Complex In Cancer Cells Research Articles

DNA-templated spatially controlled proteolysis targeting chimeras for CyclinD1-CDK4/6 complex protein degradation.

Constraining proximity-based drugs, such as proteolysis-targeting chimeras (PROTACs), into its bioactive conformation can significantly impact their selectivity and potency. However, traditional methods for achieving this often involve complex and time-consuming synthetic procedures. Here, we introduced an alternative approach by demonstrating DNA-templated spatially controlled PROTACs (DTACs), which leverage the programmability of nucleic-acid based self-assembly for efficient synthesis, providing precise control over inhibitors' spacing and orientation. The resulting constructs revealed distance- and orientation-dependent selectivity and degradation potency for the CyclinD1-CDK4/6 protein complex in cancer cells. Notably, an optimal construct DTAC-V1 demonstrated the unprecedented synchronous degradation of entire CyclinD1-CDK4/6 complex. This resulted in the effective cell cycle arrest in G1 phase, and further therapeutic studies showed its potent anti-tumor effects compared to inhibitors alone. These findings present a novel framework for PROTACs design, offering critical insights that may inform the development of other proximity-induced therapeutic modalities.

Multiomics approach towards characterization of tumor cell plasticity and its significance in precision and personalized medicine.

Cellular plasticity refers to the ability of cells to change their identity or behavior, which can be advantageous in some cases (e.g., tissue regeneration) but detrimental in others (e.g., cancer metastasis). With a better understanding of cellular plasticity, the complexity of cancer cells, their heterogeneity, and their role in metastasis is being unraveled. The plasticity of the cells could also prove as a nemesis to their characterization. In this review, we have attempted to highlight the possibilities and benefits of using multiomics approach in characterizing the plastic nature of cancer cells. There is a need to integrate fragmented evidence at different levels of cellular organization (DNA, RNA, protein, metabolite, epigenetics, etc.) to facilitate the characterization of different forms of plasticity and cell types. We have discussed the role of cellular plasticity in generating intra-tumor heterogeneity. Different omics level evidence is being provided to highlight the variety of molecular determinants discovered using different techniques. Attempts have been made to integrate some of this information to provide a quantitative assessment and scoring of the plastic nature of the cells. However, there is a huge gap in our understanding of mechanisms that lead to the observed heterogeneity. Understanding of these mechanism(s) is necessary for finding targets for early detection and effective therapeutic interventions in metastasis. Targeting cellular plasticity is akin to neutralizing a moving target. Along with the advancements in precision and personalized medicine, these efforts may translate into better clinical outcomes for cancer patients, especially in metastatic stages.

N-Indolyl diiron vinyliminium complexes exhibit antiproliferative effects in cancer cells associated with disruption of mitochondrial homeostasis, ROS scavenging, and antioxidant activity

The indole scaffold has been established as a key organic moiety for developing new drugs; on the other hand, a range of diiron bis-cyclopentadienyl complexes have recently emerged for their promising anticancer potential. Here, we report the synthesis of novel diiron complexes with an indole-functionalized vinyliminium ligand (2–5) and an indole-lacking analogue for comparative purposes (6), which were characterized by analytical and spectroscopic techniques. Complexes 2–6 are substantially stable in DMSO‑d6 and DMEM-d solutions at 37 °C (8% average degradation after 48 h) and display a balanced hydrophilic/lipophilic behaviour (LogPow values in the range −0.32 to 0.47), associated with appreciable water solubility. The complexes display selective antiproliferative potency towards several cancer cells in monolayer cultures, mainly in the low micromolar range, with reduced toxicity towards noncancerous epithelial cells. Thus, the cytotoxicity of the complexes is comparable to or better than clinically used metallopharmaceutical cisplatin. Comparing the antiproliferative activity obtained for complexes containing different ligands, we confirmed the importance of the indolyl group in the mechanism of antiproliferative activity of these complexes. Cell-based mechanistic studies suggest that the investigated diiron vinyliminium complexes (DVCs) show cytostatic rather than cytotoxic effects and subsequently induce a population of cells to undergo apoptosis. Furthermore, the molecular mechanism of action involves interactions with mitochondrial DNA and proteins, the reactive oxygen species (ROS)-scavenging properties and antioxidant activity of these complexes in cancer cells. This study highlights the importance of DVCs to their cancer cell activity and reinforces their prospective therapeutic potential as anticancer agents.

