A Phase I/II Trial of Carfilzomib, Pegylated Liposomal Doxorubicin, and Dexamethasone for the Treatment of Relapsed/Refractory Multiple Myeloma.

PURPOSE: Pegylated liposomal doxorubicin (PLD) combined with bortezomib is an effective salvage regimen for relapsed refractory multiple myeloma (RRMM). Carfilzomib, a second-generation proteasome inhibitor, has clinical efficacy even among bortezomib-refractory patients. PATIENTS AND METHODS: We performed a phase I/II trial of carfilzomib, PLD, and dexamethasone (KDD) with the primary endpoints being safety and efficacy (NCT01246063). Twenty-three patients were enrolled in the phase I portion and the MTD of carfilzomib was determined to be 56 mg/m2 (days 1, 2, 8, 9, 15, and 16) when combined with PLD (30 mg/m2 on day 8) and dexamethasone (20 mg on days 1, 2, 8, 9, 15, and 16). Seventeen additional patients were enrolled in the phase II portion. RESULTS: KDD was determined to be well tolerated with the only common grade 3/4 nonhematologic adverse events of infection. Grade 3/4 hematologic toxicity included lymphopenia (63%), thrombocytopenia (40%), anemia (40%), and neutropenia (28%). In the cohort of patients treated at the MTD, where median prior therapies were 2% and 42% were refractory to bortezomib, the overall response rate was 83% (20/24) with 54% (13/24) having a very good partial response or better. The median progression-free survival was 13.7 months (95% CI, 5.0-21.7). CONCLUSIONS: This trial is the first to report outcomes using a triplet regimen of high-dose carfilzomib. KDD was well tolerated and appears efficacious in RRMM. Additional study is needed to more precisely determine patient outcomes with this regimen and its utility compared with other carfilzomib containing salvage regimens.

Programmable Metal/Semiconductor Nanostructures for mRNA-Modulated Molecular Delivery.

Cytotoxic chemotherapeutics are important tools for the clinical treatment of a variety of solid tumors. However, their use is often complicated by multidrug resistance that can develop in patients, limiting the potencies of these agents. New strategies are needed to provide versatile systems that can respond to and disable resistance mechanisms. We demonstrate the use of a new family of materials, programmable metal/semiconductor nanostructures, for drug delivery and mRNA sensing in drug-resistant cells. These materials are composed of a central core gold nanoparticle surrounded by a layer of DNA-capped quantum dots. The modularity of these "core-satellite" assemblies allows for the construction of superstructures with controlled size and the incorporation of multiple functionalities for drug delivery. The DNA sequence within the nanoparticle specifically binds to an mRNA encoding an important drug resistance factor, MRP1, inside cancer cells, releasing a potent anticancer drug doxorubicin. This event triggers a turn-on fluorescence emission along with a downregulation of the MRP1 drug efflux pump, a main resistance factor for doxorubicin, yielding a remarkable improvement in therapeutic efficacy against drug-resistant cancer cells. This work paves the way for the development of programmable materials with multiple synergistic functionalities for biomedical applications.

Author Correction: Mitochondrial DNA repair and replication proteins revealed by targeted chemical probes.

In the version of this article initially published, Sanduni Liyanage and Aaron Schimmer were not properly acknowledged as co-authors. Both authors have now been included in the current author list, and their contributions are now specified in the author contributions statement. The error has been corrected in the PDF and HTML versions of this article.

Mitochondrial tyrosyl-DNA phosphodiesterase 2 and its TDP2S short isoform.

Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs abortive topoisomerase II cleavage complexes. Here, we identify a novel short isoform of TDP2 (TDP2S) expressed from an alternative transcription start site. TDP2S contains a mitochondrial targeting sequence, contributing to its enrichment in the mitochondria and cytosol, while full-length TDP2 contains a nuclear localization signal and the ubiquitin-associated domain in the N-terminus. Our study reveals that both TDP2 isoforms are present and active in the mitochondria. Comparison of isogenic wild-type (WT) and TDP2 knockout (TDP2-/-/-) DT40 cells shows that TDP2-/-/- cells are hypersensitive to mitochondrial-targeted doxorubicin (mtDox), and that complementing TDP2-/-/- cells with human TDP2 restores resistance to mtDox. Furthermore, mtDox selectively depletes mitochondrial DNA in TDP2-/-/- cells. Using CRISPR-engineered human cells expressing only the TDP2S isoform, we show that TDP2S also protects human cells against mtDox. Finally, lack of TDP2 in the mitochondria reduces the mitochondria transcription levels in two different human cell lines. In addition to identifying a novel TDP2S isoform, our report demonstrates the presence and importance of both TDP2 isoforms in the mitochondria.

Multifunctional quantum dot DNA hydrogels.

