Wee1 Inhibition by MK-1775 Leads to Tumor Inhibition and Enhances Efficacy of Gemcitabine in Human Sarcomas

Sarcomas are rare and heterogeneous mesenchymal tumors affecting both pediatric and adult populations with more than 70 recognized histologies. Doxorubicin and ifosfamide have been the main course of therapy for treatment of sarcomas; however, the response rate to these therapies is about 10–20% in metastatic setting. Toxicity with the drug combination is high, response rates remain low, and improvement in overall survival, especially in the metastatic disease, remains negligible and new agents are needed. Wee1 is a critical component of the G2/M cell cycle checkpoint control and mediates cell cycle arrest by regulating the phosphorylation of CDC2. Inhibition of Wee1 by MK1775 has been reported to enhance the cytotoxic effect of DNA damaging agents in different types of carcinomas. In this study we investigated the therapeutic efficacy of MK1775 in various sarcoma cell lines, patient-derived tumor explants ex vivo and in vivo both alone and in combination with gemcitabine, which is frequently used in the treatment of sarcomas. Our data demonstrate that MK1775 treatment as a single agent at clinically relevant concentrations leads to unscheduled entry into mitosis and initiation of apoptotic cell death in all sarcomas tested. Additionally, MK1775 significantly enhances the cytotoxic effect of gemcitabine in sarcoma cells lines with different p53 mutational status. In patient-derived bone and soft tissue sarcoma samples we showed that MK1775 alone and in combination with gemcitabine causes significant apoptotic cell death. Magnetic resonance imaging (MRI) and histopathologic studies showed that MK1775 induces significant cell death and terminal differentiation in a patient-derived xenograft mouse model of osteosarcoma in vivo. Our results together with the high safety profile of MK1775 strongly suggest that this drug can be used as a potential therapeutic agent in the treatment of both adult as well as pediatric sarcoma patients.     

Facile measurement of protein stability and folding kinetics using a nano differential scanning fluorimeter

With advancements in high‐throughput generation of phenotypic data on mutant proteins, it has become important to individually characterize different proteins or their variants rapidly and with minimal sample consumption. We have made use of a nano differential scanning fluorimetric device, from NanoTemper technologies, to rapidly carry out isothermal chemical denaturation and measure folding/unfolding kinetics of proteins and compared these to corresponding data obtained from conventional spectrofluorimetry. We show that using sample volumes 10‐50‐fold lower than with conventional fluorimetric techniques, one can rapidly and accurately measure thermodynamic and kinetic stability, as well as folding/unfolding kinetics. This method also facilitates characterization of proteins that are difficult to express and purify.     

The structure-specific nicking of small heteroduplexes by the RAG complex: implications for lymphoid chromosomal translocations

During V(D)J recombination, the RAG complex binds at recombination signal sequences and creates double-strand breaks. In addition to this sequence-specific recognition of the RSS, the RAG complex has been shown to be a structure-specific nuclease, cleaving 3' overhangs and 3' flaps, and, more recently, 10 nucleotides (nt) bubble (heteroduplex) structures. Here, we assess the smallest size heteroduplex that core and full-length RAGs can cleave. We also test whether bubbles adjacent to a partial RSS are nicked any differently or any more efficiently than bubbles that are surrounded by random sequence. These points are important in considering what types and what size of non-B DNA structure that the RAG complex can nick, and this helps assess the role of the RAG complex in mediating lymphoid chromosomal translocations. We find that the smallest bubble nicked by the RAG complex is 3nt, and proximity to a partial or full RSS sequence does not affect the nicking by RAGs. RAG nicking efficiency increases with the size of the heteroduplex and is only about two-fold less efficient than an RSS when the bubble is 6nt. We consider these findings in the context of RAG nicking at non-B DNA structures in lymphoid chromosomal translocations.     

Combinatorial library of indinavir analogues: replacement for the aminoindanol at P2'

A 1X22X41 combinatorial library or 902 compounds of indinavir analogues was synthesized on the solid support to identify a replacement for the aminoindanol moiety at P2'. 2,6-Dimethyl-4-hydroxy phenol was discovered to be a good replacement for aminoindanol.     

