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Abstract The essential G1-cyclin, CCND1, is frequently overexpressed in cancer, contributing to tumorigenesis by driving cell-cycle progression. D-type cyclins are rate-limiting regulators of G1-S progression in mammalian cells via their ability to bind and activate CDK4 and CDK6. In addition, cyclin D1 conveys kinase-independent transcriptional functions of cyclin D1. Here we report that cyclin D1 associates with H2BS14 via an intrinsically disordered domain (IDD). The same region of cyclin D1 was necessary for the induction of aneuploidy, induction of the DNA damage response, cyclin D1-mediated recruitment into chromatin, and CIN gene transcription. In response to DNA damage H2BS14 phosphorylation occurs, resulting in co-localization with γH2AX in DNA damage foci. Cyclin D1 ChIP seq and γH2AX ChIP seq revealed ~14% overlap. As the cyclin D1 IDD functioned independently of the CDK activity to drive CIN, the IDD domain may provide a rationale new target to complement CDK-extinction strategies.

A report of the small molecule Carbazole Blue demonstrates inhibition of breast cancer growth and metastasis without adverse effects on normal tissue.

Abstract Tumor cell‐intrinsic programmed death‐ligand 1 (PD‐L1) signals mediate immunopathologic effects in breast, colon, and ovarian cancers and in melanomas, but bladder cancer (BC) effects are unreported. We show here that BC cell‐intrinsic PD‐L1 signals in mouse MB49 and human RT4, UM‐UC3, and UM‐UC‐14 BC cells regulate important pathologic pathways and processes, including effects not reported in other cancers. α‐PD‐L1 antibodies reduced BC cell proliferation in vitro, demonstrating direct signaling effects. BC cell‐intrinsic PD‐L1 promoted mammalian target of rapamycin complex 1 (mTORC1) signals in vitro and augmented in vivo immune‐independent cell growth and metastatic cancer spread, similar to effects we reported in melanoma and ovarian cancer. BC cell‐intrinsic PD‐L1 signals also promoted basal and stress‐induced autophagy, whereas these signals inhibited autophagy in melanoma and ovarian cancer cells. BC cell‐intrinsic PD‐L1 also mediated chemotherapy resistance to the commonly used BC chemotherapy agents cis‐platinum and gemcitabine and to the mTORC1 inhibitor, rapamycin. Thus, BC cell‐intrinsic PD‐L1 signals regulate important virulence and treatment resistance pathways that suggest novel, actionable treatment targets meriting additional studies. As a proof‐of‐concept, we showed that the autophagy inhibitor chloroquine improved cis‐platinum treatment efficacy in vivo, with greater efficacy in PD‐L1 null versus PD‐L1‐replete BC.

OBJECTIVES/GOALS: Glioblastoma (GBM) patients face a poor prognosis. Glioma stem cells (GSCs), a chemo resistant GBM subpopulation, possess enhanced DNA repair and elevated levels of epigenetic modifier KDM1A. This study aims to establish the significance of KDM1A in DNA repair and determine the potential of novel KDM1A inhibitor NCD38 to enhance TMZ efficacy in GSCs. METHODS/STUDY POPULATION: Patient derived GSCs were obtained via IRB-approved protocol from patient samples at UT Health San Antonio. KDM1A knockdown and knockout cells were generated by transduction of validated KDM1A-specific shRNA or gRNA, respectively. Brain bioavailability of KDM1A inhibitor NCD38 was established using LS-MS/MS. Effect of combination of KDM1A knockdown, knockout, or inhibition with TMZ was studied using cell viability, neurosphere, and self-renewal assays. Mechanistic studies were conducted using CUT&Tag-seq, RNA-seq, immunofluorescence, comet, Western blotting, RT-qPCR, homologous recombination (HR) or non-homologous end-joining (NHEJ) DNA repair reporter assays. In vivo efficacy of KDM1A knockdown or inhibitor alongside TMZ treatment was determined using orthotopic murine GBM models. RESULTS/ANTICIPATED RESULTS: KDM1A knockdown, knockout, or inhibition increased efficacy of TMZ in reducing cell viability and self-renewal of GSCs. Pharmacokinetic studies demonstrated KDM1A inhibitor NCD38 is readily brain penetrable. CUT&Tag-seq studies revealed KDM1A is enriched at DNA repair gene promoters. RNA-seq studies suggest KDM1A inhibition reduces DNA double strand break repair gene expression, with these findings validated using RT-qPCR and Western blotting. Knockdown, knockout, or inhibition of KDM1A attenuated HR and NHEJ-mediated DNA repair capacity. Immunofluorescence and comet assay support findings of increased DNA damage in NCD38/TMZ combination treated GSCs. Importantly, KDM1A knockdown or inhibition enhanced efficacy of TMZ and significantly improved survival of orthotopic GBM tumor-bearing mice. DISCUSSION/SIGNIFICANCE: Our results show compelling evidence that KDM1A is essential for DNA repair in GSCs and that KDM1A inhibition sensitizes GBM to TMZ via attenuation of DNA repair pathways. These findings suggest combination of KDM1A inhibitor NCD38 with TMZ could serve as a promising novel therapeutic strategy that can be translated to improve GBM patient outcomes.

The radiation and chemotherapy used for treating medulloblastoma patients cause debilitating side effects. Here, the authors show that miR-584 acts as a therapeutic adjuvant as it sensitizes medulloblastoma to radiation and chemotherapy by targeting HDAC1 or eIF4E3 to enhance spindle defects and DNA damage.

OBJECTIVES/SPECIFIC AIMS: To further elucidate the role of estrogen receptor β (ER-β) in the early endometriotic lesion attachment. METHODS/STUDY POPULATION: EECs were immortalized using a telomerase vector. Immortalized cells and parental cells were characterized by genotyping, and expression of ER-β as well as other epithelial cell markers. ER-β was knocked-down in immortalized EECs using lentivirus-mediated shRNA transduction. ER-β knockdown was confirmed by RT-qPCR and Western analysis. EEC cells with or without ER-β knockdown were used to assess their attachment to PMCs in an established in vitro assay (Lucidi, 2005). Results were analyzed with Student t-test. RESULTS/ANTICIPATED RESULTS: Genotyping using karyotype assay confirmed a normal chromosomal profile. Also positive staining for cytokeratin and lack of any staining with vimentin confirms the epithelial origin of these cells. ER-β knockdown has a significant decrease in attachment compared to control (p=0.02). DISCUSSION/SIGNIFICANCE OF IMPACT: Primary and immortalized cells were 46XX, cytokeratin positive, and vimentin negative confirming their epithelial origin. ER-β knockdown has a significant decrease in attachment compared with control.