Trametinib sensitizes KRAS-mutant lung adenocarcinoma tumors to PD-1/PD-L1 axis blockade via Id1 downregulation.

BACKGROUND: The identification of novel therapeutic strategies to overcome resistance to the MEK inhibitor trametinib in mutant KRAS lung adenocarcinoma (LUAD) is a challenge. This study analyzes the effects of trametinib on Id1 protein, a key factor involved in the KRAS oncogenic pathway, and investigates the role of Id1 in the acquired resistance to trametinib as well as the synergistic anticancer effect of trametinib combined with immunotherapy in KRAS-mutant LUAD. METHODS: We evaluated the effects of trametinib on KRAS-mutant LUAD by Western blot, RNA-seq and different syngeneic mouse models. Genetic modulation of Id1 expression was performed in KRAS-mutant LUAD cells by lentiviral or retroviral transductions of specific vectors. Cell viability was assessed by cell proliferation and colony formation assays. PD-L1 expression and apoptosis were measured by flow cytometry. The anti-tumor efficacy of the combined treatment with trametinib and PD-1 blockade was investigated in KRAS-mutant LUAD mouse models, and the effects on the tumor immune infiltrate were analyzed by flow cytometry and immunohistochemistry. RESULTS: We found that trametinib activates the proteasome-ubiquitin system to downregulate Id1 in KRAS-mutant LUAD tumors. Moreover, we found that Id1 plays a major role in the acquired resistance to trametinib treatment in KRAS-mutant LUAD cells. Using two preclinical syngeneic KRAS-mutant LUAD mouse models, we found that trametinib synergizes with PD-1/PD-L1 blockade to hamper lung cancer progression and increase survival. This anti-tumor activity depended on trametinib-mediated Id1 reduction and was associated with a less immunosuppressive tumor microenvironment and increased PD-L1 expression on tumor cells. CONCLUSIONS: Our data demonstrate that Id1 expression is involved in the resistance to trametinib and in the synergistic effect of trametinib with anti-PD-1 therapy in KRAS-mutant LUAD tumors. These findings suggest a potential therapeutic approach for immunotherapy-refractory KRAS-mutant lung cancers.

Presence of Activated (Phosphorylated) STAT3 in Radiation Necrosis Following Stereotactic Radiosurgery for Brain Metastases.

Brain radiation necrosis (RN) is a subacute or late adverse event following radiotherapy, involving an exacerbated inflammatory response of the brain tissue. The risk of symptomatic RN associated with stereotactic radiosurgery (SRS) as part of the treatment of brain metastases (BMs) has been a subject of recent investigation. The activation of the signal transducer and activator of transcription 3 (STAT3) was shown in reactive astrocytes (RA) associated with BMs. Given that the pathophysiological mechanisms behind RN are not fully understood, we sought to investigate the role of STAT3 among other inflammatory markers in RN development. A mouse model of RN using clinical LINAC-based SRS was designed to induce brain necrosis with the administration of 50 Gy in a single fraction to the left hemisphere using a circular collimator of 5 mm diameter. Immunohistochemistry and multiplex staining for CD4, CD8, CD68, GFAP, and STAT3 were performed. For validation, eleven patients with BMs treated with SRS who developed symptomatic RN and required surgery were identified to perform staining for CD68, GFAP, and STAT3. In the mouse model, the RN and perinecrotic areas showed significantly higher staining for F4/80+ and GFAP+ cells, with a high infiltration of CD4 and CD8 T-lymphocytes, when compared to the non-irradiated cerebral hemisphere. A high number of GFAP+pSTAT3+ and F4/80+pSTAT3+ cells was found in the RN areas and the rest of the irradiated hemisphere. The analysis of human brain specimens showed that astrocytes and microglia were actively phosphorylating STAT3 in the areas of RN and gliosis. Phosphorylated STAT3 is highly expressed in the microglia and RA pertaining to the areas of brain RN. Targeting STAT3 via inhibition represents a promising strategy to ameliorate symptomatic RN in BM patients undergoing SRS.

Evaluation of trefoil factor 3 as a non-invasive biomarker of gastric intestinal metaplasia and gastric cancer in a high-risk population.

