Survival with Axicabtagene Ciloleucel in Large B-Cell Lymphoma.

BACKGROUND: In an analysis of the primary outcome of this phase 3 trial, patients with early relapsed or refractory large B-cell lymphoma who received axicabtagene ciloleucel (axi-cel), an autologous anti-CD19 chimeric antigen receptor T-cell therapy, as second-line treatment had significantly longer event-free survival than those who received standard care. Data were needed on longer-term outcomes. METHODS: In this trial, we randomly assigned patients with early relapsed or refractory large B-cell lymphoma in a 1:1 ratio to receive either axi-cel or standard care (two to three cycles of chemoimmunotherapy followed by high-dose chemotherapy with autologous stem-cell transplantation in patients who had a response). The primary outcome was event-free survival, and key secondary outcomes were response and overall survival. Here, we report the results of the prespecified overall survival analysis at 5 years after the first patient underwent randomization. RESULTS: A total of 359 patients underwent randomization to receive axi-cel (180 patients) or standard care (179 patients). At a median follow-up of 47.2 months, death had been reported in 82 patients in the axi-cel group and in 95 patients in the standard-care group. The median overall survival was not reached in the axi-cel group and was 31.1 months in the standard-care group; the estimated 4-year overall survival was 54.6% and 46.0%, respectively (hazard ratio for death, 0.73; 95% confidence interval [CI], 0.54 to 0.98; P = 0.03 by stratified two-sided log-rank test). This increased survival with axi-cel was observed in the intention-to-treat population, which included 74% of patients with primary refractory disease and other high-risk features. The median investigator-assessed progression-free survival was 14.7 months in the axi-cel group and 3.7 months in the standard-care group, with estimated 4-year percentages of 41.8% and 24.4%, respectively (hazard ratio, 0.51; 95% CI, 0.38 to 0.67). No new treatment-related deaths had occurred since the primary analysis of event-free survival. CONCLUSIONS: At a median follow-up of 47.2 months, axi-cel as second-line treatment for patients with early relapsed or refractory large B-cell lymphoma resulted in significantly longer overall survival than standard care. (Funded by Kite; ZUMA-7 ClinicalTrials.gov number, NCT03391466.).

Axicabtagene ciloleucel vs standard of care in second-line large B-cell lymphoma: outcomes by metabolic tumor volume.

ABSTRACT: Metabolic tumor volume (MTV) assessed using 2-deoxy-2-[18F]fluoro-d-glucose positron emission tomography, a measure of tumor burden, is a promising prognostic indicator in large B-cell lymphoma (LBCL). This exploratory analysis evaluated relationships between baseline MTV (categorized as low [median or less] vs high [greater than median]) and clinical outcomes in the phase 3 ZUMA-7 study (NCT03391466). Patients with LBCL relapsed within 12 months of or refractory to first-line chemoimmunotherapy were randomized 1:1 to axicabtagene ciloleucel (axi-cel; autologous anti-CD19 chimeric antigen receptor T-cell therapy) or standard care (2-3 cycles of chemoimmunotherapy followed by high-dose chemotherapy with autologous stem cell transplantation in patients who had a response). All P values are descriptive. Within high- and low-MTV subgroups, event-free survival (EFS) and progression-free survival (PFS) were superior with axi-cel vs standard care. EFS in patients with high MTV (vs low MTV) was numerically shorter with axi-cel and was significantly shorter with standard care. PFS was shorter in patients with high MTV vs low MTV in both the axi-cel and standard-care arms, and median MTV was lower in patients in ongoing response at data cutoff vs others. Median MTV was higher in patients treated with axi-cel who experienced grade ≥3 neurologic events or cytokine release syndrome (CRS) than in patients with grade 1/2 or no neurologic events or CRS, respectively. Baseline MTV less than or equal to median was associated with better clinical outcomes in patients receiving axi-cel or standard care for second-line LBCL. The trial was registered at www.clinicaltrials.gov as #NCT03391466.

Axicabtagene Ciloleucel as Second-Line Therapy for Large B-Cell Lymphoma.

