Proteasome Inhibitors Block DNA Repair and Radiosensitize Non-Small Cell Lung Cancer

Despite optimal radiation therapy (RT), chemotherapy and/or surgery, a majority of patients with locally advanced non-small cell lung cancer (NSCLC) fail treatment. To identify novel gene targets for improved tumor control, we performed whole genome RNAi screens to identify knockdowns that most reproducibly increase NSCLC cytotoxicity. These screens identified several proteasome subunits among top hits, including the topmost hit PSMA1, a component of the core 20 S proteasome. Radiation and proteasome inhibition showed synergistic effects. Proteasome inhibition resulted in an 80–90% decrease in homologous recombination (HR), a 50% decrease in expression of NF-κB-inducible HR genes BRCA1 and FANCD2, and a reduction of BRCA1, FANCD2 and RAD51 ionizing radiation-induced foci. IκBα RNAi knockdown rescued NSCLC radioresistance. Irradiation of mice with NCI-H460 xenografts after inducible PSMA1 shRNA knockdown markedly increased murine survival compared to either treatment alone. Proteasome inhibition is a promising strategy for NSCLC radiosensitization via inhibition of NF-κB-mediated expression of Fanconi Anemia/HR DNA repair genes.American Society for Radiation Oncology (Junior Faculty Career Research Training Award)Harvard University. Joint Center for Radiation Therapy (Foundation Grant)Dana-Farber/Harvard Cancer Center (SPORE Developmental Research Project Award in Lung Cancer Research)National Cancer Institute (U.S.) (Award K08CA172354

A922 Sequential measurement of 1 hour creatinine clearance (1-CRCL) in critically ill patients at risk of acute kidney injury (AKI)

Meeting abstrac

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

Proteasome inhibition delays DNA repair <i>in vivo</i>.

<p>(A) FANCD2 immunofluorescence in 10 Gy irradiated vs. unirradiated NCI-H460 xenografts recovered from mice with or without doxycycline-induced <i>PSMA1</i> shRNA expression in tumor cells. Bar = 10 µm. (B) γ-H2AX immunohistochemistry in NCI-H460 xenograft tumors with or without doxycycline-induced <i>PSMA1</i> shRNA knockdown, recovered from mice 1, 6, and 24 hours after 10 Gy irradiation. Bar = 10 µm. (C) Quantification of immunohistochemistry for γ-H2AX in (B). Cells with ≥5 foci were scored as positive (n>400 cells). All results are mean ± SEM. P values were calculated using a two-tailed Student’s t test.</p

Role of proteasome inhibition in modulating NF-κB pathway-mediated expression of Fanconi Anemia (FA)/homologous recombination (HR) genes.

<p>Proteasome inhibition by bortezomib or <i>PSMA1</i> knockdown results in an increase in IκBα, which in turn decreases NF-κB binding to the promoters of FA/HR genes including <i>FANCD2</i> and <i>BRCA1</i>. This reduces the availability of these DNA repair proteins for recruitment to DNA damage sites, resulting in decreased RAD51 focus formation and HR following induction of DNA double strand breaks by ionizing radiation.</p

Proteasome inhibition sensitizes NSCLC cells to radiation.

<p>(A) Western blot showing protein levels of PSMA1 and PSMB5 in A549 and NCI-H460 NSCLC cells after <i>PSMA1</i> shRNA knockdown compared to non-silencing shRNA control. (B) Chymotrypsin-like (CTL) proteasome activity assay in A549 (left) and NCI-H460 (right) NSCLC cells after treatment with bortezomib, or <i>PSMA1</i> siRNA knockdown. All results are mean ± SEM and normalized to DMSO vehicle control. (C) Clonogenic survival assay of A549 (left) and NCI-H460 (right) following IR and bortezomib. Marked bars show the percent kill of bortezomib-treated samples compared to DMSO vehicle control at each IR dose. All results are mean ± SEM and normalized to DMSO vehicle control. (D) Apoptosis detection assay of NCI-H460 following 2 and 4 Gy IR and 50 nM bortezomib. Bars show percentage of cells in early apoptosis (left) or late apoptosis (right) via Annexin V and propidum iodide staining, respectively. All results are mean ± SD and P values were calculated using a two-tailed Student’s <i>t</i> test.</p

Proteasome inhibition blocks expression of Fanconi Anemia/Homologous Recombination genes.

<p>(A) Diagram of NF-kB promoters on <i>FANCD2</i> and <i>BRCA1</i> genes. (B) Expression by qPCR of <i>FANCD2</i> following proteasome inhibition ±30 nM (A549) or 50 nM (NCI-H460) bortezomib or ± inducible <i>PSMA1</i> shRNA, and ±10 Gy IR. All values are normalized to ACTB and all results are mean ± SEM. (C) As in (B) but with BRCA1. (D) NSCLC cell survival following bortezomib and IR is partially rescued by <i>IkBα</i> siRNA knockdown when performed in advance. Cell viability was assayed using an ATP luminescence-based assay measuring relative luminescent units (RLU). All results are mean ± SEM.</p

Knockdown of individual proteasome subunits results in NSCLC cytotoxicity.

<p>Diagram of the 26 S proteasome showing multiple whole genome shRNA screen hits with the following color code: top hit (red), strong hit (>1 shRNA sequence per gene in both cell lines, dark orange), minor hit (1 shRNA sequence per gene in both cell lines, light orange), chymotrypsin-like proteolytic catalytic site (not a hit but highlighted for illustrative purposes, green). Each hit is labeled using the last two alphanumeric characters of the gene’s HUGO nomenclature; e.g., A1 = PSMA1, B5 = PSMB5, M1 = SHFM1.</p