Adenine-adenine, adenine-cytosine and cytosine-cytosine intrastrand crosslinks induced by a photoactivatable Pt(IV) anticancer prodrug.

Four trinucleotides 5'-ATA-3' (I), 5'-ATC-3' (II), 5'-CTA-3' (III) and 5'-CTC-3' (IV) were introduced to interact with a diazido-based photoactivatable anticancer prodrug trans,trans,trans-[PtIV(N3)2(OH)2(py)2] (py = pyridine; 1) upon light irradiation. Using electrospray ionization mass spectrometry (ESI-MS), we aimed to investigate the possibility of 1,3-intrastrand crosslinks at adenine and/or cytosine in the trinucleotides via the bi-functional trans-[PtII(py)2]2+ species generated by photodecomposition of complex 1. The primary mass spectrometry results showed that although mono- and di-platinated trinucleotides bound by mono-functional trans-[PtII(N3)(py)2]+ species were the major platinated adducts, comparable amounts of bifunctional trans-[PtII(py)2]2+-bound trinucleotides were also observed. Further tandem mass spectrometry of the trans-[PtII(py)2]2+-bound trinucleotides showed the formation of 1,3-crosslinks between adenine-adenine, adenine-cytosine and cytosine-cytosine bases in the trinucleotides. The formation of such unique structures is not only distinct from the action modes of cisplatin with DNA but also an important complement to the acknowledged 1,3-GNG intrastrand crosslink by trans-Pt species, which may support the promising and distinct anticancer activities of such photoactivatable diazido Pt(IV) anticancer prodrugs and deserve further studies.

Single VHH-directed BCMA CAR-NK cells for multiple myeloma.

Natural killer (NK) cells are promising alternatives for the production of "off-the-shelf" CAR products, posing a lower risk of cytokine release syndrome (CRS) than CAR-T cells. We synthesized four single VHH-directed anti-BCMA CARs, incorporating various intracellular regions (2B4 versus CD28) and hinge domains (CD28 versus IgG1) and ectopically producing IL-15. NK cells derived from peripheral blood (PB) were expanded ex vivo by K562-mbIL21 feeder cells. Stable CAR transduction was obtained through lentiviral transduction with the BaEV-Rless pseudotyped lentiviral vector. BCMA-CD28-IL15 CAR-NK cells with ectopic expression of IL-15 exhibited superior cytotoxicity were compared to BCMA-CD28 CAR-NK cells lacking IL-15 and BCMA-hIgG1-IL15 CAR-NK cells with an IgG1 hinge domain. We further assessed the cytotoxic capabilities of BCMA-2B4-IL15 CAR-NK cells with 2B4 intracellular domain. The BCMA-CD28-IL15 CAR-NK cells revealed stronger cytotoxicity and higher cytokine secretion against BCMA+ tumor cells than BCMA-2B4-IL15 CAR-NK cells in vitro. In the MM.1S-Luc mouse model, BCMA-CD28-IL15 CAR-NK inhibited the growth of tumor cells and prolonged mouse survival. These results show that the single VHH-directed BCMA CAR-NK cells exhibited remarkable specific killing ability, making them a potential candidate for immunotherapy in multiple myeloma treatment.

Reaction of human telomeric unit TTAGGG and a photoactivatable Pt(IV) anticancer prodrug.

The interaction of a photoactivatable diazidodihydroxido Pt(IV) prodrug, trans,trans,trans-[Pt(N3)2(OH)2(py)2] (py = pyridine; 1), with a hexamer straight human telomeric DNA unit sequence (5'-T1T2A3G4G5G6-3', I) upon light irradiation was investigated by electrospray ionization mass spectroscopy (ESI-MS). In the primary mass spectrum, two major mono-platinated I adducts with the bound Pt moieties, trans-[PtII(N3)(py)2]+ (1') and trans-[PtII(py)2]2+ (1''), respectively, were detected. It is rare to observe such high abundance and nearly equal intensity platinated DNA adducts formed by these two PtII species because 1' is usually the only major reduced Pt(II) species produced by the photodecomposition of complex 1 in the presence of DNA while 1'' was rarely detected as the major reduced PtII species reported previously. Subsequent tandem mass spectrometric analysis by collision-induced dissociation (CID) showed that in the former adduct {I + 1'}2+, G6 and A3 were the platination sites. While in the latter adduct {I + 1''}2+, a potential intrastrand crosslink was speculated after G4 and G6 sites were identified. Additionally, other minor platinated adducts like di-platinated I adduct by 1' with platination sites at G4 and G6 and mono-platinated I adducts containing base oxidation were also detected by mass spectrometry. Due to the rich guanines and their sensitivity to oxidation, the oxidation induced by 1 most probably occurred at guanine. The oxidation adducts were proposed as 8-hydroxyl guanine, spiroiminodihydantoin (Sp), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG), 5-guanidinohydantoin (Gh), and/or dehydroguanidinohydantoin (DGh) referring to previous reports. The obtained results provide useful chemical information about the photoreaction between photoactivatable Pt(IV) anticancer prodrugs and human telomeric DNA. Such special damages of Pt(IV) prodrugs on human telomeric DNA implicate its active role in the mechanism of Pt(IV) prodrugs and further support the unique sequence-dependent photointeraction profile of complex 1 reacting with DNA.