BACKGROUND: The prognosis of patients with early relapsed or refractory large B-cell lymphoma after the receipt of first-line chemoimmunotherapy is poor. METHODS: In this international, phase 3 trial, we randomly assigned, in a 1:1 ratio, patients with large B-cell lymphoma that was refractory to or had relapsed no more than 12 months after first-line chemoimmunotherapy to receive axicabtagene ciloleucel (axi-cel, an autologous anti-CD19 chimeric antigen receptor T-cell therapy) or standard care (two or three cycles of investigator-selected, protocol-defined chemoimmunotherapy, followed by high-dose chemotherapy with autologous stem-cell transplantation in patients with a response to the chemoimmunotherapy). The primary end point was event-free survival according to blinded central review. Key secondary end points were response and overall survival. Safety was also assessed. RESULTS: A total of 180 patients were randomly assigned to receive axi-cel and 179 to receive standard care. The primary end-point analysis of event-free survival showed that axi-cel therapy was superior to standard care. At a median follow-up of 24.9 months, the median event-free survival was 8.3 months in the axi-cel group and 2.0 months in the standard-care group, and the 24-month event-free survival was 41% and 16%, respectively (hazard ratio for event or death, 0.40; 95% confidence interval, 0.31 to 0.51; P<0.001). A response occurred in 83% of the patients in the axi-cel group and in 50% of those in the standard-care group (with a complete response in 65% and 32%, respectively). In an interim analysis, the estimated overall survival at 2 years was 61% in the axi-cel group and 52% in the standard-care group. Adverse events of grade 3 or higher occurred in 91% of the patients who received axi-cel and in 83% of those who received standard care. Among patients who received axi-cel, grade 3 or higher cytokine release syndrome occurred in 6% and grade 3 or higher neurologic events in 21%. No deaths related to cytokine release syndrome or neurologic events occurred. CONCLUSIONS: Axi-cel therapy led to significant improvements, as compared with standard care, in event-free survival and response, with the expected level of high-grade toxic effects. (Funded by Kite; ZUMA-7 ClinicalTrials.gov number, NCT03391466.).

Farnesyltransferase Inhibition Exacerbates Eosinophilic Inflammation and Airway Hyperreactivity in Mice with Experimental Asthma: The Complex Roles of Ras GTPase and Farnesylpyrophosphate in Type 2 Allergic Inflammation.

Ras, a small GTPase protein, is thought to mediate Th2-dependent eosinophilic inflammation in asthma. Ras requires cell membrane association for its biological activity, and this requires the posttranslational modification of Ras with an isoprenyl group by farnesyltransferase (FTase) or geranylgeranyltransferase (GGTase). We hypothesized that inhibition of FTase using FTase inhibitor (FTI)-277 would attenuate allergic asthma by depleting membrane-associated Ras. We used the OVA mouse model of allergic inflammation and human airway epithelial (HBE1) cells to determine the role of FTase in inflammatory cell recruitment. BALB/c mice were first sensitized then exposed to 1% OVA aerosol or filtered air, and half were injected daily with FTI-277 (20 mg/kg per day). Treatment of mice with FTI-277 had no significant effect on lung membrane-anchored Ras, Ras protein levels, or Ras GTPase activity. In OVA-exposed mice, FTI-277 treatment increased eosinophilic inflammation, goblet cell hyperplasia, and airway hyperreactivity. Human bronchial epithelial (HBE1) cells were pretreated with 5, 10, or 20 µM FTI-277 prior to and during 12 h IL-13 (20 ng/ml) stimulation. In HBE1 cells, FTase inhibition with FTI-277 had no significant effect on IL-13-induced STAT6 phosphorylation, eotaxin-3 peptide secretion, or Ras translocation. However, addition of exogenous FPP unexpectedly augmented IL-13-induced STAT6 phosphorylation and eotaxin-3 secretion from HBE1 cells without affecting Ras translocation. Pharmacological inhibition of FTase exacerbates allergic asthma, suggesting a protective role for FTase or possibly Ras farnesylation. FPP synergistically augments epithelial eotaxin-3 secretion, indicating a novel Ras-independent farnesylation mechanism or direct FPP effect that promotes epithelial eotaxin-3 production in allergic asthma.

