Direct Targeting Strategy Research Articles

Mitochondrial DNA-targeted therapy: A novel approach to combat cancer.

Mitochondrial DNA (mtDNA) encodes proteins and RNAs that are essential for mitochondrial function and cellular homeostasis, and participates in important processes of cellular bioenergetics and metabolism. Alterations in mtDNA are associated with various diseases, especially cancers, and are considered as biomarkers for some types of tumors. Moreover, mtDNA alterations have been found to affect the proliferation, progression and metastasis of cancer cells, as well as their interactions with the immune system and the tumor microenvironment (TME). The important role of mtDNA in cancer development makes it a significant target for cancer treatment. In recent years, many novel therapeutic methods targeting mtDNA have emerged. In this study, we first discussed how cancerogenesis is triggered by mtDNA mutations, including alterations in gene copy number, aberrant gene expression and epigenetic modifications. Then, we described in detail the mechanisms underlying the interactions between mtDNA and the extramitochondrial environment, which are crucial for understanding the efficacy and safety of mtDNA-targeted therapy. Next, we provided a comprehensive overview of the recent progress in cancer therapy strategies that target mtDNA. We classified them into two categories based on their mechanisms of action: indirect and direct targeting strategies. Indirect targeting strategies aimed to induce mtDNA damage and dysfunction by modulating pathways that are involved in mtDNA stability and integrity, while direct targeting strategies utilized molecules that can selectively bind to or cleave mtDNA to achieve the therapeutic efficacy. This study highlights the importance of mtDNA-targeted therapy in cancer treatment, and will provide insights for future research and development of targeted drugs and therapeutic strategies.

Enhanced hydrogen evolution reaction in alkaline solution by constructing strong metal-support interaction on Pd-CeO2-x-NC hybrids

Diminishing the size of metal nanostructures can significantly improve the performance of catalysts. However, the self-aggregation of small particles is still an insurmountable obstacle, resulting in the unfavorable stability and recyclability. Herein, we designed and fabricated the Pd-CeO2-x-NC catalyst though an accurate deposition strategy to downsize the Pd particle to sub-nanometer level and enhance its running stability. The CeO2-x nanoclusters were firstly dispersed on the nitrogen-doped carbon nanosheets. Further, the active Pd sub-nanoclusters were accurately scattered on the surface of CeO2-x ascribing to the strong metal-support interaction (SMSI) between Pd and CeO2-x, which was beneficial to promote the catalytic activity. Subsequently, the high oxidation state Pdn+ species were formed due to the electron transfer from Pd to CeO2-x caused by the SMSI effect. Strikingly, the HER performance of Pd-CeO2-x-NC was surprisingly correlated with the ratio of Pdn+, suggesting Pdn+ acted as the dominant active species. Besides, the SMSI effect stabilized the valence state of active Pdn+ species and prevented the sub-nanometer Pd clusters from aggregation, which played a vital role for the enhanced stability of the hybrid catalyst. This synthetic process described here is contributed to prepare various nanostructured catalysts with satisfactory stability through the direct targeting strategy.

Phage display targeting identifies EYA1 as a regulator of glioblastoma stem cell maintenance and proliferation.

Glioblastoma (GBM) ranks among the most lethal of human malignancies with GBM stem cells (GSCs) that contribute to tumor growth and therapeutic resistance. Identification and isolation of GSCs continue to be a challenge, as definitive methods to purify these cells for study or targeting are lacking. Here, we leveraged orthogonal in vitro and in vivo phage display biopanning strategies to isolate a single peptide with GSC-specific binding properties. In silico analysis of this peptide led to the isolation of EYA1 (Eyes Absent 1), a tyrosine phosphatase and transcriptional coactivator. Validating the phage discovery methods, EYA1 was preferentially expressed in GSCs compared to differentiated tumor progeny. MYC is a central mediator of GSC maintenance but has been resistant to direct targeting strategies. Based on correlation and colocalization of EYA1 and MYC, we interrogated a possible interaction, revealing binding of EYA1 to MYC and loss of MYC expression upon targeting EYA1. Supporting a functional role for EYA1, targeting EYA1 expression decreased GSC proliferation, migration, and self-renewal in vitro and tumor growth in vivo. Collectively, our results suggest that phage display can identify novel therapeutic targets in stem-like tumor cells and that an EYA1-MYC axis represents a potential therapeutic paradigm for GBM.

