bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2746: An evaluation of poly (ADP-ribose) polymerase inhibitor efficacy in head and neck cancer
Authors: Heitmann Jana , Geeleher Paul , Keck Michaela , Zuo Zhixiang , Khattri Arun , Tepper Susanne , Beckett Michael , Weichselbaum Ralph R. , Fetscher Sebastian , Vokes Everett E. , Seiwert Tanguy
Published: May 2015

Background: Synthetic lethality induced by poly (ADP-ribose) polymerase (PARP) inhibitors can occur in tumors without BRCA mutations. This “BRCAness” phenomenon has been observed in ovarian and triple negative breast cancers, but its role in other malignancies is not known. This study aims to evaluate the potency of PARP inhibitors in head and neck cancer, where BRCA mutations are rare.

Methods: First, we compared three PARP inhibitors (veliparib, olaparib and rucaparib). We subsequently established dose response curves for rucaparib for ten head and neck cancer cell lines and compared with two BRCA deficient breast cancer cell lines. Furthermore, we used immunofluorescent staining for γH2AX and RAD51 to study the capability for the DNA repair mechanism homologous recombination.

Results: We identified rucaparib as the most potent of the three PARP inhibitors tested and found a subset of tumors that show high rucaparib sensitivity (IC50 values: 7.0µM, 10.3µM and 11.7µM) comparable to a BRCA deficient breast cancer cell line (IC50 value: 8.9µM). Foci formation of the homologous recombination marker RAD51 did not serve as a reliable post-treatment biomarker.

Conclusion: In conclusion, we demonstrate that PARP inhibitors are effective in a subset of head and neck cancer cell lines, suggesting that these compounds could play a role in the treatment of a subset of head and neck tumors that exhibit the “BRCAness” phenotype. Further studies regarding the underlying mechanism of this phenotype are warranted.

Citation Format: Jana Heitmann, Paul Geeleher, Michaela Keck, Zhixiang Zuo, Arun Khattri, Susanne Tepper, Michael Beckett, Ralph R. Weichselbaum, Sebastian Fetscher, Everett E. Vokes, Tanguy Seiwert. An evaluation of poly (ADP-ribose) polymerase inhibitor efficacy in head and neck cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2746. doi:10.1158/1538-7445.AM2014-2746
Abstract 2749: SRPK1 inhibition and modulation of VEGF alternative splicing as a potential therapeutic strategy in prostate cancer
Authors: Mavrou Athina , Bates Dave , Oltean Sebastian
Published: May 2015

Angiogenesis is required for tumor growth and is induced principally by VEGF-A. VEGF-A pre-mRNA is alternatively spliced at the terminal exon to produce two families of isoforms, pro- and anti-angiogenic, only the former of which is upregulated in prostate cancer. In renal epithelial cells and colon cancer cells, the choice of VEGF splice isoforms is controlled by the splicing factor SRSF1, phosphorylated by SRPK1. Immunohistochemistry staining of human samples revealed a significant increase in SRPK1 expression both in prostate intra-epithelial neoplasia lesions as well as malignant adenocarcinoma compared to benign prostate tissue. We therefore tested the hypothesis that the selective upregulation of pro-angiogenic VEGF in prostate cancer may be under control of SRPK1 activity. SRPK1 levels were up-regulated in several prostate cancer cell lines in comparison with normal prostate epithelial cells. A switch in the expression of VEGF165 towards the anti-angiogenic isoform, VEGF165b, was seen in PC-3 cells with SRPK1 knock-down (KD). PC-3 -SRPK1 KD cells resulted in tumors that grew more slowly in xenografts, with decreased microvessel density. No effect was seen as a result of SRPK1 KD on growth, proliferation, migration and invasion capabilities of PC-3 cells in vitro. Moreover, VEGF over-expression from a splicing-insensitive construct in PC-3/SRPK1 KD cells rescued tumor growth. Small molecule inhibitors of SRPK1 are able to switch splicing towards the anti-angiogenic isoform, VEGF165b in PC3 cells in vitro and decrease tumor growth when administered intraperitoneally in vivo in a mouse model of orthotopic prostate cancer. Our study suggests that modulation of SRPK1 and subsequent inhibition of tumor angiogenesis by regulation of VEGF splicing can alter prostate tumor growth and supports further studies into the use of SRPK1 inhibition as a potential anti-angiogenic therapy in prostate cancer.

Note: This abstract was not presented at the meeting.

Citation Format: Athina Mavrou, Dave Bates, Sebastian Oltean. SRPK1 inhibition and modulation of VEGF alternative splicing as a potential therapeutic strategy in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2749. doi:10.1158/1538-7445.AM2014-2749
Abstract 2756: Ceramide analog targets xIAP and cIAP1 to sensitize metastatic colon carcinoma cells to apoptosis induction to suppress tumor progression
Authors: Paschall Amy , Zimmerman Mary , Torres Christina , Yang Dafeng , Chen May , Li Xia , Bieberich Erhard , Bai Aiping , Bielawski Jacek , Bielawska Alicja , Liu Kebin
Published: May 2015

Sphingolipids are bioeffectors that mediate various cellular processes. We aimed to identify molecular targets of ceramide in the apoptosis pathway and to determine the efficacy of a ceramide analog in overcoming cancer cell resistance to apoptosis induction. We observed that exposure of human colon carcinoma cells to ceramide analog LCL85 results in apoptosis in a dose-dependent manner. Interestingly, a sublethal dose of LCL85 increased C16 ceramide content and overcame tumor cell resistance to FasL-induced apoptosis. Subsequently, treatment of tumor cells with exogenous C16 ceramide resulted in increased tumor cell sensitivity to FasL-induced apoptosis. LCL85 resembles Smac mimetic in sensitization of colon carcinoma cells to FasL-induced apoptosis by inducing proteasomal degradation of cIAP1 and xIAP proteins. Consistent with its apoptosis sensitization activity, LCL85 suppressed colon carcinoma cell metastatic potential in an experimental colon carcinoma lung metastasis mouse model. Taken together, we determined that a sublethal dose of LCL85 is effective as a sensitizer in overcoming apoptosis resistance of metastatic human colon carcinoma cells, and LCL85 is effective in suppressing tumor metastasis in vivo.