Src regulates cigarette smoke-induced ceramide generation via neutral sphingomyelinase 2 in the airway epithelium.

We previously demonstrated that the neutral sphingomyelinase (nSMase) 2 is the sole sphingomyelinase activated during cigarette smoke (CS)-induced oxidative stress of human airway epithelial cells, leading to ceramide generation and subsequent apoptosis of affected cells. Since then, we reported that nSMase2 is a phosphoprotein, the degree of enzymatic activity and stability of which are dictated by its degree of phosphorylation. Simultaneously, the non-receptor tyrosine kinase and proto-oncogene Src has increasingly become a target of interest in both smoking-related lung injury, such as chronic obstructive pulmonary disease, and lung cancer. Within this context, we tested and now present Src as a regulator of ceramide generation via modulation of nSMase2 phosphorylation and activity during CS-induced oxidative stress. Specifically, we provide evidence that Src activity is necessary for both CS-induced ceramide accumulation in vivo (129/Sv mice) and in vitro (human airway epithelial cells) and for nSMase2 activity during CS-induced oxidative stress. Moreover, because nSMase2 is exclusively phosphorylated on serines, we show that this occurs through Src-dependent activation of the serine/threonine kinase p38 mitogen-activated protein kinase during oxidative stress. Finally, we provide evidence that Src and p38 mitogen-activated protein kinase activities are critical for regulating nSMase2 phosphorylation. This study provides insights into a molecular target involved in smoking-related lung injury, represented here as nSMase2, and its modulation by the oncogene Src.

Product Attributes of CAR T-cell Therapy Differentially Associate with Efficacy and Toxicity in Second-line Large B-cell Lymphoma (ZUMA-7).

Treatment resistance and toxicities remain a risk following chimeric antigen receptor (CAR) T-cell therapy. Herein, we report pharmacokinetics, pharmacodynamics, and product and apheresis attributes associated with outcomes among patients with relapsed/refractory large B-cell lymphoma (LBCL) treated with axicabtagene ciloleucel (axi-cel) in ZUMA-7. Axi-cel peak expansion associated with clinical response and toxicity, but not response durability. In apheresis material and final product, a naive T-cell phenotype (CCR7+CD45RA+) expressing CD27 and CD28 associated with improved response durability, event-free survival, progression-free survival, and a lower number of prior therapies. This phenotype was not associated with high-grade cytokine release syndrome (CRS) or neurologic events. Higher baseline and postinfusion levels of serum inflammatory markers associated with differentiated/effector products, reduced efficacy, and increased CRS and neurologic events, thus suggesting targets for intervention. These data support better outcomes with earlier CAR T-cell intervention and may improve patient care by informing on predictive biomarkers and development of next-generation products. SIGNIFICANCE: In ZUMA-7, the largest randomized CAR T-cell trial in LBCL, a naive T-cell product phenotype (CCR7+CD45RA+) expressing CD27 and CD28 associated with improved efficacy, decreased toxicity, and a lower number of prior therapies, supporting earlier intervention with CAR T-cell therapy. In addition, targets for improvement of therapeutic index are proposed. This article is featured in Selected Articles from This Issue, p. 4.

Impact of tumor microenvironment on efficacy of anti-CD19 CAR T cell therapy or chemotherapy and transplant in large B cell lymphoma.

The phase 3 ZUMA-7 trial in second-line large B cell lymphoma demonstrated superiority of anti-CD19 CAR T cell therapy (axicabtagene ciloleucel (axi-cel)) over standard of care (SOC; salvage chemotherapy followed by hematopoietic transplantation) ( NCT03391466 ). Here, we present a prespecified exploratory analysis examining the association between pretreatment tumor characteristics and the efficacy of axi-cel versus SOC. B cell gene expression signature (GES) and CD19 expression associated significantly with improved event-free survival for axi-cel (P = 0.0002 for B cell GES; P = 0.0165 for CD19 expression) but not SOC (P = 0.9374 for B cell GES; P = 0.5526 for CD19 expression). Axi-cel showed superior event-free survival over SOC irrespective of B cell GES and CD19 expression (P = 8.56 × 10-9 for B cell GES high; P = 0.0019 for B cell GES low; P = 3.85 × 10-9 for CD19 gene high; P = 0.0017 for CD19 gene low). Low CD19 expression in malignant cells correlated with a tumor GES consisting of immune-suppressive stromal and myeloid genes, highlighting the inter-relation between malignant cell features and immune contexture substantially impacting axi-cel outcomes. Tumor burden, lactate dehydrogenase and cell-of-origin impacted SOC more than axi-cel outcomes. T cell activation and B cell GES, which are associated with improved axi-cel outcome, decreased with increasing lines of therapy. These data highlight differences in resistance mechanisms to axi-cel and SOC and support earlier intervention with axi-cel.

