Small Molecule Functional Converter of B-Cell Lymphoma-2 (Bcl-2) Suppresses Breast Cancer Lung Metastasis.

The B-cell lymphoma-2 (Bcl-2) family of proteins plays a vital role in tumorigenesis. Cancer cells utilize the expression of Bcl-2 to evade therapy and develop resistance. Bcl-2 overexpression also causes cancer cells to be more invasive and metastatic. About 80% of cancer deaths are due to metastases, and yet targeted therapies for metastatic cancers are scarce. We discovered a small molecule, BFC1103, which changes the conformation of Bcl-2 to convert the antiapoptotic protein to a proapoptotic protein. BFC1103-induced apoptosis is dependent on the expression levels of Bcl-2, with higher levels causing more apoptosis. BFC1103 suppressed the growth of breast cancer lung metastasis. BFC1103 has the potential for further optimization and development for clinical testing in metastatic cancers that express Bcl-2. This study demonstrates a new approach to target Bcl-2 using a small molecule, BFC1103, to suppress metastatic disease.

Identification and Characterization of a Small Molecule Bcl-2 Functional Converter.

Cancer cells exploit the expression of anti-apoptotic protein Bcl-2 to evade apoptosis and develop resistance to therapeutics. High levels of Bcl-2 leads to sequestration of pro-apoptotic proteins causing the apoptotic machinery to halt. In this study, we report discovery of a small molecule, BFC1108 (5-chloro-N-(2-ethoxyphenyl)-2-[(4-methoxybenzyol)amino]benzamide), which targets Bcl-2 and converts it into a pro-apoptotic protein. The apoptotic effect of BFC1108 is not inhibited, but rather potentiated, by Bcl-2 overexpression. BFC1108 induces a conformational change in Bcl-2, resulting in the exposure of its BH3 domain both in vitro and in vivo. BFC1108 suppresses the growth of triple-negative breast cancer xenografts with high Bcl-2 expression and inhibits breast cancer lung metastasis. This study demonstrates a novel approach to targeting Bcl-2 using BFC1108, a small molecule Bcl-2 functional converter that effectively induces apoptosis in Bcl-2-expressing cancers. SIGNIFICANCE: We report the identification of a small molecule that exposes the Bcl-2 killer conformation and induces death in Bcl-2-expressing cancer cells. Selective targeting of Bcl-2 and elimination of cancer cells expressing Bcl-2 opens up new therapeutic avenues.

11-Cl-BBQ, a select modulator of AhR-regulated transcription, suppresses lung cancer cell growth via activation of p53 and p27Kip1.

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor and functions as a tumour suppressor in different cancer models. In the present study, we report detailed characterization of 11-chloro-7H-benzimidazo[2,1-a]benzo[de]iso-quinolin-7-one (11-Cl-BBQ) as a select modulator of AhR-regulated transcription (SMAhRT) with anti-cancer actions. Treatment of lung cancer cells with 11-Cl-BBQ induced potent and sustained AhR-dependent anti-proliferative effects by promoting G1 phase cell cycle arrest. Investigation of 11-Cl-BBQ-induced transcription in H460 cells with or without the AhR expression by RNA-sequencing revealed activation of p53 signalling. In addition, 11-Cl-BBQ suppressed multiple pathways involved in DNA replication and increased expression of cyclin-dependent kinase inhibitors, including p27Kip1 , in an AhR-dependent manner. CRISPR/Cas9 knockout of individual genes revealed the requirement for both p53 and p27Kip1 for the AhR-mediated anti-proliferative effects. Our results identify 11-Cl-BBQ as a potential lung cancer therapeutic, highlight the feasibility of targeting AhR and provide important mechanistic insights into AhR-mediated-anticancer actions.

Beyond Programmed Death-Ligand 1: B7-H6 Emerges as a Potential Immunotherapy Target in SCLC.

INTRODUCTION: The programmed death-ligand 1 (PD-L1) immune checkpoint inhibitors, atezolizumab and durvalumab, have received regulatory approval for the first-line treatment of patients with extensive-stage SCLC. Nevertheless, when used in combination with platinum-based chemotherapy, these PD-L1 inhibitors only improve overall survival by 2 to 3 months. This may be due to the observation that less than 20% of SCLC tumors express PD-L1 at greater than 1%. Evaluating the composition and abundance of checkpoint molecules in SCLC may identify molecules beyond PD-L1 that are amenable to therapeutic targeting. METHODS: We analyzed RNA-sequencing data from SCLC cell lines (n = 108) and primary tumor specimens (n = 81) for expression of 39 functionally validated inhibitory checkpoint ligands. Furthermore, we generated tissue microarrays containing SCLC cell lines and patient with SCLC specimens to confirm expression of these molecules by immunohistochemistry. We annotated patient outcomes data, including treatment response and overall survival. RESULTS: The checkpoint protein B7-H6 (NCR3LG1) exhibited increased protein expression relative to PD-L1 in cell lines and tumors (p < 0.05). Higher B7-H6 protein expression correlated with longer progression-free survival (p = 0.0368) and increased total immune infiltrates (CD45+) in patients. Furthermore, increased B7-H6 gene expression in SCLC tumors correlated with a decreased activated natural killer cell gene signature, suggesting a complex interplay between B7-H6 expression and immune signature in SCLC. CONCLUSIONS: We investigated 39 inhibitory checkpoint molecules in SCLC and found that B7-H6 is highly expressed and associated with progression-free survival. In addition, 26 of 39 immune checkpoint proteins in SCLC tumors were more abundantly expressed than PD-L1, indicating an urgent need to investigate additional checkpoint targets for therapy in addition to PD-L1.

