MYC activation impairs cell-intrinsic IFNγ signaling and confers resistance to anti-PD1/PD-L1 therapy in lung cancer.

Elucidating the adaptive mechanisms that prevent host immune response in cancer will help predict efficacy of anti-programmed death-1 (PD1)/L1 therapies. Here, we study the cell-intrinsic response of lung cancer (LC) to interferon-γ (IFNγ), a cytokine that promotes immunoresponse and modulates programmed death-ligand 1 (PD-L1) levels. We report complete refractoriness to IFNγ in a subset of LCs as a result of JAK2 or IFNGR1 inactivation. A submaximal response affects another subset that shows constitutive low levels of IFNγ-stimulated genes (IγSGs) coupled with decreased H3K27ac (histone 3 acetylation at lysine 27) deposition and promoter hypermethylation and reduced IFN regulatory factor 1 (IRF1) recruitment to the DNA on IFNγ stimulation. Most of these are neuroendocrine small cell LCs (SCLCs) with oncogenic MYC/MYCL1/MYCN. The oncogenic activation of MYC in SCLC cells downregulates JAK2 and impairs IγSGs stimulation by IFNγ. MYC amplification tends to associate with a worse response to anti-PD1/L1 therapies. Hence alterations affecting the JAK/STAT pathway and MYC activation prevent stimulation by IFNγ and may predict anti-PD1/L1 efficacy in LC.

SMARCA4 deficient tumours are vulnerable to KDM6A/UTX and KDM6B/JMJD3 blockade.

Despite the genetic inactivation of SMARCA4, a core component of the SWI/SNF-complex commonly found in cancer, there are no therapies that effectively target SMARCA4-deficient tumours. Here, we show that, unlike the cells with activated MYC oncogene, cells with SMARCA4 inactivation are refractory to the histone deacetylase inhibitor, SAHA, leading to the aberrant accumulation of H3K27me3. SMARCA4-mutant cells also show an impaired transactivation and significantly reduced levels of the histone demethylases KDM6A/UTX and KDM6B/JMJD3, and a strong dependency on these histone demethylases, so that its inhibition compromises cell viability. Administering the KDM6 inhibitor GSK-J4 to mice orthotopically implanted with SMARCA4-mutant lung cancer cells or primary small cell carcinoma of the ovary, hypercalcaemic type (SCCOHT), had strong anti-tumour effects. In this work we highlight the vulnerability of KDM6 inhibitors as a characteristic that could be exploited for treating SMARCA4-mutant cancer patients.

Determinants of immunological evasion and immunocheckpoint inhibition response in non-small cell lung cancer: the genetic front.

The incorporation into clinical practice of immune-checkpoint inhibitors (ICIs), such as those targeting the cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and the programmed cell death 1 (PD-1) and its ligand (PD-L1), has represented a major breakthrough in non-small cell lung cancer (NSCLC) treatment, especially in cases where the cancer has no druggable genetic alterations. Despite becoming the standard of care in certain clinical settings, either alone or in combination with chemotherapy, a proportion of patients do not respond while others actually progress during treatment. Therefore, there is a clinical need to identify accurate predictive biomarkers and to develop novel therapeutic strategies based on ICIs. Although they have limitations, the current markers evaluated to select which patients will undergo ICI treatment are the levels of PD-L1 and the tumor mutational burden. In this paper we describe what is currently known about the dynamic interaction between the cancer cell and the immune system during carcinogenesis, with a particular focus on the description of the functions and gene alterations that preclude the host immunoresponse in NSCLC. We emphasize the deleterious gene alterations in components of the major histocompatibility complex (HLA-I or B2M) and of the response to IFNγ (such as JAK2) which are mutually exclusive and can affect up to one fifth of the NSCLCs. The participation of other gene alterations, such as those of common oncogenes and tumor suppressors, and of the epigenetic alterations will also be discussed, in detail. Finally, we discuss the potential use of the tumor's genetic profile to predict sensitivity to ICIs.

MET-Oncogenic and JAK2-Inactivating Alterations Are Independent Factors That Affect Regulation of PD-L1 Expression in Lung Cancer.