Differentiated oxidation modes of guanine between CpG and 5mCpG by a photoactivatable Pt(IV) anticancer prodrug.

CpG and its cytosine-methylated counterpart (5mCpG) are a unique reversible pair of sequences in regulating the expression of genes epigenetically. As DNA is the potential target of Pt-based anticancer metallodrugs, herein, we comparatively investigate the interactions of 5'-CpG and 5'-5mCpG with a photoactivatable anticancer Pt(IV) prodrug, trans,trans,trans-[PtIV(N3)2(OH)2(py)2] (1; py = pyridine), to explore the effects of methylation on the platination and ROS-induced oxidation of the CpG motif. Mono-platinated dinucleotides were demonstrated by ESI-MS to be the main products for both 5'-CpG and 5'-5mCpG with the bound Pt moiety as [PtII(N3)(py)2] generated by the photodecomposition of complex 1 under irradiation with blue light, accompanied by the formation of less abundant di-platinated adducts. G-N7 and C-N3/5mC-N3 were shown to be the major and minor platination sites, respectively, with G-N1 as the third and weakest platination site, in particular, in di-platinated products. Moreover, platinated dinucleotides associated with guanine and/or cytosine oxidation were also observed. Apart from 8-oxo-guanine (oxG) and N-formylamidoiminohydantoin (RedSp) reported previously, novel oxidation adducts 5-guanidinohydantoin (Gh) derived from guanine and 1-carbamoyl-4,5-dihydroxy-2-oxoimidazolidine (ImidCyt) derived from cytosine in CpG, and diimino imidazole (DIz) and 2,5-diaminoimidazol-4-one (imidazolone, Iz) derived from guanine and Imid5mCyt derived from 5mC in 5mCpG were proposed according to MS information. These results showed that methylation exerted little effects on the platination modes of CpG, but triggered distinct oxidation pathways of CpG, perhaps causing discriminated DNA damage to CpG-rich genes. This work provides novel insights into the role of the anticancer photoactivatable Pt(IV) prodrug through damaging the epigenetically modified DNA sequences.

Case Report: Successful engraftment of allogeneic hematopoietic stem cells using CAR-T cell therapy as the conditioning regimen in R/R Ph+ B cell acute lymphoblastic leukemia.

Background: Consolidative allogeneic hematopoietic stem cells (allo-HSCs) after chimeric antigen receptor T cells (CAR-T) therapy is an emerging modality in hematologic malignancies. Knowledge about the success of allogeneic hematopoietic stem cell transplantation (allo-HSCT) after CAR-T therapy without a conditioning regimen is limited. Case presentation: We report a patient with relapsed/refractory (R/R) Ph+ B-cell acute lymphoblastic leukemia (ALL) who underwent anti-CD19 CAR-T immunotherapy. After 1 month of treatment, bone marrow hyperplasia remained reduced with no hematopoietic improvements. In line with this, allogeneic hematopoietic stem cells (HSCs) were extracted from an HLA-matched sibling donor and administered to the patient on day 33 after CAR-T cell therapy to support hematopoiesis. On day 40, the level of immature bone marrow lymphocytes was at 0% and minimal residual disease-negative, and the fusion gene BCR/ABL 190 was negative. Chimerism analysis showed full donor chimerism. Three months after CAR-T cells infusion, the patient was still in complete remission with full donor chimerism. However, decreased liver function with skin pigmentation and festering, indicative of acute graft versus host disease, was noted. The treatment was halted owing to financial reasons. Conclusion: We report the successful engraftment of allogeneic HSCs using CAR-T cell therapy as a conditioning regimen for R/R B-ALL patients.