Abstract 705: A cell-based screening assay to identify agents interfering protein-protein interaction of MAPKAPK2 and OCT4

Abstract c-MYC overexpression plays an important role in tumorigenesis as well as in the progression of tumors. Historically, direct targeting strategies have been challenging due to c-MYC being a transcription factor. Our observation demonstrated that MAPKAPK2 as one of the two major protein kinases involved in binding and phosphorylating OCT4 at serine 111 residue to induce c-MYC activation in progressive disease neuroblastoma. In targeting this novel pathway of c-MYC activation, we considered two options: direct inhibition of MAPKAPK2 or inhibition of MAPKAPK2-OCT4 protein-protein interaction. Given the limitations of direct targeting kinases, namely resistance and off-target effect, we pursued the inhibition of the protein-protein interaction. The purpose of this study is to introduce a novel cell-based assay for compound screening and validation steps to identify compounds that inhibit OCT4-MAPKAPK2 interaction. First, OCT4 (NTD and POUs: the domains critical for c-MYC activation) and MAPKAPK2 were exogenously expressed by a single vector with CMV promoter using a lentiviral vector system in NCI-H82, a small cell lung cancer cell line. Upon the optimization of the assay, we screened approximately 100,000 compounds to identify “hits” by statistically analyzing the reduction in luminescence. Seventy-six compounds were found to inhibit the interaction of MAPKAPK2-OCT4. Subsequently, we have used a two-step hit validation for the 76 compounds: 1) the decrease in pOCT4S111 expression along with the reduction in c-MYC expression by immunoblotting: 2) and the inhibition of binding between MAPKAPK2 and OCT4 by co-immunoprecipitation. The validation step further narrowed down the hits to three compounds that reduced the phosphorylation of OCT4 at S111 and inhibited the MAPKAPK2-OCT4 interaction. To verify the compounds interfering the MAPKAPK2-OCT4 interaction, we used in vitro assay using recombinant human MAPKAPK2 kinase and OCT4 and all three compounds inhibited phosphorylation of OCT4S111. These all compounds also tested minimally affected ATP-dependent MAPKAPK2 kinase relative to the known MAPKAPK2 inhibitor (PF3644022). In conclusion, our screening assay and validation experiments was successfully implemented in identifying hits and the validation step confirmed three novel compounds that demonstrated: 1) impairment of MAPKAPK2-mediated phosphorylation of OCT4; and 2) inhibition of the MAPKAPK2-OCT4 kinase-substrate interaction. These compounds may be further optimized to minimize systemic toxicities and improve the anti-cancer activity for the treatment of neuroblastoma or small cell lung cancer with high c-MYC expression. Citation Format: Inhyoung Yang, Sung-Jen Wei, Ismail Mohiuddin, Gloria M. Martinez, Min H. Kang. A cell-based screening assay to identify agents interfering protein-protein interaction of MAPKAPK2 and OCT4 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 705.

Nanoswimmer-oriented Direct Targeting Strategy Inspired by Momentum-based Gradient Optimization.

This paper considers the advantage of knowledgeaided direct targeting strategy (DTS) over systemic targeting for tumor homing in complex human vasculature. Tumor location in the body can be estimated by closely observing the tumortriggered bio-physical gradients in its vicinity, helping drugloaded magnetic nanoswimmers to accumulate at the disease location. These nanoswimmers are assembled by magnetic nanoparticles (MNPs), which act as contrast agents increasing the diagnostic capability of different medical imaging techniques. We propose a novel DTS inspired by the iterative gradient descent (GD) with momentum optimization for tumor targeting amplification. We show by computational experiments that the MNPs accumulate at the disease location at a faster rate and the probability of tumor detection is higher for the proposed knowledge-aided DTS as compared to the knowledgeless systemic targeting. We believe that our work for tumor targeting amplification based on such nanosystem will open new horizons in the field of diagnosing tumor at its early stage.