Purpose: The blockade of immune checkpoints such as PD-L1 and PD-1 is being exploited therapeutically in several types of malignancies. Here, we aimed to understand the contribution of the genetics of lung cancer to the ability of tumor cells to escape immunosurveillance checkpoints.Experimental Design: More than 150 primary non-small cell lung cancers, including pulmonary sarcomatoid carcinomas, were tested for levels of the HLA-I complex, PD-L1, tumor-infiltrating CD8+ lymphocytes, and alterations in main lung cancer genes. Correlations were validated in cancer cell lines using appropriate treatments to activate or inhibit selected pathways. We also performed RNA sequencing to assess changes in gene expression after these treatments.Results:MET-oncogenic activation tended to associate with positive PD-L1 immunostaining, whereas STK11 mutations were correlated with negative immunostaining. In MET-altered cancer cells, MET triggered a transcriptional increase of PD-L1 that was independent of the IFNγ-mediated JAK/STAT pathway. The activation of MET also upregulated other immunosuppressive genes (PDCD1LG2 and SOCS1) and transcripts involved in angiogenesis (VEGFA and NRP1) and in cell proliferation. We also report recurrent inactivating mutations in JAK2 that co-occur with alterations in MET and STK11, which prevented the induction of immunoresponse-related genes following treatment with IFNγ.Conclusions: We show that MET activation promotes the expression of several negative checkpoint regulators of the immunoresponse, including PD-L1. In addition, we report inactivation of JAK2 in lung cancer cells that prevented the response to IFNγ. These alterations are likely to facilitate tumor growth by enabling immune tolerance and may affect the response to immune checkpoint inhibitors. Clin Cancer Res; 24(18); 4579-87. ©2018 AACR.

Efectos aditivos de la exenatida, la glucosa-insulina-potasio y el condicionamiento isquémico a distancia frente a las arritmias ventriculares de la reperfusión en cerdos / Additive effects of exenatide, glucose-insulin-potassium, and remote ischemic conditioning against reperfusion ventricular arrhythmias in pigs

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Effects of the Selective Stretch-Activated Channel Blocker GsMtx4 on Stretch-Induced Changes in Refractoriness in Isolated Rat Hearts and on Ventricular Premature Beats and Arrhythmias after Coronary Occlusion in Swine.

Mechanical factors may contribute to ischemic ventricular arrhythmias. GsMtx4 peptide, a selective stretch-activated channel blocker, inhibits stretch-induced atrial arrhythmias. We aimed to assess whether GsMtx4 protects against ventricular ectopy and arrhythmias following coronary occlusion in swine. First, the effects of 170-nM GsMtx4 on the changes in the effective refractory period (ERP) induced by left ventricular (LV) dilatation were assessed in 8 isolated rat hearts. Then, 44 anesthetized, open-chest pigs subjected to 50-min left anterior descending artery occlusion and 2-h reperfusion were blindly allocated to GsMtx4 (57 µg/kg iv. bolus and 3.8 µg/kg/min infusion, calculated to attain the above concentration in plasma) or saline, starting 5-min before occlusion and continuing until after reflow. In rat hearts, LV distension induced progressive reductions in ERP (35±2, 32±2, and 29±2 ms at 0, 20, and 40 mmHg of LV end-diastolic pressure, respectively, P<0.001) that were prevented by GsMTx4 (33±2, 33±2, and 32±2 ms, respectively, P=0.002 for the interaction with LV end-diastolic pressure). Pigs receiving GsMtx4 had similar number of ventricular premature beats during the ischemic period as control pigs (110±28 vs. 103±21, respectively, P=0.842). There were not significant differences among treated and untreated animals in the incidence of ventricular fibrillation (13.6 vs. 22.7%, respectively, P=0.696) or tachycardia (36.4 vs. 50.0%, P=0.361) or in the number of ventricular tachycardia episodes during the occlusion period (1.8±0.7 vs. 5.5±2.6, P=0.323). Thus, GsMtx4 administered under these conditions does not suppress ventricular ectopy following coronary occlusion in swine. Whether it might protect against malignant arrhythmias should be tested in studies powered for these outcomes.

Number and distribution of mouse retinal cone photoreceptors: differences between an albino (Swiss) and a pigmented (C57/BL6) strain.

We purpose here to analyze and compare the population and topography of cone photoreceptors in two mouse strains using automated routines, and to design a method of retinal sampling for their accurate manual quantification. In whole-mounted retinas from pigmented C57/BL6 and albino Swiss mice, the longwave-sensitive (L) and the shortwave-sensitive (S) opsins were immunodetected to analyze the population of each cone type. In another group of retinas both opsins were detected with the same fluorophore to quantify all cones. In a third set of retinas, L-opsin and Brn3a were immunodetected to determine whether L-opsin+cones and retinal ganglion cells (RGCs) have a parallel distribution. Cones and RGCs were automatically quantified and their topography illustrated with isodensity maps. Our results show that pigmented mice have a significantly higher number of total cones (all-cones) and of L-opsin+cones than albinos which, in turn, have a higher population of S-opsin+cones. In pigmented animals 40% of cones are dual (cones that express both opsins), 34% genuine-L (cones that only express the L-opsin), and 26% genuine-S (cones that only express the S-opsin). In albinos, 23% of cones are genuine-S and the proportion of dual cones increases to 76% at the expense of genuine-L cones. In both strains, L-opsin+cones are denser in the central than peripheral retina, and all-cones density increases dorso-ventrally. In pigmented animals S-opsin+cones are scarce in the dorsal retina and very numerous in the ventral retina, being densest in its nasal aspect. In albinos, S-opsin+cones are abundant in the dorsal retina, although their highest densities are also ventral. Based on the densities of each cone population, we propose a sampling method to manually quantify and infer their total population. In conclusion, these data provide the basis to study cone degeneration and its prevention in pathologic conditions.