A novel CD19/CD22/CD3 trispecific antibody enhances therapeutic efficacy and overcomes immune escape against B-ALL.

The bispecific T-cell engager (BiTE) blinatumomab against CD19 and CD3 has emerged as the most successful bispecific antibody (bsAb) to date; however, a significant proportion of patients do not respond to the treatments or eventually experience relapse after an initial response, and the recurrence rate increases significantly due to escape or downregulation of the CD19 antigen. To enhance antitumor efficacy and overcome potential immune escape, we developed a novel approach to design a CD19/CD22/CD3 trispecific antibody (tsAb) by site-specifically fusing anti-CD19 scFv (FMC63) and anti-CD22 nanobody (Nb25) to the defined sites of the CD3 antigen-binding fragment (Fab, SP34). This strategy allows for the optimal formation of immune synapses mediated by CD19/CD22/CD3 between target cells and T cells. Optimized tsAb can be superior for inducing T-cell-specific cytotoxicity and cytokine production against CD19+ and/or CD22+ tumor cells compared to other tsAb formats, and demonstrated significantly enhanced antitumor efficacy and the ability to overcome immune escape compared with the corresponding bsAbs alone or in combination, as well as with blinatumomab. In addition, tsAb treatment can lead to the long-term elimination of primary B-ALL patient samples in the PDX model and significantly prolong survival. This novel approach provides unique insight into the structural optimization of T-cell-redirected multispecific antibodies using site-specific recombination, and may be broadly applicable to heterogeneous and resistant tumor populations as well as solid tumors.

Dual-platination and induced oxidation of uridine by a photoactivatable diazido Pt(IV) anticancer prodrug.

Photoactivatable Pt(IV) anticancer prodrug trans,trans,trans-[Pt(N3)2(OH)2(pyridine)2] (1) has been shown to bind to and induce oxidation of all four DNA nucleobases. Herein, to further render the binding spectrum of complex 1 to nucleic acids, the interaction between complex 1 and uridine, an exclusive RNA component, was investigated by electrospray ionization mass spectrometry (ESI-MS) and NMR spectroscopy. The results showed that complex 1 can bind to uridine through the N3 (major) and O4 (minor) sites upon light irradiation to form the major mono-platinated uridine adduct and the minor di-platinated uridine adduct. Moreover, mono-platinated uridine associated with the oxidation of uridine to 5-hydroxyuridine and 6-hydroxyuridine was also observed. This is the first report that the photoactivatable Pt(IV) prodrug binds to and induces the oxidation of uridine, and also the last piece of the puzzle for the interactions of complex 1 with nucleobases. Combined with our previous results about the interactions between complex 1 and DNA bases, these data showed a wide interaction spectrum of this kind of photoactivatable diazido Pt(IV) prodrugs with nucleobases through such dual-action modes, strongly suggesting that RNA may be a potential target of Pt(IV) prodrugs.

A Novel Mitochondrial-Related Nuclear Gene Signature Predicts Overall Survival of Lung Adenocarcinoma Patients.

Background: Lung cancer is the leading cause of cancer-related death worldwide, of which lung adenocarcinoma (LUAD) is one of the main histological subtypes. Mitochondria are vital for maintaining the physiological function, and their dysfunction has been found to be correlated with tumorigenesis and disease progression. Although, some mitochondrial-related genes have been found to correlate with the clinical outcomes of multiple tumors solely. The integrated relationship between nuclear mitochondrial genes (NMGs) and the prognosis of LUAD remains unclear. Methods: The list of NMGs, gene expression data, and related clinical information of LUAD were downloaded from public databases. Bioinformatics methods were used and obtained 18 prognostic related NMGs to construct a risk signature. Results: There were 18 NMGs (NDUFS2, ATP8A2, SCO1, COX14, COA6, RRM2B, TFAM, DARS2, GARS, YARS2, EFG1, GFM1, MRPL3, MRPL44, ISCU, CABC1, HSPD1, and ETHE1) identified by LASSO regression analysis. The mRNA expression of these 18 genes was positively correlated with their relative linear copy number alteration (CNA). Meanwhile, the established risk signature could effectively distinguish high- and low-risk patients, and its predictive capacity was validated in three independent gene expression omnibus (GEO) cohorts. Notably, a significantly lower prevalence of actionable EGFR alterations was presented in patients with high-risk NMGs signature but accompanied with a more inflame immune tumor microenvironment. Additionally, multicomponent Cox regression analysis showed that the model was stable when risk score, tumor stage, and lymph node stage were considered, and the 1-, 3-, and 5-year AUC were 0.74, 0.75, and 0.70, respectively. Conclusion: Together, this study established a signature based on NMGs that is a prognostic biomarker for LUAD patients and has the potential to be widely applied in future clinical settings.

