Organotypic cultures of primary human airway epithelial cells have been used to investigate the morphology, ion and fluid transport, innate immunity, transcytosis, infection, inflammation, signaling, ...cilia, and repair functions of this complex tissue. However, we do not know how closely these cultures resemble the airway surface epithelium in vivo. In this study, we examined the genome-wide expression profile of tracheal and bronchial human airway epithelia in vivo and compared it with the expression profile of primary cultures of human airway epithelia grown at the air-liquid interface. For comparison, we also investigated the expression profile of Calu-3 cells grown at the air-liquid interface and primary cultures of human airway epithelia submerged in nutrient media. We found that the transcriptional profile of differentiated primary cultures grown at the air-liquid interface most closely resembles that of in vivo airway epithelia, suggesting that the use of primary cultures and the presence of an air-liquid interface are important to recapitulate airway epithelia biology. We describe a high level of similarity between cells of tracheal and bronchial origin within and between different human donors, which suggests a very robust expression profile that is specific to airway cells.
T cell Ig and mucin domain (Tim) 3 is a surface molecule expressed throughout the immune system that can mediate both stimulatory and inhibitory effects. Previous studies have provided evidence that ...Tim-3 functions to enforce CD8 T cell exhaustion, a dysfunctional state associated with chronic stimulation. In contrast, the role of Tim-3 in the regulation of CD8 T cell responses to acute and transient stimulation remains undefined. To address this knowledge gap, we examined how Tim-3 affects CD8 T cell responses to acute Listeria monocytogenes infection. Analysis of wild-type (WT) mice infected with L. monocytogenes revealed that Tim-3 was transiently expressed by activated CD8 T cells and was associated primarily with acquisition of an effector phenotype. Comparison of responses to L. monocytogenes by WT and Tim-3 knockout (KO) mice showed that the absence of Tim-3 significantly reduced the magnitudes of both primary and secondary CD8 T cell responses, which correlated with decreased IFN-γ production and degranulation by Tim-3 KO cells stimulated with peptide Ag ex vivo. To address the T cell-intrinsic role of Tim-3, we analyzed responses to L. monocytogenes infection by WT and Tim-3 KO TCR-transgenic CD8 T cells following adoptive transfer into a shared WT host. In this setting, the accumulation of CD8 T cells and the generation of cytokine-producing cells were significantly reduced by the lack of Tim-3, demonstrating that this molecule has a direct effect on CD8 T cell function. Combined, our results suggest that Tim-3 can mediate a stimulatory effect on CD8 T cell responses to an acute infection.
The coxsackie and adenovirus receptor (CAR) plays a role in viral infection, maintenance of the junction adhesion complex in polarized epithelia, and modulation of cellular growth properties. As a ...viral receptor, the C-terminus appears to play no role indicating that the major function of CAR is to tether the virus to the cell. By contrast, the C-terminus is known to play a role in cellular localization and probably has a significant function in CAR-mediated adhesion and cell growth properties. We hypothesized that the CAR PDZ (PSD-95/Disc-large/ZO-1) binding motif interacts with PDZ-domain-containing proteins to modulate the cellular phenotype. CAR was modified by deleting the last four amino acids (CARDeltaGSIV) and evaluated for cell-cell adhesion in polarized primary human airway epithelia and growth characteristics in stably transfected L-cells. Although ablation of the CAR PDZ-binding motif did not affect adenoviral infection, it did have a significant effect both on cell-cell adhesion and on cell growth. Expression of CARDeltaGSIV failed to increase the transepithelial resistance in polarized epithelia to the same degree as wild-type CAR and failed to act as a growth modulator in L-cells. Furthermore, we provide evidence for three new CAR interacting partners, including MAGI-1b, PICK1 and PSD-95. CAR appears to interact with several distinct PDZ-domain-containing proteins and may exert its biological function through these interactions.
