Rapid and sustained decline in CXCL-10 (IP-10) annotates clinical outcomes following TNFα-antagonist therapy in hospitalized patients with severe and critical COVID-19 respiratory failure.

BACKGROUND: A feedforward pathological signaling loop generated by TNFα and IFN-γ synergy in the inflamed lung, driving CXCL-10 (IP-10) and CXCL-9 chemokine-mediated activated T-cell and monocyte/macrophage tissue recruitment, may define the inflammatory biology of lethal COVID-19 respiratory failure. METHODS: To assess TNFα-antagonist therapy, 18 hospitalized adults with hypoxic respiratory failure and COVID-19 pneumonia received single-dose infliximab-abda therapy 5 mg/kg intravenously between April and December 2020. The primary endpoint was time to increase in oxygen saturation to fraction of inspired oxygen ratio (SpO2/FiO2) by ≥50 compared to baseline and sustained for 48 h. Secondary endpoints included 28-day mortality, dynamic cytokine profiles, secondary infections, duration of supplemental oxygen support, and hospitalization. FINDINGS: Patients were predominantly in critical respiratory failure (15/18, 83%), male (14/18, 78%), above 60 years (median 63 years, range 31-80), race-ethnic minorities (13/18, 72%), lymphopenic (13/18, 72%), steroid-treated (17/18, 94%), with a median ferritin of 1953 ng/ml. Sixteen patients (89%) met the primary endpoint within a median of 4 days; 14/18 (78%) were discharged in a median of 8 days and were alive at 28-day follow-up. Three deaths were attributed to secondary lung infection. Mean plasma IP-10 levels declined sharply from 9183 to 483 pg/ml at Day 3 and 146 pg/ml at Day 14/discharge. Significant Day 3 declines in IFN-, TNFα, IL-27, CRP, and ferritin occurred. IP-10 and CXCL-9 declines were strongly correlated among patients with lymphopenia reversal (Day 3, Pearson r: 0.98, P-value 0.0006). INTERPRETATION: Infliximab-abda may rapidly abrogate pathological inflammatory signaling to facilitate clinical recovery in severe and critical COVID-19.

Rapid and sustained decline in CXCL-10 (IP-10) annotates clinical outcomes following TNF-α antagonist therapy in hospitalized patients with severe and critical COVID-19 respiratory failure.

BACKGROUND: A feed-forward pathological signaling loop generated by TNFα and IFN-γ in inflamed lung tissue, driving CXCL-10 (IP-10) and CXCL-9 chemokine-mediated activated T-cell and monocyte/macrophage tissue recruitment, may define, sustain and amplify the inflammatory biology of lethal COVID-19 respiratory failure. METHODS: To assess TNFα-antagonist therapy, 18 hospitalized adults with hypoxic respiratory failure and COVID-19 pneumonia received single-dose infliximab-abda therapy 5mg/kg intravenously between April and December 2020. The primary endpoint was time to increase in oxygen saturation to fraction of inspired oxygen ratio (SpO2/FiO2) by ≥ 50 compared to baseline and sustained for 48 hours. Secondary endpoints included 28-day mortality, dynamic cytokine profiles (Human Cytokine 48-Plex Discovery Assay, Eve Technologies), secondary infections, duration of supplemental oxygen support and hospitalization. FINDINGS: Patients were predominantly in critical respiratory failure (15/18, 83%), male (14/18, 78%), above 60 years (median 63 yrs, range 31-80), race-ethnic minorities (13/18, 72%), lymphopenic (13/18, 72%), steroid-treated (17/18, 94%), with a median ferritin of 1953ng/ml. Sixteen patients (89%) met the primary endpoint within a median of 4 days, 15/18 (83%) recovered from respiratory failure, and 14/18 (78%) were discharged in a median of 8 days and were alive at 28-day follow-up. Deaths among three patients ≥ 65yrs age with pre-existing lung disease or multiple comorbidities were attributed to secondary lung infection. Mean plasma IP-10 levels declined sharply from 9183 pg/ml to 483 pg/ml at Day 3 and further to 146 pg/ml at Day 14/discharge. Significant declines in IFN- γ , TNFα, IL-27, CRP and ferritin were specifically observed at Day 3 whereas other cytokines were unmodified. IL-6 levels declined sharply among patients with baseline levels >10 pg/ml. Among 13 lymphopenic patients, six (46%) had resolution of lymphopenia by day 3, and 11 by day 14. CXCR3-ligand (IP-10 and CXCL-9) declines were strongly correlated among patients with lymphopenia reversal (Day 3, Pearson r: 0.98, p-value: 0.0006). INTERPRETATION: Consistent with a pathophysiological role of TNFα, the clinical and cytokine data indicate that infliximab-abda may rapidly abrogate pathological inflammatory signaling to facilitate clinical recovery in severe and critical COVID-19. Randomized studies are required to formally assess mortality outcomes. Funding: National Center for Advancing Translational Sciences.