The Functionalities and Clinical Significance of Tumor-Infiltrating Immune Cells in Esophageal Squamous Cell Carcinoma.

Tumor-infiltrating immune cells have been implicated in the tumorigenesis and progression of esophageal squamous cell carcinoma (ESCC). However, the functionalities and clinical significance of immune cells remain largely unveiled. In this study, the gene expression data from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) were extracted. The relative infiltrating levels were estimated by single-sample gene set enrichment analysis. Some cytotoxic immune cells were attenuated, and resting cytotoxic immune cells were accumulated in ESCC. Remarkably, we also observed that infiltrating levels of macrophage M2 and resting natural killer (NK) cells were increased in nonresponders of CRT, and T cells that had anticancer activities such as activated memory CD4 and T helper 2 (Th2) cells were significantly reduced in ESCC tissues of the nonresponders. Moreover, the high infiltrations of the resting natural killer (NK) and dendritic cell (DC) were observed to result in a shorter overall survival in ESCC. Consistently, high expression of immune checkpoint genes, CTLA4 and HAVCR2, was associated with poor prognosis. Furthermore, STAT5B, a key transcription factor, as well as its target genes, involved in the regulation of T cells, was significantly downregulated in ESCC, especially subgroup I, indicating that downregulation of STAT5B might be associated with reduced T cell-mediated anticancer activity. In conclusion, the present study significantly improved our understanding of the regulatory roles of immune cells in ESCC.

Loss of Smad4 promotes aggressive lung cancer metastasis by de-repression of PAK3 via miRNA regulation.

SMAD4 is mutated in human lung cancer, but the underlying mechanism by which Smad4 loss-of-function (LOF) accelerates lung cancer metastasis is yet to be elucidated. Here, we generate a highly aggressive lung cancer mouse model bearing conditional KrasG12D, p53fl/fl LOF and Smad4fl/fl LOF mutations (SPK), showing a much higher incidence of tumor metastases than the KrasG12D, p53fl/fl (PK) mice. Molecularly, PAK3 is identified as a downstream effector of Smad4, mediating metastatic signal transduction via the PAK3-JNK-Jun pathway. Upregulation of PAK3 by Smad4 LOF in SPK mice is achieved by attenuating Smad4-dependent transcription of miR-495 and miR-543. These microRNAs (miRNAs) directly bind to the PAK3 3'UTR for blockade of PAK3 production, ultimately regulating lung cancer metastasis. An inverse correlation between Smad4 and PAK3 pathway components is observed in human lung cancer. Our study highlights the Smad4-PAK3 regulation as a point of potential therapy in metastatic lung cancer.

Culturing adequate CAR-T cells from less peripheral blood to treat B-cell malignancies.

OBJECTIVE: Chimeric antigen receptor-modified T (CAR-T) cells have shown impressive results against relapsed/refractory B cell malignancies. However, the traditional manufacture of CAR-T cells requires leukapheresis to isolate large amounts of peripheral blood T cells, thus making some patients ineligible for the procedure. METHODS: We developed a simple method for CAR-T cell preparation requiring small volumes of peripheral blood. First, CD3+ T cells isolated from 50 mL peripheral blood from patients (B-cell malignancies) were stimulated with immobilized anti-CD3/RetroNectin in 6-well plates and then transduced with CAR-expressing lentiviral vector. After 4 d, the T cells were transferred to culture bags for large-scale CAR-T cell expansion. In vitro and animal experiments were performed to evaluate the activity of the manufactured CAR-T cells. Finally, 29 patients with B-cell acute lymphoblastic leukemia (B-ALL) and 9 patients with B-cell lymphoma were treated with the CAR-T cells. RESULTS: The CAR-T cells were expanded to 1-3 × 108 cells in 8-10 d and successfully killed B cell-derived malignant tumor cells in vitro and in vivo. For patients with B-ALL, the complete remission rate was 93% 1 month after CAR-T cell infusion; after 12 months, the overall survival (OS) and leukemia-free survival rates were 69% and 31%, respectively. For patients with lymphoma, the objective response rate (including complete and partial remission) was 78% 2 months after CAR-T cell infusion, and after 12 months, the OS and progression-free survival rates were 71% and 43%, respectively. Cytokine-release syndrome (CRS) occurred in 65.51% and 55.56% of patients with B-ALL and B-cell lymphoma, respectively; severe CRS developed in 20.69% of patients with B-ALL and in no patients with lymphoma. CONCLUSIONS: Our novel method can generate sufficient numbers of CAR-T cells for clinical use from 50-100 mL peripheral blood, thus providing an alternative means of CAR-T cell generation for patients ineligible for leukapheresis.

