay and divide their chromosomes on multipolar spindles, resulting in chromosome missegregation and cell death. Consistent with these cell culture data, most mitotic cells in primary human breast cancers contain multipolar spindles after paclitaxel treatment. Contrary to the previous hypothesis, we find that mitotic arrest is dispensable for tumor regression in patients. These results demonstrate that mitotic arrest is not responsible for the efficacy of paclitaxel, which occurs because of chromosome missegregation on highly abnormal, multipolar spindles. This mechanistic insight may be used to improve selection of future antimitotic drugs and to identify a biomarker with which to select patients likely to benefit from paclitaxel.

Simulation of exchanges of multiple gases in bubbles in the body

This communication introduces a system of equations, for numerical solution, which simulates the generation, growth, and decay of bubbles. The system is an advance over previous works because it allows for simultaneous diffusion of any number of gases. Our purpose for developing the system is to gain insight into the bubbles that occur in the body in decompression sickness (DCS). We validate the calculation system by matching observed data of DCS bubbles and of large subcutaneous gas pockets in rats. We demonstrate how a temporary supersaturation and bubble formation can occur without change of ambient pressure when there is a change in the inert gas being breathed. With exposures to hypobaric environments, such as when astronauts work in space, simulations show that O2, CO2, and water vapor add appreciably to volume of bubbles and affect the diffusion of inert gas.

Behavior of bubbles of slowly permeating gas used for ultrasonic imaging contrast.

RATIONALE AND OBJECTIVES.The authors predict behavior of blood cell-sized bubbles containing a foreign gas that slowly crosses a gas-liquid interface. Such bubbles are being developed for ultrasonic contrast. METHODS.Using appropriate coefficients for N2, O2, CO2, and foreign gases, the authors simulate diffusion between bubbles and blood with a numerically solved equation system. RESULTS.Within 30 seconds after intravenous injection of bubbles, entrance of endogenous gases more than doubles the radius. In vivo size and duration of the bubbles are quantitatively related to the amount of the foreign gas inside. Size and ultrasonic imaging effectiveness of the circulating bubbles become larger in lungs than in the rest of the circulation. As the foreign gas is lost, imaging effectiveness diminishes more rapidly than bubble radius. CONCLUSIONS.Bubbles of slowly permeating gas change size and composition after injection and also during their passage through different parts of the circulation.

The energetics of small internal loops in RNA

The energetics of small internal loops are important for prediction of RNA secondary and tertiary structure, selection of drug target sites, and understanding RNA structure–function relationships. Hydrogen bonding, base stacking, electrostatic interactions, backbone distortion, and base‐pair size compatibility all contribute to the energetics of small internal loops. Thus, the sequence dependence of these energetics are idiosyncratic. Current approximations for predicting the free energies of internal loops consider size, asymmetry, closing base pairs, and the potential to form GA, GG, or UU pairs. The database of known three‐dimensional structures allows for comparison with the models used for predicting stability from sequence.

High mitotic activity of Polo-like Kinase 1 is required for chromosome segregation and genomic integrity in human epithelial cells

