Inhibition of antiapoptotic BCL-2 proteins with ABT-263 induces fibroblast apoptosis, reversing persistent pulmonary fibrosis.

Patients with progressive fibrosing interstitial lung diseases (PF-ILDs) carry a poor prognosis and have limited therapeutic options. A hallmark feature is fibroblast resistance to apoptosis, leading to their persistence, accumulation, and excessive deposition of extracellular matrix. A complex balance of the B cell lymphoma 2 (BCL-2) protein family controlling the intrinsic pathway of apoptosis and fibroblast reliance on antiapoptotic proteins has been hypothesized to contribute to this resistant phenotype. Examination of lung tissue from patients with PF-ILD (idiopathic pulmonary fibrosis and silicosis) and mice with PF-ILD (repetitive bleomycin and silicosis) showed increased expression of antiapoptotic BCL-2 family members in α-smooth muscle actin-positive fibroblasts, suggesting that fibroblasts from fibrotic lungs may exhibit increased susceptibility to inhibition of antiapoptotic BCL-2 family members BCL-2, BCL-XL, and BCL-W with the BH3 mimetic ABT-263. We used 2 murine models of PF-ILD to test the efficacy of ABT-263 in reversing established persistent pulmonary fibrosis. Treatment with ABT-263 induced fibroblast apoptosis, decreased fibroblast numbers, and reduced lung collagen levels, radiographic disease, and histologically evident fibrosis. Our studies provide insight into how fibroblasts gain resistance to apoptosis and become sensitive to the therapeutic inhibition of antiapoptotic proteins. By targeting profibrotic fibroblasts, ABT-263 offers a promising therapeutic option for PF-ILDs.

Saracatinib, a Selective Src Kinase Inhibitor, Blocks Fibrotic Responses in Preclinical Models of Pulmonary Fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and often fatal disorder. Two U.S. Food and Drug Administration-approved antifibrotic drugs, nintedanib and pirfenidone, slow the rate of decline in lung function, but responses are variable and side effects are common. Objectives: Using an in silico data-driven approach, we identified a robust connection between the transcriptomic perturbations in IPF disease and those induced by saracatinib, a selective Src kinase inhibitor originally developed for oncological indications. Based on these observations, we hypothesized that saracatinib would be effective at attenuating pulmonary fibrosis. Methods: We investigated the antifibrotic efficacy of saracatinib relative to nintedanib and pirfenidone in three preclinical models: 1) in vitro in normal human lung fibroblasts; 2) in vivo in bleomycin and recombinant Ad-TGF-ß (adenovirus transforming growth factor-ß) murine models of pulmonary fibrosis; and 3) ex vivo in mice and human precision-cut lung slices from these two murine models as well as patients with IPF and healthy donors. Measurements and Main Results: In each model, the effectiveness of saracatinib in blocking fibrogenic responses was equal or superior to nintedanib and pirfenidone. Transcriptomic analyses of TGF-ß-stimulated normal human lung fibroblasts identified specific gene sets associated with fibrosis, including epithelial-mesenchymal transition, TGF-ß, and WNT signaling that was uniquely altered by saracatinib. Transcriptomic analysis of whole-lung extracts from the two animal models of pulmonary fibrosis revealed that saracatinib reverted many fibrogenic pathways, including epithelial-mesenchymal transition, immune responses, and extracellular matrix organization. Amelioration of fibrosis and inflammatory cascades in human precision-cut lung slices confirmed the potential therapeutic efficacy of saracatinib in human lung fibrosis. Conclusions: These studies identify novel Src-dependent fibrogenic pathways and support the study of the therapeutic effectiveness of saracatinib in IPF treatment.

PTPα promotes fibroproliferative responses after acute lung injury.

The acute respiratory distress syndrome (ARDS) is a major healthcare problem, accounting for significant mortality and long-term disability. Approximately 25% of patients with ARDS will develop an overexuberant fibrotic response, termed fibroproliferative ARDS (FP-ARDS) that portends a poor prognosis and increased mortality. The cellular pathological processes that drive FP-ARDS remain incompletely understood. We have previously shown that the transmembrane receptor-type tyrosine phosphatase protein tyrosine phosphatase-α (PTPα) promotes pulmonary fibrosis in preclinical murine models through regulation of transforming growth factor-ß (TGF-ß) signaling. In this study, we examine the role of PTPα in the pathogenesis of FP-ARDS in a preclinical murine model of acid (HCl)-induced acute lung injury. We demonstrate that although mice genetically deficient in PTPα (Ptpra-/-) are susceptible to early HCl-induced lung injury, they exhibit markedly attenuated fibroproliferative responses. In addition, early profibrotic gene expression is reduced in lung tissue after acute lung injury in Ptpra-/- mice, and stimulation of naïve lung fibroblasts with the BAL fluid from these mice results in attenuated fibrotic outcomes compared with wild-type littermate controls. Transcriptomic analyses demonstrate reduced extracellular matrix (ECM) deposition and remodeling in mice genetically deficient in PTPα. Importantly, human lung fibroblasts modified with a CRISPR-targeted deletion of PTPRA exhibit reduced expression of profibrotic genes in response to TGF-ß stimulation, demonstrating the importance of PTPα in human lung fibroblasts. Together, these findings demonstrate that PTPα is a key regulator of fibroproliferative processes following acute lung injury and could serve as a therapeutic target for patients at risk for poor long-term outcomes in ARDS.

Role of mTOR As an Essential Kinase in SCLC.