New Insights into the Behavior of NHC-Gold Complexes in Cancer Cells

Among the non-platinum antitumor agents, gold complexes have received increased attention owing to their strong antiproliferative effects, which generally occur through non-cisplatin-like mechanisms of action. Several studies have revealed that many cytotoxic gold compounds, such as N-heterocyclic carbene (NHC)-gold(I) complexes, are potent thioredoxin reductase (TrxR) inhibitors. Many other pathways have been supposed to be altered by gold coordination to protein targets. Within this frame, we have selected two gold(I) complexes based on aromatic ligands to be tested on cancer cells. Differently from bis [1,3-diethyl-4,5-bis(4-methoxyphenyl)imidazol-2-ylidene]gold(I) bromide (Au4BC), bis [1-methyl-3-acridineimidazolin-2-ylidene]gold(I) tetrafluoroborate (Au3BC) inhibited TrxR1 activity in vitro. Treatment of Huh7 hepatocellular carcinoma (HCC) cells, and MDA-MB-231 triple-negative breast cancer (TNBC) cells, with Au4BC inhibited cell viability, increased reactive oxygen species (ROS) levels, caused DNA damage, and induced autophagy and apoptosis. Notably, we found that, although Au3BC inhibited TrxR1 activity, no effect on the cell viabilities of HCC and BC cells was observed. At the molecular level, Au3BC induced a protective response mechanism in Huh7 and MDA-MB-231 cells, by inducing up-regulation of RAD51 and p62 protein expression, two proteins involved in DNA damage repair and autophagy, respectively. RAD51 gene knock-down in HCC cells increased cell sensitivity to Au3BC by significant reduction of cell viability, induction of DNA damage, and induction of apoptosis and autophagy. All together, these results suggest that the tested NHC-Gold complexes, Au3BC and Au4BC, showed different mechanisms of action, either dependent or independent of TrxR1 inhibition. As a result, Au3BC and Au4BC were found to be promising candidates as anticancer drugs for the treatment of HCC and BC.

Assessing the role of membrane lipids in the action of ruthenium(III) anticancer compounds.

This work addresses the possible role of the cell membrane in the molecular mechanism of action of two salan-type ruthenium complexes that were previously shown to be active against human tumor cells, namely [Ru(III)(L1)(PPh3)Cl] and [Ru(III)(L2)(PPh3)Cl] (where L1 is 6,6'-(1R,2R)-cyclohexane-1,2-diylbis(azanediyl)bis(methylene)bis(3-methoxyphenol); and L2 is 2,2'-(1R,2R)-cyclohexane-1,2-diylbis(azanediyl)bis(methylene)bis(4-methoxyphenol)). One-component membrane models were first used, a disordered fluid bilayer of dioleoylphosphatodylcholine (DOPC), and an ordered rigid gel bilayer of dipalmitoylphosphatidylcholine. In addition, two quaternary mixtures of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin and cholesterol were used to mimic the lipid composition either of mammalian plasma membrane (1:1:1:1mol ratio) or of a cancer cell line membrane (36.2:23.6:6.8:33.4mol ratio). The results show that both salan ligands L1 and L2 bind relatively strongly to DOPC bilayers, but without significantly affecting their structure. The ruthenium complexes have moderate affinity for DOPC. However, their impact on the membranes was notable, leading to a significant increase in the permeability of the lipid vesicles. None of the compounds compromised liposome integrity, as revealed by dynamic light scattering. Fluorescence spectroscopy studies revealed changes in the biophysical properties of all membrane models analyzed in the presence of the two complexes, which promoted an increased fluidity and water penetration into the lipid bilayer in the one-component systems. In the quaternary mixtures, one of the complexes had an analogous effect (increasing water penetration), whereas the other complex reorganized the liquid ordered and liquid disordered domains. Thus, small structural differences in the metal ligands may lead to different outcomes. To better understand the effect of these complexes in cancer cells, the membrane dipole potential was also measured. For both Ru complexes, an increase in the dipole potential was observed for the cancer cell membrane model, while no alteration was detected on the non-cancer plasma membrane model. Our results show that the action of the Ru(III) complexes tested involves changes in the biophysical properties of the plasma membrane, and that it also depends on membrane lipid composition, which is frequently altered in cancer cells when compared to their normal counterparts.

