31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Activation of Ras in the Vascular Endothelium Induces Brain Vascular Malformations and Hemorrhagic Stroke

Summary: Cerebrovascular malformations (CVMs) affect approximately 3% of the population, risking hemorrhagic stroke, seizures, and neurological deficits. Recently Ras mutations have been identified in a majority of brain arterio-venous malformations. We generated an endothelial-specific, inducible HRASV12 mouse model, which results in dilated, proliferative blood vessels in the brain, blood-brain barrier breakdown, intracerebral hemorrhage, and rapid lethality. Organoid morphogenesis models revealed abnormal cessation of proliferation, abnormalities in expression of tip and stalk genes, and a failure to properly form elongating tubes. These defects were influenced by both hyperactive PI-3′ kinase signaling and altered TGF-β signaling. Several phenotypic changes predicted by the in vitro morphogenesis analysis were validated in the mouse model. These data provide a model of brain vascular malformations induced by mutant Ras and reveal insights into intersecting molecular mechanisms in the pathogenesis of brain vascular malformations. : Li et al. generate a mouse model of vascular malformations induced by Ras mutation. Vascular anomalies arise in the brain and involve a failure in proper tip or stalk cell definition and elongation. These changes are linked to PI3K and TGF-β signaling, revealing molecular intersections with other genetically defined malformations. Keywords: HRAS, vascular malformation, endothelial, proliferation, angiogenesis, ERK, PI-3′ kinase, TGF-β, ALK1, ALK

Loss of NF1 Expression in Human Endothelial Cells Promotes Autonomous Proliferation and Altered Vascular Morphogenesis

<div><p>Neurofibromatosis is a well known familial tumor syndrome, however these patients also suffer from a number of vascular anomalies. The loss of NFl from the endothelium is embryonically lethal in mouse developmental models, however little is known regarding the molecular regulation by NF1 in endothelium. We investigated the consequences of losing NF1 expression on the function of endothelial cells using shRNA. The loss of NF1 was sufficient to elevate levels of active Ras under non-stimulated conditions. These elevations in Ras activity were associated with activation of downstream signaling including activation of ERK, AKT and mTOR. Cells knocked down in NF1 expression exhibited no cellular senescence. Rather, they demonstrated augmented proliferation and autonomous entry into the cell cycle. These proliferative changes were accompanied by enhanced expression of cyclin D, phosphorylation of p27^KIP, and decreases in total p27^KIP levels, even under growth factor free conditions. In addition, NF1-deficient cells failed to undergo normal branching morphogenesis in a co-culture assay, instead forming planar islands with few tubules and branches. We find the changes induced by the loss of NF1 could be mitigated by co-expression of the GAP-related domain of NF1 implicating Ras regulation in these effects. Using doxycycline-inducible shRNA, targeting NF1, we find that the morphogenic changes are reversible. Similarly, in fully differentiated and stable vascular-like structures, the silencing of NF1 results in the appearance of abnormal vascular structures. Finally, the proliferative changes and the abnormal vascular morphogenesis are normalized by low-dose rapamycin treatment. These data provide a detailed analysis of the molecular and functional consequences of NF1 loss in human endothelial cells. These insights may provide new approaches to therapeutically addressing vascular abnormalities in these patients while underscoring a critical role for normal Ras regulation in maintaining the health and function of the vasculature.</p> </div

mTORC1 is critical for enhanced proliferation following the loss of NF1 (pSIREN).

<p>Control and NF1 knockdown primary endothelial cells were serum deprived for 24 h prior. Inhibitors were added was added at the time of serum starvation at the following concentrations (U0126, 1 µM; LY29002, 100 nM; Rapamycin, 0.1 ng/ml). At 21 h, some cells were pulsed with BrdU for 3 h. At 24 h cell lysates were made and probed for changes in cellular signaling (A) using phospho-specific antibodies or changes in cellular proliferation (B), visualized with an anti-BrdU antibody. (A) Results from three independent experiments were quantified from western blots similar to those shown in Supplemental 2. Data represent band average response under each condition. Error bars represent standard error of the mean and relevant statistical relationships are shown (*p<0.05 compared to Control; **p<0.01 compared to NF-kd. (B) BrdU positive cells were quantified and the data was graphed as the % positive cells compared to total cell number. The error bars represent standard error of the mean (**p<0.01 compared to control; ^#p<0.01 compared to NF1-kd). In a similar experiment, cells was serum starved 24 h in the absence and presence of rapamycin (0.1 ng/ml) and lysates were made and probed with antibodies against cyclin D1, phospho-p27 and ERK2, the latter used as a loading control. (D) Results from three experiments similar to those performed in (C) were quantified. Data represents the mean result. Error bars represent standard error of the mean (*p<0.05 compared to control; **p<0.01 compared to NF1-kd).</p

Loss of NF1 expression results in abnormal vascular morphogenesis.

