The AKT inhibitor AZD5363 elicits synthetic lethality in ARID1A-deficient gastric cancer cells via induction of pyroptosis.

BACKGROUND: Gastric cancer (GC) is a deadly disease with poor overall survival and limited therapeutic options. Genetic alterations such as mutations and/or deletions in chromatin remodeling gene AT-rich interactive domain 1 A (ARID1A) occur frequently in GC. Although ARID1A mutations/deletions are not a druggable target for conventional treatments, novel therapeutic strategies based on a synthetic lethal approach may be effective for the treatment of ARID1A-deficient cancers. METHODS: A kinase inhibitor library containing 551 compounds was screened in ARID1A isogenic GC cells for the ability to induce synthetic lethality effect. Selected hits' activity was validated, and the mechanism of the most potent candidate drug, AKT inhibitor AD5363 (capivasertib), on induction of the synthetic lethality with ARID1A deficiency was investigated. RESULTS: After robust vulnerability screening of 551 diverse protein kinase inhibitors, we identified the AKT inhibitor AZD5363 as being the most potent lead compound in inhibiting viability of ARID1A-/- cells. A synthetic lethality between loss of ARID1A expression and AKT inhibition by AZD5363 was validated in both GC cell model system and xenograft model. Mechanistically, AZD5363 treatment induced pyroptotic cell death in ARID1A-deficient GC cells through activation of the Caspase-3/GSDME pathway. Furthermore, ARID1A occupied the AKT gene promoter and regulated its transcription negatively, thus the GC cells deficient in ARID1A showed increased expression and phosphorylation of AKT. CONCLUSIONS: Our study demonstrates a novel synthetic lethality interaction and unique mechanism between ARID1A loss and AKT inhibition, which may provide a therapeutic and mechanistic rationale for targeted therapy on patients with ARID1A-defective GC who are most likely to be beneficial to AZD5363 treatment.

Luxeptinib interferes with LYN-mediated activation of SYK and modulates BCR signaling in lymphoma.

Luxeptinib (LUX) is a novel oral kinase inhibitor that inhibits FLT3 and also interferes with signaling from the BCR and cell surface TLRs, as well as activation of the NLRP3 inflammasome. Ongoing clinical trials are testing its activity in patients with lymphoma and AML. This study sought to refine understanding of how LUX modulates the earliest steps downstream of the BCR following its activation by anti-IgM in lymphoma cells in comparison to ibrutinib (IB). LUX decreased anti-IgM-induced phosphorylation of BTK at Y551 and Y223 but its ability to reduce phosphorylation of kinases further upstream suggests that BTK is not the primary target. LUX was more effective than IB at reducing both steady state and anti-IgM-induced phosphorylation of LYN and SYK. LUX decreased phosphorylation of SYK (Y525/Y526) and BLNK (Y96) which are necessary regulators of BTK activation. Further upstream, LUX blunted the anti-IgM-induced phosphorylation of LYN (Y397) whose activation is required for phosphorylation of SYK and BLNK. These results indicate that LUX is targeting autophosphorylation of LYN or a step further upstream of LYN in the cascade of signal generated by BCR and that it does so more effectively than IB. The fact that LUX has activity at or upstream of LYN is important because LYN is an essential signaling intermediate in multiple cellular signaling processes that regulate growth, differentiation, apoptosis, immunoregulation, migration and EMT in normal and cancer cells.

Exploiting the Receptor-Binding Domains of R-Spondin 1 to Target Leucine-Rich Repeat-Containin G-Coupled Protein Receptor 5-Expressing Stem Cells in Ovarian Cancer.