IGF2BP2 promotes cancer progression by degrading the RNA transcript encoding a v-ATPase subunit

IGF2BP2 binds to a number of RNA transcripts and has been suggested to function as a tumor promoter, although little is known regarding the mechanisms that regulate its roles in RNA metabolism. Here we demonstrate that IGF2BP2 binds to the 3' untranslated region of the transcript encoding ATP6V1A, a catalytic subunit of the vacuolar ATPase (v-ATPase), and serves as a substrate for the NAD+-dependent deacetylase SIRT1, which regulates how IGF2BP2 affects the stability of the ATP6V1A transcript. When sufficient levels of SIRT1 are expressed, it catalyzes the deacetylation of IGF2BP2, which can bind to the ATP6V1A transcript but does not mediate its degradation. However, when SIRT1 expression is low, the acetylated form of IGF2BP2 accumulates, and upon binding to the ATP6V1A transcript recruits the XRN2 nuclease, which catalyzes transcript degradation. Thus, the stability of the ATP6V1A transcript is significantly compromised in breast cancer cells when SIRT1 expression is low or knocked-down. This leads to a reduction in the expression of functional v-ATPase complexes in cancer cells and to an impairment in their lysosomal activity, resulting in the production of a cellular secretome consisting of increased numbers of exosomes enriched in ubiquitinated protein cargo and soluble hydrolases, including cathepsins, that together combine to promote tumor cell survival and invasiveness. These findings describe a previously unrecognized role for IGF2BP2 in mediating the degradation of a messenger RNA transcript essential for lysosomal function and highlight how its sirtuin-regulated acetylation state can have significant biological and disease consequences.

A Pyrazolate Osmium(VI) Nitride Exhibits Anticancer Activity through Modulating Protein Homeostasis in HepG2 Cells.

Interest in the third-row transition metal osmium and its compounds as potential anticancer agents has grown in recent years. Here, we synthesized the osmium(VI) nitrido complex Na[OsVI(N)(tpm)2] (tpm = [5-(Thien-2-yl)-1H-pyrazol-3-yl]methanol), which exhibited a greater inhibitory effect on the cell viabilities of the cervical, ovarian, and breast cancer cell lines compared with cisplatin. Proteomics analysis revealed that Na[OsVI(N)(tpm)2] modulates the expression of protein-transportation-associated, DNA-metabolism-associated, and oxidative-stress-associated proteins in HepG2 cells. Perturbation of protein expression activity by the complex in cancer cells affects the functions of the mitochondria, resulting in high levels of cellular oxidative stress and low rates of cell survival. Moreover, it caused G2/M phase cell cycle arrest and caspase-mediated apoptosis of HepG2 cells. This study reveals a new high-valent osmium complex as an anticancer agent candidate modulating protein homeostasis.