The Coxsackievirus B and Adenovirus Receptor (CAR) plays a dual role as a homotypic junctional adhesion protein and as a viral receptor. CAR is a transmembrane protein and a member of the ...Immunoglobulin (Ig) superfamily with two extracellular Ig-like domains. The most distal Ig-like domain (D1) mediates the homophilic interaction and is also responsible for the high-affinity binding of the adenovirus (Ad) fiber protein. Currently, no activity has been ascribed to the proximal Ig-like domain (D2). To further understand the function of the extracellular domain in the biological activities of CAR, we created extracellular deletion mutants and evaluated cellular localization, adhesion, and viral infection. Deletion of any segment of the extracellular domain results in loss of adhesion and mislocalization as explained by a model, termed "diffusion trapping," that suggests adhesion is the driving force in junctional localization. Loss of junctional localization and adhesion was particularly apparent in polarized human airway epithelia, where mutant CAR expression was basolateral but not limited to the lateral junctions between cells. Surprisingly, the D2 domain was required for adenovirus fiber-knob binding and infection. In summary, the entire extracellular domain of CAR is of vital importance to the biology of this highly conserved and important protein.
The development of radiation-induced pulmonary fibrosis represents a critical clinical issue limiting delivery of therapeutic doses of radiation to non-small cell lung cancer. Identification of the ...cell types whose injury initiates a fibrotic response and the underlying biological factors that govern that response are needed for developing strategies that prevent or mitigate fibrosis. C57BL/6 mice (wild type, Nrf2 null, Nrf2flox/flox, and Nrf2Δ/Δ; SPC-Cre) were administered a thoracic dose of 12Gy and allowed to recover for 250 days. Whole slide digital and confocal microscopy imaging of H&E, Masson's trichrome and immunostaining were used to assess tissue remodeling, collagen deposition and cell renewal/mobilization during the regenerative process. Histological assessment of irradiated, fibrotic wild type lung revealed significant loss of alveolar type 2 cells 250 days after irradiation. Type 2 cell loss and the corresponding development of fibrosis were enhanced in the Nrf2 null mouse. Yet, conditional deletion of Nrf2 in alveolar type 2 cells in irradiated lung did not impair type 2 cell survival nor yield an increased fibrotic phenotype. Instead, radiation-induced ΔNp63 stem/progenitor cell mobilization was inhibited in the Nrf2 null mouse while the propensity for radiation-induced myofibroblasts derived from alveolar type 2 cells was magnified. In summary, these results indicate that Nrf2 is an important regulator of irradiated lung's capacity to maintain alveolar type 2 cells, whose injury can initiate a fibrotic phenotype. Loss of Nrf2 inhibits ΔNp63 stem/progenitor mobilization, a key event for reconstitution of injured lung, while promoting a myofibroblast phenotype that is central for fibrosis.
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•Loss of Nrf2 inhibits the alveolar type 2 cell reparative process in irradiated lung.•Radiation-induced ΔNp63 stem cell mobilization is inhibited in the Nrf2 null mouse.•Loss of Nrf2 promotes alveolar type 2 cell EMT.
IL-4 signaling promotes IgE class switching through STAT6 activation and the induction of Ig germ-line ε (GLε) transcription. Previously, we and others identified a transcription factor, Nfil3, as a ...gene induced by IL-4 stimulation in B cells. However, the precise roles of nuclear factor, IL-3-regulated (NFIL3) in IL-4 signaling are unknown. Here, we report that NFIL3 is important for IgE class switching. NFIL3-deficient mice show impaired IgE class switching, and this defect is B-cell intrinsic. The induction of GLε transcripts after LPS and IL-4 stimulation is significantly reduced in NFIL3-deficient B cells. Expression of NFIL3 in NFIL3-deficient B cells restores the impairment of IgE production, and overexpression of NFIL3 in the presence of cycloheximide induces GLε transcripts. Moreover, NFIL3 binds to Iε promoter in vivo. Together, these results identify NFIL3 as a key regulator of IL-4-induced GLε transcription in response to IL-4 and subsequent IgE class switching.
