Intratumoral Copper Modulates PD-L1 Expression and Influences Tumor Immune Evasion.

Therapeutic checkpoint antibodies blocking programmed death receptor 1/programmed death ligand 1 (PD-L1) signaling have radically improved clinical outcomes in cancer. However, the regulation of PD-L1 expression on tumor cells is still poorly understood. Here we show that intratumoral copper levels influence PD-L1 expression in cancer cells. Deep analysis of the The Cancer Genome Atlas database and tissue microarrays showed strong correlation between the major copper influx transporter copper transporter 1 (CTR-1) and PD-L1 expression across many cancers but not in corresponding normal tissues. Copper supplementation enhanced PD-L1 expression at mRNA and protein levels in cancer cells and RNA sequencing revealed that copper regulates key signaling pathways mediating PD-L1-driven cancer immune evasion. Conversely, copper chelators inhibited phosphorylation of STAT3 and EGFR and promoted ubiquitin-mediated degradation of PD-L1. Copper-chelating drugs also significantly increased the number of tumor-infiltrating CD8+ T and natural killer cells, slowed tumor growth, and improved mouse survival. Overall, this study reveals an important role for copper in regulating PD-L1 and suggests that anticancer immunotherapy might be enhanced by pharmacologically reducing intratumor copper levels. SIGNIFICANCE: These findings characterize the role of copper in modulating PD-L1 expression and contributing to cancer immune evasion, highlighting the potential for repurposing copper chelators as enhancers of antitumor immunity. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/19/4129/F1.large.jpg.

Evaluation of umbilical cord blood CD34 (+) hematopoietic stem cell expansion in co-culture with bone marrow mesenchymal stem cells in the presence of TEPA.

BACKGROUND: During the last three decades hematopoietic stem cells (HSC) have become a standard protocol for the treatment of many hematologic malignancies and non-malignant disorders. Umbilical cord blood (UCB), as a source of HSCs, has many advantages compared with other sources. One major drawback in using this source in treatment of adult patients is the low HSC dose available. Ex vivo expansion of HSCs is a solution to overcome this limitation. In this study we used TEPA, as a Cu chelator, and human bone marrow (BM) mesenchymal stem cells (MSCs) to investigate expansion rate of UCB-HSCs. MATERIALS AND METHODS: CB-HSCs were isolated using miniMACS magnetic separation system. We cultured the enriched CD34(+)cells in various conditions: culture condition A, supplemented only with recombinant cytokines; culture condition B, supplemented with BM-MSCs as a cell feeder layer and recombinant cytokines; culture condition C, supplemented with recombinant cytokines and TEPA; culture condition D, supplemented with recombinant cytokines, BM-MSCs as a cell feeder layer and TEPA. In order to evaluate the HSC expansion, we performed cell count, analysis of CD34(+) expression by flow cytometry, and colony-forming cell assay on Day 10 after culture. RESULTS: The most fold increase in CD34(+) cell, total cell, and total colony numbers was observed in culture condition D (110.11 ± 15.3, 118.5 ± 21, and 172.9 ± 44.7, respectively) compared to other conditions. CONCLUSION: The results showed that co-culture of HSCs with BM-MSCs in the presence of copper chelating agent (TEPA) could dramatically increase expansion rate of UCB-HSCs. Therefore, this strategy could be useful for HSC expansion.

In the search for new anticancer drugs. 29. A study on the correlation of lipophilicities, ionization constants and anticancer activities of aminoxyl labeled TEPA congeners.

Ionization constants for a series of sterically hindered pyrrolidine, pyrroline and piperidine derivatives were determined by potentiometric titrations. The pKa values for the secondary amines as a group ranged from about 7.7 to 11.7, whereby the ring size had no decisive effect on the values. The corresponding hydroxylamine derivatives as a group had distinctly lower pKa values than the amine derivatives ranging between about 4.0 and 6.3. It was shown using the Henderson-Hasselbalch equation that at physiological pH, arbitrarily chosen 6, 7 and 8, the amine derivatives would exist mainly in the protonated form, whereas the hydroxylamine derivatives would be expected to be mainly in the unprotonated form. In contrast, the 4-hydroxy-2,2,6,6-tetramethylpiperdin-1-oxyl radical, under analogous conditions, was a neutral species, i.e. it could not be titrated in aqueous media. On the basis of these results, it was hypothesized that the alkylating anticancer drugs of TEPA (N,N:N'N':N",N"-tri-1,2-ethanediylphosphorictriamide) type, containing sterically hindered carrier moieties, would be expected to permeate across cell membranes, and, consequently, exhibit anticancer activities according to the following sequence: spin-labeled drugs containing no titratable components > hydroxylamine derivatives > secondary amine congeners. This assumption is confirmed by good correlations of anticancer activities of these drugs with their pKa values, and the partition coefficients. The conclusion was reiterated that, in the quest for a rational design of anticancer drugs, the aim should be to construct agents with partition coefficients approaching the logarithm of zero, either from the negative or positive side, and pKa values as low as practically possible and applicable.