Abstract 3592: Targeting SWI/SNF ATPases in enhancer-addicted human cancers

Abstract In mammalian cells, DNA is wrapped around histone octamers (collectively referred to as nucleosomes) which form a physical barrier to all DNA-based processes. The switch/sucrose non-fermentable (SWI/SNF) is a multi-subunit chromatin remodeling complex that uses energy from ATP hydrolysis to reposition or eject nucleosomes at non-coding regulatory elements, thereby enabling access to the underlying DNA for transcriptional activation. Notably, the SWI/SNF complex plays a crucial role in chromatin remodeling and is recurrently altered in over 20% of human cancers, with the revised complex in cancer cells enabling central oncogenic gene programs. Yet, no studies have assessed the therapeutic efficacy of complete SWI/SNF inactivation across human cancers. Here, we developed a proteolysis targeting chimera (PROTAC) degrader of ATPase subunits of the SWI/SNF complex, SMARCA2 and SMARCA4. In a panel with over 90 normal and cancer cell lines from 18 different lineages, we found MYC-driven multiple myeloma and androgen receptor (AR)/forkhead box A1 (FOXA1)-positive prostate and breast cancers to be preferentially sensitive to dual SMARCA2 and SMARCA4 degradation relative to benign prostate as well as other cancer cell lines, including cancer cell lines with inactivating SMARCA4 mutations. We found complete SWI/SNF ATPase degradation to instantaneously compact the cis-regulatory elements that are bound and activated by transcription factors that drive cancer proliferation, namely MYC, IRF4, TCF3, AR, FOXA1, and ERG. This ensued in parallel untethering of these oncogenic drivers from the chromatin, with subsequent chemical decommissioning of their core enhancer circuitry and attenuation of downstream gene programs. Furthermore, using chromatin conformation assays we found SWI/SNF inactivation to disrupt super-enhancer and promoter DNA looping interactions that wire supra-physiologic expression of the MYC, AR, ERG, IRF4, and TCF3 oncogenes themselves, thereby tempering their expression in cancer cells. Treatment with the SMARCA2/4 degrader alone induced potent inhibition of tumor growth in cell line-derived xenograft models of multiple myeloma, as well as prostate cancer, and synergized with AR antagonists, inducing disease remission in several drug-resistant disease models. Notably, no major toxicities were seen in mice upon prolonged treatment with the SMARCA2/4 degrader, including no indications of thrombocytopenia, gastrointestinal goblet cell depletion, or germ cell degeneration—all being major toxicities associated with the BRD4-targeting therapeutics. To our knowledge, this study is the first preclinical proof of concept that targeted obstruction of chromatin accessibility at non-coding regulatory elements can be a potent therapeutic strategy in enhancer-addicted tumors, warranting the safety and efficacy assessments of SWI/SNF inhibitors and degraders in human clinical trials. Citation Format: Abhijit Parolia, Lanbo Xiao, Yuanyuan Qiao, Pushpinder Bawa, Sanjana Eyunni, Eleanor Young, Rahul Mannan, Sandra E. Carson, Yu Chang, Yuping Zhang, James George, Mustapha Jaber, Fengyun Su, Rui Wang, Sanjita Sasmal, Leena Khare, Subhendu Mukerjee, Chandrasekhar AbbinenI, Kiran Aithal, Xuhong Cao, Yuzhuo Wang, Susanta Samajdar, Murali Ramachandra, Arul M. Chinnaiyan. Targeting SWI/SNF ATPases in enhancer-addicted human cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3592.

Synthesis and New Cytotoxicity Screening Approach of Novel Dirhodium Complexes

Transition metal–based chemotherapeutics have been used to treat a variety of cancers since the 1960s with cisplatin being the most prominent of these drugs used in a clinical setting. Many cancers become chemoresistant to cisplatin, so there is currently a need to synthesize and explore the anticancer abilities of other transition metal complexes. Rhodium, one of the six platinum–group metals, demonstrates cytotoxic effects in cancer cells in the form of dirhodium tetraacetate (Rh2A4). However, Rh2A4 is known to be less cytotoxic than cisplatin in HeLa cervical cancer cells. This research aims to modify Rh2A4 to increase its cytotoxicity to that of cisplatin. One of the acetate ligands of Rh2A4 was selectively replaced with 6‐maleimidohexanoic acid (M) to form a dirhodium triacetate maleimide (Rh2A3M) complex. Maleimides are known to readily react with alkylamines (R‐NH2) under basic conditions to produce amine conjugates. Alkylamines were chosen because of this reactivity and because their presence in a variety of metabolic and physiological processes may aid in the uptake of the dirhodum complex in cancer cells. The Rh2A3M complex was successfully reacted with four alkylamines: benzylamine, cyclohexylamine, ethanolamine, and glucosamine. This reaction was done in a 1:10 molar ratio in a phosphate buffer of pH 7.25 at room temperature for 72 hours to form subsequent Rh2A3M‐NH‐R complexes. The crude reaction mixture was then purified by preparative HPLC and characterized via 1H 1D NMR. A new screening method was developed using MTT reagent to compare the cellular viability of HeLa cervical cancer cells treated with the four crude reaction mixtures and the four HPLC purified Rh2A3M‐NH‐R complexes. Each pure complex had a similar percent cellular viability to its corresponding crude reaction mixture. This procedure will allow for future screens of many Rh2A3M + R‐NH2 crude reaction mixtures to indicate cytotoxicity of Rh2A3M‐NH‐R products without the need for the purification and characterization steps. Out of the four pure complexes, Rh2A3M‐cyclohexylamine was found to be the most cytotoxic at ~20 μM, suggesting it is comparable in cytotoxicity to cisplatin. Current work is focused on the determination of the IC50 value for this complex to definitively compare its cytotoxicity to cisplatin.