Background: Polo-like kinase 1 (Plk1) has multiple functions and substrates in human mitosis. Results: Plk1 functions are separable via distinct thresholds of activity, and partial functional loss leads to chromosome missegregation. Conclusion: Plk1 has several distinct mitotic roles that are separable by chemical genetics. Significance: It is possible to dissect individual functions of a multifunctional enzyme using activity thresholds. Protein kinases play key roles in regulating human cell biology, but manifold substrates and functions make it difficult to understand mechanism. We tested whether we could dissect functions of a pleiotropic mitotic kinase, Polo-like kinase 1 (Plk1), via distinct thresholds of kinase activity. We accomplished this by titrating Plk1 activity in RPE1 human epithelial cells using chemical genetics and verifying results in additional lines. We found that distinct activity thresholds are required for known functions of Plk1 including (from low to high activity) bipolar spindle formation, timely mitotic entry, and formation of a cytokinesis cleavage furrow. Subtle losses in Plk1 activity impaired chromosome congression and produced severe anaphase dysfunction characterized by poor separation of chromosome masses. These two phenotypes were separable, suggesting that they stem from distinct phosphorylation events. Impaired chromosome segregation in anaphase was the most sensitive to modest loss in Plk1 activity. Mechanistically, it was associated with unpaired sister chromatids with stretched kinetochores, suggestive of merotelic attachments. The C-terminal Polo box domain of Plk1 was required for its anaphase function, although it was dispensable for forming a bipolar spindle. The ultimate effect of partial inhibition of Plk1 was the formation of micronuclei, an increase in tetraploid progeny, and senescence. These results demonstrate that different thresholds of Plk1 activity can elicit distinct phenotypes, illustrating a general method for separating pleiotropic functions of a protein kinase even when these are executed close in time.

Enabling and disabling polo-like kinase 1 inhibition through chemical genetics.

Polo-like kinase 1 (Plk1) is a core regulator of cell division and an emerging target for cancer therapy. Pharmacologic inhibitors of Plk1 exist but affect other kinases, complicating their in vivo validation. To address this, we examined effects of two structurally unrelated Plk1 inhibitors (BI-2536 and TAL) against isogenic human cell lines that solely express wildtype (wt) or analogue-sensitive (as) Plk1 alleles. Unexpectedly, Plk1(as) cells displayed profound biochemical and functional resistance to both inhibitors. Cells that co-express Plk1(wt) and Plk1(as) exhibit loss-of-function phenotypes only when both kinase alleles are inhibited. Resistance to BI-2536 is linked to an intragenic suppressor mutation (C67V) that restores an otherwise invariant valine to the kinase active site. Structural modeling demonstrates that this mutation not only enables Plk1(as) to function in vivo but also occludes BI-2536 from the ATP-binding pocket. Our results reveal the molecular basis of Plk inhibitor selectivity and a potential mechanism for tumor cell resistance.

Phase I Study of an AKT Inhibitor (MK-2206) Combined with Lapatinib in Adult Solid Tumors Followed by Dose Expansion in Advanced HER2+ Breast Cancer

Purpose: Preclinical data support combining AKT inhibitors with HER2-targeted therapies to overcome resistance to treatment. This phase I study combined the investigational AKT inhibitor, MK-2206, with lapatinib to determine the MTD. Experimental Design: The dose escalation cohort enrolled adults with advanced solid tumors, who received MK-2206 dosed 30 to 60 mg every other day and lapatinib 1,000 to 1,500 mg daily continuously, escalated using a 3+3 design. Cycles were 28 days except cycle 1 (35 days, including an initial 8 days of MK-2206 alone to evaluate pharmacokinetic interactions). The dose expansion cohort enrolled adults with advanced HER2+ breast cancer. Results: Twenty-three participants enrolled in the dose escalation cohort. Dose-limiting toxicities were hyponatremia, fatigue, rash, hypocalcemia, and mucositis. Common toxicities included diarrhea, nausea, and rash. The MTD was reached at MK-2206 45 mg orally every other day and lapatinib 1,500 mg orally daily. Two participants maintained stable disease for >4 months, including a colorectal cancer participant with substantial carcinoembryonic antigen decrease. Of 5 participants in the dose expansion cohort, 2 maintained stable disease for >6 months, including one with prior progression on single-agent lapatinib. Plasma MK-2206 concentrations decreased after addition of lapatinib, but in vitro studies indicate lapatinib increases the intracellular levels of MK-2206. Conclusions: MK-2206 combined with lapatinib can be tolerated with both drugs above biologically active single-agent doses. Overlapping toxicities result in significant diarrhea and rash, which can be managed medically. Antitumor activity was promising and supports evaluation of AKT inhibitors combined with HER2-targeted therapies. Clin Cancer Res; 22(11); 2659–67. ©2016 AACR.