In the search for new anticancer drugs. 26. A comparison of anticancer activities of several TEPA, thio-TEPA, Seleno-TEPA, and azetidine analogs, including congeners containing an aminoxyl moiety.

A series of TEPA, Thio-TEPA, Seleno-TEPA, and azetidine analogs, including congeners containing an aminoxyl moiety, were synthesized and evaluated in vivo for anticancer activity against the murine lymphocytic leukemia P388. All aziridine derivatives were found to be active with an increase in life span ranging from 42% to 272%, and all azetidine analogs were rated as inactive with one marginal exception. An attempt was made to rationalize the results on the basis of the lipophilic properties of the compounds. The most active compound (8) possessed the most balanced lipophilic properties, corresponding to a log P value near zero.

Interaction of N,N',N''-triethylenethiophosphoramide and N,N',N''-triethylenephosphoramide with cellular DNA.

The antineoplastic agents N,N',N''-triethylenethiophosphoramide (thioTEPA) and N,N',N''-triethylenephosphoramide (TEPA) were studied for their interaction with the DNA of L1210 cells in the presence and absence of rat hepatic microsomes and NADPH. Alkaline elution was used to study 3 types of DNA lesions. When L1210 cells were incubated with thioTEPA alone, or with thioTEPA in the presence of microsomes and NADPH, no single-strand breaks were detected. However, incubation of L1210 cells for 2 h with thioTEPA, at concentrations greater than or equal to 100 microM, caused a dose-dependent increase in interstrand cross-linking that reached a maximum by 2 h after drug exposure. In the presence of rat hepatic microsomes and NADPH, this cross-linking was eliminated, but a different DNA lesion, alkali-labile sites, was produced. These alkali-labile sites were partially reparable with maximum repair achieved by 2 h after removal of drug. ThioTEPA was greater than 85% consumed by the microsomal incubation conditions employed, and TEPA was the only product of the microsomal metabolism of thioTEPA. Alkaline elution studies of L1210 cells that had been incubated with TEPA, alone or in the presence of microsomes and NADPH, demonstrated an elution pattern identical to that produced by thioTEPA in the presence of microsomes and NADPH. Lymphoblastoid cell lines derived from patients with Fanconi's anemia were far more sensitive to thioTEPA and mechlorethamine hydrochloride than were lymphoblasts derived from normal humans, but this hypersensitivity was not noted with TEPA or bleomycin. This is consistent with the known hypersensitivity of cells from patients with Fanconi's anemia to agents that produce interstrand cross-links and with the alkaline elution studies described above. In contrast, lymphoblastoid cell lines derived from patients with ataxia telangiectasia were no more sensitive to thioTEPA than were lymphoblasts derived from normal humans but were far more sensitive to bleomycin. One of these cell lines proved hypersensitive to TEPA, whereas the other was no more sensitive to TEPA than were lymphoblasts from normal humans. Our data imply that thioTEPA produces interstrand cross-links but that TEPA, the primary metabolite of thioTEPA, produces DNA lesions that are alkali labile.

The ratio of induced recessive lethals to ring-X loss has prognostic value in terms of functionality of chemical mutagens in Drosophila melanogaster.

