Pretreatment Of Tumor Cells Research Articles

Targeted tumor killing by pomegranate polyphenols: Pro-oxidant role of a classical antioxidant

One of the key biochemical features that distinguish a cancer cell from normal cells is its persistent pro-oxidative state that leads to intrinsic oxidative stress. Malignant cells have evolved sophisticated adaptation systems that involve high dependency on antioxidant functions and upregulation of pro-survival molecules to counteract the deleterious effects of reactive species and to maintain dynamic redox balance. This situation renders them vulnerable to further oxidative challenges by exogenous agents. In the present study, we advocated that pomegranate polyphenols act as pro-oxidants and trigger ROS-mediated apoptosis in cancer cells. With the help of both in vitro and in vivo models, we have established that pomegranate fruit extract (PFE) can cause a significant reduction in tumor proliferation while leaving normal tissues and cells unharmed. Administration of PFE (0.2% v/v) in Erhlich's ascites carcinoma-bearing mice for 3 weeks, inhibited the nuclear factor (erythroid-derived 2)-like 2-antioxidant response element signaling cascade, increased intracellular reactive oxygen species content, altered glutathione cycle thereby activating reactive oxygen species-induced apoptotic pathway in Erhlich's ascites carcinoma cells. Moreover, PFE mitigated epithelial to mesenchymal transition and migration in triple negative breast cancer cells (MDA-MB 231 cells) by down-regulating nuclear factor kappa light-chain-enhancer of activated B cells. Pre-treatment of tumor cells with N-acetyl cysteine protected these cells from undergoing PFE-induced apoptosis while siRNA-mediated silencing of Nuclear factor (erythroid-derived 2)-like 2 and nuclear factor kappa light-chain-enhancer of activated B cells in tumor cells increased the cytotoxic potential and pro-oxidative activity of PFE, indicating a clear role of these transcription factors in orchestrating the anticancer/pro-oxidative properties of PFE. The seminal findings provided may be exploited to develop potential therapeutic targets for selective killing of malignant cells.

The inhibition of protein translation promotes tumor angiogenic switch

The ‘angiogenic switch’ is critical for tumor progression. However, the pathological details and molecular mechanisms remain incompletely characterized. In this study, we established mammal xenografts in zebrafish to visually investigate the first vessel growth (angiogenic switch) in real-time, by inoculating tumor cells into the perivitelline space of live optically transparent Transgenic (flk1:EGFP) zebrafish larvae. Using this model, we found that hypoxia and hypoxia-inducible factor (HIF) signaling were unnecessary for the angiogenic switch, whereas vascular endothelial growth factor A gene (Vegfa) played a crucial role. Mechanistically, transcriptome analysis showed that the angiogenic switch was characterized by inhibition of translation, but not hypoxia. Phosphorylation of eukaryotic translation initiation factor 2 alpha (Eif2α) and the expression of Vegfa were increased in the angiogenic switch microtumors, and 3D tumor spheroids, and puromycin-treated tumor cells. Vegfa overexpression promoted early onset of the angiogenic switch, whereas Vegfa knockout prevented the first tumor vessel from sprouting. Pretreatment of tumor cells with puromycin promoted the angiogenic switch in vivo similarly to Vegfa overexpression, whereas Vegfa knockdown suppressed the increase. This study provides direc and dynamic in vivo evidences that inhibition of translation, but not hypoxia or HIF signaling promotes the angiogenic switch in tumor by increasing Vegfa transcription.

Abstract 6615: Peroxynitrite mediates immune escape of tumor cells from cytotoxic T cells in situ

Abstract Despite improved ability to generate strong tumor-specific T cell responses, clinical benefits remain relatively moderate. Only a small proportion of predicted neoepitopes was shown to induce specific cytotoxic T cells (CTLs) or serve as an efficient target for T cell-based therapy. We suggest here that low efficacy of CTLs could be at least partially explained by the fact that tumor microenvironment (TME) modulates antigen presentation and epitope profile expressed by tumor. As a result, tumor escapes recognition by CTLs. TME is enriched in peroxynitrite (PNT), a potent oxidant. It is mostly produced by infiltrating myeloid cells, but also by some tumors (as melanoma). We hypothesized that PNT is able to affect antigen presentation on tumor cells and facilitates tumor immune escape. To check this hypothesis, we treated murine EG7 tumor cells with PNT, isolated MHC I peptides and compared their expression on treated versus non-treated cells using SILAC mass spectrometry. We found that PNT treatment significantly decreased expression of a portion of MHC I peptides presented by tumor cells, whereas other peptides remained relatively intact. We found that PNT-sensitive and resistant peptides demonstrated similar binding capacity to MHC I. However, the off-rate of PNT-sensitive peptides was significantly higher. PNT strongly affected proteasomal activity of tumor cells suggesting the mechanism for under-representation of MHC I peptides with high off-rate level. To investigate the biological role of this effect, we generated CTLs specific to a pool of PNT-sensitive or resistant peptides. In vitro we observed that both types of CTLs effectively killed tumor cells. Pre-treatment of tumor cells with PNT significantly reduced killing only by CTLs specific to PNT-sensitive peptides. By ELISPOT we showed that most of antitumor-specific CTL responses in mice with different tumors were induced against PNT-resistant, but not PNT-sensitive species. Moreover, the adoptive transfer of CTLs specific to PNT-resistant peptides combined with immune checkpoint inhibition (ICI) was able to induce tumor rejection in 70% of mice, whereas similar setting for CTLs specific to PNT-sensitive peptides didn't affect tumor growth. PNT inhibition combined with ICI significantly delayed the tumor growth in mice. In addition, we showed that PNT had similar effect on MHC I peptide profile for human melanoma cells. Finally, we measured nitrotyrosine (NT) level (marker of PNT presence) in tumor tissues resected from melanoma patients before the start of ICI therapy. We retrospectively correlated NT levels with patient ability to respond to ICI therapy. We found that high NT level was a predictor of poor prognosis and patient un-responsiveness to ICI. Overall, our study demonstrates PNT ability to affect antigen presentation in tumor site. In addition, we suggest PNT as a promising target for ICI-combined therapy as well as a biomarker for resistance to ICI. Citation Format: Evgenii N. Tcyganov, Dmitry I. Gabrilovich. Peroxynitrite mediates immune escape of tumor cells from cytotoxic T cells in situ [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6615.