BACKGROUND: Adenocarcinoma is preceded by chronic atrophic gastritis, gastric intestinal metaplasia and dysplasia. Trefoil factor 3 (TFF3) is a peptide secreted by goblet cells, which is abundantly present in intestinal metaplasia. AIM: To evaluate the utility of serum TFF3 as a non-invasive biomarker for the diagnosis of intestinal metaplasia and gastric cancer. METHODS: Single-center, cross-sectional study of 274 patients who consecutively underwent upper gastrointestinal endoscopy with gastric biopsies (updated Sydney system). TFF3 levels were measured in serum by a commercial ELISA kit. Patients with normal histology or chronic atrophic gastritis without intestinal metaplasia comprised the control group. In addition, 14 patients with invasive gastric cancer were included as a reference group. The association between TFF3 levels and intestinal metaplasia was assessed by logistic regression. RESULTS: Patients with intestinal metaplasia (n=110) had a higher median TFF3 level as compared to controls (n=164), 13.1 vs. 11.9ng/mL, respectively (p=0.024). Multivariable logistic regression showed a no significant association between TFF3 levels and intestinal metaplasia (OR=1.20; 95%CI: 0.87-1.65; p-trend=0.273). The gastric cancer group had a median TFF3 level of 20.5ng/mL, and a significant association was found (OR=3.26; 95%CI: 1.29-8.27; p-trend=0.013). CONCLUSION: Serum levels of TFF3 do not discriminate intestinal metaplasia in this high-risk Latin American population. Nevertheless, we confirmed an association between TFF3 levels and invasive gastric cancer.

IL-6/STAT3 signaling in tumor cells restricts the expression of frameshift-derived neoantigens by SMG1 induction.

BACKGROUND: The quality and quantity of tumor neoantigens derived from tumor mutations determines the fate of the immune response in cancer. Frameshift mutations elicit better tumor neoantigens, especially when they are not targeted by nonsense-mediated mRNA decay (NMD). For tumor progression, malignant cells need to counteract the immune response including the silencing of immunodominant neoantigens (antigen immunoediting) and promoting an immunosuppressive tumor microenvironment. Although NMD inhibition has been reported to induce tumor immunity and increase the expression of cryptic neoantigens, the possibility that NMD activity could be modulated by immune forces operating in the tumor microenvironment as a new immunoediting mechanism has not been addressed. METHODS: We study the effect of SMG1 expression (main kinase that initiates NMD) in the survival and the nature of the tumor immune infiltration using TCGA RNAseq and scRNAseq datasets of breast, lung and pancreatic cancer. Different murine tumor models were used to corroborate the antitumor immune dependencies of NMD. We evaluate whether changes of SMG1 expression in malignant cells impact the immune response elicited by cancer immunotherapy. To determine how NMD fluctuates in malignant cells we generated a luciferase reporter system to track NMD activity in vivo under different immune conditions. Cytokine screening, in silico studies and functional assays were conducted to determine the regulation of SMG1 via IL-6/STAT3 signaling. RESULTS: IL-6/STAT3 signaling induces SMG1, which limits the expression of potent frameshift neoantigens that are under NMD control compromising the outcome of the immune response. CONCLUSION: We revealed a new neoantigen immunoediting mechanism regulated by immune forces (IL-6/STAT3 signaling) responsible for silencing otherwise potent frameshift mutation-derived neoantigens.

Tumor-Intrinsic Nuclear ß-Catenin Associates with an Immune Ignorance Phenotype and a Poorer Prognosis in Head and Neck Squamous Cell Carcinomas.

Activation of WNT/ß-catenin signaling has been associated with a non-T-cell-inflamed tumor microenvironment (TME) in several cancers. The aim of this work was to investigate the relationship between ß-catenin signaling and TME inflammation in head and neck squamous cell carcinomas (HNSCCs). Membrane and nuclear ß-catenin expression, PD-L1 expression, and CD8+ tumor-infiltrating lymphocyte (TIL) density were jointly evaluated by immunohistochemistry in a series of 372 HPV-negative HNSCCs. Membrane ß-catenin levels decreased in carcinomas compared to the normal epithelium. Positive nuclear ß-catenin was detected in 50 tumors (14.3%) and was significantly associated with a low CD8+ TIL density (168 cells/mm2 versus 293 cells/mm2 in nuclear-ß-catenin-negative cases; p = 0.01) and a tendency for a lower expression of PD-L1, resulting in association with a noninflamed TME (i.e., type II, immunological ignorance). Multivariate Cox analysis further demonstrated that low infiltration by CD8+ TILs (HR = 1.6, 95% CI = 1.19-2.14, p = 0.002) and nuclear ß-catenin expression (HR = 1.47, 95% CI = 1.01-2.16, p = 0.04) were both independently associated with a poorer disease-specific survival. In conclusion, tumor-intrinsic nuclear ß-catenin activation is associated with a non-inflamed TME phenotype and a poorer prognosis, thereby suggesting a possible implication as an immune exclusion mechanism for a subset of HNSCC patients.

Strategies for the Biofunctionalization of Straining Flow Spinning Regenerated Bombyx mori Fibers.

