bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2670: Natural compound tetrocarcin-A downregulates junctional adhesion molecule-A in conjunction with erα, her2 and inhibitor of apoptosis proteins and inhibits tumor cell growth
Authors: Vellanki Sri Harikrishna , Hudson Lance , Jahns Hanne , Cruz Rodrigo G.B. , Sette Giovanni , Eramo Adriana , Hopkins Ann M.
Published: August 2018

Overexpression of the tight junction protein Junctional Adhesion Molecule-A (JAM-A) has been linked to aggressive disease in breast and several other cancers, but JAM-targeting drugs remain elusive. Screening of a natural compound library identified the cytotoxic antibiotic Tetrocarcin-A as a novel downregulator of JAM-A protein expression in breast cancer cells. Specifically, Tetrocarcin-A-induced cytotoxicity was accompanied by downregulation of JAM-A protein expression in conjunction with that of estrogen receptor alpha (ERα) and human epidermal growth factor receptor 2 (HER2) in MCF7-HER2-overexpressing breast cells. Lysosomal inhibition partially rescued the downregulation of JAM-A, HER2 and ERα caused by Tetrocarcin-A, and attenuated its cytotoxicity. Interestingly, Tetrocarcin-A repressed the transcriptional activity of c-FOS by inhibiting ERK1/2-mediated phosphorylation at Threonine-232, and downregulated protein expression of the inhibitor of apoptosis proteins (IAP). Investigation of cell death pathways revealed that Tetrocarcin-A induced caspase-dependent apoptosis. To begin testing the potential clinical relevance of our findings, we extended our studies into various other models. Encouragingly, Tetrocarcin-A downregulated JAM-A expression and caused cytotoxicity in primary breast cancer and lung cancer stem cells, and inhibited the growth of model human breast tumors in an in vivo model involving invasion across the chicken egg chorioallantoic membrane. Taken together, our data suggest that Tetrocarcin-A merits future evaluation as a novel cancer therapeutic by virtue of its ability to downregulate JAM-A expression, induce apoptosis and reduce tumorigenic signalling. This work was financially supported by Science Foundation Ireland (grant 13/IA/1994 to AMH). Tetrocarcin-A was provided by the NCI/DTP Open Chemical Repository https://dtp.cancer.gov. (NSC 333856)

Citation Format: Sri Harikrishna Vellanki, Lance Hudson, Hanne Jahns, Rodrigo G.B. Cruz, Giovanni Sette, Adriana Eramo, Ann M. Hopkins. Natural compound tetrocarcin-A downregulates junctional adhesion molecule-A in conjunction with erα, her2 and inhibitor of apoptosis proteins and inhibits tumor cell growth [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 2670.
Abstract 2672: Phyllanthusmins induce apoptosis and reduce tumor burden in high grade serous ovarian cancer by late-stage autophagy inhibition
Authors: Young Alexandria N. , Herrera Denisse , Huntsman Andrew , Lantvit Daniel D. , Korkmaz Melissa A. , Aldrich Leslie N. , Kinghorn A. Douglas , Fuchs James R. , Burdette Joanna E.
Published: August 2018

High grade serous ovarian cancer (HGSOC) is a lethal gynecological malignancy with a need for new therapeutics. Many of the most widely used chemotherapeutic drugs are derived from natural products or their semi-synthetic derivatives. We developed potent synthetic analogues of a class of compounds known as the phyllanthusmins, inspired by natural products isolated from Phyllanthus poilanei Beille. The most potent analogue, PHY34, had the highest potency in HGSOC cell lines in vitro and displayed cytotoxic activity through activation of apoptosis. PHY34 exerts its effects by initially inhibiting autophagy at a late stage in the pathway, involving the disruption of lysosomal function. The autophagy activator, rapamycin, combined with PHY34 eliminated apoptosis, suggesting that autophagy inhibition was required for apoptosis. PHY34 was readily bioavailable through intraperitoneal administration in vivo where it significantly reduced cancer cell lines grown in hollow fibers as well as ovarian tumor burden. We show that PHY34 is a new late-stage autophagy inhibitor with nanomolar potency and significant antitumor efficacy as a single-agent against HGSOC in vivo. This class of compounds holds promise as a potential, novel chemotherapeutic and demonstrates the effectiveness of targeting the autophagic pathway as a viable strategy for combating the disease.