Neutral sphingomyelinase 2 activity and protein stability are modulated by phosphorylation of five conserved serines.

We previously presented that the neutral sphingomyelinase 2 (nSMase2) is the only SMase activated in human airway epithelial (HAE) cells following exposure to oxidative stress (ox-stress), yielding ceramide accumulation and thereby inducing apoptosis. Furthermore, we reported that nSMase2 is a phospho-protein in which the level of phosphorylation controls nSMase2 activation induced by ox-stress. Here we identify five specific serines that are phosphorylated in nSMase2 and demonstrate that their phosphorylation controls the nSMase2 activity upon ox-stress exposure in an interdependent manner. Furthermore, we show that the nSMase2 protein stability and thus its level of expression is also post-translationally regulated by these five serine phosphorylation sites. This study provides initial structure/function insights regarding nSMase2 phosphorylation sites and offers some new links for future studies aiming to fully elucidate nSMase2 regulatory machinery.

Simvastatin inhibits smoke-induced airway epithelial injury: implications for COPD therapy.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death. The statin drugs may have therapeutic potential in respiratory diseases such as COPD, but whether they prevent bronchial epithelial injury is unknown. We hypothesised that simvastatin attenuates acute tobacco smoke-induced neutrophilic lung inflammation and airway epithelial injury. Spontaneously hypertensive rats were given simvastatin (20 mg·kg(-1) i.p.) daily for either 7 days prior to tobacco smoke exposure and during 3 days of smoke exposure, or only during tobacco smoke exposure. Pretreatment with simvastatin prior to and continued throughout smoke exposure reduced the total influx of leukocytes, neutrophils and macrophages into the lung and airways. Simvastatin attenuated tobacco smoke-induced cellular infiltration into lung parenchymal and airway subepithelial and interstitial spaces. 1 week of simvastatin pretreatment almost completely prevented smoke-induced denudation of the airway epithelial layer, while simvastatin given only concurrently with the smoke exposure had no effect. Simvastatin may be a novel adjunctive therapy for smoke-induced lung diseases, such as COPD. Given the need for statin pretreatment there may be a critical process of conditioning that is necessary for statins' anti-inflammatory effects. Future work is needed to elucidate the mechanisms of this statin protective effect.

Lung cancer and lung injury: the dual role of ceramide.