Citation Format: Amy Paschall, Mary Zimmerman, Christina Torres, Dafeng Yang, May Chen, Xia Li, Erhard Bieberich, Aiping Bai, Jacek Bielawski, Alicja Bielawska, Kebin Liu. Ceramide analog targets xIAP and cIAP1 to sensitize metastatic colon carcinoma cells to apoptosis induction to suppress tumor progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2756. doi:10.1158/1538-7445.AM2014-2756
Abstract 2758: Development of small molecule Bax agonists for lung cancer treatment
Authors: Xin Meiguo , Li Rui , Park Dongkyoo , Owonikoko Taofeek K. , Sica Gabriel L. , Corsino Patrick E. , Zhou Jia , Ding Chunyong , Magis Andrew T. , Ramalingam Suresh S. , Curran Walter J. , Khuri Fadlo R. , Deng Xingming
Published: May 2015

Bax, a central death regulator, is required at the decisional stage of apoptosis. We recently discovered that the serine(S) 184 site on Bax is a critical functional switch for controlling the proapoptotic activity of Bax. The structural pocket around the S184 residue holds great promise as an ideal target for small molecule docking. We therefore employed the S184 residue as a docking site for screening small molecule therapeutic agents using the UCSF DOCK program suite and the NCI library of small molecules. Three compounds named small molecule Bax agonists (i.e. SMBA1, SMBA2 and SMBA3) were found to induce a conformational change of Bax by blocking phosphorylation of its S184 site, facilitating Bax insertion into mitochondrial membranes and formation of Bax oligomers leading to cytochrome c release and apoptosis in human lung cancer cells. Intriguingly, SMBA1 potently suppresses lung tumor growth via apoptosis in vivo without significant normal tissue toxicity. Survival outcomes remain very poor for lung cancer patients due to resistance to standard therapeutic interventions with radiation and systemic chemotherapy. Development of novel Bax agonists as an entirely new class of anti-cancer drugs has translational value to foster novel strategies for the treatment of lung cancer and other Bax expressing malignancies.

Citation Format: Meiguo Xin, Rui Li, Dongkyoo Park, Taofeek K. Owonikoko, Gabriel L. Sica, Patrick E. Corsino, Jia Zhou, Chunyong Ding, Andrew T. Magis, Suresh S. Ramalingam, Walter J. Curran, Fadlo R. Khuri, Xingming Deng. Development of small molecule Bax agonists for lung cancer treatment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2758. doi:10.1158/1538-7445.AM2014-2758
Abstract 2763: EGFR endocytosis is a rational target in lung cancer with wild-type EGFR
Authors: Jo Ukhyun , Park Kyong Hwa , Whang Young M. , Sung Jae Sook , Won Nam Hee , Park Jong Kuk , Kim Yeul Hong
Published: May 2015

Oncogenic alterations of epidermal growth factor receptor (EGFR) signaling are frequently observed in lung cancer patients with worse differentiation and poor prognosis. However, the therapeutic efficacy of EGFR-tyrosine kinase inhibitors (TKIs) is currently limited in selected patients with EGFR mutations. Therefore, in this study, we investigated the potential molecular mechanism that contributes to cell viability and the response of gefitinib, one of the EGFR-TKIs, in lung cancer models with wide-type EGFR (wtEGFR). Interestingly, we found that EGF-induced EGFR endocytosis is existed differently between gefitinib-sensitive and -insensitive lung cancer cell lines. Suppressing EGFR endocytos decreased cell viability and increased apoptotic cell death in gefitinib-insensitive lung cancer with wtEGFR in vitro and in vivo. In addition, we found that Rab25 was differentially expressed in between gefitinib-sensitive and -insensitive lung cancer cells. Rab25 knockdown caused the changed EGFR endocytosis and reverted the gefitinib response in gefitinib-sensitive lung cancer with wtEGFR in vitro and in vivo. Taken together, our findings suggest a novel insight that EGFR endocytosis is a rational therapeutic target in lung cancer with wtEGFR, in which the combined efficacy with gefitinib is expected. Furthermore, we demonstrated that Rab25 plays an important role in EGFR endocytosis and gefitinib therapy.