Abstract
NPM1 is a chaperone involved in base excision, translesion synthesis and homologous recombination (HR). In the presence of DNA DSBs pT199 NPM1 co-localizes with H2AX foci, a consequence of ...binding to K63-linked ubiquitinated histones that surround DSBs via its pT199 domain comprised of an acidic tract and adjacent ubiquitin interacting motif-like domain (1-3). SUMOlyated K263 NPM1 binds to and promotes Rad51 loading onto resected 3-prime DNA at the DSB. Failure to SUMOlyate K263NPM or inhibition of NPM1 reduces Rad51 filament formation, thus inhibiting HR (4). A major effort is underway to develop DNA damage response inhibitors. The novel chemical entity YTR107 is a 5-((N-benzyl-1H-indol-3-yl) methylene) pyrimidine-2, 4,6(1H,3H,5H) trione developed using forward chemical genetics and structure/function screening (5). Colony formation assays revealed that YTR107 radiosensitized 7 NSCLC lines (dose modifying factors of 1.5). Radiosensitization was NPM1-dependent as YTR107 did not radiosensitize an NPM1 null mouse embryo fibroblast cell line (3). Here we show that YTR107 inhibits radiation-induced NPM1 binding to Rad51, Rad 51 foci formation and repair of DNA DSBs.Tumor initiating cells (TICs) were radiosensitized by YTR107. YTR107 inhibited tumor growth (P = 0.027, 2-way ANOVA) and increased the survival of A549 tumor-bearing mice, determined 70 days after the last treatment. Survival of tumor-bearing mice was 60% for mice treated with YTR107and 2.2 Gy(q.d. x 7) vs 20% for mice treated with 2.2 Gy alone (q.d.x 7). Cox Proportional log rank hazard ratio for irradiated vs unirradiated tumor-bearing mice was 0.24 (P = 0.0034) while the log rank hazard ratio for YTR107 plus irradiation vs unirradiated (± YTR107) tumor-bearing mice was 0.17 (P = 0.0023). Thus, these data provide support for development of YTR107 as a therapeutic radiation sensitizer. Supported in part by R44CA228756.
1.Koike A, Nishikawa H, Wu W, Okada Y, Venkitaraman AR, Ohta T.Cancer Res 2010;70:6746-56 2. Sekhar KR, Benamar M, Venkateswaran A, Sasi S, Penthala NR, Crooks PA, et al. Int J Radiat Oncol Biol Phys 2014;89:1106-14 3. Sekhar KR, Reddy YT, Reddy PN, Crooks PA, Venkateswaran A, McDonald WH, et al. Clin Cancer Res 2011;17:6490-9 4. Xu R, Yu S, Zhu D, Huang X, Xu Y, Lao Y, et al. Nat Commun 2019;10:3812 5. Reddy YT, Sekhar KR, Sasi N, Reddy PN, Freeman ML, Crooks PA. Bioorg Med Chem Lett 2010;20:600-2
Citation Format: Michael L. Freeman, Geri Traver, Konjeti R. Sekhar, Peter A. Crooks. Radiosensitization by targeting the NPM1/RAD51 axis abstract. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PR-003.
