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

Characterization of carfilzomib-resistant non-small cell lung cancer cell lines.

We previously showed that carfilzomib (CFZ) has potent anti-proliferative and cytotoxic activity in a broad range of lung cancer cell lines. Here we investigate possible mechanisms of CFZ acquired resistance in lung cancer cell lines. CFZ-resistant non-small cell lung cancer (NSCLC) cell lines were developed by exposing A549 and H520 cells to stepwise increasing concentrations of CFZ. Resistance to CFZ and cross-resistance to bortezomib and other chemotherapy drugs was measured using the MTT assay. Cytotoxicity to CFZ was determined using a CytoTox assay. Western blot was used to measure apoptosis, autophagy, and drug efflux transporter-related proteins. Quantitative targeted whole transcriptome sequencing and quantitative RT-PCR was used to measure gene expression. Flow cytometry was used to analyze intracellular accumulation of doxorubicin. The CFZ IC Upregulation of Pgp appears to be an important, but not the only, mechanism of CFZ resistance in NSCLC cell lines.Onyx Pharmaceuticals, Inc.; Basic/Clinical Translational Partnership Pilot Grant award from the Arizona Cancer Center Support Grant [P30CA023074]; Basic/Clinical Translational Partnership Pilot Grant award from National Cancer Institute (NCI) [P30CA023074]12 month embargo; published online 15 May 2018This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Structure–Activity Relationships for Withanolides as Inducers of the Cellular Heat-Shock Response

To understand the relationship between the structure and the remarkably diverse bioactivities reported for withanolides, we obtained withaferin A (WA; <b>1</b>) and 36 analogues (<b>2</b>–<b>37</b>) and compared their cytotoxicity to cytoprotective heat-shock-inducing activity (HSA). By analyzing structure–activity relationships for the series, we found that the ring A enone is essential for both bioactivities. Acetylation of 27-OH of 4-<i>epi</i>-WA (<b>28</b>) to <b>33</b> enhanced both activities, whereas introduction of β-OH to WA at C-12 (<b>29</b>) and C-15 (<b>30</b>) decreased both activities. Introduction of β-OAc to 4,27-diacetyl-WA (<b>16</b>) at C-15 (<b>37</b>) decreased HSA without affecting cytotoxicity, but at C-12 (<b>36</b>), it had minimal effect. Importantly, acetylation of 27-OH, yielding <b>15</b> from <b>1</b>, <b>16</b> from <b>14</b>, and <b>35</b> from <b>34</b>, enhanced HSA without increasing cytotoxicity. Our findings demonstrate that the withanolide scaffold can be modified to enhance HSA selectively, thereby assisting development of natural product-inspired drugs to combat protein aggregation-associated diseases by stimulating cellular defense mechanisms