Biosensing-by-Learning Direct Targeting Strategy for Enhanced Tumor Sensitization.

We propose a novel iterative-optimization-inspired direct targeting strategy (DTS) for smart nanosystems, which harness swarms of externally manipulable nanoswimmers assembled by magnetic nanoparticles (MNPs) for knowledge-aided tumor sensitization and targeting. We aim to demonstrate through computational experiments that the proposed DTS can significantly enhance the accumulation of MNPs in the tumor site, which serve as a contrast agent in various medical imaging modalities, by using the shortest possible physiological routes and with minimal systemic exposure. The epicenter of a tumor corresponds to the global maximum of an externally measurable objective function associated with an in vivo tumor-triggered biological gradient; the domain of the objective function is the tissue region at a high risk of malignancy; swarms of externally controllable magnetic nanoswimmers for tumor sensitization are modeled as the guess inputs. The objective function may be resulted from a passive phenomenon such as reduced blood flow or increased kurtosis of microvasculature due to tumor angiogenesis; otherwise, the objective function may involve an active phenomenon such as the fibrin formed during the coagulation cascade activated by tumor-targeted "activator" nanoparticles. Subsequently, the DTS can be interpreted from the iterative optimization perspective: guess inputs (i.e., swarms of nanoswimmers) are continuously updated according to the gradient of the objective function in order to find the optimum (i.e., tumor) by moving through the domain (i.e., tissue under screening). Along this line of thought, we propose the computational model based on the gradient descent (GD) iterative method to describe the GD-inspired DTS, which takes into account the realistic in vivo propagation scenario of nanoswimmers. By means of computational experiments, we show that the GD-inspired DTS yields higher probabilities of tumor sensitization and more significant dose accumulation compared to the "brute-force" search, which corresponds to the systemic targeting scenario where drug nanoparticles attempt to target a tumor by enumerating all possible pathways in the complex vascular network. The knowledge-aided DTS has potential to enhance the tumor sensitization and targeting performance remarkably by exploiting the externally measurable, tumor-triggered biological gradients. We believe that this work motivates a novel biosensing-by-learning framework facilitated by externally manipulable, smart nanosystems.

Abstract B21: Epigenetic modifiers MLL1 and JMJD3 regulate neuroblastoma tumorigenicity by maintaining a cancer stem cell-like population

Abstract High-risk neuroblastoma (NB) represents a major clinical challenge in pediatric oncology. Despite significant dose escalation of intense therapies, long-term survival for NB patients remains poor (<45%), and the disease accounts for almost 15% of all pediatric cancer deaths. Relapse of metastatic, drug-resistant disease and treatment-related toxicities mandates the development of new therapeutic strategies. Recently, we discovered a highly tumorigenic, chemoresistant, and self-renewing subpopulation in NB with features similar to cancer stem cells (CSCs). This G-CSF receptor (CD114)-expressing subpopulation can escape initial therapy and cause aggressively invasive relapsed disease. Therefore, developing direct targeting strategies and understanding for the molecular mechanisms maintaining this NB CSC-like subpopulation is essential to delineate effective therapeutics for NB patients. By using siRNA screening approach and low-density pathway arrays, we found that the epigenetic regulators mixed-lineage leukemia-1 (MLL1; KMT2A; a H3K4me3 methyltransferase) and Jumonji D3 (JMJD3; KDM6B; a H3K27me3 demethylase) are important epigenetic regulators for NB proliferation and growth. Interestingly, we found that epigenetic regulators MLL1 and JMJD3 are aberrantly expressed in CD114+ subpopulation and regulate the expression of the G-CSF receptor gene (CSF3R) itself, by maintaining active histone modifications at the promoter locus. Inhibition of MLL1 and JMJD3 with specific small-molecule inhibitors reverses the histone patterns at CSF3R promoter and blocks gene expression, induces apoptosis selectively in CD114+ cells, and inhibits overall NB proliferation in vitro. Furthermore, inhibiting MLL1 and JMJD3 leads to dramatic tumor regression (p<0.001) and reduction in incidences of metastasis (p<0.001) in vivo. Most interestingly, both of these inhibitors increase the overall survival of the treated mice in vivo. As expected, reduction in tumor size was significantly correlated with the reduction in tumor CD114+ cells. Taken together, these data highlight that: a) neuroblastoma is maintained by the complex interplay of epigenetic modifiers, and b) direct targeting of these epigenetic modifiers and combining this approach with current therapy is a novel therapeutic approach to high-risk NB. Citation Format: Saurabh Agarwal, Julie Tomolonis, Sanjeev Vasudevan, Jason Shohet. Epigenetic modifiers MLL1 and JMJD3 regulate neuroblastoma tumorigenicity by maintaining a cancer stem cell-like population [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B21.