A steroidal saponin isolated from Allium chinense simultaneously induces apoptosis and autophagy by modulating the PI3K/Akt/mTOR signaling pathway in human gastric adenocarcinoma

Allium chinense, as a side dish on Asian table, is often used in folk medicine for its health benefits. (25R)-5α-spirostan-3β-yl-3-O-acetyl-O-β-d-glucopyranosyl-(1 → 2)-O-[β-d-glucopyranosyl-(1 → 3)]-O-β-d-glucopyranosyl-(1 → 4)-β-d-galactopyranoside (A-24) is a bioactive steroidal saponin isolated from Allium chinense. Previously, we have shown that A-24 has cytotoxic effects on cancer cells, but not on normal cells. To further explore the underlying mechanisms, in this study, we investigated the anticancer activity of A-24 in human gastric cancer cell lines in terms of cell proliferation, colony formation, cell cycle, induction of apoptosis/autophagy, and PI3K/Akt/mTOR pathway. A-24 showed dose-dependent cytotoxicity in SGC-7901 and AGS cell lines, it induced intrinsic mitochondrial pathway of apoptosis as well as autophagy, G2/M phase arrest and modulation of cyclinB1, p-cdc2, p-wee1 and p-Histone H3 expression. Furthermore, A-24 downregulated the phosphorylation of Akt at Ser473 and mTOR at Ser2448 in PI3K/Akt/mTOR pathway, and its downstream substrates p-p70S6K and p-4EBP1 in a dose-dependent manner. In addition, the pre-treatment of tumor cells with 3-methyladenine (3-MA) and LY294002 increased A-24-induced apoptosis. Collectively, these findings highlight the significance of downregulation of PI3K/Akt/mTOR pathway in A-24-induced apoptosis and autophagy, and the potential application of A-24 as a novel candidate in the treatment of human gastric adenocarcinoma.

Adoptive therapy with TCR gene-engineered T cells to treat patients with MAGE-C2-positive melanoma and head and neck cancer

Background & Aim We are scheduling a patient study, in which we will target the Cancer Germline Antigen MAGE-C2 (MC2), and use T cells with a young phenotype. MC2 is highly expressed in, amongst others, melanoma, head-and-neck squamous cell carcinoma (HNSSC), bladder, and triple-negative breast cancers but not in healthy adult tissues. Methods, Results & Conclusion We isolated MC2-specific TCRs from melanoma patients who showed clinical responses following vaccination that were accompanied by significant frequencies of anti-MC2 CD8 T cells in blood and tumor without apparent side effects. Following extensive evaluation of in vitro anti-tumor and self-reactivities, we have selected a TCR that recognizes the ALK epitope in the context of HLA-A2 for clinical development. Furthermore our preclinical studies showed that epigenetic pretreatment of tumor cells, but not normal cells, up-regulated MC2 gene expression and resulted in enhanced recognition of MC2 by the selected TCR. In parallel to the above studies, we renewed our GMP protocol to process T cells, using stimulating antibodies and cytokines, to generate T cells with a young phenotype. In a phase I/II study, we will explore the safety and anti-tumor efficacy of T cells engineered with the selected TCR in patients with MC2-positive melanoma and HNSSC. The study contains the following unique elements: - CT antigen not targeted before by T cell therapy - a new T cell processing method to generate young T cells - pretreatment of patients with epigenetic drugs to increase MC2 expression in the tumor - no chemotherapy prior to T cell infusion. All preparations have been successfully completed - including production and testing of the clinical vector batch and the clinical scale process validations. To date regulatory approvals have been obtained - including license for use of the vector and approval for the clinical trial. Patient treatment will start Q1 2020.

MAIT Cells Promote Tumor Initiation, Growth, and Metastases via Tumor MR1

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that require MHC class I-related protein 1 (MR1) for their development. The role of MAIT cells in cancer is unclear, and to date no study has evaluated these cells in vivo in this context. Here, we demonstrated that tumor initiation, growth, and experimental lung metastasis were significantly reduced in Mr1 -/- mice, compared with wild-type mice. The antitumor activity observed in Mr1 -/- mice required natural killer (NK) and/or CD8+ T cells and IFNγ. Adoptive transfer of MAIT cells into Mr1 -/- mice reversed metastasis reduction. Similarly, MR1-blocking antibodies decreased lung metastases and suppressed tumor growth. Following MR1 ligand exposure, some, but not all, mouse and human tumor cell lines upregulated MR1. Pretreatment of tumor cells with the stimulatory ligand 5-OP-RU or inhibitory ligand Ac-6-FP increased or decreased lung metastases, respectively. MR1-deleted tumors resulted in fewer metastases compared with parental tumor cells. MAIT cell suppression of NK-cell effector function was tumor-MR1-dependent and partially required IL17A. Our studies indicate that MAIT cells display tumor-promoting function by suppressing T and/or NK cells and that blocking MR1 may represent a new therapeutic strategy for cancer immunotherapy. SIGNIFICANCE: Contradicting the perception that MAIT cells kill tumor cells, here MAIT cells promoted tumor initiation, growth, and metastasis. MR1-expressing tumor cells activated MAIT cells to reduce NK-cell effector function, partly in a host IL17A-dependent manner. MR1-blocking antibodies reduced tumor metastases and growth, and may represent a new class of cancer therapeutics.This article is highlighted in the In This Issue feature, p. 1.