IGF2BP2 Promotes Cancer Progression by Degrading the RNA Transcript Encoding a v‐ATPase Subunit

IGF2BP2 binds to a number of RNA transcripts and has been suggested to function as a tumor promoter, although little is known regarding the mechanisms that regulate its roles in RNA metabolism. Here we demonstrate that IGF2BP2 binds to the 3’ untranslated region of the transcript encoding ATP6V1A, a catalytic subunit of the v‐ATPase, and serves as a novel substrate for the NAD+‐dependent deacetylase SIRT1, which regulates how IGF2BP2 affects the stability of the ATP6V1A transcript. When sufficient levels of SIRT1 are expressed, it catalyzes the deacetylation of IGF2BP2, which can bind to the ATP6V1A transcript but does not mediate its degradation. However, when SIRT1 expression is low, the acetylated form of IGF2BP2 accumulates, and upon binding to the ATP6V1A transcript, recruits the XRN2 nuclease which catalyzes transcript degradation. Thus, the stability of the ATP6V1A transcript is significantly compromised in breast cancer cells when SIRT1 expression is low or knocked‐down. This leads to a reduction in the expression of functional v‐ATPase complexes in cancer cells and to an impairment in their lysosomal activity, resulting in the production of a cellular secretome consisting of increased numbers of exosomes enriched in ubiquitinated protein cargo and soluble hydrolases including cathepsins, that together combine to promote tumor cell survival and invasiveness. These findings describe a previously unrecognized role for IGF2BP2 in mediating the degradation of an mRNA transcript essential for lysosomal function and highlight how its sirtuin‐regulated acetylation state can have significant biological and disease consequences.

Possible new mitochondrial mechanism of vitamin C anticancer activity

The main feature of fast-growing malignant tumors is the Warburg-type metabolism, which is directly related to an extremely high quantity of hexokinase (HK) bound to the voltage-dependent anion channels (VDACs) in the mitochondrial outer membrane. Previously, we explained the Warburg type metabolism as a result of the electrical suppression of mitochondria due to the closure of free VDACs that are not bound to HK. Here, we propose a possible new mechanism of high-dose vitamin C (ascorbate) anticancer activity estimated with a simplified computational model. According to the proposed hypothesis and the model, ascorbate oxidation in mitochondria leads to the generation of the negative outer membrane potential (OMP) of opposite sign to the positive OMP generated by the VDAC-HK complexes in cancer cells. The model demonstrates that negative OMP generated by any mechanism, even of relatively low magnitudes, leads tothe reopening of the electrically closed VDACs, thus reprogramming cell energy metabolism.According to the hypothesis, redox mediators, which increase the rate of ascorbate oxidation in mitochondria, should synergistically increase anticancer effects of high-dose ascorbate in accordance with experimental data recorded in the literature. The model shows that even small changes in the VDAC-voltage sensitivity and/or quantity of the VDAC-HK complexes, known to be caused by various physiological factors, might strongly influence the proposed mitochondrial mechanism of ascorbate anticancer activity.

Cytosolic localization and in vitro assembly of human de novo thymidylate synthesis complex.