Sphingolipids play key roles in cancer, yet our current understanding of sphingolipid function in lung cancer is limited to a few key players. The best characterized of these are sphingosine-1-phosphate and ceramide which are described for their opposing roles in cell fate. However, because sphingolipids as a whole are readily interconverted by a complex enzymatic machinery, no single sphingolipid appears to have exactly one role. Instead, the roles of specific sphingolipids appear to be context specific as demonstrated by findings that ceramide-1-phosphate has both proliferative and apoptotic effects depending on its concentration. Therefore, we present herein several years of research on ceramide, a sphingolipid linked to apoptotic signaling, that is emerging in cancer research for its potential roles in proliferation and cell-to-cell communication via exosomes.Ceramide is a well-studied sphingolipid in both normal and pathological conditions ranging from skin development to lung cancer. Interestingly, several groups have previously reported its increased levels in emphysema patients who are smokers, a patient subpopulation greatly susceptible to lung cancer. However, the molecular mechanisms through which cigarette smoke (CS) and ceramide accumulation lead to lung cancer, non-small cell lung cancer (NSCLC) specifically, are unknown.Interestingly, recent studies clearly establish that two signaling pathways are activated during CS exposure in the lung airway. One centers on the activation of neutral sphingomyelinase2 (nSMase2), an enzyme that hydrolyzes sphingomyelin to ceramide. The other pathway focuses on the oncogenic EGF receptor (EGFR), which becomes aberrantly activated but not degraded, leading to prolonged proliferative signaling. Recent studies show that these two signaling pathways may actually converge and integrate. Specifically, Goldkorn et al. demonstrated that during CS exposure, EGFR is favorably co-localized in ceramide-enriched regions of the plasma membrane, proposing that nSMase2/ceramide plays a role in the aberrant EGFR activation, leading to augmented tumorigenic signaling. Moreover, new findings indicate that CS exposure may induce resistance to the tyrosine kinase inhibitors (TKIs), used for treatment of NSCLC, merely through posttranslational molecular alterations. Furthermore, structural anomalies of the CS-activated EGFR appear to be supported by the excess ceramide produced by the CS-activated nSMase2 in the plasma membrane of lung epithelial cells.We present in this chapter the progression of the sphingolipid field in lung cancer using ceramide as an example. However, many crucial questions remain to be answered regarding the role of sphingolipids in lung cancer because of the glut of promising observations.

Axicabtagene Ciloleucel in Combination with the 4-1BB Agonist Utomilumab in Patients with Relapsed/Refractory Large B-Cell Lymphoma: Phase 1 Results from ZUMA-11.

PURPOSE: Chimeric antigen receptor (CAR) T-cell therapies have shown clinical benefit for patients with relapsed/refractory (R/R) large B-cell lymphoma (LBCL), yet approximately 60% of patients do not respond or eventually relapse. We investigated the safety and feasibility of the CD19-directed CAR T-cell therapy axicabtagene ciloleucel (axi-cel) in combination with the 4-1BB agonist antibody utomilumab as an approach to improve efficacy of CAR T-cell therapy. PATIENTS AND METHODS: In phase 1 of the single-arm ZUMA-11 trial, patients with R/R LBCL received a single axi-cel infusion (target dose, 2 × 106 cells/kg) plus utomilumab 10 to 200 mg intravenously every 4 weeks for up to 6 months in a dose-escalation design. The primary endpoint was incidence of dose-limiting toxicities (DLT) with utomilumab. Key secondary endpoints were safety, antitumor activity, pharmacokinetics, and pharmacodynamics. RESULTS: No DLTs were observed among patients treated with axi-cel and utomilumab (n = 12). Grade ≥3 adverse events occurred in 10 patients (83%); none were Grade ≥3 cytokine release syndrome or neurologic events. The objective response rate was 75% and seven patients (58%) had a complete response. Peak CAR T-cell levels increased in a utomilumab dose-dependent manner up to 100 mg. Patients who received utomilumab 100 mg had persistently increased CAR T cells on days 57 to 168 compared with other dose levels. Utomilumab was associated with dose-dependent increases in IL2, IFNγ, and IL10. CONCLUSIONS: Utomilumab-mediated 4-1BB agonism combined with axi-cel therapy had a manageable safety profile. Dual 4-1BB and CD28 costimulation is a feasible therapeutic approach that may enhance CAR T-cell expansion in patients with LBCL.

Safety and Efficacy of Axicabtagene Ciloleucel versus Standard of Care in Patients 65 Years of Age or Older with Relapsed/Refractory Large B-Cell Lymphoma.