The Effect of Apoptosis Inhibitor Blockade Agents on the Third Generation CD19 CAR T Cells

Background: Chimeric antigen receptor (CAR) engineered T cell therapy is currently revolutionizing the field of cancer immunotherapy. However, further enhancement of the function of CAR T cells either through modification of CAR structure or through combination with other therapies is intensively investigated. Resistance to apoptosis is one of the hallmarks of cancer biology. Overexpression of anti-apoptotic proteins of the Bcl-2 family proteins, such as Bcl-2, Mcl-1 and Bcl-xl is considered primarily responsible for the increased apoptosis resistance and prolonged survival of hematological tumor cells. Apoptosis inhibitor blockade agents (AIBAs) such as Bcl-2, Mcl-1 and COX inhibitors could sensitize malignant cells to apoptosis. Therefore, combining CAR T cell therapy with AIBAs might be a promising approach to increase the therapeutic response. However, the third generation of CAR T cells including a 4-1BB co-stimulatory domain which triggers a signaling cascade with the upregulation of anti-apoptotic molecules might be hampered by AIBAs. Therefore, in this study we analyzed the influence of AIBAs on third generation CD19 CAR T cells. Materials and methods: CD19 CAR T cells were manufactured using a 3rd generation CAR vector containing both CD28 and 4-1BB co-stimulatory domains. The expression of anti-apoptotic Bcl-2 family members in leukemia/lymphoma cell lines and in CD19 CAR T cells have been assessed by quantitative real time polymerase chain reaction (qRT-PCR), western blot (WB) and flow cytometery. The optimal concentrations of inhibitors have been determined by CellTiter Glo™ assay. The effect of inhibitors on CAR T cells and tumor cells has been evaluated by chromium-51 (51Cr) release assay, Calcein AM™ assay and FACS-based apoptosis assay. Intracellular cytokine staining and surface marker staining were performed to determine the function of CAR T cells. Results: qRT-PCR and WB showed that leukemia/lymphoma cell lines 380 and U698M had the highest Bcl-2 (qRT-PCR: 1.73 ± 0.04, p = 0.001; WB: 1.82 ± 0.54, p = 0.016) and Mcl-1 (qRT-PCR: 12.39 ± 1.37, p = 0.000; WB: 1.92 ± 1.08, p = 0.142) expression levels, respectively,. These findings were confirmed by anti-apoptotic BCL-2 family protein intracellular staining. Elevated protein expression of Mcl-1, Bcl-2 and Bcl-xl was observed in CAR T cells upon 380 cell stimulation. The manufactured CD19 CAR T cells exhibited specific killing on CD19+ tumor cells. A slightly enhanced killing efficiency of CAR T cells was observed when 380 cells were co-cultured with CAR T cells in the presence of ABT199 (0 µM vs. 1 µM: 54.21 ± 3.23 vs. 69.06 ± 2.17, p = 0.0041). This result could be explained by a higher sensitivity of 380 cells to ABT199 than CART cells and by an increased activation of CAR T cells. Even though the killing efficiency of CAR T cells was not improved by S63845, the CD107a+TNF-α+IFN-γ+ CAR T cells were increased as well as the proliferation and persistence of CAR T cells were strengthened. Of note, pretreatment of tumor cells with inhibitors could significantly enhance the killing efficiency of CAR T cells via upregulation of CD19 antigen on tumor cells (ABT199-pretreated 380 cells: 0 µM vs.1 µM, 42.59 ± 5.82 vs. 54.58 ± 4.87, p = 0.0493; S63845-pretreated-U698M cells: 0 µM vs. 0.3 µM, 31.88 ± 4.77 vs. 66.35 ± 8.09, p = 0.000). Conclusion: The quantity of CAR T cells can be negatively affected by Apoptosis inhibitor blockade agents. However, the quality of CAR T cells could be improved. The cytotoxic effect of CAR T cells can be further enhanced by pretreatment of tumor cells with inhibitors. Disclosures Müller-Tidow: MSD: Membership on an entity's Board of Directors or advisory committees. Dreger:Neovii, Riemser: Research Funding; MSD: Membership on an entity's Board of Directors or advisory committees, Other: Sponsoring of Symposia; AbbVie, AstraZeneca, Gilead, Janssen, Novartis, Riemser, Roche: Consultancy; AbbVie, Gilead, Novartis, Riemser, Roche: Speakers Bureau. Schmitt:MSD: Membership on an entity's Board of Directors or advisory committees, Other: Sponsoring of Symposia; Therakos Mallinckrodt: Other: Financial Support. Schmitt:Therakos Mallinckrodt: Other: Financial Support.