The MCL-1 inhibitor S63845: an exciting new addition to the armoury of anti-cancer agents

Defects in apoptotic machinery have long been recognised as both a significant contributor to cancer development, and as an important mechanism by which tumour cells develop chemotherapeutic resistance. The resistance of multiple malignancies to apoptosis has been attributed to increases in a number of pro-survival BCL-2 family members (e.g., BCL-2, BCL-XL, MCL-1, BCL-W, BFL-1 and BCL-B), which prevent BAX/BAK-mediated mitochondrial outer membrane permeabilisation. Inhibitors targeting these BCL-2 family members have garnered significant interest with the most promising lead being the BH3 mimetic venetoclax (also known as ABT-199, and marketed as Venclexta™ and Venclyxto™), a selective inhibitor of the BCL-2 protein recently approved for 17p deletion chronic lymphocytic leukemia (CLL). In a phase I trial in relapsed or refractory CLL, venetoclax induced a 79% response rate (1) which has subsequently prompted further trials in other haematological malignancies. Despite this success in CLL, venetoclax used as a monotherapy in other haematological malignancies have shown poor response rates (2), mainly due to the reliance of other BCL-2 family members such as MCL-1 for cell survival in these cancers. Indeed, studies using genetic knockout models and RNA interference have demonstrated MCL-1 to be crucial for disease development and progression in acute myeloid leukaemia (AML) (3), MYC-driven lymphomas (4), and multiple myeloma (5), and a mechanism of venetoclax resistance in these cancers (6). Indirect approaches to target MCL-1 through transcriptional repression (7,8) or post-translational degradation (9) have recently been developed. However, direct targeting strategies with obatoclax, an inhibitor of MCL-1 and also BCL-2 and BCL-XL, induced neuronal toxicity (10,11). More recently, a reported MCL-1-selective inhibitor termed A-1210477 (12) displayed in vitro activity against multiple myeloma cells (13); however, these anti-cancer effects appear likely to result from combination of both targeting MCL-1 and off-target effects (14).

Use of Morpholino Oligomers for Pretargeting.

Differing from the conventional direct-targeting strategy in which a probe or payload is directly loaded onto a targeting molecule that binds to the native target, pretargeting is an improved targeting strategy. It converts the native target to an artificial target specific for a secondary targeting molecule loaded with the probe or payload (effector). The effector is small and does not accumulate in normal tissues, which accelerates the targeting process and generates high target to nontarget ratios. DNA/cDNA analogs can serve as the recognition pair, i.e., the artificial target and the secondary targeting effector. Morpholino oligomers are so far the most investigated and the most successful DNA/cDNA analog recognition pairs for pretargeting. Herein, we describe the pretargeting principles, the pretargeting strategy using Morpholino oligomers, and the preclinical success so far achieved.