PURPOSE: Older patients with relapsed/refractory (R/R) large B-cell lymphoma (LBCL) may be considered ineligible for curative-intent therapy including high-dose chemotherapy with autologous stem-cell transplantation (HDT-ASCT). Here, we report outcomes of a preplanned subgroup analysis of patients ≥65 years in ZUMA-7. PATIENTS AND METHODS: Patients with LBCL refractory to or relapsed ≤12 months after first-line chemoimmunotherapy were randomized 1:1 to axicabtagene ciloleucel [axi-cel; autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy] or standard of care (SOC; 2-3 cycles of chemoimmunotherapy followed by HDT-ASCT). The primary endpoint was event-free survival (EFS). Secondary endpoints included safety and patient-reported outcomes (PROs). RESULTS: Fifty-one and 58 patients aged ≥65 years were randomized to axi-cel and SOC, respectively. Median EFS was greater with axi-cel versus SOC (21.5 vs. 2.5 months; median follow-up: 24.3 months; HR, 0.276; descriptive P < 0.0001). Objective response rate was higher with axi-cel versus SOC (88% vs. 52%; OR, 8.81; descriptive P < 0.0001; complete response rate: 75% vs. 33%). Grade ≥3 adverse events occurred in 94% of axi-cel and 82% of SOC patients. No grade 5 cytokine release syndrome or neurologic events occurred. In the quality-of-life analysis, the mean change in PRO scores from baseline at days 100 and 150 favored axi-cel for EORTC QLQ-C30 Global Health, Physical Functioning, and EQ-5D-5L visual analog scale (descriptive P < 0.05). CAR T-cell expansion and baseline serum inflammatory profile were comparable in patients ≥65 and <65 years. CONCLUSIONS: Axi-cel is an effective second-line curative-intent therapy with a manageable safety profile and improved PROs for patients ≥65 years with R/R LBCL.

Neutral sphingomyelinase 2: a novel target in cigarette smoke-induced apoptosis and lung injury.

Chronic obstructive pulmonary disease (COPD) is caused by exposure to cigarette smoke (CS). One mechanism of CS-induced lung injury is aberrant generation of ceramide, which leads to elevated apoptosis of epithelial and endothelial cells in the alveolar spaces. Recently, we discovered that CS-induced ceramide generation and apoptosis in pulmonary cells is governed by neutral sphingomyelinase (nSMase) 2. In the current experiments, we expanded our studies to investigate whether nSMase2 governs ceramide generation and apoptosis in vivo using rodent and human models of CS-induced lung injury. We found that exposure of mice or rats to CS leads to colocalizing elevations of ceramide levels and terminal deoxynucleotidyl transferase mediated X-dUTP nick end labeling-positive cells in lung tissues. These increases are nSMase2 dependent, and are abrogated by treatment with N-acetyl cysteine or anti-nSMase2 small interfering RNA (siRNA). We further showed that mice that are heterozygous for nSMase2 demonstrate significant decrease in ceramide generation after CS exposure, whereas acidic sphingomyelinase (aSMase) knockout mice maintain wild-type ceramide levels, confirming our previous findings (in human airway epithelial cells) that only nSMase2, and not aSMase, is activated by CS exposure. Lastly, we found that lung tissues from patients with emphysema (smokers) display significantly higher levels of nSMase2 expression compared with lung tissues from healthy control subjects. Taken together, these data establish the central in vivo role of nSMase2 in ceramide generation, aberrant apoptosis, and lung injury under CS exposure, underscoring its promise as a novel target for the prevention of CS-induced airspace destruction.

Neutral sphingomyelinase 2 (nSMase2) is a phosphoprotein regulated by calcineurin (PP2B).

We previously reported that exposure of human airway epithelial cells to oxidative stress increased ceramide generation via specific activation of neutral sphingomyelinase2 (nSMase2). Here we show that nSMase2 is a phosphoprotein exclusively phosphorylated at serine residues. The level of nSMase2 phosphorylation can be modulated by treatment with anisomycin or phorbol 12-myristate 13-acetate (PMA/12-O-tetradecanoylphorbol-13-acetate), suggesting that p38 mitogen-activated protein kinase (MAPK) and protein kinases Cs are upstream of nSMase2 phosphorylation. Oxidative stress enhances both the activity and phosphorylation of nSMase2. Strikingly, we show here that nSMase2 is bound directly by the phosphatase calcineurin (CaN), which acts as an on/off switch for nSMase2 phosphorylation in the presence or absence of oxidative stress. Specifically, CaN is being inhibited/degraded and therefore does not bind nSMase2 under oxidative stress, and a mutant nSMase2 that lacks the CaN binding site exhibits constitutively elevated phosphorylation and increased activity relative to wild type nSMase2. Importantly, the phosphorylation and activity of the mutant no longer responds to oxidative stress, confirming that CaN is the critical link that allows oxidative stress to modulate nSMase2 phosphorylation and function.