Biotemplated nanomaterials offer versatile functionality for multimodal imaging, biosensing, and drug delivery. There remains an unmet need for traceable and biocompatible nanomaterials that can be synthesized in a precisely controllable manner. Here, we report self-assembled quantum dot DNA hydrogels that exhibit both size and spectral tunability. We successfully incorporate DNA-templated quantum dots with high quantum yield, long-term photostability, and low cytotoxicity into a hydrogel network in a single step. By leveraging DNA-guided interactions, we introduce multifunctionality for a variety of applications, including enzyme-responsive drug delivery and cell-specific targeting. We report that quantum dot DNA hydrogels can be used for delivery of doxorubicin, an anticancer drug, to increase potency 9-fold against cancer cells. This approach also demonstrated high biocompatibility, trackability, and in vivo therapeutic efficacy in mice bearing xenografted breast cancer tumors. This work paves the way for the development of new tunable biotemplated nanomaterials with multiple synergistic functionalities for biomedical applications.The development of nanomaterials for imaging and drug delivery has been of great interest to the field. Here, the authors synthesized multifunctional enzyme-responsive hydrogels with self-assembling quantum dots for nucleic acid and drug delivery as well as having imaging capability.

Mitochondrial Chemical Biology: New Probes Elucidate the Secrets of the Powerhouse of the Cell.

Mitochondria are energy-producing organelles with essential functions in cell biology, and mitochondrial dysfunction is linked to a wide range of human diseases. Efforts to better understand mitochondrial biology have been limited by the lack of tools for manipulating and detecting processes occurring within the organelle. Here, we highlight recent significant advances in mitochondrial chemical biology that have produced new tools and techniques for studying mitochondria. Specifically, we focus on the development of chemical tools to perturb mitochondrial biochemistry, probes allowing precise measurement of mitochondrial function, and new techniques for high-throughput characterization of the mitochondrial proteome. Taken together, these advances in chemical biology will enable exciting new directions in mitochondrial research.

Peptide-Mediated Delivery of Chemical Probes and Therapeutics to Mitochondria.

Mitochondria are organelles with critical roles in key processes within eukaryotic cells, and their dysfunction is linked with numerous diseases including neurodegenerative disorders and cancer. Pharmacological manipulation of mitochondrial function is therefore important both for basic science research and eventually, clinical medicine. However, in comparison to other organelles, mitochondria are difficult to access due to their hydrophobic and dense double membrane system as well as their negative membrane potential. To tackle the challenge of targeting these important subcellular compartments, significant effort has been put forward to develop mitochondria-targeted systems capable of transporting bioactive cargo into the mitochondrial interior. Systems now exist that utilize small molecule, peptide, liposome, and nanoparticle-based transport. The vectors available vary in size and structure and can facilitate transport of a variety of compounds for mitochondrial delivery. Notably, peptide-based delivery scaffolds offer attractive features such as ease of synthesis, tunability, biocompatibility, and high uptake both in cellulo and in vivo. Owing to their simple and modular synthesis, these peptides are highly adaptable for delivering chemically diverse cargo. Key design features of mitochondria-targeted peptides include cationic charge, which allows them to harness the negative membrane potential of mitochondria, and lipophilicity, which permits favorable interaction with hydrophobic membranes of mitochondria. These peptides have been covalently tethered to target therapeutic agents, including anticancer drugs, to enhance their drug properties, and to provide probes for mitochondrial biology. Interestingly, mitochondria-targeted DNA damaging agents demonstrate high potency and the ability to evade resistance mechanisms and off-target effects. Moreover, a combination of mitochondria-targeted DNA damaging agents was applied to an siRNA screen for the elucidation of poorly understood mitochondrial DNA repair and replication pathways. In this work, a variety of novel proteins were identified that are essential for the maintenance of mitochondrial nucleic acids. Mitochondria-targeted peptides have also been used to increase the therapeutic window of antibacterial drugs with significant mammalian toxicity. Given the evolutionary similarity of mitochondria and bacteria, peptides are effective transporters that can target both of these entities. These antimicrobial peptides are highly effective even in difficult to target intracellular bacteria which reside within host cells. This peptide-based approach to targeting mitochondria has provided a variety of insights into the "druggability" of mitochondria and new biological processes that could be future drug targets. Nevertheless, the mitochondrial-targeting field is quite nascent and many exciting applications of organelle-specific conjugates remain to be explored. In this Account, we highlight the development and optimization of the mitochondria-penetrating peptides that our laboratory has developed, the unique applications of mitochondria-targeted bioactive cargo, and offer a perspective on important directions for the field.

Mitochondria-Targeted Doxorubicin: A New Therapeutic Strategy against Doxorubicin-Resistant Osteosarcoma.