Citation Format: Alexandria N. Young, Denisse Herrera, Andrew Huntsman, Daniel D. Lantvit, Melissa A. Korkmaz, Leslie N. Aldrich, A. Douglas Kinghorn, James R. Fuchs, Joanna E. Burdette. Phyllanthusmins induce apoptosis and reduce tumor burden in high grade serous ovarian cancer by late-stage autophagy inhibition [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 2672.
Abstract 2673: Securidaca1 and 2 induced apoptosis: A PI3K-AKT dependent pathway in cervical cancer
Authors: Obasi Titus C. , Braicu Cornelia , Noack Heiko , Berindan-Neagoe Ioana , Junker Bjorn , Oprean Radu N.
Published: August 2018

Background: Securidaca1 and 2 are plant-derived substances isolated from African medicinal plant S. longipedunculata, using new bioactivity-guided method partly described in Obasi et al. (2017). Our previous report suggested that certain fractions from this plant root were able to activate apoptosis in cervical cancer cell lines. Generally, over 90% of cervical cancer has been associated with high-risk human papilloma virus (HPV), whose etiologic molecule E6 oncoprotein is involved in the process of carcinogenesis. The purpose of this study, therefore, is to investigate the proapoptotic potentials of these substances and their ability to block cell survival mechanism, via antiapoptotic effectors of PI3K-AKT downstream.

Experimental Procedure: Two cervical cancer cell lines (Caski and BU25TK), positive with HPV were used for the investigation. Cells were treated with the plant-derived substances and the IC50 was determined by MTT assay. Fluorescence microscopy (Annexin V-FITC, MPC and PI staining) was used to investigate autophagy/apoptosis, while relevant genes were analyzed by RT-qPCR.

Results: Reduced cell proliferation was earlier achieved in dose- and time-dependent manner by fractions retaining Securidaca1 and 2, with IC50 of 7.03 and 16.39 (μg/mL) respectively on Caski cells. Late apoptosis was also activated on cells treated with securidaca1 fraction, while the securidaca2 in contrast indicated early apoptosis. Analysis by RT-qPCR revealed a fold-change expression of antiapoptotic proteins, MCL-1 and BCL2L1, more pronounced in securidaca1. Interestingly, the AKT-3 was significantly inhibited, correlating with low expression of MCL-1, BCL2L1, VEGFA and CDH-1. Data analysis suggests the most likely activation of apoptosis via PTEN regulated PI3K-AKT pathway and respondent downstream (mTOR/ NF-kB).

Conclusion: The ability of Securidaca1 and 2 to kill tumor cells and induce apoptosis in manner involving multiple regulatory pathways enhances their potential against possible resistance and makes them attractive as candidates for anticancer development.

Citation Format: Titus C. Obasi, Cornelia Braicu, Heiko Noack, Ioana Berindan-Neagoe, Bjorn Junker, Radu N. Oprean. Securidaca1 and 2 induced apoptosis: A PI3K-AKT dependent pathway in cervical cancer [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 2673.
Abstract 2686: Polyphenolic triptorelin and leuprorelin derivatives as anticancer prodrugs
Authors: Beni Yousef , Alhamed Samiyah , Alnakhli Jawzah , Karim Kaleh , Boadi William
Published: August 2018