Aberrant integrin αv and α5 expression in prostate adenocarcinomas and bone-metastases is consistent with a bone-colonizing phenotype.

BACKGROUND: Collaborative signaling between fibronectin-binding αv and α5 integrins has been implicated in the lethal dissemination of prostate cancer in the bone-metastatic niche, the major source of morbidity and mortality in the disease. METHODS: We assessed the frequency and pattern of expression of these integrins in primary high-grade adenocarcinomas and bone metastases compared to the physiological gland. Formalin-fixed paraffin-embedded (FFPE) radical prostatectomy (RP) samples (n=25) containing ≥ Gleason grade 4 cancer and decalcified surgical or diagnostic bone metastatic samples from 10 patients were stained for integrin αv (ITGAV) and integrin α5 (ITGA5) expression. Antibody optimization and antigen-retrieval was performed beforehand. RESULTS: ITGAV was exclusively expressed in the basal layer of physiological prostate glands whereas αv expression was invariably recapitulated in the malignant gland and bone metastases (100%) in multiple distinct patterns: epithelial membranous, basilar/luminal membranous, punctate cytoplasmic, intense foci as single cells or clusters, and rim stromal layers. The luminal/basilar layer of ITGAV expression was striking in cribriform carcinomas, suggestive of a role in molecular pathogenesis. ITGA5 infrequently highlighted the basal layer of the physiological gland, was absent in primary adenocarcinoma, but was expressed with ITGAV exclusively in bone metastases (71%). CONCLUSIONS: We conclude that ITGAV expression is aberrantly expressed in high frequency in high-grade prostatic adenocarcinomas in patterns suggestive of recapitulated basal cell functions, consistent with a stem-regulatory role that has been proposed. Co-expression and enrichment of αv and α5 in osseous metastases supports their proposed collaborative role in colonization of the bone microenvironment and as candidate targets for therapy.

A Bispecific Antibody Targeting the αv and α5ß1 Integrins Induces Integrin Degradation in Prostate Cancer Cells and Is Superior to Monospecific Antibodies.

Fibronectin-binding integrins α5ß1 and αv collaborate in prostate cancer-bone stromal interactions relevant to the colonization of the bone marrow microenvironment. Combinatorial inactivation of these integrins on prostate cancer cells was assessed. Monospecific antibodies to α5ß1and αv integrins alone (MAb) and in combination (cMAb), and a bispecific antibody that simultaneously targets α5ß1and αv integrins (BsAbα5ß1/αv) were compared in assays of chemotaxis, clonogenic survival, and induction of endothelial migration. Cellular expression of integrins, their transcription, translation, and degradation fate was compared. The BsAbα5ß1/αv was superior to MAbs and cMAbs in abrogating adhesion, migration, clonogenic survival, and induction of endothelial migration responses by prostate cancer cells. Integrin upregulation observed with MAbs or cMAbs was abrogated with the BsAbα5ß1/αv. Loss of integrin expression was uniquely induced by the BsAbα5ß1/αv and blocked by lysosomal inhibition. IMPLICATIONS: A novel and effective combinatorial strategy to target α5ß1and αv integrins is defined for translational studies. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/18/1/27/F1.large.jpg.