Abstract 2918: Molecular analysis of the mechanism of action of AsiDNATM brings new clues on DNA damage response regulation

Abstract Purpose: Our laboratory succeeded at developing an original DNA repair inhibitor, called AsiDNATM, developed and tested in clinic by Onxeo. AsiDNATM is a small modified double-stranded DNA molecule vectorised by a covalently bound cholesterol. It acts by inhibiting DNA repair and sensitizes tumors to genotoxic treatments. Here we investigate the different steps involved in its activity. Experimental design: SK28, MRC-5 and MDA-MB-231 cells were treated by AsiDNATM. Cellular uptake of AsiDNATM was monitored by FACS and electron microscopy. AsiDNATM interacting proteins were identified by mass spectrometry in cell extracts pooled down with AsiDNA. Kinetics of activation of the two main targeted enzyme, DNA-PK and PARP, were monitored by measuring phosphorylation of H2AX and formation of poly-adenyl-ribose polymers (PAR) and NAD consumption. Inhibition of DNA repair enzymes recruitment at damage site (53BP1, XRCC4, BRCA1, Rad51 and NBS1) was analyzed by confocal microscopy. DNA damage were induced at different times after AsiDNATM addition. The role of PARP, DNA-PK and ATM enzymes in the recruitment inhibition was tested using specific inhibitors or deficient cells. DNA repair activity was analyzed by alcalin comet assay and survival curves were established. Transcriptomes were monitored 24h after AsiDNATMtreatment. Results: AsiDNATM enters the cells via cholesterol route using LDL receptors. It binds DNA-PK and PARP and triggers their activation for more than 24h. The levels of DNA-PK and PARP activation as well as NAD consumption were always higher than those induced by a 10 Gy irradiation. Survival rate to AsiDNATM alone was between 90% and 70%. No significant effect was observed on cell cycle or proliferation. Pretreatment of tumor cells with AsiDNATM inhibits DNA repair and increase cell death induced by irradiation. The earliest event observed after irradiation was a transitory phosphorylation of ATM(ser1981) disappearing after 2h. It was followed by the inhibition of 53BP1and XRCC4 recruitment in a PARP dependent manner. Then, concomitantly with γ-H2AX and PAR increase, recruitment of BRCA1 and RAD51 was inhibited in cells with high level of DNA-PK activation. This inhibition was dependent of DNA-PK and not of PARP or ATM. Transcriptome analysis showed large change in the expression of genes involved in metabolism. Conclusion: AsiDNATM inhibits NHEJ and HR double-strand break DNA repair by preventing the recruitment of key enzymes at break sites. The inhibition of NHEJ proteins recruitment is the earliest event and requires PARP activity. The inhibition of HR proteins appears lately and is dependent upon DNA-PK activation. PARP activation induces metabolism change that might participate to the antitumoral activity of AsiDNATM. These results highlight the unique mechanism of action of AsiDNATM through the activation of two complementary key enzymes involved in DNA damage response. Citation Format: Nathalie Berthault, Kozlac Maria, Sergey Alekseev, Pierre-Marie Girard, Marie Dutreix. Molecular analysis of the mechanism of action of AsiDNATM brings new clues on DNA damage response regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2918.

Abstract LB-082: ONC201 sensitizes resistant breast cancer cells to TRAIL through death receptor 5 upregulation

Abstract ONC201 is a potent inducer of cancer cell death through the tumor necrosis factor related apoptosis inducing ligand (TRAIL) pathway. ONC201 and its structural analogs comprise a novel class known as the imipridones. The compound is being tested clinically against a range of solid tumors and hematological malignancies. We found that both triple negative and non-triple negative breast cancer cells are sensitive to ONC201. This was initially surprising, as the majority of non-triple negative breast cancer cells are resistant to TRAIL. In TRAIL-sensitive cells, ONC201 induced cell death in a TRAIL-dependent manner. In TRAIL-resistant cells, the effects of ONC201 are not pro-apoptotic but are rather anti-proliferative. Here we further investigate the effects of ONC201 in TRAIL-resistant breast cancers from multiple molecular subtypes. ONC201 has been previously shown to induce transcriptional upregulation of TRAIL receptor death receptor 5 (DR5). A known mechanism of TRAIL resistance in breast cancer is low cell surface DR5 expression. We hypothesized that pre-treatment with ONC201 would upregulate DR5 and lead to TRAIL sensitization in resistant cells. Our results confirm our hypothesis and show that treatment with ONC201 transcriptionally upregulates DR5 and increases its expression at the cell surface. In addition, pre-treatment with ONC201 sensitizes TRAIL-resistant breast cancer cells to the pro-apoptotic effects of TRAIL. Caspase-8 and PARP are cleaved when cells are treated with the combination of ONC201 and TRAIL, but not when treated with either compound alone. Annexin-V PI staining and quantitation of subG1 DNA content via flow cytometry further confirmed these results. Knockdown of DR5 abrogated TRAIL sensitization by ONC201. Natural killer (NK) cells are known to use TRAIL to kill targets like tumor cells. We are exploring the hypothesis that pre-treatment of tumor cells with ONC201 would increase their killing by NK cells. We are currently investigating the importance of the TRAIL pathway and DR5 upregulation by imipridone compounds in this sensitization to NK cell induced apoptosis. In summary, these results describe a novel mechanism by which ONC201 sensitizes breast cancer cells to the effects of apoptosis inducing ligand TRAIL. They also provide preclinical rationale for the testing of the compound against breast cancers in the clinic. Citation Format: Marie D. Ralff, Jessica Wagner, Wafik S. El-Deiry. ONC201 sensitizes resistant breast cancer cells to TRAIL through death receptor 5 upregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-082.

Inflammatory cytokine production in tumor cells upon chemotherapy drug exposure or upon selection for drug resistance.