Rapidly fatal pneumonitis from immunotherapy and concurrent SARS-CoV-2 infection in a patient with newly diagnosed lung cancer.

Immune checkpoint inhibitors (ICIs) are used for the treatment of numerous cancers, but risks associated with ICI-therapy during the COVID-19 pandemic are poorly understood. We report a case of acute lung injury in a lung cancer patient initially treated for ICI-pneumonitis and later found to have concurrent SARS-CoV-2 infection. Post-mortem analyses revealed diffuse alveolar damage in both the acute and organizing phases, with a predominantly CD68+ inflammatory infiltrate. Serum was positive for anti-SARS-CoV-2 IgG, suggesting that viral infection predated administration of ICI-therapy and may have contributed to a more fulminant clinical presentation. These data suggest the need for routine SARS-CoV-2 testing in cancer patients, where clinical and radiographic evaluations may be non-specific.

Blood-Based Surveillance Monitoring of Circulating Tumor DNA From Patients With SCLC Detects Disease Relapse and Predicts Death in Patients With Limited-Stage Disease.

INTRODUCTION: Most patients (70%) with limited-stage SCLC (LS-SCLC) who are treated with curative-intent therapy suffer disease relapse and cancer-related death. We evaluated circulating tumor DNA (ctDNA) as a predictor of disease relapse and death after definitive therapy in patients with LS-SCLC. METHODS: In our previous work, we developed a plasma-based ctDNA assay to sequence 14 genes (TP53, RB1, BRAF, KIT, NOTCH1-4, PIK3CA, PTEN, FGFR1, MYC, MYCL1, and MYCN) that are frequently mutated in SCLC. In this work, we evaluated 177 plasma samples from 23 patients with LS-SCLC who completed definitive chemoradiation (n = 21) or surgical resection (n = 2) and had an end-of-treatment blood collection (median 4 d, range 0-40 d from treatment completion) plus monthly surveillance blood sampling. Median overall survival (OS) and progression-free survival (PFS) were compared using a Wilcoxon test. RESULTS: The median OS among patients in whom we ever detected ctDNA after definitive treatment (n = 15) was 18.2 months compared with a median OS of greater than 48 months among patients in whom we never detected ctDNA after definitive treatment (n = 8; p = 0.081). The median PFS among patients in whom we ever detected ctDNA after definitive treatment was 9.1 months compared with a median PFS of greater than 48 months among patients in whom we never detected ctDNA after definitive treatment (p < 0.001). CONCLUSIONS: Detection of ctDNA in patients with LS-SCLC after curative-intent therapy predicts disease relapse and death. Prospective trials using ctDNA as an integral biomarker for therapeutic selection should be considered in SCLC.

Induction of apoptosis and suppression of tumor growth by Nur77-derived Bcl-2 converting peptide in chemoresistant lung cancer cells.

Resistance to chemotherapy is a major cause of treatment failure and poor overall survival in patients with lung cancer. Identification of molecular targets present in resistant cancer cells is essential for addressing therapeutic resistance and prolonging lung cancer patient survival. Members of the B-cell lymphoma 2 (Bcl-2) family of proteins are associated with chemotherapeutic resistance. In this study, we found that pro-survival protein Bcl-2 is upregulated in paclitaxel resistant cells, potentially contributing to chemotherapy resistance. To exploit the increase in Bcl-2 expression for targeting therapy resistance, we investigated the effects of a peptide derived from the nuclear receptor Nur77 that converts Bcl-2 from an anti-apoptotic protein to a pro-apoptotic protein. The Nur77 derived peptide preferentially induced apoptosis in paclitaxel-resistant cancer cells with high expression of Bcl-2. This peptide also induced apoptosis of multidrug resistant H69AR lung cancer cells that express Bcl-2 and inhibited their growth in 3D spheroids. The Nur77 peptide strongly suppressed the growth of paclitaxel-resistant lung cancer cells in a zebrafish xenograft tumor model. Taken together, our data supports a new strategy for treating lung cancers that acquire resistance to chemotherapy through overexpression of Bcl-2.

The anti-inflammatory drug leflunomide is an agonist of the aryl hydrocarbon receptor.

BACKGROUND: The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxicity and biological activity of dioxins and related chemicals. The AhR influences a variety of processes involved in cellular growth and differentiation, and recent studies have suggested that the AhR is a potential target for immune-mediated diseases. METHODOLOGY/PRINCIPAL FINDINGS: During a screen for molecules that activate the AhR, leflunomide, an immunomodulatory drug presently used in the clinic for the treatment of rheumatoid arthritis, was identified as an AhR agonist. We aimed to determine whether any biological activity of leflunomide could be attributed to a previously unappreciated interaction with the AhR. The currently established mechanism of action of leflunomide involves its metabolism to A771726, possibly by cytochrome P450 enzymes, followed by inhibition of de novo pyrimidine biosynthesis by A771726. Our results demonstrate that leflunomide, but not its metabolite A771726, caused nuclear translocation of AhR into the nucleus and increased expression of AhR-responsive reporter genes and endogenous AhR target genes in an AhR-dependent manner. In silico Molecular Docking studies employing AhR ligand binding domain revealed favorable binding energy for leflunomide, but not for A771726. Further, leflunomide, but not A771726, inhibited in vivo epimorphic regeneration in a zebrafish model of tissue regeneration in an AhR-dependent manner. However, suppression of lymphocyte proliferation by leflunomide or A771726 was not dependent on AhR. CONCLUSIONS: These data reveal that leflunomide, an anti-inflammatory drug, is an agonist of the AhR. Our findings link AhR activation by leflunomide to inhibition of fin regeneration in zebrafish. Identification of alternative AhR agonists is a critical step in evaluating the AhR as a therapeutic target for the treatment of immune disorders.