Proteolytic fragments of fibronectin function as matrikines driving the chemotactic affinity of prostate cancer cells to human bone marrow mesenchymal stromal cells via the α5ß1 integrin.

The haematopoietic niche is contributed to by bone marrow-resident mesenchymal stromal cells (BM-MSCs) and subverted by prostate cancer cells. To study mechanisms by which BM-MSCs and prostate cancer cells may interact, we assessed the migration, invasion, adhesion and proliferation of bone-derived prostate cancer cells (PC-3) in co-culture with pluripotent human BM-MSCs. We observed a strong adhesive, migratory and invasive phenotype of PC-3 cells with BM- MSC-co-culture and set out to isolate and characterize the bioactive principle. Initial studies indicated that chemotaxis was secondary to a protein residing in the >100kDa fraction. Size-exclusion chromatography (SEC) recovered peak activity in a high-molecular weight fraction containing thrombospondin-1 (TSP1). While TSP1 immunodepletion decreased activity, put-back with purified TSP1 did not reproduce bioactivity. Further purification of the TSP1-containing high-molecular weight fraction of the BM-MSC secretome with heparin-affinity chromatography recovered bioactivity with highly restricted bands on polyacrylamide gel electrophoresis, determined by mass spectroscopy to be proteolytic fragments of fibronectin (FN). Put-back experiments with full-length FN permitted adhesion but failed to induce migration. Monospecific antibodies to FN blocked adhesion. Proteolytic cleavage of FN generated FN fragments which now induced migration. Neutralizing monoclonal antibodies to FN receptors α5 and ß1 integrins, and α5 knockdown specifically blocked migration and adhesion. CONCLUSION: Fibronectin fragments (FNFr) function as matrikines driving the chemotactic affinity of prostate cancer cells via the α5ß1 integrin. Taken together with the high-frequency of α5ß1 expression in disseminated prostate cancer cells in bone marrow aspirates from patients, the FNFr/FN-α5ß1 interaction warrants further study as a therapeutic target.

Synthetic Lethality in PTEN-Mutant Prostate Cancer Is Induced by Combinatorial PI3K/Akt and BCL-XL Inhibition.

The bone-conserved metastatic phenotype of prostate cancer is a prototype of nonrandom metastatic behavior. Adhesion of prostate cancer cells to fibronectin via the integrin α5 (ITGA5) has been proposed as a candidate bone marrow niche localization mechanism. We hypothesized that the mechanisms whereby ITGA5 regulates the adhesion-mediated survival of prostate cancer cells will define novel therapeutic approaches. ITGA5 shRNA reduced expression of BCL-2 family members and induced apoptosis in PC-3 cells. In these PTEN-mutant cells, pharmacologic inhibition of the PI3K signaling pathway in combination with ITGA5 knockdown enhanced apoptosis. Chemical parsing studies with BH3 mimetics indicated that PI3K/Akt inhibition in combination with BCL-XL-specific inhibition induces synergistic apoptosis specifically in PTEN-mutant prostate cancer cells, whereas single-agent PI3K/Akt inhibitors did not. Given the importance of PTEN loss in the progression of prostate and other cancers, synthetic lethality induced by combinatorial PI3K/Akt and BCL-XL inhibition represents a valuable therapeutic strategy. IMPLICATIONS: Activation of the PI3K pathway through PTEN loss represents a major molecular pathway in the progression of prostate and other cancers. This study defines a synthetic lethal therapeutic combination with significant translational potential. OVERVIEW: Synthetic lethality in PTEN-mutant prostate cancer cells with combined PI3K/Akt and BCL-XL inhibition. PTEN-mutant prostate cancer cells expressing ITGA5 bind to fibronectin in the putative bone marrow niche and transduce survival signals to BCL-XL Additional PTEN-regulated signals independent of the PI3K/Akt pathway likely feed into the BCL-XL-regulated survival program to explain synthetic lethality observed with the combination.Visual Overview: http://mcr.aacrjournals.org/content/early/2016/12/02/1541-7786.MCR-16-0202/F1.large.jpg. Mol Cancer Res; 14(12); 1176-81. ©2016 AACR.