Leucine-rich repeat-containing G-protein-coupled receptor (LGR5) and LGR6 mark epithelial stem cells in normal tissues and tumors. They are expressed by stem cells in the ovarian surface and fallopian tube epithelia from which ovarian cancer arises. High-grade serous ovarian cancer is unique in expressing unusually high levels of LGR5 and LGR6 mRNA. R-spondins are the natural ligands for LGR5 and LGR6 to which they bind with nanomolar affinity. To target stem cells in ovarian cancer, we used the sortase reaction to site-specifically conjugate the potent cytotoxin monomethyl auristatin E (MMAE) via a protease sensitive linker to the two furin-like domains of RSPO1 (Fu1-Fu2) that mediate its binding to LGR5 and LGR6 and their co-receptors Zinc And Ring Finger 3 and Ring Finger Protein 43 via a protease-cleavable linker. An immunoglobulin Fc domain added to the N-terminal end served to dimerize the receptor-binding domains so that each molecule carries two MMAE. The resulting molecule, FcF2-MMAE, demonstrated: 1) selective LGR5-dependent low nanomolar cytotoxicity against ovarian cancer cells in vitro; 2) selectivity that was dependent on binding to both the LGR receptors and ubiquitin ligase co-receptors; 3) favorable stability and plasma pharmacokinetic properties when administered intravenously with an elimination half-life of 29.7 hours; 4) selective inhibition of LGR5-rich as opposed to isogenic LGR5-poor tumors in vivo; and, 5) therapeutic efficacy in three aggressive wild-type human ovarian cancer xenograft models. These results demonstrate the successful use of the Fu1-Fu2 domain of RSPO1 as a drug carrier and the ability of FcF2-MMAE to target cells in tumors that express stem cell markers. SIGNIFICANCE STATEMENT: FcF2-MMAE is a novel cancer therapeutic that exploits the high-affinity binding domains of RSPO1 to target monomethyl auristatin E to tumor stem cells that express LGR5. FcF2-MMAE has low nanomolar LGR5-dependent cytotoxicity in vitro, favorable pharmacokinetics, and differential efficacy in an isogenic LGR5-poor versus LGR5-rich ovarian cancer xenograft model when given on a weekly schedule.

Single-cell characterization of step-wise acquisition of carboplatin resistance in ovarian cancer.

The molecular underpinnings of acquired resistance to carboplatin are poorly understood and often inconsistent between in vitro modeling studies. After sequential treatment cycles, multiple isogenic clones reached similar levels of resistance, but significant transcriptional heterogeneity. Gene-expression based virtual synchronization of 26,772 single cells from 2 treatment steps and 4 resistant clones was used to evaluate the activity of Hallmark gene sets in proliferative (P) and quiescent (Q) phases. Two behaviors were associated with resistance: (1) broad repression in the P phase observed in all clones in early resistant steps and (2) prevalent induction in Q phase observed in the late treatment step of one clone. Furthermore, the induction of IFNα response in P phase or Wnt-signaling in Q phase were observed in distinct resistant clones. These observations suggest a model of resistance hysteresis, where functional alterations of the P and Q phase states affect the dynamics of the successive transitions between drug exposure and recovery, and prompts for a precise monitoring of single-cell states to develop more effective schedules for, or combination of, chemotherapy treatments.

Luxeptinib (CG-806) Targets FLT3 and Clusters of Kinases Operative in Acute Myeloid Leukemia.

Luxeptinib (CG-806) simultaneously targets FLT3 and select other kinase pathways operative in myeloid malignancies. We investigated the range of kinases it inhibits, its cytotoxicity landscape ex vivo with acute myeloid leukemia (AML) patient samples, and its efficacy in xenograft models. Luxeptinib inhibits wild-type (WT) and many of the clinically relevant mutant forms of FLT3 at low nanomolar concentrations. It is a more potent inhibitor of the activity of FLT3-internal tandem duplication, FLT3 kinase domain and gatekeeper mutants than against WT FLT3. Broad kinase screens disclosed that it also inhibits other kinases that can drive oncogenic signaling and rescue pathways, but spares kinases known to be associated with clinical toxicity. In vitro profiling of luxeptinib against 186 AML fresh patient samples demonstrated greater potency relative to other FLT3 inhibitors, including cases with mutations in FLT3, isocitrate dehydrogenase-1/2, ASXL1, NPM1, SRSF2, TP53, or RAS, and activity was documented in a xenograft AML model. Luxeptinib administered continuously orally every 12 hours at a dose that yielded a mean Cmin plasma concentration of 1.0 ± 0.3 µmol/L (SEM) demonstrated strong antitumor activity but no myelosuppression or evidence of tissue damage in mice or dogs in acute toxicology studies. On the basis of these studies, luxeptinib was advanced into a phase I trial for patients with AML and myelodysplastic/myeloproliferative neoplasms.