Citation Format: Ukhyun Jo, Kyong Hwa Park, Young Mi Whang, Jae Sook Sung, Nam Hee Won, Jong Kuk Park, Yeul Hong Kim. EGFR endocytosis is a rational target in lung cancer with wild-type EGFR. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2763. doi:10.1158/1538-7445.AM2014-2763
Abstract 2775: Development and characterization of a MEK1 inhibitor (AZD6244) sensitive childhood astrocytoma cell line
Authors: Studebaker Adam W. , Bid Hemant K. , Phelps Doris A. , Houghton Peter J.
Published: May 2015

We previously characterized the development and reversal of acquired resistance to the MEK1 inhibitor AZD6244 in an in vivo model of childhood astrocytoma. The BT-40 astrocytoma xenograft model expresses mutated BRAFV600E and is highly sensitive to AZD6244, but acquires resistance, which can be overcome with the addition of the STAT3 inhibitor LLL12. The purpose of this current study was to establish and characterize a cell line derived from the xenograft model as well as develop an orthotopic mouse model. Sensitivity to AZD6244 and LLL12, expression-profiling assessment of MEK signature and compensatory pathways, and cytokine levels were assessed in the newly developed BT-40 cell line. The BT-40 cell line exhibited sensitivity to AZD6244 and LLL12 with IC50 values of 350nM and 1µM, respectively. Combination treatment was additive. Treatment of the cells with AZD6244 inhibited p-Erk and the mTOR downstream signaling molecule p-S6, while p-Akt, p-STAT3, p-4E-BP1 increased. Kinase expression arrays performed on cells treated with AZD6244 displayed a decrease in IL-1α, G-CSF, and IL-6 and an increase in CXCL10. The decrease in IL-6 was restored to baseline levels with the addition of LLL12. Interestingly, the JAK2 inhibitor AZD1480 failed to restore the IL-6 levels. The IL-6 results observed in the expression arrays were confirmed by ELISA, where AZD6244 decreased soluble IL-6 levels and LLL12, but not AZD1480, restored IL-6 levels to baseline. The BT-40 cell line, along with a cell line stably expressing luciferase (BT-40Luc), were implanted into the caudate putamen using stereotaxic guidance. The BT-40Luc cells exhibited exponential growth over time as evaluated with bioluminescent imaging. In conclusion, a cell line derived from a previously described in vivo model of childhood astrocytoma was developed that closely recapitulated our original findings in the xenograft model following treatment with the MEK1 inhibitor AZD6244. Furthermore, an orthotopic model of these cells was developed, which will allow us to characterize response to established and novel therapeutic agents. Supported by PHS award CA169368.

Citation Format: Adam W. Studebaker, Hemant K. Bid, Doris A. Phelps, Peter J. Houghton. Development and characterization of a MEK1 inhibitor (AZD6244) sensitive childhood astrocytoma cell line. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2775. doi:10.1158/1538-7445.AM2014-2775
Abstract 2778: Lapatinib inhibits CIP2A/PP2A/p-Akt signaling and induces apoptosis in triple negative breast cancer cells
Authors: Tseng Ling-Min , Liu Chun-Yu , Hsu Chia-Jung , Wang Duen-Shian , Su Jung-Chen , Shiau Chung-Wai , Chen Kuen-Feng
Published: May 2015

Background: Triple-negative breast cancer (TNBC) is a subtype with aggressive behaviors and poor prognosis. Our previous study has shown that CIP2A, cancerous inhibitor of PP2A, may be a novel target in breast cancer cells (Breast Cancer Research 2012). Here, we tested efficacy of lapatinib, a dual tyrosine kinase inhibitor which interrupts the HER2/neu and epidermal growth factor receptor (EGFR) pathways, in a panel of TNBC cell lines and examined the drug mechanism.

Methods: TNBC cell lines (HCC-1937, MDA-MB-468, MDA-MB-231) were used for in vitro studies. Apoptosis was examined by both flow cytometry and Western blot. Signal transduction pathways in cells were assessed by Western Blot. Quantitative RT-PCR was used for assessing gene transcription. In vivo efficacy of lapatinib was tested in nude mice with breast cancer xenografts.

Results: Lapatinib showed anti-proliferative effect in HCC 1937, MDA-MB-468, and MDA-MB-231 cell lines. Lapatinib induced significant apoptosis in dose-dependent and time-dependent manners in the three tested TNBC cell lines. Importantly, lapatinib inhibited CIP2A and p-Akt in a dose-dependent manner in all sensitive TNBC cells. Overexpression of CIP2A protected MDA-MB-468 cells from lapatinib-induced apoptosis. In addition, lapatinib increased PP2A activity (in relation to CIP2A inhibition) in sensitive cells. Moreover, lapatinib-induced apoptosis as well as lapatinib-induced p-Akt downregulation, was attenuated by PP2A antagonist okadaic acid in sensitive cells. Lapatinib downregulated CIP2A mRNA. Importantly, lapatinib showed anti-tumor activity in mice bearing MDA-MB-468 xenograft tumors, and downregulated CIP2A as well as p-Akt in these xenografted tumors.

Conclusions: Inhibition of CIP2A determines the effects of lapatinib-induced apoptosis in TNBC cells. Our data suggest that in addition to a dual tyrosine kinase inhibitor of HER2/neu and EGFR, lapatinib can be a novel CIP2A inhibitor and that CIP2A can be a potential target for TNBC.

(Supported by Yen Tjing Ling Medical Foundation; NSC 101-2325-B-075-006, NSC-102-2325-B-075-006 and NSC 100-2325-B-010-007; and V100-D-005-4)

Citation Format: Ling-Min Tseng, Chun-Yu Liu, Chia-Jung Hsu, Duen-Shian Wang, Jung-Chen Su, Chung-Wai Shiau, Kuen-Feng Chen. Lapatinib inhibits CIP2A/PP2A/p-Akt signaling and induces apoptosis in triple negative breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2778. doi:10.1158/1538-7445.AM2014-2778
Abstract 2781: Elimination of PanIn2/3 in a GEMM for pancreatic cancer with disrupted p16 and activated Kras
Authors: Dinnen Richard D. , Qiu Wanglong , Su Gloria H. , Petrylak Daniel P. , Fine Robert L.
Published: May 2015