For 25 mutagens in Drosophila the ratio was determined between the induction of sex-linked recessive lethals (SLRL) and the induction of ring-X loss in male adults. For small monofunctional alkylating agents this ratio increases with decreasing s-value from 1.8 for methyl methanesulfonate (MMS) to 27 for ethylnitrosourea (ENU). For multifunctional cross-linking agents, however, the ratio varies within relatively narrow limits, ranging from 0.15 for cisplatin to 0.07 for tris-(1-aziridinyl)phosphineoxide (TEPA), while for most agents the ratio is around 0.12. The number of reactive groups seems to be of minor importance for compounds with more than one functionality as bi- and tri-functional agents show similar ratios. The systemic difference in the ratios between mono- and multi-functional agents suggests that different mechanisms are involved in the induction of SLRLs and ring-X loss. For ethyleneimine (EI) and ethyleneoxide (EO) low ratios of 0.32 and 0.60 respectively were observed which do not correlate with their s-values. An alternative chromosome-breaking mechanism may be responsible for this deviation, possibly alkylation of the phosphate backbone of DNA, followed by an intramolecular displacement of one of the deoxyribose groups by the beta-amino or the beta-hydroxy group. It is felt that the considerable difference between the ratios for monofunctional and multifunctional agents may be of prognostic value and can be used to obtain information on the mechanisms of mutagens with 'unknown' action, provided that structural features are taken into account. Hexamethylphosphoramide (HMPA), hexamethylmelamine (HEMEL), tetramethylurea (TMU) and dimethylpropyleneurea (DMPU) all show SLRL: ring-X loss ratios that match those of multifunctional agents, 0.08, 0.12, 0.08, and 0.16, respectively. The ratios for the pyrrolizidine alkaloids monocrotalin and seniciphilline, 0.053 and 0.24 respectively, also correspond with this group of mutagens. The low ratios for formaldehyde, 2-chloro-acetaldehyde and 2-chloroethyl methanesulfonate, 0.30, 0.052 and 0.36 respectively, are indicative that cross-linking may attribute considerably to their mutagenic action in Drosophila. On the other hand, not all mutagens containing 2 reactive groups act as cross-linking agents. The ratio for 1,2-dibromoethane, 2.7, indicates that it may act as a monofunctional agent. This is in accordance with the proposed activation mechanism by glutathione S-transferase, producing a monofunctional half-mustard derivative (Rannug, 1980; van Bladeren et al., 1981).

In the search for new anticancer drugs. XX: A comparison of the in vitro growth inhibition of P388 cells by TEPA and N,N;N',N'-bis (1,2-ethanediyl)-N''-(1-oxyl-2,2,6,6-tetramethyl-4- piperidinylaminocarbonyl) phosphoric triamide and congeners.

We tested the in vitro growth inhibitory activity of TEPA, and three analogs against P388 murine lymphocytic leukemia cells in culture. The analogs consist of spin-labeled TEPA and two reduced forms containing the NH and NOH groups instead of the nitroxyl function. Spin label TEPA was obtained by replacing one of the aziridine groups in TEPA with spin-labeled urea. In a concentration range of 10(-6)-10(-5) M, only the reduced analog containing the NH group was active. That is, to achieve a 50% inhibition of cell growth, a five-fold excess in concentration of this analog (IC50 = 10 X 10(-6) M) was needed as compared to the parent compound TEPA (IC50 = 2 X 10(-6) M). These results are in contrast with those obtained in vivo against the same leukemia cell line, indicating inherent discrepancies between in vivo and in vitro evaluation of antitumor agents.

In the search for new anticancer drugs. 19. A predictive design of N,N:N',N':N'',N''-tri-1,2-ethanediylphosphorothioic triamide (TEPA) analogues.

The nitroxyl-labeled analogues of N,N:N',N':N",N"-tri-1,2-ethanediylphosphoric triamide (TEPA), N,N:N',N'-bis(1,2-ethanediyl)-N"-[[(2,2,6,6-tetramethyl-1-oxypiperidi n-4- yl)amino]carbonyl]phosphoric triamide (5a) and N,N:N',N'-bis(1,2-ethanediyl)-N"-[[(2,2,5,5-tetramethyl-1-oxypyrrolid in-3- yl)amino]carbonyl]phosphoric triamide (11a), possess therapeutic indexes that are 8-12 times higher than those of thio-TEPA (1) and TEPA (2). The introduction of methyl groups into the aziridine ring, or the replacement of the nitroxyl moiety with hydroxylamine or amine derivatives, or with an adamantane moiety, results in compounds of lesser activity. An attempt is made to rationalize these results using a lipophilicity scale. A predictive design pattern is established.

In the search for new anticancer drugs. X. N,N; N',N'-bis(1,2-ethanediyl)-N''-(1-oxyl-2,2,6,6-tetramethyl- 4-piperidinylaminocarbonyl) phosphoric triamide--a new potential anticancer drug of high activity and low toxicity.

A new nitroxyl labeled TEPA derivative 5 containing the urea bridge between the phosphorus and the nitroxyl moiety, and the congeners containing the NOH and NH groups instead of the nitroxyl function were synthesized, and tested in vivo on CD2F1 mice for anticancer activity against P388 and L1210. The nitroxyl compound is more active than the reduced forms. The nitroxyl compound 5 elicits 170% ILS at 90 mg/kg after 30 days and 439% ILSmax after 60 days against P388, and has a higher therapeutic ratio (26.4) than the clinically used Thio-TEPA (2.75). The LD50 of 5 is 270 mg/kg, while that of Thio-TEPA is 18 mg/kg. Consequently, the nitroxyl compound 5 is a promising new anticancer drug.