De novo thymidylate synthesis is a crucial pathway for normal and cancer cells. Deoxythymidine monophosphate (dTMP) is synthesized by the combined action of three enzymes: serine hydroxymethyltransferase (SHMT1), dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS), with the latter two being targets of widely used chemotherapeutics such as antifolates and 5‐fluorouracil. These proteins translocate to the nucleus after SUMOylation and are suggested to assemble in this compartment into the thymidylate synthesis complex. We report the intracellular dynamics of the complex in cancer cells by an in situ proximity ligation assay, showing that it is also detected in the cytoplasm. This result indicates that the role of the thymidylate synthesis complex assembly may go beyond dTMP synthesis. We have successfully assembled the dTMP synthesis complex in vitro, employing tetrameric SHMT1 and a bifunctional chimeric enzyme comprising human thymidylate synthase and dihydrofolate reductase. We show that the SHMT1 tetrameric state is required for efficient complex assembly, indicating that this aggregation state is evolutionarily selected in eukaryotes to optimize protein–protein interactions. Lastly, our results regarding the activity of the complete thymidylate cycle in vitro may provide a useful tool with respect to developing drugs targeting the entire complex instead of the individual components.

Harnessing the hERG1/β1 Integrin Complex via a Novel Bispecific Single-chain Antibody: An Effective Strategy against Solid Cancers.

mAbs, either mono- or bispecific (bsAb), represent one of the most successful approaches to treat many types of malignancies. However, there are certain limitations to the use of full length mAbs for clinical applications, which can be overcome by engineered antibody fragments. The aim of this study was to develop a small bsAb, in the format of a single-chain diabody (scDb), to efficiently target two proteins, the hERG1 potassium channel and the β1 subunit of integrin receptors, which specifically form a macromolecular complex in cancer cells. We provide evidence that the scDb we produced binds to the hERG1/β1 complex in cancer cells and tissues, but does not bind to the hERG1 channel in nonpathologic tissues, in particular the heart. The scDb-hERG1-β1 (i) downregulates the formation of the hERG1/β1 complex, (ii) inhibits Akt phosphorylation and HIF-1α expression, and (iii) decreases cell survival, proliferation, and migration in vitro These effects only occur in cancer cells (either colon, pancreatic, or breast), but not in normal cells. In vivo, the scDb-hERG1-β1 shows a good pharmacokinetic profile, with a half-life of 13.5 hours and no general, cardiac, or renal toxicity when injected intravenously up to the dose of 8 mg/kg. The scDb-hERG1-β1 accumulates into subcutaneous xenografted tumors, arising from either colon or pancreatic human cancer cells, and induces a reduction of tumor growth and vascularization. Overall, the scDb-hERG1-β1 represents an innovative single-chain bispecific antibody for therapeutic applications in solid cancers that overexpress the hERG1/β1 integrin signaling complex.

G protein-coupled receptor kinase 2 modifies the cellular reaction to cisplatin through interactions with NADPH oxidase 4.

G protein-coupled receptor kinases (GRKs), in addition to their role in modulating signal transduction mechanisms associated with activated G protein-coupled receptors (GPCRs), can also interact with many non-GPCR proteins to mediate cellular responses to chemotherapeutics. The rationale for this study is based on the presumption that GRK2 modulates the responses of cancer cells to the chemotherapeutic cisplatin. In this report, we show that GRK2 modulates the responses of cancer cells to cisplatin. Cervical cancer HeLa cells stably transfected with GRK2 shRNA, to decrease GRK2 protein expression, show increased sensitivity to cisplatin. Of interest, these cells also show increased accumulation of NADPH, associating with decreased NADP buildup, at low concentrations of cisplatin tested. These changes in NADPH and NADP levels are also observed in the breast cancer MDA MB 231 cells, which has lower endogenous GRK2 protein expression levels, but not BT549, a breast cancer cell line with higher GRK2 protein expression. This effect of NADPH accumulation may be associated with a decrease in NADPH oxidase 4 (NOX4) protein expression, which is found to correlate with GRK2 protein expression in cancer cells-a relationship which mimics that observed in cardiomyocytes. Furthermore, like in cardiomyocytes, GRK2 and NOX4 interact to form complexes in cancer cells. Collectively, these results suggest that GRK2 interacts with NOX4 to modify cisplatin sensitivity in cancer cells and may also factor into the success of cisplatin-based regimens.

Elemental mapping of half-sandwich azopyridine osmium arene complexes in cancer cells

Nanofocused synchrotron X-ray fluorescence and inductively coupled plasma-mass spectrometry provide insights into time-dependent ligand exchange reactions of organo-osmium anticancer complexes in cancer cells. Created with Biorender.com.