The Application of Nanobody in CAR-T Therapy.

Chimeric antigen receptor (CAR) T therapy represents a form of immune cellular therapy with clinical efficacy and a specific target. A typical chimeric antigen receptor (CAR) construct consists of an antigen binding domain, a transmembrane domain, and a cytoplasmic domain. Nanobodies have been widely applied as the antigen binding domain of CAR-T due to their small size, optimal stability, high affinity, and manufacturing feasibility. The nanobody-based CAR structure has shown a proven function in more than ten different tumor-specific targets. After being transduced in Jurkat cells, natural killer cells, or primary T cells, the resulting nanobody-based CAR-T or CAR-NK cells demonstrate anti-tumor effects both in vitro and in vivo. Interestingly, anti-BCMA CAR-T modulated by a single nanobody or bi-valent nanobody displays comparable clinical effects with that of single-chain variable fragment (scFv)-modulated CAR-T. The application of nanobodies in CAR-T therapy has been well demonstrated from bench to bedside and displays great potential in forming advanced CAR-T for more challenging tasks.

Photoactivatable diazido Pt(IV) anticancer complex can bind to and oxidize all four nucleosides.

Photoactivatable diazidodihydroxido Pt(iv) complex trans,trans,trans-[Pt(N3)2(OH)2(py)2] (1; py = pyridine) is a promising anticancer agent which can be activated by visible light to induce cancer cell death. DNA has been thought to be involved in the mechanism of action of this kind of Pt(iv) prodrug. However, the detailed photodecomposition pathways of complex 1 and its interaction modes with DNA are complex. Herein we report that upon light irradiation complex 1 can bind to all four nucleosides covalently with the reduced Pt(ii) species. Moreover, apart from the covalent coordination, various oxidation adducts of these four nucleosides induced by the reactive oxidative species (ROS) generated during the photoactivation of the complex 1 have also been identified, especially the induced oxidation of adenosine and cytidine which was firstly reported for this kind of photoactivatable Pt(iv) prodrug. Such dual-action may contribute to the highly potent photo-antiproliferativity of complex 1 towards cancer cells, which may account for the unique mechanism of action of the photoactivatable diazido Pt(iv) anticancer complexes.

Reactions of a photoactivatable diazido Pt(iv) anticancer complex with a single-stranded oligodeoxynucleotide.

Platinum based anticancer agents are widely applied in clinic and their major target is believed to be DNA. Herein, the interaction of a photoactivatable diazido Pt(iv) anticancer prodrug trans,trans,trans-[Pt(N3)2(OH)2(py)2] (py = pyridine; 1) with a 15-mer single-G-containing oligodeoxynucleotide (ODN I: 5'-CT2CTCTTG8T9CT11TCTC-3') was investigated by mass spectrometric methods. Up to penta-platinated ODN I adducts were identified from primary mass spectra while the mono- and di-platinated adducts had the highest intensity. Fragmentation of mono-, di- and tri-platinated I adducts in tandem MS revealed that T2, G8, T11 and T9 are binding sites. No cytosine sites were identified which may be due to the facile loss of Pt adducts from cytosine during CID. The intensity of {Pt(py)2}-bound adducts was comparable to that of {Pt(N3)(py)2}-bound adducts, indicating that the photo-reduction pathway of complex 1 from Pt(iv) to Pt(ii) through two one-electron donations from two azides was substantial. Moreover, no transformation of N3 to NH3 on the {Pt(N3)(py)2}-bound adducts was observed, whereas it is very popular during the reactions of complexes with short ODNs or mono-nucleotides. The oxidation on I induced by the reactive oxygen species (ROS) formed by the photodecomposition of complex 1 was significant, and the oxidation of G8 to 8-hydroxyguanine (8-OH-G), spiroiminodihydantoin (Sp) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) was discovered. These results unambiguously revealed a sequence-length-dependent photochemical reactivity of complex 1 when it interacted with different ODNs, providing deeper understanding in the reactivity of photoactivatable diazido anticancer Pt(iv) prodrugs to DNA.