Luxeptinib disables NLRP3 inflammasome-mediated IL-1ß release and pathways required for secretion of inflammatory cytokines IL-6 and TNFα.

Luxeptinib (CG-806) is an orally bioavailable multikinase inhibitor with nanomolar potency against select clusters of kinases including the BTK, FLT3, TRK, STE/MAPK and aurora kinase clusters. It is cytotoxic to primary malignant cells obtained from patients with AML, ALL, and CLL at lower concentrations than other BTK and FLT3 inhibitors, and has activity in AML and lymphoma xenografts at concentrations attainable in patients. Exposure of macrophages and monocytes to endotoxin triggers the release of IL-1ß through activation of the NLRP3 inflammasome and IL-6 and TNFα through transcriptional up-regulation. These cytokines are key components of the innate immune signaling network that plays a central role in the pathogenesis of multiple human diseases including cancer. Drugs that concurrently inhibit proliferation and inflammatory signaling pathways may provide better therapeutic efficacy. The aim of this study was to determine the extent to which luxeptinib interferes with the release of IL-1ß, IL-6 and TNFα from THP-1 monocytes and bone marrow-derived macrophages following endotoxin exposure and priming of the NLRP3 inflammasome. Luxeptinib inhibited the release of all 3 cytokines from THP-1 monocytes and macrophages at concentrations of 0.1 µM and above. Investigation of the mechanism disclosed that luxeptinib does not inhibit the assembly of the NLRP3 inflammasome but disables its ability to cleave and activate caspase-1 that is required for IL-1ß release. It also inhibits the kinases p38MAPK, ERK1/2, SAPK/JNK and activation of transcription factor NF-κBp65 with a concentration profile similar to its inhibition of cytokine release. IMPLICATIONS: The ability of luxeptinib to inhibit the NLRP3-mediated release of IL-1ß and pathways involved in the release of IL-6 and TNFα at concentrations which are well-tolerated in patients makes it a candidate for the treatment of inflammatory diseases and inflammation-associated resistance in cancer.

Therapeutic Targeting of Tumor Cells Rich in LGR Stem Cell Receptors.

LGR5 and LGR6 mark epithelial stem cells in many niches including the ovarian surface and fallopian tube epithelia from which ovarian cancer arises. Human ovarian cancers express these receptors at high levels and express one of their ligands, RSPO1, at levels uniquely higher than all other tumor types except mesothelioma. Reasoning that these receptors are also important to tumor stem cells, arming the LGR binding domain of RSPO1 with a cytotoxin may permit depletion of the tumor stem cells. The Fu1-Fu2 receptor binding domain of RSPO1 (R1FF), containing a sortase recognition sequence at the C-terminal end, was produced in bacteria and a single molecule of MMAE was attached to each R1FF through a val-cit-PAB linker using the sortase reaction, thus producing a homogeneous population of armed molecules. R1FF-MMAE demonstrated (1) selective LGR-dependent binding, uptake, and cytotoxicity; (2) low nM cytotoxicity to multiple types of human tumor cell lines in vitro; (3) favorable plasma pharmacokinetic properties when administered iv with an elimination half-life of 27.8 h; (4) favorable absorption from the peritoneal cavity; and (5) therapeutic activity in aggressive xenograft models of ovarian cancer in the absence of any weight loss or other adverse events. These results demonstrate that the Fu1-Fu2 domain of RSPO1 can be exploited to deliver a potent cytotoxin to tumor cells that express the LGR4-6 family of stem cell receptors.

WNT Signaling Driven by R-spondin 1 and LGR6 in High-grade Serous Ovarian Cancer.