Pathway Engineering of Anthracyclines: Blazing Trails in Natural Product Glycodiversification

The anthracyclines are structurally diverse anticancer natural products that bind to DNA and poison the topoisomerase II-DNA complex in cancer cells. Rational modifications in the deoxysugar functionality are especially advantageous for synthesizing drugs with improved potency. Combinatorial biosynthesis of glycosyltransferases and deoxysugar synthesis enzymes is indispensable for the generation of glycodiversified anthracyclines. This Synopsis considers recent advances in glycosyltransferase structural biology and site-directed mutagenesis, pathway engineering, and deoxysugar combinatorial biosynthesis with a focus on the generation of "new-to-nature" anthracycline analogues.

Synthesis, antiproliferative activity in cancer cells and DNA interaction studies of [Pt(cis-1,3-diaminocycloalkane)Cl2] analogs

The search for more effective platinum anticancer drugs has led to the design, synthesis, and preclinical testing of hundreds of new platinum complexes. This search resulted in the recognition and subsequent FDA approval of the third-generation Pt(II) anticancer drug, [Pt(1,2-diaminocyclohexane)(oxalate)], oxaliplatin, as an effective agent in treating colorectal and gastrointestinal cancers. Another promising example of the class of anticancer platinum(II) complexes incorporating the Pt(1,n-diaminocycloalkane) moiety is kiteplatin ([Pt(cis-1,4-DACH)Cl2], DACH = diaminocyclohexane). We report here our progress in evaluating the role of the cycloalkyl moiety in these complexes focusing on the synthesis, characterization, evaluation of the antiproliferative activity in tumor cells and studies of the mechanism of action of new [Pt(cis-1,3-diaminocycloalkane)Cl2] complexes wherein the cis-1,3-diaminocycloalkane group contains the cyclobutyl, cyclopentyl, and cyclohexyl moieties. We demonstrate that [Pt(cis-1,3-DACH)Cl2] destroys cancer cells with greater efficacy than the other two investigated 1,3-diamminocycloalkane derivatives, or cisplatin. Moreover, the investigated [Pt(cis-1,3-diaminocycloalkane)Cl2] complexes show selectivity toward tumor cells relative to non-tumorigenic normal cells. We also performed several mechanistic studies in cell-free media focused on understanding some early steps in the mechanism of antitumor activity of bifunctional platinum(II) complexes. Our data indicate that reactivities of the investigated [Pt(cis-1,3-diaminocycloalkane)Cl2] complexes and cisplatin with glutathione and DNA binding do not correlate with antiproliferative activity of these platinum(II) complexes in cancer cells. In contrast, we show that the higher antiproliferative activity in cancer cells of [Pt(cis-1,3-DACH)Cl2] originates from its highest hydrophobicity and most efficient cellular uptake.

Abstract A09: CD4 T cells mediated protumorigenic pathway in pancreatic cancer

Abstract A hallmark of pancreatic ductal adenocarcinoma (PDAC) is an exuberant stroma comprised of diverse cell types that enable or suppress tumor progression. This Kras*-driven heterotypic signaling circuit in the early and advance tumor microenvironment enables cooperative protumorigenic interactions in early steps of the neoplastic transformation process as well as in promoting growth of established tumors, providing novel therapeutic targets in the Kras* pathway for this intractable disease. Here, we explored the role of Kras* in protumorigenic signaling interactions between cancer cells and host cells in tumor microenvironment. Early-stage malignant lesions and advanced tumors show close proximity of Kras* expressing cancer cells and abundant invading TH2 (CD4+ Gata-3+) cells, which can produce IL-4 and IL-13. Single-cell analysis of low- and high-grade IPMN and PDAC patient samples revealed CD4+ T-cell infiltration and higher percentage of the CD4+ T cells are Gata3+ TH2 subtypes compared to T-bet+ TH1 cells. Similarly, imaging mass spectrometry analysis showed presence of CD4+ T cells in close proximity to cancer cells. Mechanistic studies identified Jak1-Stat6 as the primary signaling pathway downstream of IL-4/IL-13 activated IL-4 receptor complexes in cancer cells. Integrated transcriptomic and chromatin immunoprecipitation sequencing (ChIP-seq) profiling identified cMyc, specifically its upstream enhancer element, as a direct target of activated Stat6. Metabolomic analysis demonstrated that IL-4/13STAT6cMyc upregulation drives increased glycolysis along with upregulated expression of key glycolysis enzymes, forging a link between Kras*-driven glycolysis via paracrine activation of cMyc. Citation Format: Prasenjit Dey, Jun Li, Jianhua Zhang, Huamin Wang, Wen-Ting Liao, Vincent P Bernard, Giannicola Genovese, Anirban Maitra, Anders Strom, Ronald A. DePinho. CD4 T cells mediated protumorigenic pathway in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A09.