Tumor Necrosis Factor alpha (TNF-α) has been shown to be released by tumor cells in response to docetaxel, and lipopolysaccharides (LPS), the latter through activation of toll-like receptor 4 (TLR4). However, it is unclear whether the former involves TLR4 receptor activation through direct binding of the drug to TLR4 at the cell surface. The current study was intended to better understand drug-induced TNF-α production in tumor cells, whether from short-term drug exposure or in cells selected for drug resistance. ELISAs were employed to measure cytokine release from breast and ovarian tumor cells in response to several structurally distinct chemotherapy agents and/or TLR4 agonists or antagonists. Drug uptake and drug sensitivity studies were also performed. We observed that several drugs induced TNF-αrelease from multiple tumor cell lines. Docetaxel-induced cytokine production was distinct from that of LPS in both MyD88-positive (MCF-7) and MyD88-deficient (A2780) cells. The acquisition of docetaxel resistance was accompanied by increased constitutive production of TNF-αand CXCL1, which waned at higher levels of resistance. In docetaxel-resistant MCF-7 and A2780 cell lines, the production of TNF-α could not be significantly augmented by docetaxel without the inhibition of P-gp, a transporter protein that promotes drug efflux from tumor cells. Pretreatment of tumor cells with LPS sensitized MyD88-positive cells (but not MyD88-deficient) to docetaxel cytotoxicity in both drug-naive and drug-resistant cells. Our findings suggest that taxane-induced inflammatory cytokine production from tumor cells depends on the duration of exposure, requires cellular drug-accumulation, and is distinct from the LPS response seen in breast tumor cells. Also, stimulation of the LPS-induced pathway may be an attractive target for treatment of drug-resistant disease.

Abstract 3735: A novel ALDH1A selective inhibitor induces necroptosis in ovarian cancer stem-like cells

Abstract Background: The high relapse rate in ovarian cancer patients is highly consistent with a cancer stem cell (CSC) model in which rare, inherently chemoresistant CSC are capable of proliferating and differentiating to cause disease. We recently demonstrated ovarian CSC (OvCSC) can be defined by Aldehyde Dehydrogenase (ALDH) enzymatic activity. ALDH expression is increased in chemotherapy resistant OvCSC and ALDH KO can reverse chemotherapy resistance. While ALDH inhibitors exist, they are not isoform specific. We therefore identified and screened numerous putative ALDH inhibitors, based on molecular homology to the known ALDH inhibitor DEAB, and assessed their impact on OvCSC. Methods and Results: While most ALDH inhibitors, including DEAB, had no impact on CSC, we identified a novel compound, 673A which specifically depletes OvCSC in vitro. Supporting functional depletion of OvCSC, treatment of primary tumor ascites resulted in 4-20 fold decrease tumor sphere formation. ALDH inhibition is highly synergistic with cisplatin both in vitro and in vivo as assessed by cell growth curves and tumor growth. Pre-treatment of tumor cells with 673 significantly reduced tumor in tumor initiation and growth rates. Unlike non-CSC depleting ALDH inhibitors, 673A is a pan-ALDH1A isoform specific inhibitor with IC50<230 nM for ALDH1A1, 1A2, and 1A3. OvCSC Caspase independent cell death was accompanied by negative Annexin V stain and HMB1 translocation to cytoplasm. IF demonstrated nuclear swelling and loss of DNA in a manner consistent with karyolysis and programmed cell necrosis. TEM of treated cells demonstrated clear cellular, nuclear, and organelle swelling and severe vacuolation of cytoplasm consistent with necrosis. Consistent with the induction of programmed cell necrosis, 673A treatment resulted in increased intracellular calcium. Furthermore, the intracellular calcium chelator BAPTA was able to rescue ALDH inhibition-induced necrosis. As RIP1 and RIP3 kinase activity is known to be a major mediator of necroptosis, we next assessed the impact of the RIP1 kinase inhibitor, Nec-1, and down-regulation of RIP1 and RIP3 by siRNA. Inhibition or depletion of RIP Kinase 1 did not prevent 673A mediated necroptosis of OvCSC. 673A initiates necroptotic cell death through translocation of RIPK3 downstream target, MLKL, to membrane and activation of mitochondria fission protein DRP1 and its translocation from cytoplasm to mitochondria. Conclusions: We have identified a novel ALDH inhibitor, 673A, which selective inhibits three ALDH1A isoforms. 673A mediated ALDH1A isoform inhibition triggers programmed cell necrosis of OvCSC. Our data suggest that pan ALDH1A1 targeted therapy this may be a powerful new therapeutic approach to target OvCSC and bypass their resistance to apoptosis inducing drugs. Future studies will elucidate the importance of mitochondria metabolic pathways (Glycolysis, OXPHOS, and TCA cycle) in ALDH inhibition-induced necroptosis. Citation Format: Ilana Chefetz-Menaker, Kun Yang, Ron Buckanovich. A novel ALDH1A selective inhibitor induces necroptosis in ovarian cancer stem-like cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3735.

Adoptive transfer of natural killer cells promotes the anti-tumor efficacy of T cells