BACKGROUND/AIM: R-spondins control WNT signaling and RSPO1 and LGR6, two of its receptors, are uniquely expressed at high levels in high-grade serous ovarian cancer (HGSOC). The aim of this study was to assess the interrelations between the expression of the RSPOs and LGRs in HGSOC and in the ovarian surface (OSE) and fallopian tube surface epithelium (FTSE) from which HGSOC arises. MATERIALS AND METHODS: Analysis of TCGA (HGSOC), CCLE (ovary), and other publicly accessed RNA-Seq data using UC San Diego Computational Cancer Analysis Library (CCAL) to perform differential expression analysis, association studies, and gene set inspection using the single-sample GSEA method. Additionally, we employed multiple publicly available databases including StringDB, Human Protein Atlas, and cBioPortal to aid the investigation. RESULTS: Among normal tissues, expression of RSPO1, LGR5 and LGR6 was highest in the fallopian tube. The relative levels of expression of the RSPOs and LGRs in the OSE and FTSE matched those in HGSOC. RSPO1 and LGR6 were highly co-expressed in all three tissues. Gene set enrichment analysis (GSEA) showed that expression of RSPO1 was strongly linked to the enrichment of three separate WNT-driven GO pathways. Analysis of genes that impacted overall survival identified two other immediately adjacent genes that control WNT signaling, KREMEN1 and ZNRF3 whose expression and copy number were coordinately linked. CONCLUSION: RSPO1 and LGR6 are coordinately expressed in HGSOC and the two normal tissues from which this tumor arises, and their expression is linked to WNT signaling pathways known the control cell fate and proliferation.

Securing the Payload, Finding the Cell, and Avoiding the Endosome: Peptide-Targeted, Fusogenic Porous Silicon Nanoparticles for Delivery of siRNA.

Despite the promise of ribonucleic acid interference therapeutics, the delivery of oligonucleotides selectively to diseased tissues in the body, and specifically to the cellular location in the tissues needed to provide optimal therapeutic outcome, remains a significant challenge. Here, key material properties and biological mechanisms for delivery of short interfering RNAs (siRNAs) to effectively silence target-specific cells in vivo are identified. Using porous silicon nanoparticles as the siRNA host, tumor-targeting peptides for selective tissue homing, and fusogenic lipid coatings to induce fusion with the plasma membrane, it is shown that the uptake mechanism can be engineered to be independent of common receptor-mediated endocytosis pathways. Two examples of the potential broad clinical applicability of this concept in a mouse xenograft model of ovarian cancer peritoneal carcinomatosis are provided: silencing the Rev3l subunit of polymerase Pol ζ to impair DNA repair in combination with cisplatin; and reprogramming tumor-associated macrophages into a proinflammatory state.

Tumor-Targeting, MicroRNA-Silencing Porous Silicon Nanoparticles for Ovarian Cancer Therapy.

Silencing of aberrantly expressed microRNAs (miRNAs or miRs) has emerged as one of the strategies for molecular targeted cancer therapeutics. In particular, miR-21 is an oncogenic miRNA overexpressed in many tumors, including ovarian cancer. To achieve efficient administration of anti-miR therapeutics, delivery systems are needed that can ensure local accumulation in the tumor environment, low systemic toxicity, and reduced adverse side effects. In order to develop an improved anti-miR therapeutic agent for the treatment of ovarian cancer, a nanoformulation is engineered that leverages biodegradable porous silicon nanoparticles (pSiNPs) encapsulating an anti-miR-21 locked nucleic acid payload and displaying a tumor-homing peptide for targeted distribution. Targeting efficacy, miR-21 silencing, and anticancer activity are optimized in vitro on a panel of ovarian cancer cell lines, and a formulation of anti-miR-21 in a pSiNP displaying the targeting peptide CGKRK is identified for in vivo evaluation. When this nanoparticulate agent is delivered to mice bearing tumor xenografts, a substantial inhibition of tumor growth is achieved through silencing of miR-21. This study presents the first successful application of tumor-targeted anti-miR porous silicon nanoparticles for the treatment of ovarian cancer in a mouse xenograft model.

Tumor Retention of Enzyme-Responsive Pt(II) Drug-Loaded Nanoparticles Imaged by Nanoscale Secondary Ion Mass Spectrometry and Fluorescence Microscopy.