Thermodynamic effects of replacements of Pro residues in helix interiors of maltose-binding protein

Introduction of Pro residues into helix interiors results in protein destabilization. It is currently unclear if the converse substitution (i.e., replacement of Pro residues that naturally occur in helix interiors would be stabilizing). Maltose-binding protein is a large 370-amino acid protein that contains 21 Pro residues. Of these, three nonconserved residues (P48, P133, and P159) occur at helix interiors. Each of the residues was replaced with Ala and Ser. Stabilities were characterized by differential scanning calorimetry (DSC) as a function of pH and by isothermal urea denaturation studies as a function of temperature. The P48S and P48A mutants were found to be marginally more stable than the wild-type protein. In the pH range of 5-9, there is an average increase in T(m) values of P48A and P48S of 0.4 degrees C and 0.2 degrees C, respectively, relative to the wild-type protein. The other mutants are less stable than the wild type. Analysis of the effects of such Pro substitutions in MBP and in three other proteins studied to date suggests that substitutions are more likely to be stabilizing if the carbonyl group i-3 or i-4 to the mutation site is not hydrogen bonded in the wild-type protein.     

Conditional ablation of beta1 integrin in skin. Severe defects in epidermal proliferation, basement membrane formation, and hair follicle invagination

The major epidermal integrins are alpha3beta1 and hemidesmosome-specific alpha6beta4; both share laminin 5 as ligand. Keratinocyte culture studies implicate both integrins in adhesion, proliferation, and stem cell maintenance and suggest unique roles for alphabeta1 integrins in migration and terminal differentiation. In mice, however, whereas ablation of alpha6 or beta4 results in loss of hemidesmosomes, epidermal polarity, and basement membrane (BM) attachment, ablation of alpha3 only generates microblistering due to localized internal shearing of BM. Using conditional knockout technology to ablate beta1 in skin epithelium, we have uncovered biological roles for alphabeta1 integrins not predicted from either the alpha3 knockout or from in vitro studies. In contrast to alpha3 null mice, beta1 mutant mice exhibit severe skin blistering and hair defects, accompanied by massive failure of BM assembly/organization, hemidesmosome instability, and a failure of hair follicle keratinocytes to remodel BM and invaginate into the dermis. Although epidermal proliferation is impaired, a spatial and temporal program of terminal differentiation is executed. These results indicate that beta1's minor partners in skin are important, and together, alphabeta1 integrins are required not only for extracellular matrix assembly but also for BM formation. This, in turn, is required for hemidesmosome stability, epidermal proliferation, and hair follicle morphogenesis. However, beta1 downregulation does not provide the trigger to terminally differentiate.     

Homology and enzymatic requirements of microhomology-dependent alternative end joining