The density of NK cells in tumors correlates positively with prognosis in many types of cancers. The average number of infiltrating NK cells is, however, quite modest (approximately 30 NK cells/sq.mm), even in tumors deemed to have a “high” density of infiltrating NK cells. It is unclear how such low numbers of tumor-infiltrating NK cells can influence outcome. Here, we used ovalbumin-expressing tumor cell lines and TCR transgenic, OVA-specific cytotoxic T lymphocytes (OT-I-CTLs) to determine whether the simultaneous attack by anti-tumor CTLs and IL-2-activated NK (A-NK) cells synergistically increases the overall tumor cell kill and whether upregulation of tumor MHC class-I by NK cell-derived interferon-gamma (IFNγ) improves tumor-recognition and kill by anti-tumor CTLs.At equal E:T ratios, A-NK cells killed OVA-expressing tumor cells better than OT-I-CTLs. The cytotoxicity against OVA-expressing tumor cells increased by combining OT-I-CTLs and A-NK cells, but the increase was additive rather than synergistic. A-NK cells adenovirally-transduced to produce IL-12 (A-NKIL-12) produced high amounts of IFNγ. The addition of a low number of A-NKIL-12 cells to OT-I-CTLs resulted in a synergistic, albeit modest, increase in overall cytotoxicity. Pre-treatment of tumor cells with NK cell-conditioned medium increased tumor MHC expression and sensitivity to CTL-mediated killing. Pre-treatment of CTLs with NK cell-conditioned medium had no effect on CTL cytotoxicity. In vivo, MHC class-I expression by OVA-expressing B16 melanoma lung metastases increased significantly within 24–48h after adoptive transfer of A-NKIL-12 cells. OT-I-CTLs and A-NKIL-12 cells localized selectively and equally well into OVA-expressing B16 lung metastases and treatment of mice bearing 7-days-old OVA-B16 lung metastases with both A-NKIL-12 cells and OT-I-CTLs lead to a significant prolongation of survival.Thus, an important function of tumor-infiltrating NK cells may be to increase tumor cell expression of MHC class-I through secretion of IFNγ, to prepare them for recognition by tumor-specific CTLs.

Abstract A42: MK-1775, a WEE1 inhibitor alleviates resistance to immune attack of tumor cells undergoing an epithelial-mesenchymal transition

Abstract The phenomenon of epithelial-mesenchymal transition (EMT) is a phenotypic switch that allows epithelial tumor cells to acquire features of mesenchymal cells, including the ability to migrate and invade and, therefore, to disseminate from the primary site. In addition to promoting tumor dissemination, various reports have demonstrated that acquisition of features of EMT associates with tumor resistance to the cytotoxic effects of chemotherapy, radiation, and some targeted therapies. It is not well understood, however, the contribution of EMT to the escape of tumors from host immune-surveillance and immune-mediated rejection. Our laboratory has characterized the T-box transcription factor brachyury as a tumor-associated antigen and a regulator of EMT in human carcinomas. By generating isogenic cancer cell lines with various levels of brachyury expression, we demonstrated that high levels of brachyury significantly reduce the susceptibility of cancer cells to lysis by both antigen-specific T cells and natural killer cells, and render tumor cells less responsive to a cancer vaccine in syngeneic murine tumor models. This resistance was due to inefficient caspase-dependent apoptosis, manifested as inefficient nuclear lamin degradation in the presence of activated effector caspases. We correlated this phenomenon to loss of cell cycle kinase CDK1, which mediates lamin phosphorylation. In support of a causal connection, pre-treatment of tumor cells with MK-1775, a specific inhibitor of WEE1, which is known to be a negative regulator kinase of CDK1 activity, could counter the defective apoptosis of tumor cells expressing high levels of brachyury. Thus, our findings suggest reconstituting CDK1 activity to threshold levels might be sufficient to elevate immune-mediated lysis of mesenchymal-like cancer cells, and improve the efficacy of anti-tumor vaccines. Citation Format: Duane H. Hamilton, Romaine I. Fernando, Kwong-Yok Tsang, Claudia Palena. MK-1775, a WEE1 inhibitor alleviates resistance to immune attack of tumor cells undergoing an epithelial-mesenchymal transition. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A42.

In vivo Effects in Melanoma of ROCK Inhibition-Induced FasL Overexpression.

Ectopic Fas-ligand (FasL) expression in tumor cells is responsible for both tumor escape through tumor counterattack of Fas-positive infiltrating lymphocytes and tumor rejection though inflammatory and immune responses. We have previously shown that RhoA GTPase and its effector ROCK negatively control FasL membrane expression in murine melanoma B16F10 cells. In this study, we found that B16F10 treatment with the ROCK inhibitor H1152 reduced melanoma development in vivo through FasL membrane overexpression. Although H1152 treatment did not reduce tumor growth in vitro, pretreatment of tumor cells with this inhibitor delayed tumor appearance, and slowed tumor growth in C57BL/6 immunocompetent mice. Thanks to the use of mice-bearing mutated Fas receptors (B6/lpr), we found that reduced tumor growth, observed in immunocompetent mice, was linked to FasL overexpression induced by H1152 treatment. Tumor growth analysis in immunosuppressed NUDE and IFN-γ-KO mice highlighted major roles for T lymphocytes and IFN-γ in the H1152-induced tumor growth reduction. Histological analyses of subcutaneous tumors, obtained from untreated versus H1152-treated B16F10 cells, showed that H1152 pretreatment induced a strong intratumoral infiltration of leukocytes. Cytofluorometric analysis showed that among these leukocytes, the number of activated CD8 lymphocytes was increased. Moreover, their antibody-induced depletion highlighted their main responsibility in tumor growth reduction. Subcutaneous tumor growth was also reduced by repeated intravenous injections of a clinical ROCK inhibitor, Fasudil. Finally, H1152-induced ROCK inhibition also reduced pulmonary metastasis implantation independently of T cell-mediated immune response. Altogether, our data suggest that ROCK inhibitors could become interesting pharmacological molecules for melanoma immunotherapy.

Cross-Presentation of the Oncofetal Tumor Antigen 5T4 from Irradiated Prostate Cancer Cells--A Key Role for Heat-Shock Protein 70 and Receptor CD91.