When treated with clinically achievable concentrations of arsenic trioxide, ascorbic acid and antabuse (disulfiram) (AAA), we found that four human pancreatic cancer cell lines underwent robust cell death associated with ATP decline and ROS accumulation. In these cells, early apoptosis shifted to aponecrosis, and was dependent on VDAC since shRNA antagonistic to VDAC-1 abrogated the ROS accumulation and blocked cell death. In this study, assessment of AAA was performed in Pdx1-Cre transgenic mice with a conditional p16 knockout (p16flox/flox), and oncogenic KrasG12D. The KrasG12D, p16flox/flox; LSL-KrasG12D; Pdx1-Cre mice develop a full spectrum of pancreatic intraepithelial neoplasia (PanIN) lesions, pancreatic ductal adenocarcinoma (PDA), and frequent metastases. Eight week old mice were treated for 8 weeks, 3 times/week with AAA, sacrificed, and assessed for the development of pancreatic cancer. Little or no toxicity was observed in the AAA-treated mice. The development of frank pancreatic tumors was decreased from 37% in controls to 0% in AAA-treated mice. The mean number of PanIN 3 lesions/mouse declined from 14.2 in controls to 1.6 in AAA-treated mice (p=0.004); the mean number of PanIN 2 lesions declined from 5.8 in controls to 1.7 in AAA-treated mice (p=0.029), and the mean number of PanIN 1 lesions in control mice was 14.2 and in AAA-treated mice was 16.0 (p=0.82), indicating no difference in PanIN 1 occurrence between control and AAA-treated mice. The data also suggests that PanIN 2 and PanIN 3 lesions are killed after exposure to AAA, since mice prior to treatment had high levels of PanIN 2 (8.1 lesions/mouse) and PanIN 3 (11.4 lesions/mouse), similar to controls at 4 months of age. Therefore, AAA may have eliminated pre-existing PanINs rather than blocking their development. Results from immunohistochemical staining of the PanIN lesions for VDAC, indicated that PanIN 1 lesions have low, and PanIN 2 and 3 lesions have high, VDAC expression. This result parallels the in vitro human cell line data showing VDAC expression is critical to AAA sensitivity, and suggests a similar mechanism of action in the murine model, as we see in vitro. This is the first treatment that has shown potential to kill existing PanIN lesions and arrest the development of tumors in this pancreatic cancer murine model.

Citation Format: Richard D. Dinnen, Wanglong Qiu, Gloria H. Su, Daniel P. Petrylak, Robert L. Fine. Elimination of PanIn2/3 in a GEMM for pancreatic cancer with disrupted p16 and activated Kras. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2781. doi:10.1158/1538-7445.AM2014-2781
Abstract 2789: Iron chelation inhibits mTOR activity in cancer cells
Authors: Shang Chaowei , Zhou Hongyu , Huang Shile
Published: May 2015

Mammalian target of rapamycin (mTOR) plays a critical role in regulating cell proliferation and survival through sensing environmental cues. mTOR functions as at least two complexes (mTORC1/2). In cancer cells, mTOR activity is often deregulated. Intracellular iron levels are higher in cancer cells than in normal cells mainly due to the increased requirement of iron for proliferation. Thus, cancer cells are more sensitive to iron chelation than normal cells. Iron chelators have been used as traditional treatment for iron overload diseases. Recent studies have shown that iron chelators possess potent anticancer activity. However, the underlying mechanism is not well understood. In this study, we found that ciclopirox olamine (CPX), an iron chelator and off-patent antifungal drug, inhibited cell proliferation and induced cell death in human head and neck cancer cells (PCI-13 and FaDu), breast adenocarcinoma (MDA-MB-231), human rhabdomyosarcoma (Rh30), and skin squamous cell carcinoma (SRB-M7) cells. Similar results were observed using Dp44mT, another iron chelator. Concurrently, CPX and Dp44mT inhibited mTORC1 signaling in the cells. This is evidenced by the findings that treatment with the iron chelators inhibited phosphorylation of 4E-BP1 and S6K1, two best known effector molecules of mTORC1, which could be blocked by addition of ferrous sulfate, but not cuprous sulfate. At the same time, the addition of ferrous sulfate prevented cancer cell death. The findings suggest that the inhibitory effect of CPX or Dp44mT on mTORC1 function was attributed to selective chelation of iron in the cells. Mechanistically, chelation of iron did not activate protein phosphatase 2A (PP2A), but induced raptor (S792) phosphorylation and disrupted mTORC1 integrity. Of note, the effect of iron chelation on mTORC2 was cell line dependent. Further research may enhance our understanding the role of iron in the regulation of mTOR signaling.

Citation Format: Chaowei Shang, Hongyu Zhou, Shile Huang. Iron chelation inhibits mTOR activity in cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2789. doi:10.1158/1538-7445.AM2014-2789
Abstract 2792: Engineering T lymphocytes with protein nanogels for cancer immunotherapy
Authors: Tang Li , Zheng Yiran , Irvine Darrell J.
Published: May 2015