<p><i>Top Panel</i> - Primary endothelial cells infected with non-targeting pSIREN lentivirus (Control) or one directed toward knockdown of NF1 (NF1-KD). These cells were plated with primary fibroblasts in an admixed co-culture. After 14 days, endothelial cells were visualized by the vector expressed GFP. <i>Bottom Panel</i> - Endothelial cells infected with TRIPZ-Control or NF1miR-shRNA, were plated in co-culture with primary fibroblasts in the presence of doxycycline to induce expression of shRNA and RFP. Vascular structures were visualized at day 14 using RFP co-expressed in the endothelial cells.</p

Loss of NF1 expression is sufficient to induce endothelial cell proliferation.

<p>(A) pSIREN Control and <i>NF1</i> knockdown primary endothelial cells were grown in complete media under sub-confluent conditions and cumulative population doublings were recorded over the course of 25 days as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049222#pone.0049222-Bajaj1" target="_blank">[10]</a>. (B) Control or NF1 knockdown endothelial cells were serum deprived for 24 h prior to cells being pulsed with BrdU for 3 h. BrdU positive cells were quantified and the data was graphed as the % positive cells compared to total cell number. Data are presented as the mean with error bars representing the standard error (**p<0.01). (C) In parallel to the experiments performed in (B), serum starved cell lysates were made after 24 h and probed with antibodies against cyclin D1, p27, phospho p27, and ERK2. ERK2 is used to insure equal loading of lysate. (D) In three independent experiments similar to those performed in (C), results were quantified and data expressed as fold-change from control. Error bars represent standard error of the mean (*p<0.05).</p

Changes in vascular morphogenesis are reversible and dependent upon active Ras.

<p>Cells for these experiments were co-infected with TRIPZ-NF1KD and pSLIK virus that was either empty or expressing the GRD domain of NF1 and sorted by FACS for double positive populations. These cells were then plated in co-culture with primary fibroblasts in the presence or absence of doxycycline as indicated. (A) Admixed cultures were allowed to form structures for 14 days. Representative fields were photographed using expressed RFP at this time and doxycycline was removed from the culture medium. Co-cultures were allowed to persist for an additional 14 days, after which time representative fields were again photographed following visualization of endothelial cells with FITC-labeled UEA-1 lectin. (B) Double positive cells were plated in the absence of doxycycline for 14 days and endothelial cells were visualized by staining live cultures with FITC labeled UEA-1 lectin and representative fields were photographed. Doxycycline was then added to cultures and they were incubated for an additional 14 days followed by visualization again using UEA-1 lectin. Arrows in both (A) and (B) highlight representative changes in morphology.</p

Abnormal vascular morphogenesis is normalized by Rapamycin.

<p>Endothelial cells infected with pSIREN expressing either non-targeting (control) or shRNA directed against NF1 (NF1-kd) were plated in co-culture with primary fibroblasts. Co-cultures were incubated in the presence of vehicle (DMSO) or Rapamycin (0.1 ng/ml) from the time of plating. Vascular structures are shown at day 14, visualized by expression of GFP in the endothelial cells by the pSIREN vector.</p

Knockdown of NF1 in Human Endothelial Cells is Sufficient to Activate Ras and Induce Cellular Signaling.

<p>(A) Early passage HUVECs were infected with a pSIREN-GFP retroviral vector carrying either a control or a NF1 shRNA and sorted for GFP expression. Western blot analysis confirmed knockdown of neurofibromin. (B) Early passage endothelial cells were infected with a pTRIPZ lentiviral vector expressing either a non-silencing control or a NF1 miR-based shRNA. The infected cells were induced with 1 µg/mL doxycycline for 48 h to induce expression of the microRNA along with red fluorescent protein (expressed in tandem) and sorted for RFP expression. Western blot confirms knockdown of neurofibromin in the presence of doxycycline while no knockdown was seen in the uninduced cells. (C) Uninduced (−Dox) and induced (+Dox) endothelial cells were serum starved for 24 h and levels of active Ras (RasGTP) were determined by using GST-Raf pull-down assay (Pierce), according to the manufacturer’s protocol. Total Ras in the total cell lysates confirmed similar amounts of protein input and was used to normalize GTP-Ras quantification. (D) Cells were treated as described above but western analysis was performed to measure activation of several key signaling proteins including phospho-Akt, phospho-S6, and phospho-ERK. Equal lysate loading was confirmed by monitoring total ERK2 levels. All experiments were performed in at least three independent sets of control and NF1 knockdown HUVECs and quantification results were averaged. (Identical results were seen with both knockdown vectors (see for example <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049222#pone-0049222-g006" target="_blank">figure 6</a>) Error bars represent standard error of the mean (**p<0.01, *p<0.05).</p