Telomerase promotes formation of a telomere protective complex in cancer cells.

Telomerase is a ribonucleoprotein complex that catalyzes addition of telomeric DNA repeats to maintain telomeres in replicating cells. Here, we demonstrate that the telomerase protein hTERT performs an additional role at telomeres that is independent of telomerase catalytic activity yet essential for telomere integrity and cell proliferation. Short-term depletion of endogenous hTERT reduced the levels of heat shock protein 70 (Hsp70-1) and the telomere protective protein Apollo at telomeres, and induced telomere deprotection and cell cycle arrest, in the absence of telomere shortening. Short-term expression of hTERT promoted colocalization of Hsp70-1 with telomeres and Apollo and reduced numbers of deprotected telomeres, in a manner independent of telomerase catalytic activity. These data reveal a previously unidentified noncanonical function of hTERT that promotes formation of a telomere protective complex containing Hsp70-1 and Apollo and is essential for sustained proliferation of telomerase-positive cancer cells, likely contributing to the known cancer-promoting effects of both hTERT and Hsp70-1.

Triazolyl RuII, RhIII, OsII, and IrIII Complexes as Potential Anticancer Agents: Synthesis, Structure Elucidation, Cytotoxicity, and DNA Model Interaction Studies

Novel conjugated ruthenium(II), rhodium(III), and iridium(III) organometallic complexes of triazoles 1 and 2 synthesized and evaluated for anticancer activity against cervical (HeLa), kidney (HEK293), nonsmall lung cancer (A549), and leukemia (MT4) cancer cell lines are reported herein. The complexes are κ2-N,C coordinated and have the formula [ML(Ar)Cl] (where L is 1-benzyl-4-phenyl-1H-1,2,3-triazole for 1 and 1-benzyl-4-hydroxymethyl-1H-1,2,3-triazole for 2, Ar is p-cymene for RuII and OsII and Cp* for RhIII and IrIII, and M is metal). NMR studies, including HMBC and NOESY, were employed to unambiguously elucidate their structures and provide their conformational information in solution. Single-crystal X-ray diffraction data have been used to establish the solid-state structures of selected complexes, which further confirm the structural elucidation by NMR. Dynamic NMR studies, such as differential transferred NOE, have been employed to distinguish between isomers 1a_I and 1a_II of ruthenium(II) complexes of triazole 1. The rhodium(III) (1b) and iridium(III) (1c) complexes exhibited good cytotoxic activities (CC50 = 4–6 μM) comparable to that of the drug auranofin against lung cancer A549 cell lines (CC50 = 4.69 μM). While triazole 1 based ruthenium(II) (1a) and osmium(II) (1d) complexes displayed modest anticancer activities against HeLa and HEK293 cell lines, the analogous rhodium(III) and iridium(III) complexes exhibited good potential (CC50 = 9–54 μM versus auranofin (3–9 μM)) against these cancer cell lines. Insightful NMR studies on the interaction between the DNA model guanosine 5′-GMP and the complexes 1b,c reveal a possible mode of action of the aquated complexes involving carbenylation with DNA bases or purines through the triazolyl proton H-5. From the findings, these complexes could possibly confer their cytotoxic activities through intercalation with the DNA of pathological cells. Therefore, carbenylation of the triazolylrhodium(III) and iridium(III) complexes by DNA guanosine 5′-GMP is proposed as a novel mode of DNA intercalation of these complexes in cancer cells.