Adoptive cell transfer (ACT) with autologous tumor-reactive T-lymphocytes is a promising strategy in cancer immunotherapy, but treatment of solid tumors is limited by the rapid decline in function of the transplanted T cells. In order to maintain high numbers of viable tumor-specific cytotoxic T cells in tumors, co-administration of supporting immunostimulant agents together with transferred cells is typically necessary in clinical practice. When given systemically at high doses, these agents could enhance the in vivo viability of donor cells, improve their therapeutic function and lead to overall improved efficacy against cancer. However, high doses of such agents can also result in serious side effects. For example, the use of Interleukin-2 (IL-2) as an adjuvant drug significantly enhances the efficacy of ACT in melanoma, where IL-2 provides key adjuvant signals to donor T cells but also elicits severe dose-limiting inflammatory toxicity and expands regulatory T cells (Tregs). One approach to focus adjuvant drug action on the transferred cells and lower systemic toxicity is to genetically engineer donor cells to secrete their own supporting factors. However, the technical difficulty of engineering safe autocrine production of potent adjuvant molecules such as cytokines is a challenge. Thus, an inexpensive technology platform that would allow simple, safe and efficient delivery of adjuvant drugs specifically to therapeutic cells over a defined window of time could be broadly useful in ACT and other cell therapies for cancer. Here, we developed a novel strategy to deliver therapeutic proteins in T cell therapies, via the chemical conjugation of protein-polyethylene glycol (PEG) nanogels (NGs) directly onto the plasma membrane of donor cells, enabling continuous pseudoautocrine release of cargo proteins for stimulation of transferred T-cells. These PEG NGs loaded with IL-2 contained very high level of drug (∼80 wt% of NG was the protein cargo) and released native IL-2 in physiological conditions in a sustained manner through breakdown of a degradable linker between the IL-2 cargo and PEG chains of the NG. T-cells with optimized number of NGs conjugated per cell maintained their cellular functions and cancer targeting and killing capability. We demonstrated that this IL2-PEG NG-T cell conjugation strategy could augment T-cell expansion in vivo and result in markedly improved efficacy against metastatic B16F10 melanoma in mice. This approach provides a readily generalizable strategy to provide autocrine protein drug support to donor cells to enhance the safety and efficacy of ACT.

Citation Format: Li Tang, Yiran Zheng, Darrell J. Irvine. Engineering T lymphocytes with protein nanogels for cancer immunotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2792. doi:10.1158/1538-7445.AM2014-2792
Abstract 2798: Targeting IL13R alpha2 heterogeneity in malignant glioma with engineered T cells
Authors: Sengupta Sadhak , Balyasnikova Irina , Naheed Seema , Lesniak Maciej S. , Junghans Richard P. , Sampath Prakash
Published: May 2015

A majority of malignant glioma exclusively expresses Interleukin 13 receptor alpha chain variant 2 (IL13Rα2), making it an ideal candidate for targeted immunotherapy. A number of immunotherapeutic molecules have been developed against IL13Rα2 with varied levels of therapeutic efficacy. However, the importance of IL13Rα2 as a successful immunotherapy candidate is under scrutiny due to its heterogeneous expression on the tumor. We have used an IL13-chimeric antigen receptor (IL13CAR) expressing T cells to target the IL13Rα2 heterogeneity on malignant glioma. IL13Rα2 was over-expressed on U251MG human glioma cell line by stable transfection (IL13Rα2Hi) to evaluate the effects of its increased expression in tumor progression and immunotherapeutic efficacy, compared to the regular U251 cells (IL13Rα2Lo) and IL13Rα2-knocked down U251 cells (IL13Rα2KD). Tumor cells readily formed neurospheres under established culture conditions. Increased expression of IL13Rα2 was observed in IL13Rα2Hi-neurosphere derived tumor cells when compared to those derived from IL13Rα2Lo neurospheres, as well as the parental IL13Rα2Hi cells. IL13Rα2Hi-neurosphere derived tumor cells were also resistant to radiation therapy and temozolomide chemotherapy when compared to those IL13Rα2Lo neurospheres-derived tumor cells, as well as parental cell lines. Higher levels of IFN gamma and TNF alpha in the culture supernatants, increased T cell proliferation, and CAR+ TCR enrichment were observed when IL13CAR T cells were co-cultured with IL13Rα2Hi-neurosphere derived tumor cells, in comparison to co-culture with IL13Rα2Lo -neurosphere derived tumor cells, as well as the parental cells. IL13CAR T cells, upon co-culture with IL13Rα2Hi-neurosphere derived tumor cells exerted highest levels of cytotoxicity. Together, these results predict that IL13Rα2 heterogeneity can be addressed by a combination of established therapeutic regimen and targeted immunotherapy. While those cells expressing lower levels of IL13Rα2 are susceptible to radiation and chemotherapy, the high IL13Rα2-expressing tumor cells that escape these treatments can be candidates for IL13Rα2-targeted immunotherapy.

Note: This abstract was not presented at the meeting.

Citation Format: Sadhak Sengupta, Irina Balyasnikova, Seema Naheed, Maciej S. Lesniak, Richard P. Junghans, Prakash Sampath. Targeting IL13R alpha2 heterogeneity in malignant glioma with engineered T cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2798. doi:10.1158/1538-7445.AM2014-2798
Abstract 28: The influence of PDGF and VEGF on tumor proliferation in colon cancer
Authors: Mönch Romana , Grimmig Tanja , Kannen Vinicius , Germer Christoph T. , Waaga-Gasser Ana Maria , Gasser Martin
Published: May 2015

Background: The platelet derived growth factor (PDGF) and Vascular Endothelial Growth Factor (VEGF) are known to play a crucial role in different tumor entities. Involved in cell migration and proliferation of stromal cells and tumor cells they represent key targets in cancer therapy. The aim of the study was to analyse the specific role of PDGF and VEGF stimulation on tumor cell proliferation and metabolism in colorectal cancer (CRC).

Methods: The human colon cancer cell line HT-29 and Caco-2 were cultured and stimulated with PDGF and VEGF in a time-dependent manner. Whole cell extracts and RNA extracts were analyzed by Western Blot and RT-q-PCR for PDGF- and VEGF receptors and for components of cellular metabolism. To discover effects of PDGF and VEGF on proliferation MTS proliferation assays were performed.