Cytochemical changes induced by apholate, tepa and hempa during vitellogenesis in Locusta migratoria.

Proteins (--SS, --SH, and NH2 groups; tyrosine, tryptophan and arginine), DNA, phospholipids and triglycerides showed a progressive decline in the oocytes of Locusta migratoria with increase in doses of apholate, tepa and hempa. Carbohydrates showed a decline only after higher doses of apholate and tepa whereas they were not affected at all with hempa. The pyroninophilia due to RNA in the oocytes decreased in the ooplasm of oocytes slightly, but increased in the cytoplasm and nucleoplasm of follicular epithelial cells with all the chemosterilants. The follicular epithelial cells degenerated in the mature oocytes, and yolk formation was inhibited.

[Effect of diiodobenzotefa on the functionally different subpopulations of T-lymphocytes]. / Deistvie diiodbenzotéfa na funktsional'no razlichnye subpopuliatsii T-limfotsitov.

Diiodobenzo-tepa (DIB) was given orally to CBA mice in a dose of 25 mg/kg for 3 successive days. The number of nucleus-containing cells decreased 3.9 fold in the thymus and 1.4 fold in the bone marrow. In experiments on transplantation of lymphoid cells to intact or lethally irradiated (CBA X C57BL/6J)F1 mice treated with DIB this substance did not influence the helper activity of T lymphocytes but inhibited the activity or B and T lymphocytes, inducing "graft-versus-host" and T cell-suppressor functions.

Effects of alkylating agents on lymphocytes from controls and from patients with Fanconi's anemia. Studies of sister chromatid exchanges, chromosome aberrations, and kinetics of cell division.

The frequency of sister chromatid exchanges (SCE) and chromosome aberrations and the dynamics of cell division in peripheral blood lymphocytes of four patients with Fanconi's anemia were studied after in vitro exposure to alkylating agents TEPA and mitomycin. SCE frequency was significantly increased even after very low doses of mutagens, while chromosome aberrations were significantly increased only after high doses (0.160 micrograms/ml mitomycin and 10(-5) M TEPA). The responses of Fanconi's anemia cells and control cells did not differ significantly. The increased frequency of both SCE and chromosome aberrations was accompanied by gradual delay of cell division, which was most conspicuous in cells from patients with Fanconi's anemia.

[Effect of iodobenzoteph and cytostasan on several immune responses in animals]. / Vliianie iodbenzotefa i tsitostazana na nekotorye immunnye reaktsii zhivotnykh.

Tests staged on line-bred mice immunized with sheep erythrocytes brought evidence that cystostasan and iodobenzoteph cut down the number of antibody-forming cells both with the primary and secondary immune response. The maximal effect becomes manifest following introduction of the drugs on the 1--2nd day after immunization of the animals. Iodobenzoteph brings down the number of hemopoietic stem cells colonies while cytostosan leaves them unchanged.

Cytogenetic and other effects of the chemosterilants tepa, metepa, apholate an hempa in insects (a review).

A review of the literature revealed that the chemosterilants tepa (tris(1-aziridinyl)phosphine oxide), metepa (tris(2-methyl-1-aziridinyl) phosphineoxide), apholate (2,2,4,4,6,6-hexakis(1-aziridinyl)-2,2,4,4,6,6-hexahydro-1,3,5,2,4,6-triazatriphosphorine), and hempa (hexamethylmelamine) affected both reproductive and somatic tissues in over 65 species of insects. The effects were cytological, physiological, and genetic and varied from slight to severe. In some cases the deleterious effects may have been species-specific, but in general, they appeared to be dose-dependent. More than 150 publications are cited.

Potential genetic risks from stationary magnetic field.

Cytogenetic analysis of Dede cell lines (Chinese hamster) was used to study the mutagenic effect of stationary magnetic field (SMF) and the combined effect of SMF and TEPA. Cytogenetic analysis showed a moderate mutagenic effect after exposure to SMF. In combination with TEPA, only the additive effect of the two mutagens was observed.

Cytogenetic analysis of human chromosomes and its value for the estimation of genetic risk.

A short review of present-day contradictory opinions on the usefulness of human chromosomal analysis in the system of chemical mutagen testing is illustrated by examples of the results achieved by both conventional and banding techniques. The results include exposures of human chromosomes to ECHH and TEPA in vitro, and to ECHH, vinyl chloride and Imuran in vivo. Exposures of human lymphocytes in vitro to the chemical to be tested for mutagenicity are recommended as one of the tests to be included in the system of mutagenicity testing, parallel with all other tests on mammalian and submammalian levels. The testing of human chromosomes of people exposed to chemicals in vivo is considered essential.