Abstract
Nucleophosmin1 (NPM1) is rapidly recruited to sites of DNA double-strand breaks and is required for radiation-induced Rad51 foci formation. Inhibition of NPM1 or Rad51 is a radiosensitizing ...event. YTR107 is a small molecule, identified in a forward chemical genetics functional structure/activity screening, that binds to the amino terminus of NPM1 and prevents NPM1 recruitment to damaged chromatin. In silico docking studies of YTR107 and the crystal structure of the NPM1 N-terminal oligomerization domain revealed that YTR107 docked to monomeric NPM1 in a pocket crucial for forming an interface between neighboring subunits of the NPM1 pentamer. YTR107 inhibited radiation-induced Rad51 foci formation and repair of DNA DSBs, as measured by a neutral comet assay. Colony formation assays revealed that YTR107 radiosensitized 7 NSCLC lines, HT29 colorectal cancer cells, D54 glioblastoma cells, PANC1 pancreatic cancer cells, and 2 breast cancer cell lines (dose modifying factors of > 1.5; cells irradiated with 300 kVp/10mA X-rays @ 2 Gy/min). Radiosensitization was found to be NPM1-dependent as YTR107 did not radiosensitize an NPM1 null mouse embryo fibroblast cell line. YTR107 produced a 4.7 fold increase in HT29 xenograft growth delay (time required for the tumor to increase 4-fold in volume (p = 0.001) following 10 mg/kg YTR107 administered 30 min prior to administering 3 Gy to the tumor for 7 consecutive days). YTR107 increased the survival of A549 tumor-bearing mice determined 70 days after the last treatment. Survival of tumor-bearing mice was 60% for mice treated with YTR107 and 3 Gy vs 20% for mice treated with 3 Gy alone, p = 0.0012 (0 or 20 mg/kg YTR107 administered 1 hr prior to 0 or 2.2 Gy being administered to the tumor for 7 consecutive days). Female C57BL/6J mice were administered 0 or 10 mg/kg i.p. YTR107 for 5 consecutive days. Thirty five days after injection mice were euthanized and subjected to necropsy by a veterinarian trained in veterinary pathology. Gross and histological examination of liver, lung, thymus, heart, spleen, cerebellum, pancreas, small intestine, kidney, and adrenal gland did not reveal evidence of toxicity. Both a complete blood count and a white blood cell differential count were performed. No significant differences were noted. Mice were subjected to five daily injections of 30 mg/kg YTR107. This dose level was also tolerated without overt toxicity after 35 days. Mice were injected with 0 or 30 mg/kg YTR107 (i.p.) and 30 min later administered 0 or 16-Gy to the thorax. Pulmonary fibrotic lesions were quantified using respiratory-gated micro CT 136 days later. YTR107 did not increase the severity of radiation-induced lung fibrosis and did not affect body weights or life span of unirradiated or irradiated mice (p > 0.05) over the 136-day study. Thus, this study provides support for development of YTR107 as a therapeutic radiation sensitizer and was supported in part by R44CA228756-02
Citation Format: Konjeti R. Sekhar, Geri Traver, Peter A. Crooks, Michael L. Freeman. Small-molecule targeting of the NPM1/Rad51 repair pathway for the radiosensitization of cancer abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6164.
The ability of chemo-radiation therapy to control locally advanced stage III non-small cell lung cancer (NSCLC) is poor. While addition of consolidation immunotherapy has improved outcomes in subsets ...of patients there is still an urgent need for new therapeutic targets. Emerging research indicates that nucleophosmin1 (NPM1) is over-expressed in NSCLC, promotes tumor growth and that over-expression correlates with a lower survival probability. NPM1 is critical for APE1 base excision activity and for RAD51-mediated repair of DNA double strand breaks (DSBs). YTR107 is a small molecule radiation sensitizer that has been shown to bind to NPM1, suppressing pentamer formation. Here we show that in irradiated cells YTR107 inhibits SUMOylated NPM1 from associating with RAD51, RAD51 foci formation and repair of DSBs. YTR107 acts synergistically with the PARP1/2 inhibitor ABT 888 to increase replication stress and radiation-induced cell lethality. YTR107 was found to radiosensitize tumor initiating cells. Congruent with this knowledge, adding YTR107 to a fractionated irradiation regimen diminished NSCLC xenograft growth and increased overall survival. These data support the hypothesis that YTR107 represents a therapeutic target for control of NSCLC.
•The small molecule YTR107 inhibits NPM1 from binding to RAD51.•YTR107 inhibits RAD51 recruitment to DNA DSBs.•YTR107 inhibits repair of DNA DSBs.•YTR107 radiosensitizes tumor initiating cells and tumor xenografts.
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