Immune responses contribute to the success of radiotherapy of solid tumors; however, the mechanism of triggering CD8(+) T-cell responses is poorly understood. Antigen cross-presentation from tumor cells by dendritic cells (DC) is a likely dominant mechanism to achieve CD8(+) T-cell stimulation. We established a cross-presentation model in which DCs present a naturally expressed oncofetal tumor antigen (5T4) from irradiated DU145 prostate cancer cells to 5T4-specific T cells. The aim was to establish which immunogenic signals are important in radiation-induced cross-presentation. Radiation (12 Gy) caused G2-M cell-cycle arrest and cell death, increased cellular 5T4 levels, high-mobility protein group-B1 (HMGB1) release, and surface calreticulin and heat-shock protein-70 (Hsp70) expression in DU145 cells. DCs phagocytosed irradiated tumor cells efficiently, followed by upregulation of CD86 on phagocytic DCs. CD8(+) 5T4-specific T cells, stimulated with these DCs, proliferated and produced IFNγ. Inhibition of HMGB1 or the TRIF/MyD88 pathway only had a partial effect on T-cell stimulation. Unlike previous investigators, we found no evidence that DCs carrying Asp299Gly Toll-like receptor-4 (TLR4) single-nucleotide polymorphism had impaired ability to cross-present tumor antigen. However, pretreatment of tumor cells with Hsp70 inhibitors resulted in a highly statistically significant and robust prevention of antigen cross-presentation and CD86 upregulation on DCs cocultured with irradiated tumor cells. Blocking the Hsp70 receptor CD91 also abolished cross-presentation. Together, the results from our study demonstrate that irradiation induces immunologically relevant changes in tumor cells, which can trigger CD8(+) T-cell responses via a predominantly Hsp70-dependent antigen cross-presentation process.

WEE1 Inhibition Alleviates Resistance to Immune Attack of Tumor Cells Undergoing Epithelial–Mesenchymal Transition

Aberrant expression of the T-box transcription factor brachyury in human carcinomas drives the phenomenon of epithelial-mesenchymal transition (EMT), a phenotypic modulation that facilitates tumor dissemination and resistance to conventional therapies, including chemotherapy and radiotherapy. By generating isogenic cancer cell lines with various levels of brachyury expression, we demonstrate that high levels of brachyury also significantly reduce the susceptibility of cancer cells to lysis by both antigen-specific T cells and natural killer cells. Our results indicated that resistance of brachyury-high tumor cells to immune-mediated attack was due to inefficient caspase-dependent apoptosis, manifested as inefficient nuclear lamin degradation in the presence of activated effector caspases. We correlated this phenomenon with loss of cell-cycle-dependent kinase 1 (CDK1), which mediates lamin phosphorylation. In support of a causal connection, pretreatment of tumor cells with a specific inhibitor of WEE1, a negative regulator kinase of CDK1, could counter the defective apoptosis of tumor cells expressing high levels of brachyury. Thus, our findings suggested that reconstituting CDK1 activity to threshold levels may be sufficient to restore immunosurveillance of mesenchymal-like cancer cells that have escaped previous immune detection or eradication.

Abstract 3731: Targeting ovarian cancer stem cells using a novel ALDH inhibitor.

Abstract Background: Cancer cells with stem cell-like properties (CSC) are resistant to chemotherapy and are believed to be the source of cancer recurrence. Aldehyde dehydrogenase enzymatic activity (ALDH), alone and in combination with the stem cell marker CD133, can be used to define a hierarchy of Ovarian Cancer Stem Cells (OvCSC). ALDH is functionally important for stem cells, regulating motile cellular metabolic functions and the biosynthesis of retinoic acid. ALDH agonists have been demonstrated to enhance ‘stemness’ in some tissues. We therefore sought to determine the impact of ALDH antagonist on OvCSC. Methods and Results: We screened a variety of novel derivatives with confirmed or potential ALDH activity based on the molecular homology to the known ALDH inhibitor DEAB. Using the ALDEFLUOR assay and A2780 cancer cell line as a source of ALDH+CD133+ cells, we identified a lead compound 673 which effectively inhibits ALDH activity. Interestingly this compound also potently depletes CD133+ ovarian cancer cells. Supporting functional depletion of OvCSC, treatment of primary human ovarian tumor ascites cells with 673 resulted in 4-20 fold decrease tumor sphere formation. 673 treatment is highly synergistic with cisplatin both in-vitro and in-vivo as assessed by cell growth curves and tumor growth. Importantly, pre-treatment of tumor cells with 673 significantly reduced tumor in tumor initiation and growth rates. Finally, 673 is non-toxic to normal stem cells. Conclusions: We have identified a novel ALDH inhibitor, 673, which depletes the ovarian cancer cells expressing CSC markers. CSC marker independent functional assays, with both cell lines and primary human ovarian cancer cells, indicate 673 is depleting OvCSC activity. Thus this may be a powerful new therapeutic approach to target CSC. Future studies will elucidate the molecular mechanism that underlies 673 OvCSC depletion activities as well as to identify a specific ALDH isoforms targeted in ovarian cancer. Citation Format: Ilana Chefetz, Kun Yang, Scott D. Larsen, Ronald J. Buckanovich. Targeting ovarian cancer stem cells using a novel ALDH inhibitor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3731. doi:10.1158/1538-7445.AM2013-3731

Doxorubicin sensitizes human tumor cells to NK cell‐ and T‐cell‐mediated killing by augmented TRAIL receptor signaling