High-performance regenerated silkworm (Bombyx mori) silk fibers can be produced efficiently through the straining flow spinning (SFS) technique. In addition to an enhanced biocompatibility that results from the removal of contaminants during the processing of the material, regenerated silk fibers may be functionalized conveniently by using a range of different strategies. In this work, the possibility of implementing various functionalization techniques is explored, including the production of fluorescent fibers that may be tracked when implanted, the combination of the fibers with enzymes to yield fibers with catalytic properties, and the functionalization of the fibers with cell-adhesion motifs to modulate the adherence of different cell lineages to the material. When considered globally, all these techniques are a strong indication not only of the high versatility offered by the functionalization of regenerated fibers in terms of the different chemistries that can be employed, but also on the wide range of applications that can be covered with these functionalized fibers.

YES1 Drives Lung Cancer Growth and Progression and Predicts Sensitivity to Dasatinib.

Rationale: The characterization of new genetic alterations is essential to assign effective personalized therapies in non-small cell lung cancer (NSCLC). Furthermore, finding stratification biomarkers is essential for successful personalized therapies. Molecular alterations of YES1, a member of the SRC (proto-oncogene tyrosine-protein kinase Src) family kinases (SFKs), can be found in a significant subset of patients with lung cancer.Objectives: To evaluate YES1 (v-YES-1 Yamaguchi sarcoma viral oncogene homolog 1) genetic alteration as a therapeutic target and predictive biomarker of response to dasatinib in NSCLC.Methods: Functional significance was evaluated by in vivo models of NSCLC and metastasis and patient-derived xenografts. The efficacy of pharmacological and genetic (CRISPR [clustered regularly interspaced short palindromic repeats]/Cas9 [CRISPR-associated protein 9]) YES1 abrogation was also evaluated. In vitro functional assays for signaling, survival, and invasion were also performed. The association between YES1 alterations and prognosis was evaluated in clinical samples.Measurements and Main Results: We demonstrated that YES1 is essential for NSCLC carcinogenesis. Furthermore, YES1 overexpression induced metastatic spread in preclinical in vivo models. YES1 genetic depletion by CRISPR/Cas9 technology significantly reduced tumor growth and metastasis. YES1 effects were mainly driven by mTOR (mammalian target of rapamycin) signaling. Interestingly, cell lines and patient-derived xenograft models with YES1 gene amplifications presented a high sensitivity to dasatinib, an SFK inhibitor, pointing out YES1 status as a stratification biomarker for dasatinib response. Moreover, high YES1 protein expression was an independent predictor for poor prognosis in patients with lung cancer.Conclusions: YES1 is a promising therapeutic target in lung cancer. Our results provide support for the clinical evaluation of dasatinib treatment in a selected subset of patients using YES1 status as predictive biomarker for therapy.

Identification of mutations associated with acquired resistance to sunitinib in renal cell cancer.

Sunitinib is one of the most widely used targeted therapeutics for renal cell carcinoma (RCC), but acquired resistance against targeted therapies remains a major clinical challenge. To dissect mechanisms of acquired resistance and unravel reliable predictive biomarkers for sunitinib in RCC, we sequenced the exons of 409 tumor-suppressor genes and oncogenes in paired tumor samples from an RCC patient, obtained at baseline and after development of acquired resistance to sunitinib. From newly arising mutations, we selected, using in silico prediction models, six predicted to be deleterious, located in G6PD, LRP1B, SETD2, TET2, SYNE1, and DCC. Consistently, immunoblotting analysis of lysates derived from sunitinib-desensitized RCC cells and their parental counterparts showed marked differences in the levels and expression pattern of the proteins encoded by these genes. Our further analysis demonstrates essential roles for these proteins in mediating sunitinib cytotoxicity and shows that their loss of function renders tumor cells resistant to sunitinib in vitro and in vivo. Finally, sunitinib resistance induced by continuous exposure or by inhibition of the six proteins was overcome by treatment with cabozantinib or a low-dose combination of lenvatinib and everolimus. Collectively, our results unravel novel markers of acquired resistance to sunitinib and clinically relevant approaches for overcoming this resistance in RCC.

Scaling Laws of an Exploding Liquid Column under an Intense Ultrashort X-Ray Pulse.

A general formulation of the partial destruction of a liquid object in vacuum after the sudden deposition of a very large amount of energy is proposed. That energy instantaneously raises the pressure of a portion of the liquid to extreme values and changes its state, which causes its explosive expansion into vacuum and against the rest of the liquid object. When the deformable object is a liquid capillary column, the model reduces to a universal equation for the evolution of the expanding gap between the two sides of the exploding liquid column. The theoretical analysis contemplates two asymptotic stages for small and large times from the initiation of the blast, whose asymptotic solutions are fitted to available experimental data. A universal approximate analytical solution is obtained. A complete dimensional analysis of the problem and an optimal collapse of experimental data reveal that the proposed solution is in remarkable agreement with experiments of a jet exploding after being irradiated by an ultrashort and intense x-ray pulse from an x-ray free electron laser.