Data generated from the clinical trials, CITN-10 (NCT02243579) and CITN-13 (NCT03063632), analyzing the in vivo responses to anti-PD-1 monotherapy or anti-PD-1 interferon-gamma combination therapy in patients with advanced mycosis fungoides and sezary syndrome. Data include mass cytometry files of PBMCs, Olink serum targeted proteomics, Nanostring targeted bulk transcriptomics of tumor biopsies, CODEX imaging of tumor biopsies, and TCR sequencing from peripheral blood and tumor biopsies. Please direct correspondences to DRG or EWN.
In this study, we further characterize a mutant P-glycoprotein (P-gp) that has a deletion of Phe335 and is resistant to inhibition by cyclosporins. Photoaffinity labeling with [3H]cyclosporine and [3H]azidopine revealed markedly decreased binding to the mutant P-gp compared with wild-type P-gp. Expression of the mutant P-gp in multidrug-resistant variant cell line MES-SA/DxP (DxP) cells was associated with a 2-fold higher basal ATPase activity relative to multidrug-resistant cell line MES-SA/Dx5 (Dx5) cells with wild-type P-gp. Cyclosporine inhibited ATPase activity in both cell types, whereas the cyclosporin D analog valspodar (PSC 833), vinblastine, and dactinomycin stimulated ATPase activity in Dx5 but not in mutant DxP cells. Moreover, the cell lines differed in their responses to verapamil, which produced greater stimulation of ATPase in Dx5 than DxP cells. Verapamil significantly reversed the [3H]daunorubicin accumulation defect in wild-type Dx5 cells, but it had no significant effect on [3H]daunorubicin accumulation in the mutant DxP cells. Verapamil was not transported by cells expressing either mutant or wild-type P-gp. Vanadate trapping of azido-ATP was markedly impaired in mutant P-gp. In conclusion, our data demonstrate that Phe335 of transmembrane 6 is an important amino acid residue for the formation of cyclosporine and azidopine drug-binding site(s). Phe335 also plays a role in the coupling of verapamil binding and modulation of daunorubicin intracellular accumulation in wild-type P-gp. In addition, Phe335 in transmembrane 6 may play a role in coupling drug binding to ATPase activity. The deletion of Phe335 results in a significant increase in the basal ATPase activity with a concomitant decrease in its ability to trap ATP and transport some P-gp substrates.
The PD-1 inhibitor pembrolizumab is effective in treating Sézary syndrome, a leukemic variant of cutaneous T-cell lymphoma. Our purpose was to investigate the effects of pembrolizumab on healthy and malignant T cells in Sézary syndrome and to discover characteristics that predict pembrolizumab response. Samples were analyzed before and after 3 weeks of pembrolizumab treatment using single-cell RNA-sequencing of 118,961 peripheral blood T cells isolated from six Sézary syndrome patients. T-cell receptor clonotyping, bulk RNA-seq signatures, and whole-exome data were integrated to classify malignant T-cells and their underlying subclonal heterogeneity. We found that responses to pembrolizumab were associated with lower KIR3DL2 expression within Sézary T cells. Pembrolizumab modulated Sézary cell gene expression of T-cell activation associated genes. The CD8 effector populations included clonally expanded populations with a strong cytotoxic profile. Expansions of CD8 terminal effector and CD8 effector memory T-cell populations were observed in responding patients after treatment. We observed intrapatient Sézary cell heterogeneity including subclonal segregation of a coding mutation and copy number variation. Our study reveals differential effects of pembrolizumab in both malignant and healthy T cells. These data support further study of KIR3DL2 expression and CD8 immune populations as predictive biomarkers of pembrolizumab response in Sézary syndrome.