Results: Western Blot analysis and RT-qPCR showed no relevant PDGF-receptor α and β expression in HT-29 cells but varying expression in Caco-2 cells. Stimulation with PDGF or VEGF resulted in increased proliferation compared to untreated controls while simultaneous stimulation with both growth factors did not result in intensified proliferation. Additionally, under stimulation altered expression of various metabolic components was detected.

Conclusion: Our results demonstrate a promoting effect on tumor cell proliferation of both growth factors VEGF and PDGF. Simultaneous stimulation did not show synergistic effects. PDGF in HT-29 cells resulted in proliferation in the absence of PDGF receptors with changes in tumor cell metabolism after stimulation. Moreover in HT-29 cells glucose metabolism changed during stimulation. In conclusion, our data give evidence for direct receptor/ligand signalling of PDGF not only on stromal cells but also on colon cancer cells even in the absence of the PDGF receptor.

Citation Format: Romana Mönch, Tanja Grimmig, Vinicius Kannen, Christoph T. Germer, Ana Maria Waaga-Gasser, Martin Gasser. The influence of PDGF and VEGF on tumor proliferation in colon cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 28. doi:10.1158/1538-7445.AM2014-28
Abstract 2802: Fine-tuning T cell receptors for adoptive T cell therapy
Authors: Jakobsen Bent
Published: May 2015

Adoptive T cell therapy (ACT) with gene-modified T cells is emerging as a highly promising strategy for the treatment of many types of cancer. Therapies associated with the induction of anti-tumor immunity, such as the administration of vaccines or the ex-vivo expansion of patient-derived TILs, have historically had limited success. This lack of efficacy is almost certainly brought about as a consequence of thymic selection, leading to a paucity of T cells, and T cell receptors, in the circulating repertoire, with a sufficient degree of sensitivity towards tumor-associated antigens. An alternative approach, which has generated promising results, relies on the adoptive transfer of patient-derived T cells that have been first modified to express a chimeric antigen receptor (CAR). Nevertheless, a lack of tumor-specific antibody targets limits the wider applicability of CAR T cells.

To overcome these issues we have developed a strategy that involves the transfer of T cells expressing engineered TCRs with optimised affinity for a defined tumor antigen. Since TCRs recognize peptide antigens presented on the cell surface in complex with human leukocyte antigen (HLA), they give access to the large pool of intracellular proteins. Furthermore, the use of TCRs means that the natural interaction between a T cell and the corresponding antigen presenting cell is preserved. Importantly, for any given antigen, engineered TCRs require individual optimization, to ensure the appropriate balance between potency and specificity.

Recent and emerging clinical data using engineered TCRs for ACT are beginning to corroborate promising pre-clinical findings, with evidence of both potent and durable responses in vivo. However, experiences have also highlighted the dangers of this approach. The main risks associated with TCR adoptive therapy are target presentation on healthy tissue and TCR cross-reactivity to a look-alike peptide. Understanding these risks and ensuring comprehensive and robust safety testing mechanisms are in place is pivotal to mitigate these risks.

Citation Format: Bent Jakobsen. Fine-tuning T cell receptors for adoptive T cell therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2802. doi:10.1158/1538-7445.AM2014-2802
Abstract 2803: Preclinical models for the development of HER2-specific T-cell therapy
Authors: Kreutzer Christiane , Conrad Heinke , Meyerhuber Peter , Uckert Wolfgang , Beckhove Philipp , Bernhard Helga
Published: May 2015

The adoptive transfer of autologous, tumor-reactive T cells to treat cancer patients is a promising tool in immunotherapy. One of our clinical goals is the adoptive transfer of autologous T cells transduced with a human epidermal growth factor receptor 2 (HER2)-directed TCR in patients with advanced breast cancer. We successfully established cytotoxic T-cell clones and CD8+ T cells transduced with a TCR specific for the tumor and self-antigen HER2 and tested their lytic capability in vitro.

Since HER2 is a self antigen that is not only expressed by malignant cells but also in normal tissue, e.g. the heart, we tested the recognition of normal HER2+-cell lines by HER2-directed T cells in vitro. For this purpose HER2-directed CD8+ T cells were used to analyze the recognition and lysis of HLA-A2+ cardiomyocyte cell lines in IFNγ-ELISA and chromium-release-assay. Compared to HER2-overexpressing breast cancer cell lines recognition of the cardiomyocytes by HER2-directed T cells was much lower. From these data we hypothesize that there might be a therapeutic window, which may allow the treatment with HER2-directed T cells without having any unwanted side effects, like cardiac dysfunction.

To further analyse this, we are establishing a mouse model using HLA-A2-transgenic NOD/SCID-HHDII mice. We injected a human breast cancer cell line s.c. into NOD/SCID-HHDII mice, waited until a solid tumor had been established, and evaluated the responsiveness of tumor cells towards adoptive T cell transfer. Our data show an improvement in tumor growth inhibition after adoptive T cell transfer and IL-15 treatment; however, no significant tumor reduction was observed yet. Furthermore, in the NOD/SCID-HHDII mice we are able to detect potential autoimmune reactions. Subsequent experiments will analyze the combination therapy of transferred HER2-directed T cells with HER2-directed antibody treatment (Trastuzumab and/or Pertuzumab) in order to increase the anti-tumor response. These pre-clinical studies, as well as the in vitro tests, are important for the evaluation of possible unwanted side effects after adoptive transfer of TCR-transduced T cells specific for self-antigens like HER2.