In nanomedicine, determining the spatial distribution of particles and drugs, together and apart, at high resolution within tissues, remains a major challenge because each must have a different label or detectable feature that can be observed with high sensitivity and resolution. We prepared nanoparticles capable of enzyme-directed assembly of particle therapeutics (EDAPT), containing an analogue of the Pt(II)-containing drug oxaliplatin, an 15N-labeled monomer in the hydrophobic block of the backbone of the polymer, the near-infrared dye Cy5.5, and a peptide that is a substrate for tumor metalloproteinases in the hydrophilic block. When these particles reach an environment rich in tumor associated proteases, the hydrophilic peptide substrate is cleaved, causing the particles to accumulate through a morphology transition, locking them in the tumor extracellular matrix. To evaluate the distribution of drug and EDAPT carrier in vivo, the localization of the isotopically labeled polymer backbone was compared to that of Pt by nanoscale secondary ion mass spectrometry (NanoSIMS). The correlation of NanoSIMS with super-resolution fluorescence microscopy revealed the release of the drug from the nanocarrier and colocalization with cellular DNA within tumor tissue. The results confirmed the dependence of particle accumulation and Pt(II) drug delivery on the presence of a Matrix Metalloproteinase (MMP) substrate and demonstrated antitumor activity. We conclude that these techniques are powerful for the elucidation of the localization of cargo and carrier, and enable a high-resolution assessment of their performance following in vivo delivery.

APTO-253 Is a New Addition to the Repertoire of Drugs that Can Exploit DNA BRCA1/2 Deficiency.

APTO-253 is a small molecule with antiproliferative activity against cell lines derived from a wide range of human malignancies. We sought to determine the mechanisms of action and basis for resistance to APTO-253 so as to identify synthetic lethal interactions that can guide combination studies. The cellular pharmacology of APTO-253 was analyzed in Raji lymphoma cells and a subline selected for resistance (Raji/253R). Using LC/MS/ESI analysis, APTO-253 was found to convert intracellularly to a complex containing one molecule of iron and three molecules of APTO-253 [Fe(253)3]. The intracellular content of Fe(253)3 exceeded that of the native drug by approximately 18-fold, and Fe(253)3 appears to be the most active form. Treatment of cells with APTO-253 caused DNA damage, which led us to ask whether cells deficient in homologous recombination (i.e., loss of BRCA1/2 function) were hypersensitive to this drug. It was found that loss of either BRCA1 or BRCA2 function in multiple isogenic paired cell lines resulted in hypersensitivity to APTO-253 of a magnitude similar to the effects of PARP inhibitors, olaparib. Raji cells selected for 16-fold acquired resistance had 16-fold reduced accumulation of Fe(253)3 RNA-seq analysis revealed that overexpression of the ABCG2 drug efflux pump is a key mechanism of resistance. ABCG2-overexpressed HEK-293 cells were resistant to APTO-253, and inhibition of ABCG2 reversed resistance to APTO-253 in Raji/253R. APTO-253 joins the limited repertoire of drugs that can exploit defects in homologous recombination and is of particular interest because it does not produce myelosuppression. Mol Cancer Ther; 17(6); 1167-76. ©2018 AACR.

APTO-253 Stabilizes G-quadruplex DNA, Inhibits MYC Expression, and Induces DNA Damage in Acute Myeloid Leukemia Cells.

APTO-253 is a phase I clinical stage small molecule that selectively induces CDKN1A (p21), promotes G0-G1 cell-cycle arrest, and triggers apoptosis in acute myeloid leukemia (AML) cells without producing myelosuppression in various animal species and humans. Differential gene expression analysis identified a pharmacodynamic effect on MYC expression, as well as induction of DNA repair and stress response pathways. APTO-253 was found to elicit a concentration- and time-dependent reduction in MYC mRNA expression and protein levels. Gene ontogeny and structural informatic analyses suggested a mechanism involving G-quadruplex (G4) stabilization. Intracellular pharmacokinetic studies in AML cells revealed that APTO-253 is converted intracellularly from a monomer to a ferrous complex [Fe(253)3]. FRET assays demonstrated that both monomeric APTO-253 and Fe(253)3 stabilize G4 structures from telomeres, MYC, and KIT promoters but do not bind to non-G4 double-stranded DNA. Although APTO-253 exerts a host of mechanistic sequelae, the effect of APTO-253 on MYC expression and its downstream target genes, on cell-cycle arrest, DNA damage, and stress responses can be explained by the action of Fe(253)3 and APTO-253 on G-quadruplex DNA motifs. Mol Cancer Ther; 17(6); 1177-86. ©2018 AACR.

LGR5 and LGR6 in stem cell biology and ovarian cancer.