Lung injury and cancer: Mechanistic insights into ceramide and EGFR signaling under cigarette smoke.

Cigarette smoke has been connected to an array of chronic lung diseases and is a major source of morbidity and mortality. Active smoking is responsible for approximately 90% of lung cancer cases. In addition, cigarette smoke is associated with other chronic pulmonary diseases such as pulmonary edema, chronic bronchitis, and pulmonary emphysema, the last two also termed chronic obstructive pulmonary disease (COPD). Lung cancer and COPD are developed very frequently in chronic cigarette smokers. It has been known for some time that lung cancer incidence increases in patients with COPD. Even the existence of some low-grade emphysema without noticeable airflow obstruction is associated with significantly elevated risk of lung cancer. These recent clinical insights demand new thinking and exploration of novel mechanistic studies to fully understand these observations. Lung injury and repair involve cell death and hyperplasia of airway epithelial cells and infiltration of inflammatory cells. All of these occur simultaneously. The mechanisms of cell death and hyperplasia in the lung constitute two sides of the coin of lung injury and repair. However, most molecular studies in airway epithelial cells center on the mechanism(s) of either cell growth and proliferation or cell death and the ceramide-generating machinery that drives aberrant induction of apoptotic cell death. Very few address both sides of the coin as an outcome of cigarette smoke exposure, which is the focus of this review.

High Proliferation Rate and a Compromised Spindle Assembly Checkpoint Confers Sensitivity to the MPS1 Inhibitor BOS172722 in Triple-Negative Breast Cancers.

BOS172722 (CCT289346) is a highly potent, selective, and orally bioavailable inhibitor of spindle assembly checkpoint kinase MPS1. BOS172722 treatment alone induces significant sensitization to death, particularly in highly proliferative triple-negative breast cancer (TNBC) cell lines with compromised spindle assembly checkpoint activity. BOS172722 synergizes with paclitaxel to induce gross chromosomal segregation defects caused by MPS1 inhibitor-mediated abrogation of the mitotic delay induced by paclitaxel treatment. In in vivo pharmacodynamic experiments, BOS172722 potently inhibits the spindle assembly checkpoint induced by paclitaxel in human tumor xenograft models of TNBC, as measured by inhibition of the phosphorylation of histone H3 and the phosphorylation of the MPS1 substrate, KNL1. This mechanistic synergy results in significant in vivo efficacy, with robust tumor regressions observed for the combination of BOS172722 and paclitaxel versus either agent alone in long-term efficacy studies in multiple human tumor xenograft TNBC models, including a patient-derived xenograft and a systemic metastasis model. The current target indication for BOS172722 is TNBC, based on their high sensitivity to MPS1 inhibition, the well-defined clinical patient population with high unmet need, and the synergy observed with paclitaxel.

Environmentally relevant cadmium concentrations affect development and induce apoptosis of Paracentrotus lividus larvae cultured in vitro.

Sea urchin embryos and larvae represent suitable model systems on where to investigate the effects of heavy metals on development and cell viability. Here, we tested the toxic effects of low (10(-12 )M), medium (10(-9 )M), and high (10(-6 )M) cadmium chloride concentrations, mimicking unpolluted, moderately and highly polluted seawaters, respectively, on Paracentrotus lividus sea urchins offspring. Larvae were continuously treated from fertilization and inspected at time intervals comprised between 10 and 30 days of development. Delays and/or morphological abnormalities were firstly evident in larvae treated for 15 days with high cadmium (10(-6 )M) and for 25 days with medium cadmium (10(-9 )M). Major defects consisted in the reduction and lack of arms and skeleton elongation. No obvious differences with respect to controls were observed in embryos/larvae exposed to low cadmium (10(-12) M), even after 30 days of exposure. Using in situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay (TUNEL) assay on larvae whole mounts, we detected apoptosis after 10 days of treatment with 10(-6) and 10(-9) M CdCl(2,) when no morphological abnormalities were recognizable yet. Supernumerary apoptotic cells were found in arm buds, ciliary bands, and apex. In conclusion, echinoderm embryos and larvae represent candidates of choice for the study of stress and defense mechanisms activated by cadmium exposure.