OBJECTIVES: SCLC represents 15% of all lung cancer diagnoses in the United States and has a particularly poor prognosis. We hypothesized that kinases regulating SCLC survival pathways represent therapeutically targetable vulnerabilities whose inhibition may improve SCLC outcome. METHODS: A short-hairpin RNA (shRNA) library targeting all human kinases was introduced in seven chemonaive patient-derived xenografts (PDX) and the cells were cultured in vitro and in vivo. On harvest, lost or depleted shRNAs were considered as regulating-cell survival pathways and deemed essential kinases. RESULTS: Unsupervised hierarchical cluster analysis of recovered shRNAs separated the PDXs into two clusters, suggesting kinase-based heterogeneity among the SCLC PDXs. A total of 23 kinases were identified as essential in two or more PDXs, with mechanistic Target of Rapamycin (mTOR) a candidate essential kinase in four. mTOR phosphorylation status correlated with PDX sensitivity to mTOR kinase inhibition, and mTOR inhibition sensitized the PDX to cisplatin and etoposide. In the PDX in which mTOR was defined as essential, mTOR inhibition caused a 43% decrease in tumor volume at 21 days (p < 0.01). Combining mTOR inhibition with cisplatin and etoposide decreased PDX tumor volume 96% compared with cisplatin and etoposide alone at 70 days (p < 0.002). Chemoresistance did not develop with the combination of mTOR inhibition and cisplatin and etoposide in mTOR-essential PDX over 105 days. The prevalence of phospho-mTOR-Ser-2448 in a tissue microarray of chemonaive SCLC was 27%, thus, identifying an important SCLC subtype that might benefit from the addition of mTOR inhibition to standard chemotherapy. CONCLUSIONS: These studies reveal that kinases can define SCLC subgroups, can identify therapeutic vulnerabilities, and can potentially be used to optimize therapeutic approaches. Significance We used functional genomics to identify kinases regulating SCLC survival. mTOR was identified as essential in a subset of PDXs. mTOR inhibition decreased PDX growth, sensitized PDX to cisplatin and etoposide, and prevented chemoresistance.

Interleukin-6 is required for Neuregulin-1 induced HER2 signaling in lung epithelium.

A clear understanding of the mechanisms that regulate the alveolar epithelium's barrier is critical to develop new therapeutic strategies to mitigate lung injury. The HER2/HER3 receptor tyrosine kinase complex plays a central role in maintaining the alveolar-capillary barrier. This receptor complex is activated by its ligand, neuregulin-1 (NRG-1). Interleukin-6 (IL-6) is also known to induce HER2 signaling through HER2 transphosphorylation by the IL-6 receptor (IL-6R) complex (1). Due to this interaction, we hypothesized that NRG-1 and IL-6 cooperatively interacted to activate the HER2/HER3 complex. Studies were performed in cultured pulmonary epithelial cells measuring the HER2/IL-6/IL-6R/GP130 interaction and receptor activation by western blotting and confocal microscopy, IL-6 production by ELISA, and IL-6 inhibition using specific antibodies, small molecule inhibitors and shRNA. We found that IL-6 was required for NRG-1 induced activation of HER2 in pulmonary epithelial cells. IL-6 inhibition led to a decrease in NRG-1 induced HER2 activation. The IL-6R and GP130, a subunit of the IL-6R complex, were physically associated with HER2 and were required for NRG-1 induced HER2 activation. Inhibition of GP130, the ß-subunit of the IL-6 receptor decreased NRG-1 induced HER2 activation lower than control by 38% Finally, HER2 activation increased IL-6 secretion more than two-fold over resting cells (526 ± 131 vs 231 ± 39.7 pg/ml), and inhibition of HER2 gene expression decreased basal IL-6 secretion over 80% (89 + 4.6 vs 1.3 + 0.8 pg/ml). These findings identify a requirement for IL-6 and the IL-6R complex to allow NRG-1 mediated HER2 activation, and a HER2 driven IL-6 production feedback loop.

Ubiquitin-specific protease 9x deubiquitinates and stabilizes the spinal muscular atrophy protein-survival motor neuron.

Spinal muscular atrophy (SMA), the leading genetic disorder of infant mortality, is caused by low levels of survival motor neuron (SMN) protein. Currently it is not clear how the SMN protein levels are regulated at the post-transcriptional level. In this report, we find that Usp9x, a deubiquitinating enzyme, stably associates with the SMN complex via directly interacting with SMN. Usp9x deubiquitinates SMN that is mostly mono- and di-ubiquitinated. Knockdown of Usp9x promotes SMN degradation and reduces the protein levels of SMN and the SMN complex in cultured mammalian cells. Interestingly, Usp9x does not deubiquitinate nuclear SMNΔ7, the main protein product of the SMN2 gene, which is polyubiquitinated and rapidly degraded by the proteasome. Together, our results indicate that SMN and SMNΔ7 are differently ubiquitinated; Usp9x plays an important role in stabilizing SMN and the SMN complex, likely via antagonizing Ub-dependent SMN degradation.

Nitric oxide produced by cytochrome c oxidase helps stabilize HIF-1α in hypoxic mammalian cells.

The mitochondrial respiratory chain has been reported to play a role in the stabilization of HIF-1α when mammalian cells experience hypoxia, most likely through the generation of free radicals. Although previous studies have suggested the involvement of superoxide catalyzed by complex III more recent studies raise the possibility that nitric oxide (NO) catalyzed by cytochrome c oxidase (Cco/NO), which functions in hypoxic signaling in yeast, may also be involved. Herein, we have found that HEK293 cells, which do not express a NOS isoform, possess Cco/NO activity and that this activity is responsible for an increase in intracellular NO levels when these cells are exposed to hypoxia. By using PTIO, a NO scavenger, we have also found that the increased NO levels in hypoxic HEK293 cells help stabilize HIF-1α. These findings suggest a new mechanism for mitochondrial involvement in hypoxic signaling in mammalian cells.