Wnt signaling plays a fundamental role in patterning of the embryo and maintenance of stem cells in numerous epithelia. Epithelial stem cells are closeted in niches created by surrounding differentiated cells that express secreted Wnt and R-spondin proteins that influence proliferation rate and fate determination of stem cell daughters. R-spondins act through the LGR receptors to enhance Wnt signaling. This close association of stem cells with more differentiated regulatory cells expressing Wnt-pathway ligands is a feature replicated in all of the epithelial stem cell systems thus far examined. How the stem cell niche operates through these short-range interactions is best understood for the crypts of the gastrointestinal epithelium and skin. Less well understood are the stem cells that function in the ovarian surface epithelium (OSE) and fallopian tube epithelium (FTE). While the cuboidal OSE appears to be made up of a single cell type, the cells of the FTE progress through a life cycle that involves differentiation into ciliated and secretory subtypes that are eventually shed into the lumen in a manner similar to the gastrointestinal epithelium. Available evidence suggests that high grade serous ovarian carcinoma (HGSOC) originates most often from stem cells in the FTE and that Wnt signaling augmented by LGR6 supports tumor development and progression. This review summarizes current information on LGR5 and LGR6 in the OSE and FTE and how their niches are organized relative to that of the gastrointestinal epithelium and skin.

A phase I pharmacokinetic study of intraperitoneal bortezomib and carboplatin in patients with persistent or recurrent ovarian cancer: An NRG Oncology/Gynecologic Oncology Group study.

PURPOSE: Intraperitoneal (IP) therapy improves survival compared to intravenous (IV) treatment for women with newly diagnosed, optimally cytoreduced, ovarian cancer. However, the role of IP therapy in recurrent disease is unknown. Preclinical data demonstrated IP administration of the proteasome inhibitor, bortezomib prior to IP carboplatin increased tumor platinum accumulation resulting in synergistic cytotoxicity. We conducted this phase I trial of IP bortezomib and carboplatin in women with recurrent disease. METHODS: Women with recurrent ovarian cancer were treated with escalating doses of IP bortezomib - in combination with IP carboplatin (AUC 4 or 5) every 21days for 6cycles. Pharmacokinetics of both agents were evaluated in cycle 1. RESULTS: Thirty-three women participated; 32 were evaluable for safety. Two patients experienced dose-limiting toxicity (DLT) at the first dose level (carboplatin AUC 5, bortezomib 0.5mg/m2), prompting carboplatin reduction to AUC 4 for subsequent dose levels. With carboplatin dose fixed at AUC 4, bortezomib was escalated from 0.5 to 2.5mg/m2 without DLT. Grade 3/4 related toxicities included abdominal pain, nausea, vomiting, and diarrhea which were infrequent. The overall response rate in patients with measurable disease (n=21) was 19% (1 complete, 3 partial). Cmax and AUC in peritoneal fluid and plasma increased linearly with dose, with a favorable exposure ratio of the peritoneal cavity relative to peripheral blood plasma. CONCLUSION: IP administration of this novel combination was feasible and showed promising activity in this phase I trial of heavily pre-treated women with ovarian cancer. Further evaluation of this IP combination should be conducted.

Copper transporters and chaperones CTR1, CTR2, ATOX1, and CCS as determinants of cisplatin sensitivity.

The development of resistance to cisplatin (cDDP) is commonly accompanied by reduced drug uptake or increased efflux. Previous studies in yeast and murine embryonic fibroblasts have reported that the copper (Cu) transporters and chaperones participate in the uptake, efflux, and intracellular distribution of cDDP. However, there is conflicting data from studies in human cells. We used CRISPR-Cas9 genome editing to individually knock out the human copper transporters CTR1 and CTR2 and the copper chaperones ATOX1 and CCS. Isogenic knockout cell lines were generated in both human HEK-293T and ovarian carcinoma OVCAR8 cells. All knockout cell lines had slowed growth compared to parental cells, small changes in basal Cu levels, and varying sensitivities to Cu depending on the gene targeted. However, all of the knockouts demonstrated only modest 2 to 5-fold changes in cDDP sensitivity that did not differ from the range of sensitivities of 10 wild type clones grown from the same parental cell population. We conclude that, under basal conditions, loss of CTR1, CTR2, ATOX1, or CCS does not produce a change in cisplatin sensitivity that exceeds the variance found within the parental population, suggesting that they are not essential to the mechanism by which cDDP enters these cell lines and is transported to the nucleus.