Safety and efficacy of CAR-T cell targeting BCMA in patients with multiple myeloma coinfected with chronic hepatitis B virus.

BACKGROUND: Reactivation of hepatitis B virus (HBV) infection is a well-recognized complication in patients with chronic or resolved HBV infection undergoing anticancer therapy. There is a risk of HBV reactivation after infusion of chimeric antigen receptor (CAR) T cells for patients with refractory/relapsed (R/R) multiple myeloma (MM). METHODS: We administered B cell maturation antigen (BCMA) CAR-T cell by infusion to nine patients with R/R MM with chronic or resolved HBV infection. Patient serum was analyzed to determine the expression of five components of HBV and the copy number of HBV DNA. HBV reactivation was defined if a patient re-exhibited hepatitis B surface antigen (HBsAg) or HBV DNA regrowth after CAR-T therapy. RESULTS: In one patient who was HBsAg-positive, no HBV reactivation was observed during the follow-up period of 9.8 months after administration of anti-HBV drugs before and after CAR-T therapy. Among eight patients with MM who had resolved HBV infection, two patients administered prophylactic anti-HBV drugs did not exhibit HBV reactivation. Of the six patients who did not use prophylactic antiviral drugs, five did not exhibit HBV reactivation, while one showed recurrence of HBsAg without detection of HBV DNA or damage to liver function. The best objective response rate was 100%, and the progression-free survival (PFS) at 12 months was of 88.89% (median PFS was not observed). CONCLUSIONS: These findings showed that BCMA CAR-T cell therapy could be used in patients with R/R MM with chronic or resolved HBV infection and that antiviral drugs should be administered in these patients during CAR-T cell therapy.

Unexpected Thymine Oxidation and Collision-Induced Thymine-Pt-guanine Cross-Linking on 5'-TpG and 5'-GpT by a Photoactivatable Diazido Pt(IV) Anticancer Complex.

The photochemical products of dinucleotides 5'-TpG/5'-GpT with a photoactivatable anticancer Pt(IV) complex (trans,trans,trans-[Pt(N3)2(OH)2(py)2], py = pyridine; 1) were characterized by electrospray ionization mass spectrometry. The primary MS showed the main products were monoplatinated and diplatinated adducts for both the dinucleotides accompanied by the formation of minor triplatinated dinucleotides, indicating that T-N3 and G-N1 may be platination sites additional to the well-known G-N7 site. Surprisingly, a series of minor platinated adducts with oxidation of guanine and/or thymine were observed. Although guanine is more sensitive to oxidation than thymine, thymine can compete with guanine for complex 1-induced oxidation, of which the oxidation adducts were identified as cis- and trans-diastereomers of 5,6-dihydroxy-5,6-dihydrothymidine (cis,trans-ThdGly), 5-formyl-2'-deoxyuridine (5-FormdUrd), and 5-(hydroxymethyl)-2'-deoxyuridine (5-HMdUrd), respectively. While for guanine, apart from 8-hydroxyguanine (8-OH-G) and N-formylamidoiminohydantoin (RedSp), other guanine oxidized adducts such as spiroiminodihydantoin (Sp), dehydroguanidinohydantoin (DGh), and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) were also identified. MS/MS analysis showed that unique fragments with a Pt moiety [Pt(N3)(py)] cross-linking the G and T bases were formed during the fragmentation of monoplatinated dinucleotides. Such binding mode to and oxidative damages on DNA bases imposed by the diazido Pt(IV) complex are apparently distinct from those of cisplatin, perhaps accounting for its unique mechanism of action.

Selective binding of an organoruthenium complex to G-rich human telomeric sequence by tandem mass spectrometry.