Doxorubicin is one of the leading drugs for osteosarcoma standard chemotherapy. A total of 40% to 45% of high-grade osteosarcoma patients are unresponsive, or only partially responsive, to doxorubicin (Dox), due to the overexpression of the drug efflux transporter ABCB1/P-glycoprotein (Pgp). The aim of this work is to improve Dox-based regimens in resistant osteosarcomas. We used a chemically modified mitochondria-targeted Dox (mtDox) against Pgp-overexpressing osteosarcomas with increased resistance to Dox. Unlike Dox, mtDox accumulated at significant levels intracellularly, exerted cytotoxic activity, and induced necrotic and immunogenic cell death in Dox-resistant/Pgp-overexpressing cells, fully reproducing the activities exerted by anthracyclines in drug-sensitive tumors. mtDox reduced tumor growth and cell proliferation, increased apoptosis, primed tumor cells for recognition by the host immune system, and was less cardiotoxic than Dox in preclinical models of drug-resistant osteosarcoma. The increase in Dox resistance was paralleled by a progressive upregulation of mitochondrial metabolism. By widely modulating the expression of mitochondria-related genes, mtDox decreased mitochondrial biogenesis, the import of proteins and metabolites within mitochondria, mitochondrial metabolism, and the synthesis of ATP. These events were paralleled by increased reactive oxygen species production, mitochondrial depolarization, and mitochondria-dependent apoptosis in resistant osteosarcoma cells, where Dox was completely ineffective. We propose mtDox as a new effective agent with a safer toxicity profile compared with Dox that may be effective for the treatment of Dox-resistant/Pgp-positive osteosarcoma patients, who strongly need alternative and innovative treatment strategies. Mol Cancer Ther; 15(11); 2640-52. ©2016 AACR.

Mitochondrial DNA repair and replication proteins revealed by targeted chemical probes.

Efficient and accurate replication and repair of mitochondrial DNA is essential for cellular viability, yet only a minimal complement of mitochondrial proteins with relevant activities have been identified. Here, we describe an approach to screen for new pathways involved in the maintenance of mitochondrial DNA (mtDNA) that leverages the activities of DNA-damaging probes exhibiting specific subcellular localization. By conducting a siRNA screen of known nuclear DNA maintenance factors, and monitoring synergistic effects of gene depletion on the activity of mitochondria-specific DNA-damaging agents, we identify a series of proteins not previously recognized to act within mitochondria. These include proteins that function in pathways of oxidative DNA damage repair and dsDNA break repair, along with a novel mitochondrial DNA polymerase, POLθ, that facilitates efficient DNA replication in an environment prone to oxidative stress. POLθ expression levels affect the mutational rate of mitochondrial DNA, but this protein also appears critical for efficient mtDNA replication.

Mitochondrial Targeting of Doxorubicin Eliminates Nuclear Effects Associated with Cardiotoxicity.

The highly effective anticancer agent doxorubicin (Dox) is a frontline drug used to treat a number of cancers. While Dox has a high level of activity against cancer cells, its clinical use is often complicated by dose-limiting cardiotoxicity. While this side effect has been linked to the drug's direct activity in the mitochondria of cardiac cells, recent studies have shown that these result primarily from downstream effects of nuclear DNA damage. Our lab has developed a mitochondrially targeted derivative of Dox that enables the selective study of toxicity generated by the presence of Dox in the mitochondria of human cells. We demonstrate that mitochondria-targeted doxorubicin (mtDox) lacks any direct nuclear effects in H9c2 rat cardiomyocytes, and that these cells are able to undergo mitochondrial biogenesis. This recovery response compensates for the mitotoxic effects of Dox and prevents cell death in cardiomyocytes. Furthermore, cardiac toxicity was only observed in Dox but not mtDox treated mice. This study supports the hypothesis that mitochondrial damage is not the main source of the cardiotoxic effects of Dox.

Structural modifications of mitochondria-targeted chlorambucil alter cell death mechanism but preserve MDR evasion.

Multidrug resistance (MDR) remains one of the major obstacles in chemotherapy, potentially rendering a multitude of drugs ineffective. Previously, we have demonstrated that mitochondrial targeting of DNA damaging agents is a promising tool for evading a number of common resistance factors that are present in the nucleus or cytosol. In particular, mitochondria-targeted chlorambucil (mt-Cbl) has increased potency and activity against resistant cancer cells compared to the parent compound chlorambucil (Cbl). However, it was found that, due to its high reactivity, mt-Cbl induces a necrotic type of cell death via rapid nonspecific alkylation of mitochondrial proteins. Here, we demonstrate that by tuning the alkylating activity of mt-Cbl via chemical modification, the rate of generation of protein adducts can be reduced, resulting in a shift of the cell death mechanism from necrosis to a more controlled apoptotic pathway. Moreover, we demonstrate that all of the modified mt-Cbl compounds effectively evade MDR resulting from cytosolic GST-µ upregulation by rapidly accumulating in mitochondria, inducing cell death directly from within. In this study, we systematically elucidated the advantages and limitations of targeting alkylating agents with varying reactivity to mitochondria.