Cellular Delivery of Nanoparticles Revealed with Combined Optical and Isotopic Nanoscopy.

Direct polymerization of an oxaliplatin analogue was used to reproducibly generate amphiphiles in one pot, which consistently and spontaneously self-assemble into well-defined nanoparticles (NPs). Despite inefficient drug leakage in cell-free assays, the NPs were observed to be as cytotoxic as free oxaliplatin in cell culture experiments. We investigated this phenomenon by super-resolution fluorescence structured illumination microscopy (SIM) and nanoscale secondary ion mass spectrometry (NanoSIMS). In combination, these techniques revealed NPs are taken up via endocytic pathways before intracellular release of their cytotoxic cargo. As with other drug-carrying nanomaterials, these systems have potential as cellular delivery vehicles. However, high-resolution methods to track nanocarriers and their cargo at the micro- and nanoscale have been underutilized in general, limiting our understanding of their interactions with cells and tissues. We contend this type of combined optical and isotopic imaging strategy represents a powerful and potentially generalizable methodology for cellular tracking of nanocarriers and their cargo.

The copper transporter 1 (CTR1) is required to maintain the stability of copper transporter 2 (CTR2).

Mammalian cells have two influx Cu transporters that form trimers in membranes. CTR1 is the high affinity transporter that resides largely in the plasma membrane, and CTR2 is the low affinity transporter that is primarily associated with vesicular structures inside the cell. The major differences between CTR1 and CTR2 are that CTR1 contains a HIS/MET-rich domain N-terminal of the METS that participate in the first two stacked rings that form the pore, and a longer C-terminal tail that includes a Cu binding HIS-CYS-HIS (HCH) motif right at the end. It has been reported that CTR1 and CTR2 are physically associated with each other in the cell. We used the CRISPR-Cas9 technology to knock out either CTR1 or CTR2 in fully malignant HEK293T and OVCAR8 human ovarian cancer cells to investigate the interaction of CTR1 and CTR2. We report here that the level of CTR2 protein is markedly decreased in CTR1 knockout clones while the CTR2 transcript level remains unchanged. CTR2 was found to be highly ubiquitinated in the CTR1 knock out cells, and inhibition of the proteasome prevented the degradation of CTR2 when CTR1 was not present while inhibition of autophagy had no effect. Re-expression of CTR1 rescued CTR2 from degradation in the CTR1 knockout cells. We conclude that CTR1 is essential to maintain the stability of CTR2 and that in the absence of CTR1 CTR2 is degraded by the proteasome. This reinforces the concept that the functions of CTR1 and CTR2 are inter-dependent within the Cu homeostasis system.

Cisplatin inhibits MEK1/2.

Cisplatin (cDDP) is known to bind to the CXXC motif of proteins containing a ferrodoxin-like fold but little is known about its ability to interact with other Cu-binding proteins. MEK1/2 has recently been identified as a Cu-dependent enzyme that does not contain a CXXC motif. We found that cDDP bound to and inhibited the activity of recombinant MEK1 with an IC50 of 0.28 µM and MEK1/2 in whole cells with an IC50 of 37.4 µM. The inhibition of MEK1/2 was relieved by both Cu+1 and Cu+2 in a concentration-dependent manner. cDDP did not inhibit the upstream pathways responsible for activating MEK1/2, and did not cause an acute depletion of cellular Cu that could account for the reduction in MEK1/2 activity. cDDP was found to bind MEK1/2 in whole cells and the extent of binding was augmented by supplementary Cu and reduced by Cu chelation. Molecular modeling predicts 3 Cu and cDDP binding sites and quantum chemistry calculations indicate that cDDP would be expected to displace Cu from each of these sites. We conclude that, at clinically relevant concentrations, cDDP binds to and inhibits MEK1/2 and that both the binding and inhibitory activity are related to its interaction with Cu bound to MEK1/2. This may provide the basis for useful interactions of cDDP with other drugs that inhibit MAPK pathway signaling.