Citation Format: Christiane Kreutzer, Heinke Conrad, Peter Meyerhuber, Wolfgang Uckert, Philipp Beckhove, Helga Bernhard. Preclinical models for the development of HER2-specific T-cell therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2803. doi:10.1158/1538-7445.AM2014-2803
Abstract 2804: Expression of miR-17-92 enhances antitumor activity of T cells transduced with the anti-EGFRvIII chimeric antigen receptor in mice bearing human GBM xenografts
Authors: Ohno Masasuke , Ohkuri Takayuki , Kosaka Akemi , June Carl H. , Natsume Atsushi , Okada Hideho
Published: May 2015

Background

Expression of miR-17-92 confers type-1 phenotype and enhanced survival of T-cells. We have previously reported that miR-17-92 is down-regulated in T-cells derived from glioblastoma (GBM) patients. We hypothesized that transgene-derived co-expression of miR17-92 and chimeric antigen receptor (CAR) in T-cells improves the efficacy of adoptive transfer therapy against GBM.

Methods

We constructed novel lentiviral vectors for miR-17-92 (FG12-EF1a-miR-17/92) and a CAR consisting of epidermal growth factor receptor variant III (EGFRvIII)-specific single-chain variable fragment (scFv) coupled to the T-cell receptor CD3ζ chain signaling module and co-stimulatory motifs of CD137 (4-1BB) and CD28 in tandem (pELNS-3C10-CAR). Human T-cells were transduced with these lentiviral vectors and their anti-tumor effects were evaluated both in vitro and in vivo.

Results

In addition to antigen-specific and potent cytotoxic activities against U87 GBM cells stably expressing EGFRvIII (U87-EGFRvIII), CAR-transduced T-cells (CAR-T-cells) co-transduced with miR-17-92 exhibited improved survival in the presence of temozolomide (TMZ) compared with CAR-T-cells without miR-17-92 co-transduction. In mice bearing intracranial U87-EGFRvIII xenografts, CAR-T-cells with or without transgene-derived miR-17-92 expression demonstrated similar levels of potent therapeutic effects without demonstrating any uncontrolled growth of CAR-T-cells. However, when these mice were re-challenged with U87-EGFRvIII cells in the brains, mice receiving co-transduced CAR-T-cells exhibited improved protection compared with mice treated with CAR-T-cells without miR-17-92 co-transduction.

Conclusion

These results warrant development of novel CAR-T-cell strategies by incorporation of miR-17-92 to improve the therapeutic potency, especially in patients with GBM.

Citation Format: Masasuke Ohno, Takayuki Ohkuri, Akemi Kosaka, Carl H. June, Atsushi Natsume, Hideho Okada. Expression of miR-17-92 enhances antitumor activity of T cells transduced with the anti-EGFRvIII chimeric antigen receptor in mice bearing human GBM xenografts. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2804. doi:10.1158/1538-7445.AM2014-2804
Abstract 2805: Cancer-specific T cell receptor isolation for cancer immunotherapy
Authors: Weigand Luise U. , Paston Samantha , Hibbert Linda , Ryan Ruth K. , Baker Debbie E. , Simmons Ruth A. , Harper Jane V. , Dukes Joseph D. , Bossi Giovanna , Grand Francis , Hickman Emma , Powlesland Alex , Vuidepot Annelise , Hassan Namir J. , Jakobsen Bent K.
Published: May 2015

Malignant cells may be recognized by T cells binding cell surface Class I HLA-peptide complexes presenting disease-associated epitopes. Many cancer patients generate CD8 cytotoxic T cell responses to tumor-associated antigens, however due to the low avidity of these T cells and cancer cells developing escape mechanisms for avoiding destruction by T cells, the immune system fails to clear tumors. To overcome these issues, we have engineered novel, bi-functional protein therapeutics termed ImmTACs (Immune Mobilising monoclonal TCR Against Cancer) which re-direct the immune system to target and destroy tumor cells with a high degree of potency and specificity. An ImmTAC comprises a high affinity ‘monoclonal’ T cell Receptor (mTCR) targeting a cancer-associated HLA-peptide complex, fused to an anti-CD3 scFv domain which activates an anti-tumor T cell response.

In order to produce ImmTACs, we have developed an integrated in-house process leading to the isolation of TCRs specific for validated cancer epitopes. The critical steps in this process are: antigen selection, epitope identification, T cell cloning, TCR isolation and binding to soluble peptide:MHC on the BIAcore. We describe each of these steps in more detail and present data to illustrate the successful isolation of a number of TCRs specific for various tumor associated antigens such as prostate and cancer testis antigens leading from this procedure.

Citation Format: Luise U. Weigand, Samantha Paston, Linda Hibbert, Ruth K. Ryan, Debbie E. Baker, Ruth A. Simmons, Jane V. Harper, Joseph D. Dukes, Giovanna Bossi, Francis Grand, Emma Hickman, Alex Powlesland, Annelise Vuidepot, Namir J. Hassan, Bent K. Jakobsen. Cancer-specific T cell receptor isolation for cancer immunotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2805. doi:10.1158/1538-7445.AM2014-2805
Abstract 2806: A phase Ia/Ib trial of chemotherapy followed by infusions of activated T cells armed with OKT3 x trastuzumab bispecific antibody, IL-2 and GM-CSF for stage II/ III, Her2+ or Her2- high risk breast cancer (more than 10+ nodes)
Authors: Jagtap Deepa B. , Rathore Ritesh , Thakur Archana , Colvin Gerald , Kouttab Nicola , Maizel Abby , Deol Abhinav , Lum Lawrence G.
Published: May 2015