Nonhomologous DNA end joining (NHEJ) is one of the major double-strand break (DSB) repair pathways in higher eukaryotes. Recently, it has been shown that alternative NHEJ (A-NHEJ) occurs in the absence of classical NHEJ and is implicated in chromosomal translocations leading to cancer. In the present study, we have developed a novel biochemical assay system utilizing DSBs flanked by varying lengths of microhomology to study microhomology-mediated alternative end joining (MMEJ). We show that MMEJ can operate in normal cells, when microhomology is present, irrespective of occurrence of robust classical NHEJ. Length of the microhomology determines the efficiency of MMEJ, 5 nt being obligatory. Using this biochemical approach, we show that products obtained are due to MMEJ, which is dependent on MRE11, NBS1, LIGASE III, XRCC1, FEN1 and PARP1. Thus, we define the enzymatic machinery and microhomology requirements of alternative NHEJ using a well-defined biochemical system.DNA double-strand breaks (DSBs) are the most deleterious to the genome among various lesions. Nonhomologous end joining (NHEJ) is one of the major DSB repair pathways in higher eukaryotes.^{1, 2, 3} In the absence of key NHEJ factors, another distinct but error-prone pathway known as alternative NHEJ (A-NHEJ) has been described to have an important role in DSB repair.^{4, 5, 6, 7} It has been shown that majority of A-NHEJ-mediated repair of DSBs utilize distinct microhomology regions, hence termed microhomology-mediated end joining (MMEJ).^{4, 8, 9}A-NHEJ has been proposed as a possible cause for chromosomal translocations. Studies have shown co-amplification of c-MYC and IgH locus from pro-B lymphomas in mice deficient for p53 and NHEJ.¹⁰ A reduced level of class switch recombination (CSR) and increased number of chromosomal rearrangements at IgH locus have been shown in XRCC4- and LIGASE IV-deficient murine B cells.⁸ The occurrence of robust alternative end joining has been reported in the absence of NHEJ proteins, when murine RAG proteins were absent.¹¹Unraveling the enzymatic machinery involved in alternative end joining is currently an active area of research. Recently, it was shown that MRE11-RAD50-NBS1 complex may be involved in a subset of alternative NHEJ,^{5, 12, 13, 14} whereas ATM has a regulatory role.¹⁵ Role of PARP1 in repairing switch regions through a microhomology-mediated pathway leading to IgH/c-MYC translocations during immunoglobulin CSR has been described.¹⁶ Besides, studies have also suggested a role for DNA LIGASE IIIα and WRN in A-NHEJ.¹⁷ Interestingly, XRCC1 was shown to be dispensable in A-NHEJ during CSR, whereas functional relevance of Ligase I, III and Pol λ have been established.^{18, 19, 20} Hence, it can be concluded that canonical NHEJ (C-NHEJ) requires LIGASE IV–XRCC4 complex, while A-NHEJ is predominant in the absence of C-NHEJ proteins and is mainly characterized by joining utilizing microhomology (MMEJ). Further, it has been demonstrated that RPA, when bound to single-stranded DNA can antagonize MMEJ.²¹ Very recently, a genetic system was reported in budding yeast to detect microhomology-mediated repair.²² However, little is known whether alternative NHEJ can be operative when classical NHEJ machinery is intact.²³ A recent study suggested that MMEJ is also functional in normal mammalian cells. Besides, HR and MMEJ share the initial steps of end resection for DSB repair in mammalian cells.²⁴ However, it appears that there is not much consensus among different research groups over its presence and relevance in normal cells.²³ Therefore, several aspects of alternative NHEJ still need to be resolved. For example, its precise mechanism and microhomology length requirements are yet to be fully uncovered. Its occurrence in normal cells needs to be proved beyond doubt. Although there are independent studies showing the role of multiple proteins using gene knockdown or knockout strategies, their involvement needs to be confirmed.In the present study, we have established a cell-free repair assay system using which we show that MMEJ is operative even in the presence of classical NHEJ machinery. Further, our data suggest that MMEJ operates not only in cancer cells but also in normal cells. We show that a minimum of 5 nt microhomology is required for MMEJ and is independent of classical NHEJ proteins such as KU70, KU80 and LIGASE IV. Finally, we show that MRN complex, XRCC1, FEN1, PARP1 and LIGASE III are the factors responsible for joining mediated through microhomology.     

Efficient Imputation of Missing Markers in Low-Coverage Genotyping-by-Sequencing Data from Multiparental Crosses

We consider genomic imputation for low-coverage genotyping-by-sequencing data with high levels of missing data. We compensate for this loss of information by utilizing family relationships in multiparental experimental crosses. This nearly quadruples the number of usable markers when applied to a large rice Multiparent Advanced Generation InterCross (MAGIC) study.     

Design of disulfide-linked thioredoxin dimers and multimers through analysis of crystal contacts

Disulfide bonds play an important role in protein stability and function. Here, we describe a general procedure for generating disulfide-linked dimers and multimers of proteins of known crystal structures. An algorithm was developed to predict sites in a protein compatible with intermolecular disulfide formation with neighboring molecules in the crystal lattice. A database analysis was carried out on 46 PDB coordinates to verify the general applicability of this algorithm to predict intermolecular disulfide linkages. On the basis of the predictions from this algorithm, mutants were constructed and characterized for a model protein, thioredoxin. Of the five mutants, as predicted, in solution four formed disulfide-linked dimers while one formed polymers. Thermal and chemical denaturation studies on these mutant thioredoxins showed that three of the four dimeric mutants had similar stability to wild-type thioredoxin while one had lower stability. Three of the mutant dimers crystallized readily (in four to seven days) in contrast to the wild-type protein, which is particularly difficult to crystallize and takes more than a month to form diffraction-quality crystals. In two of the three cases, the structure of the dimer was exactly as predicted by the algorithm, while in the third case the relative orientation of the monomers in the dimer was different from the predicted one. This methodology can be used to enhance protein crystallizability, modulate the oligomerization state and to produce linear chains or ordered three-dimensional protein arrays.