Activation of RISK and SAFE pathways is not involved in the effects of Cx43 deficiency on tolerance to ischemia-reperfusion injury and preconditioning protection.

Connexin 43 (Cx43) deficiency increases myocardial tolerance to ischemia-reperfusion injury and abolishes preconditioning protection. It is not known whether modifications in baseline signaling through protective RISK or SAFE pathways or in response to preconditioning may contribute to these effects. To answer this question we used Cx43(Cre-ER(T)/fl) mice, in which Cx43 expression is abolished after 4-hydroxytamoxifen (4-OHT) administration. Isolated hearts from Cx43(Cre-ER(T)/fl) mice, or from Cx43(fl/fl) controls, treated with vehicle or 4-OHT, were submitted to global ischemia (40 min) and reperfusion. Cx43 deficiency was associated with reduced infarct size after ischemia-reperfusion (11.17 ± 3.25 % vs. 65.04 ± 3.79, 59.31 ± 5.36 and 65.40 ± 4.91, in Cx43(fl/fl) animals treated with vehicle, Cx43(fl/fl) mice treated with 4-OHT, and Cx43(Cre-ER(T)/fl) mice treated with vehicle, respectively, n = 8-9, p < 0.001). However, the ratio phosphorylated/total protein expression for Akt, ERK-1/2, GSK3ß and STAT3 was not increased in normoxic samples from animals lacking Cx43. Instead, a reduction in the phosphorylation state of GSK3ß was observed in Cx43-deficient mice (ratio: 0.15 ± 0.02 vs. 0.56 ± 0.11, 0.77 ± 0.15, and 0.46 ± 0.14, respectively, n = 5-6, p < 0.01). Furthermore, ischemic preconditioning (IPC, 4 cycles of 3.5 min of ischemia and 5 min of reperfusion) increased phosphorylation of ERK-1/2, GSK3ß, and STAT3 in all hearts without differences between groups (n = 5-6, p < 0.05), although Cx43 deficient mice were not protected by either IPC or pharmacological preconditioning with diazoxide. Our data demonstrate that modification of RISK and SAFE signaling does not contribute to the role of Cx43 in the increased tolerance to myocardial ischemia-reperfusion injury and in preconditioning protection.

Whole number, distribution and co-expression of brn3 transcription factors in retinal ganglion cells of adult albino and pigmented rats.

The three members of the Pou4f family of transcription factors: Pou4f1, Pou4f2, Pou4f3 (Brn3a, Brn3b and Brn3c, respectively) play, during development, essential roles in the differentiation and survival of sensory neurons. The purpose of this work is to study the expression of the three Brn3 factors in the albino and pigmented adult rat. Animals were divided into these groups: i) untouched; ii) fluorogold (FG) tracing from both superior colliculli; iii) FG-tracing from one superior colliculus; iv) intraorbital optic nerve transection or crush. All retinas were dissected as flat-mounts and subjected to single, double or triple immunohistofluorescence The total number of FG-traced, Brn3a, Brn3b, Brn3c or Brn3 expressing RGCs was automatically quantified and their spatial distribution assessed using specific routines. Brn3 factors were studied in the general RGC population, and in the intrinsically photosensitive (ip-RGCs) and ipsilateral RGC sub-populations. Our results show that: i) 70% of RGCs co- express two or three Brn3s and the remaining 30% express only Brn3a (26%) or Brn3b; ii) the most abundant Brn3 member is Brn3a followed by Brn3b and finally Brn3c; iii) Brn3 a-, b- or c- expressing RGCs are similarly distributed in the retina; iv) The vast majority of ip-RGCs do not express Brn3; v) The main difference between both rat strains was found in the population of ipsilateral-RGCs, which accounts for 4.2% and 2.5% of the total RGC population in the pigmented and albino strain, respectively. However, more ipsilateral-RGCs express Brn3 factors in the albino than in the pigmented rat; vi) RGCs that express only Brn3b and RGCs that co-express the three Brn3 members have the biggest nuclei; vii) After axonal injury the level of Brn3a expression in the surviving RGCs decreases compared to control retinas. Finally, this work strengthens the validity of Brn3a as a marker to identify and quantify rat RGCs.