Background: Anti-CD3 x anti-HER2Bi bispecific antibody (HER2Bi) targeted monoclonal antibody (mAb) activated T cells (ATC) exhibit anti-HER2 cytotoxicity, proliferate, and secrete immunokines upon tumor engagement. This study reports a phase I immunotherapy trial in 9 women with locally advanced breast cancer consisting of infusions of HER2Bi armed ATC (aATC) in combination with interleukin 2 (IL-2) and granulocyte-macrophage-colony stimulating factor (GM-CSF) to evaluate safety, feasibility, time to progression (TTP), overall survival (OS), T cell trafficking, and immune responses. Methods: ATC were produced by stimulating peripheral blood mononuclear cells (PBMC) obtained by leukapheresis with anti-CD3 monoclonal antibody and expanding the ATC in IL-2. ATC were harvested, armed with HER2Bi and cryopreserved in aliqouts. Groups of 3 patients received 20, 40, 80 or 160 x 109 aATC per infusion twice a week for four weeks(Table 1). Results: Eight of 9 patients were ER positive, 2 of 9 were Her 2 overexpressing tumors. The median OS for all patients was 103.5 months (14.3 to 134.7months). Six of 9 patients are alive. Four out of the six patients have no evidence of disease and 2 patients relapsed (one at 77.27 months and the other at 104.67months). It was feasible to grow up to 160 x 109 ATC and both patients assigned to this dose level were able to reach it. There were no cell-based dose limiting toxicities. aATC persisted in the blood for at least a week. aATC infusions induce cellular anti-tumor responses and cytokine responses. Interpretation: Targeting Her2 positive and negative tumors induced cytotoxic anti-tumor responses, increases in Th1 cytokines and IL-12 serum levels, clinical responses that suggest aATC infusions provided a survival benefit. These results are being confirmed in a phase II trial for metastatic breast cancer.

Table 1Planned doseActual Dose20Bill21.640Bill42.7540Bill37.640Bill42.480 Bill80.080Bill70.3580Bill96.0160Bill176.75160Bill160.0

Citation Format: Deepa B. Jagtap, Ritesh Rathore, Archana Thakur, Gerald Colvin, Nicola Kouttab, Abby Maizel, Abhinav Deol, Lawrence G. Lum. A phase Ia/Ib trial of chemotherapy followed by infusions of activated T cells armed with OKT3 x trastuzumab bispecific antibody, IL-2 and GM-CSF for stage II/ III, Her2+ or Her2- high risk breast cancer (more than 10+ nodes). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2806. doi:10.1158/1538-7445.AM2014-2806
Abstract 2808: GD2-specific genetically engineered NK cell therapy is effective in a drug-resistant neuroblastoma xenograft mouse model
Authors: Seidel Diana , Shibina Anastasia , Reynolds C. Patrick , Wels Winfried S. , Huebener Nicole , Lode Holger N.
Published: May 2015

Background:

Drug-resistant neuroblastoma remains a major challenge in pediatric oncology. A human NK cell line NK-92-scFv(ch14.18)-z engineered to express a GD2-specific chimeric antigen receptor (CAR) may help to address this problem. We investigated the cytotoxicity of NK-92-scFv(ch14.18)-z in a panel of GD2+ drug-resistant neuroblastoma cell lines and analyzed the anti-tumor efficacy of NK-92-scFv(ch14.18)-z in a drug-resistant neuroblastoma xenograft mouse model.

Methods

Cytotoxic activity of GD2-specific NK-92-scFv(ch14.18)-z towards a panel of GD2+ cell lines (CHLA-20, SK-N-BE(2), CHLA-136, CHLA-79, LA-N-1, LA-N-5), some of which exhibit partial or multidrug resistance, was analyzed in a 51Cr release assay. We investigated the impact of GD2 recognition on NK-92-scFv(ch14.18)-z-mediated lysis by blocking the CAR through the addition of an anti-idiotype antibody (anti-IdAb) and also by downregulating GD2 on target cells induced by the glucosylceramide synthase (GCS) inhibitor PPPP. We then employed ELISA to determine the production of effector molecules granzyme B and perforin in response to activation with immobilized GD2. Anti-tumor efficacy of NK-92-scFv(ch14.18)-z and IL-2 was analyzed in a drug-resistant GD2+ xenograft mouse model by peritumoral injections of the GD2-specific NK cell line.

Results

NK-92-scFv(ch14.18)-z effectively lysed GD2+ drug-resistant NB cell lines. This effect was almost completely abrogated by blocking the CAR with an anti-IdAb. Decreased GD2 expression on target cells also resulted in diminished lysis mediated by NK-92-scFv(ch14.18)-z. Quantification of granzyme B and perforin production with ELISA revealed that the plate-bound antigen GD2 alone was sufficient to induce activation of NK-92-scFv(ch14.18)-z. Importantly, repeated peritumoral subcutaneous injections of a combination of NK-92-scFv(ch14.18)-zeta and IL-2 significantly prolonged survival time of mice challenged with aggressively growing subcutaneous CHLA-20 tumors in a xenograft mouse model.

Conclusions

These encouraging results indicate that GD2-directed immunotherapy with genetically engineered NK cells is an appropriate treatment strategy especially in relapsed NB that exhibit drug resistance.

Citation Format: Diana Seidel, Anastasia Shibina, C. Patrick Reynolds, Winfried S. Wels, Nicole Huebener, Holger N. Lode. GD2-specific genetically engineered NK cell therapy is effective in a drug-resistant neuroblastoma xenograft mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2808. doi:10.1158/1538-7445.AM2014-2808