Doxorubicin (DOX) is an anthracycline antibiotic that is widely used to treat different types of malignancy. In this study, it was studied whether DOX could be used to render tumor cells susceptible to apoptosis by NK and T cells. Pretreatment with subapoptotic doses of DOX sensitized tumor cell lines of various histotypes to both NK and T cells resulting in a 3.7 to 32.7% increase in lysis (2.5 mean fold increase, p < 0.0001) and a 2.9 to 14.2% increase in lysis (3.0 mean-fold increase, p < 0.05), respectively. The sensitizing effect of the drug was primarily dependent on the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/TRAIL-receptor signaling, but not on Fas-ligand, perforin, NKG2D or DNAM-1. The central role of the TRAIL signaling pathway was further supported by an increased expression of TRAIL-R2 on DOX-treated tumor cells and by downregulation of cellular FLICE inhibitory protein, the inhibitors of death receptor-mediated apoptosis. Compared to untreated cells, pretreatment of tumor cells with DOX showed increased processing and activation of caspase-8 on coculture with NK or T cells. The significance of this treatment strategy was confirmed using a xenogeneic tumor-bearing mouse model. Tumor progression was delayed in mice that received either NK cells (p < 0.05) or T cells (p < 0.0001) following DOX treatment compared to mice receiving either cell type alone. Moreover, combined infusion of both NK and T cells following DOX treatment not only delayed tumor progression but also significantly improved the long-term survival (p < 0.01). Based on these findings, it was proposed that DOX can be used to improve the efficacy of adoptive cell therapy in patients with cancer.

A prostaglandin E (PGE) receptor EP4 antagonist protects natural killer cells from PGE2-mediated immunosuppression and inhibits breast cancer metastasis

Cyclooxygenase-2 is frequently upregulated in epithelial tumors and contributes to poor outcomes in multiple malignancies. The COX-2 product prostaglandin E2 (PGE2) promotes tumor growth and metastasis by acting on a family of four G protein-coupled receptors (EP1–4). Using a novel small molecule EP4 antagonist (RQ-15986) and a syngeneic murine model of metastatic breast cancer, we determined the effect of EP4 blockade on innate immunity and tumor biology. Natural killer (NK)-cell functions are markedly depressed in mice bearing murine mammary tumor 66.1 or 410.4 cells owing to the actions of PGE2 on NK cell EP4 receptors. The EP4 agonist PGE1-OH inhibits NK functions in vitro, and this negative regulation is blocked by RQ-15986. Likewise, the treatment of tumor-bearing mice with RQ-15986 completely protected NK cells from the immunosuppressive effects of the tumor microenvironment in vivo. RQ-15986 also has direct effects on EP4 expressed by tumor cells, inhibiting the PGE2-mediated activation of adenylate cyclase and blocking PGE2-induced tumor cell migration. The pretreatment of tumor cells with a non-cytotoxic concentration of RQ-15986 inhibited lung colonization, a beneficial effect that was lost in mice depleted of NK cells. The oral administration of RQ-15986 inhibited the growth of tumor cells implanted into mammary glands and their spontaneous metastatic colonization to the lungs, resulting in improved survival. Our findings reveal that EP4 antagonism prevents tumor-mediated NK-cell immunosuppression and demonstrates the anti-metastatic activity of a novel EP4 antagonist. These observations support the investigation of EP4 antagonists in clinical trials.

Abstract A15: IFN-gamma-inducible-protein-10 stimulates intratumoral infiltration of adoptively transferred human NK cells in a melanoma xenograft mouse model.

Abstract Aim: In the present study, we investigated whether adoptively transferred human NK cells would migrate towards IP-10 producing melanoma tumors in a xenograft mouse model. Moreover, we assessed the effect of the chemotherapeutic agent doxorubicin in increasing the sensitivity of IFNγ-treated tumors to NK cell lysis in vitro and in vivo. Background: Adoptive transfer of natural killer (NK) cells is being increasingly used in the treatment of cancer. However, clinical trials involving adoptive transfer of NK cells have to date been more successful against leukemia and other blood-borne cancers than against solid tumors. Several groups have reported that intratumoral infiltration of NK cells correlates with improved prognosis in patients with solid tumors. Recruitment and infiltration and of NK cells in the tumor is dependent on the production of IFN-gamma-inducible-protein-10 (IP-10) by tumor cells. Although tumor cells can be triggered to produce IP-10 by IFNγ stimulation, the expression of MHC class I is increased on tumor cells resulting in decreased sensitivity to NK cell lysis. Results: Following activation with IL-2, human NK cells upregulated the chemokine receptor CXCR3 resulting in significantly increased migration toward recombinant IP-10, MIG and I-TAC in a transwell migration assay (p=0.03). Expanded NK cells also migrated toward melanoma tumor cells that had been stimulated with IFNγ to induce secretion of IP-10 (p&amp;lt;0.01). In an in vivo xenograft model, an increased accumulation of adoptively infused human NK cells could be detected in subcutaneous melanoma tumors transfected with IP-10 while IP-10 negative tumors had little or no NK cell infiltration (p=0.02). Following infusion of expanded NK cells, significantly reduced tumor burden (p=0.02) and prolonged survival was observed in mice bearing IP-10 positive tumors than mice bearing IP-10 negative tumors (p=0.03). We found that in mice bearing subcutaneous melanoma tumors, intratumoral injections of IFNγ triggered IP-10 production, as confirmed by protein analysis of tumor lysates. Mice that received intravenous injections of doxorubicin prior to IFNγ-injections and adoptive transfer of NK cells had a slower tumor progression than mice that received IFNγ alone prior to NK cell transfer. Conclusion: Secretion of IP-10 from melanoma tumors stimulates intratumoral infiltration of adoptively transferred NK cells, demonstrating the importance of IP-10/CXCR3 signaling in recruitment of NK cells to solid tumors. Intratumoral IFNγ-treatment of tumors triggers an increased local production of IP-10. Pretreatment of tumor cells with doxorubicin can offset INFγ-induced resistance to NK cell lysis in vitro and in vivo. Citation Format: Erik Wennerberg, Veronika Kremer, Andreas Lundqvist. IFN-gamma-inducible-protein-10 stimulates intratumoral infiltration of adoptively transferred human NK cells in a melanoma xenograft mouse model. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A15.