Cadmium induces an apoptotic response in sea urchin embryos.

Cadmium is a heavy metal toxic for living organisms even at low concentrations. It does not have any biological role, and since it is a permanent metal ion, it is accumulated by many organisms. In the present paper we have studied the apoptotic effects of continuous exposure to subacute/sublethal cadmium concentrations on a model system: Paracentrotus lividus embryos. We demonstrated, by atomic absorption spectrometry, that the intracellular amount of metal increased during exposure time. We found, using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, that long treatments with cadmium triggered a severe DNA fragmentation. We demonstrated, by immunocytochemistry on whole-mount embryos, that treatment with cadmium causes activation of caspase-3 and cleavage of death substrates alpha-fodrin and lamin A. Incubating the embryos since fertilization with Z-DEVD FMK, a caspase-3 inhibitor, we found, by immunocytochemistry, that cleavage by caspase-3 and cleavage of death substrates were inactivated.

PIM1 kinase regulates cell death, tumor growth and chemotherapy response in triple-negative breast cancer.

Triple-negative breast cancers (TNBCs) have poor prognosis and lack targeted therapies. Here we identified increased copy number and expression of the PIM1 proto-oncogene in genomic data sets of patients with TNBC. TNBC cells, but not nonmalignant mammary epithelial cells, were dependent on PIM1 for proliferation and protection from apoptosis. PIM1 knockdown reduced expression of the anti-apoptotic factor BCL2, and dynamic BH3 profiling of apoptotic priming revealed that PIM1 prevents mitochondrial-mediated apoptosis in TNBC cell lines. In TNBC tumors and their cellular models, PIM1 expression was associated with several transcriptional signatures involving the transcription factor MYC, and PIM1 depletion in TNBC cell lines decreased, in a MYC-dependent manner, cell population growth and expression of the MYC target gene MCL1. Treatment with the pan-PIM kinase inhibitor AZD1208 impaired the growth of both cell line and patient-derived xenografts and sensitized them to standard-of-care chemotherapy. This work identifies PIM1 as a malignant-cell-selective target in TNBC and the potential use of PIM1 inhibitors for sensitizing TNBC to chemotherapy-induced apoptotic cell death.

Lung injury and lung cancer caused by cigarette smoke-induced oxidative stress: Molecular mechanisms and therapeutic opportunities involving the ceramide-generating machinery and epidermal growth factor receptor.

Chronic obstructive pulmonary disease (COPD) and lung cancer are frequently caused by tobacco smoking. However, these diseases present opposite phenotypes involving redox signaling at the cellular level. While COPD is characterized by excessive airway epithelial cell death and lung injury, lung cancer is caused by uncontrolled epithelial cell proliferation. Notably, epidemiological studies have demonstrated that lung cancer incidence is significantly higher in patients who have preexisting emphysema/lung injury. However, the molecular link and common cell signaling events underlying lung injury diseases and lung cancer are poorly understood. This review focuses on studies of molecular mechanism(s) underlying smoking-related lung injury (COPD) and lung cancer. Specifically, the role of the ceramide-generating machinery during cigarette smoke-induced oxidative stress leading to both apoptosis and proliferation of lung epithelial cells is emphasized. Over recent years, it has been established that ceramide is a sphingolipid playing a major role in lung epithelia structure/function leading to lung injury in chronic pulmonary diseases. However, new and unexpected findings draw attention to its potential role in lung development, cell proliferation, and tumorigenesis. To address this dichotomy in detail, evidence is presented regarding several protein targets, including Src, p38 mitogen-activated protein kinase, and neutral sphingomyelinase 2, the major sphingomyelinase that controls ceramide generation during oxidative stress. Furthermore, their roles are presented not only in apoptosis and lung injury but also in enhancing cell proliferation, lung cancer development, and resistance to epidermal growth factor receptor-targeted therapy for treating lung cancer.