RATIONALE: Human telomeric DNA is reported to be a potential target for anticancer organometallic ruthenium(II) complexes, however, the interaction sites were not clearly discriminated and identified. METHODS: In the current study, tandem mass spectrometry (MS/MS) using collision-induced dissociation (CID) was firstly introduced to identify the interaction sites of an organometallic ruthenium(II) complex [(η6 -biphenyl)Ru(en)Cl][PF6 ] (1; en = ethylenediamine) with 5'-T1 T2 A3 G4 G5 G6 -3' (I), the repeating unit of human telomeric DNA, in both positive- and negative-ion mode at a low reaction molar ratio (1/I = 0.2) which was applied to preserve the site selectivity. RESULTS: Mass spectrometric results showed that mono-ruthenated I was the main product under the conditions. In positive-ion mode, MS/MS results indicated that ruthenium complex 1 binds to T2 or G6 in strand I. However, in negative-ion mode, no efficient information was obtained for exact identification of ruthenation sites which may be attributed to losses of fragment ions due to charge neutralization by the coordination of the positively charged ruthenium complex to the short MS/MS fragments. CONCLUSIONS: This is the first report of using top-down MS to characterize the interactions of organometallic ruthenium(II) complexes and human telomeric DNA. Thymine can be thermodynamically competitive with guanine for binding to ruthenium complexes even at low reaction molar ratio, which inspired us to explore in greater depth the significance of thymine binding.

Unilateral congenital malformations of middle ear with intact external ear: a review of 64 cases.

OBJECTIVE: To describe the operative findings and surgical results of unilateral congenital middle ear malformations with intact external ear. METHODS: A retrospective review was performed on 64 patients with unilateral congenital middle ear malformations and intact external ear who underwent exploratory tympanotomy from 2011 to 2016. Demographic data, clinical data, high-resolution computed tomography findings, audiometric data and intraoperative findings were collected. Audiological evaluations before and 6 months after surgery were analyzed in 47 patients. RESULTS: The most common malformation were mobile stapes with missing incus long process and stapes suprastructure. The air conduction pure tone average was 58.9 ± 10.5 dB HL (range 34.4-78.1 dB HL) preoperatively and 28.8 ± 10.6 dB HL (range 9.4-55.6 dB HL) postoperatively (P = 0.000). Twenty-five cases (53.2%) acquired an air conduction hearing gain exceeding 30 dB. Mean air-bone gap (ABG) was 44.5 ± 9.4 dB (range 22.5-66.4 dB HL) before surgery and 15.6 ± 9.3 dB (range 0-35.6 dB) after surgery (P = 0.000) for an average gain of 28.8 ± 11.5 dB. Thirty-four cases (72.3%) showed a postoperative ABG of less than 20 dB, 15 had an ABG within 10 dB, and 4 had 0 dB ABG after operation. No significant difference was observed for air conduction hearing gain regarding age (P = 0.261) or types of malformations (mobile stapes footplate with or without a suprastructure anomaly, P = 0.058). CONCLUSION: Unilateral congenital middle ear malformations with intact external ear can be complex and diverse. Functional ossiculoplasty for patients with unilateral congenital middle ear malformations can achieve good hearing outcomes.

Ezh2 Acts as a Tumor Suppressor in Kras-driven Lung Adenocarcinoma.

Previous studies have suggested that enhancer zeste homolog 2 (Ezh2), a histone methyltransferase subunit of polycomb repressive complex 2 (PRC2), acts as an oncogene in lung adenocarcinoma (ADC) development. However, we found that in human lung ADC samples, deletion and mutations of EZH2 were also frequently present, with 14% of patients harboring loss-of-function EZH2 alterations. To explore the effect of Ezh2 loss on lung tumor formation, lung epithelial Ezh2 gene was deleted in Kras-driven lung ADC mouse model. Unexpectedly, Ezh2 loss dramatically promoted Kras-driven ADC formation. KrasG12D/+;Ezh2fl/fl mice exhibited shorter lifespan, more tumor lesions and higher tumor burden than KrasG12D/+ mice, suggesting the tumor-suppressive role of Ezh2 in Kras-driven ADCs. Mechanistically, Ezh2 loss amplified Akt and ERK activation through de-repressing its target insulin-like growth factor 1 (Igf1). Additionally, Ezh2 loss cooperated with Kras mutation to exacerbate the inflammatory response, as shown by massive macrophage and neutrophil infiltrates, as well as a marked increase in tumor-associated cytokines such as IL-6 and TNF-α. Taken together, our findings revealed the tumor suppressive function of Ezh2 in Kras-driven ADCs, underlining the importance of revaluating the application of EZH2 inhibitors in a variety of cancers.