La anemia de Fanconi (AF) es un síndrome de inestabilidad cromosómica, autosómico recesivo, caracterizado por una hipersensibilidad del DNA a agentes clastogénicos.Clínicamente presenta una insuficiencia medular progresiva, diversas anomalías congénitas e incremento en la predisposición a padecer enfermedades malignas.Se han definido ocho grupos de complementación y se han clonado los genes correspondientes a seis de ellos.Recientes avances en biología molecular han permitido investigar la relación entre el genotipo de AF y la naturaleza y severidad del fenotipo clínico.El tratamiento de la AF es también objeto de una intensa investigación que actualmente se centra en el trasplante de progenitores hematopoyéticos, con éxito especialmente en caso de donante hermano HLAidéntico, y en la terapia génica todavía en fase de investigación clínica
Abstract Four breast cancer cell lines (MCF-7, BT-549, MDA-MB-231 and T-47D) and four ovarian cancer cell lines (1A9/A2780, ES-2, MES-OV and OVCAR-3) were selected for taxane resistance by exposing cells to either docetaxel or paclitaxel in the presence of the P-glycoprotein inhibitor, PSC-833 (valspodar, 2 μM). All of these co-selected variants are MDR1/ABCB1(-), and the resistance to taxanes is not transporter-mediated. We have previously reported elevated class III β-tubulin (TUBB3), reduced BRCA1, elevated CDKN1A (p21), and altered epithelial-mesenchymal transition (EMT) genes in the majority of the non-MDR1 taxane variants. Inhibitors of apoptosis (IAP) proteins directly bind and inhibit several caspases, and may play a critical role in determining cell fate after exposure to chemotherapeutic agents. In this study, we profiled the expression of IAP proteins and found elevated content in this panel of taxane-resistant human breast and ovarian cancer cell lines. In both the paclitaxel- (TP) and docetaxel-selected (TxTP) ovarian cancer cell lines we observed significantly overexpressed cIAP1 (BIRC2), cIAP2 (BIRC3), XIAP (BIRC4), and Livin (BIRC7) relative to parental cell lines. Expression of cIAP2 was undetectable in OVCAR-3 and its taxane resistant cell lines, OVCAR-3/TP20 and OVCAR-3/TxTP5. Elevated cIAP1 and XIAP levels were detected in the human breast cancer variant BT-549/TxTP50, and Livin was overexpressed in all taxane-resistant breast cancer cell models. Survivin (BIRC5) is highly expressed in cancers and has been associated with taxane resistance via its effects on apoptosis and the cell cycle. We observed reduced Survivin content in the majority of the variants established in our laboratory except for the ovarian ES-2/TP80 cell line where we found slightly elevated expression resulting from paclitaxel selection relative to its parental control. In addition to these alterations in IAP content, we observed elevated Bcl-2 in ovarian (MES-OV/TP40 and MES-OV/TxTP50) and breast (MCF-7/TxTP50, BT-549/TxTP50, and MDA231/TxTP50) cancer cell lines at the transcript level by rt-PCR and confirmed by immunoblotting. Specific gene silencing by RNAi and treatment with small molecule inhibitors will test the functional significance of IAP alterations in these models of taxane resistance. Citation Format: George E. Duran, Anamaria Brozovic, Francisco J. Martinez, E Brian Francisco, Yan C. Wang, Branimir I. Sikic. Overexpression of inhibitors of apoptosis (IAP) family members in human breast and ovarian cancer models of non-MDR1 taxane resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 901. doi:10.1158/1538-7445.AM2013-901
Cutaneous T cell lymphomas (CTCLs) are skin cancers with poor survival rates and limited treatments. While immunotherapies have shown some efficacy, the immunological consequences of administering immune-activating agents to CTCL patients have not been systematically characterized. We apply a suite of high-dimensional technologies to investigate the local, cellular, and systemic responses in CTCL patients receiving either mono- or combination anti-PD-1 plus interferon-gamma (IFN-γ) therapy. Neoplastic T cells display no evidence of activation after immunotherapy. IFN-γ induces muted endogenous immunological responses, while anti-PD-1 elicits broader changes, including increased abundance of CLA
Background : The doxorubicin analogues cyanomorpholino doxorubicin (MRA-CN) and morpholino doxorubicin (MRA) were synthesized in an attempt to avoid the cardiotoxicity and drug resistance of doxorubiricn therapy. MRA-CN forms interstrand DNA cross-links without requiring microsomal metabolic activitation in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to form a product that alkylates DNA, but MRA requires metabolic activation Alkyalation produces DNA cross-links, which are associated with potentiation of the cytotoxicity of some drugs. Purpose: Our purpose was to study the DNA binding of MRA-CN and MRA with and without metabolic activation in order to better understand the mechanisms for cross linking DNA. Methods: We used ( 3 MRA and ( 3 H) MRA-CN, with the 3 H labeled at C-2 and C-6 of the morpholino ring. MRA (10 n M) was incubated with human liver microsomes with or without NADPH to measure DNA binding. In addition, a filter elution assay was used to determine the nature and extent of drug binding to DNA in the human ovarian carcionma cell line ES-2. We studied the appearence of interstrand crosslinks versus total DNA adducts in pBR322 plasmid DNA incubated with 100 n M MRA-CN in cell free medium and then subjected to denaturation and agarose gel electropohoresis. Results: Regardless of the extracellular concentration of the drug (1– 85% of intracellular MRA-CN was covalently bound to DNA, and the total amount to DNA, and the total amount of drug bound to DNA was proportional to extracellular drug concentration. No covalent binding of MRA to DNA was found in cells exposed to 10 n M MRA alone for 2 hours. In contrast, 10% of the intracellular drug was bound to DNA iof the cells were exposed to MRA preincubated with human liver microsomes and NADPH. The percentages of plasmide containing at least one interstrand cross-links rose from 35% at 15 minutes to 92% at 2 hours. We estimate that eight molecoules of MRA-CN were adducted per molecule of p BR322 DNA (or one drug adduct per 545 base pairs), with a minimum of 12% of the adducts forming interstrand cross links. Conclusions: These result suggest that the carbons at positions 2 and 6 of the morpholino ring of both MRA-CN and the activated metabolite of MRA are retained in the drug-DNA adduct. They also indicate that the formation of interstrand DNA cross links by MRA-CN is preceded by formation of drug adducts to a single strand of DNA. (J Natl Cancer Inst 1587–1592, 1992)
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