Triptorelin (TRP) and Leuprorelin (LEP) are two synthetic analogue of gonadotropin-releasing hormone (GnRH), first was approved by the FDA for treatment of advanced prostate cancer and endometriosis in mid 80s. The surge of testosterone, known as a flare effect, is the major side-effect of cancer chemotherapy using TRP and LEP in a combination regiment. Therefore, improving of the biological activity of TRP and LEP by increasing the cellular uptake and retention is a remedy to this end.In this research, polyphenolic derivatives were linked to TRP and LEP through hydrophobic linkers to enhance and optimize the hydrophobicity of these drugs with the expectation to improve the cellular uptake. In this regard, several TRP and LEP conjugates of tris(4-methoxyphenyl)methanol (TPM) derivatives with optimized hydrophobicity were synthesized by the reaction of methoxy benzenes (e.g. methyl 2-methoxybenzoate, 1,2-dimethoxybenzene, methoxy-2-methylbenzene, methoxy-2-nitrobenzene, chloro-2-methxyenzene, anisole, 2-fluoroanisole or 2-methylanisole and 1,3,5-trioxane, followed by the conjugation with TRP or LEP and decanedioic acid or dodecanedioic acid in the presence of HBTU/DIPEA/DIC in moderate yields.Comparative antiproliferative assays between TPM-TRP conjugates and the corresponding non-covalent physical mixtures of the TPM derivatives and TRP were performed against human acute lymphoblastic leukemia (CCRF-CEM), human ovarian adenocarcinoma (SK-OV-3), and mouse pre adipocytes (3T3-L1) cells. TPM-TRP conjugates inhibited the cell proliferation of CCRF-CEM, SK-OV-3 and 3T3-L1 cells by 55-92%, 24-73%, 37-56%, respectively, at a concentration of 10-50 µM after 24-72 h of incubation. Similar antiproliferative assays between TPM-LEP conjugates and the corresponding non-covalent physical mixtures of the TPM derivatives and LEP were performed against human Caucasian prostate adenocarcinoma (PC3), human breast cancer cell line (BT549) and mouse pre-adipocytes (3T3-L1) cells and indicated moderare to high inhibition of the cell proliferation at a concentration of 5-100 µM after 24-72 h of incubation. These data suggest that TPM-TRP and TPM-LEP derivatives with optimized hydrophobicity can be used to improve the biological activity of TRP or LEP.

Citation Format: Yousef Beni, Samiyah Alhamed, Jawzah Alnakhli, Kaleh Karim, William Boadi. Polyphenolic triptorelin and leuprorelin derivatives as anticancer prodrugs [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 2686.
Abstract 2700: Specific cell populations from the primary tumor invasive front and lymph nodes show protein signatures associated with local metastasis in head and neck cancer
Authors: Busso-Lopes Ariane F. , Rivera César , Macedo Carolina C. , Mello Barbara P. , Villa Luisa L. , González-Arriagada Wilfredo A. , Leme Adriana F. Paes
Published: August 2018

Lymph node metastasis is the main prognostic factor in patients with head and neck squamous cell carcinoma (HNSCC), but no molecular markers are currently used in clinical practice. Considering that the invasive front (IF) of the tumor has a prognostic value due to the invasive and metastatic potential of the cells, we explored the mechanisms associated with lymph node metastasis in the primary tumor IF and its paired lymph nodes from HNSCC patients. For that, the proteomic profile was determined for formalin-fixed, paraffin-embedded (FFPE) tissues using mass spectrometry in a 29-patient cohort. Specific cell populations from primary tumor IF (tumor and stroma cells) and lymph nodes (microenvironment cells) were microdissected and HPV genotyping was performed. In total, 27.5% of the cases were positive for HPV16 and 2,154 ± 285 proteins were identified for the three cell populations analyzed. Eighty-one proteins from tumor cells, 36 proteins of stroma cells and 149 proteins of lymph node microenvironment cells were differentially expressed between HNSCC patients positive and negative for lymph node metastasis (Student´s t-test; P-value ≤ 0.05). Specific processes were enriched for the differentially expressed proteins from tumor, stroma and lymph node cells, including post-translational modification, splicing and translation, respectively. Interestingly, differential proteins isolated from primary tumor cells are mainly transported by exosomes. Therefore, our data provide protein signatures related to lymph node metastasis that are expressed by specific cell populations and candidates as prognostic markers in HNSCC.

Financial support: FAPESP (Process number 2015/19191-6).

Citation Format: Ariane F. Busso-Lopes, César Rivera, Carolina C. Macedo, Barbara P. Mello, Luisa L. Villa, Wilfredo A. González-Arriagada, Adriana F. Paes Leme. Specific cell populations from the primary tumor invasive front and lymph nodes show protein signatures associated with local metastasis in head and neck cancer [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 2700.
Abstract 271: Diterpenoid pseudolaric acid B induces M-phase arrest and apoptosis primarily through JNK stress signaling in LNCaP prostate cancer cells
Authors: Pramanik Kartick C. , Gupta Nehal , Ye Min , Lu Junxuan , Jiang Cheng
Published: August 2018

Pseudolaric acid B (PAB) is a major diterpenoid component isolated from the root bark of the traditional Chinese medicinal plant Pseudolarix kaempferi Gordon (Pinaceae). PAB has shown potential as an anticancer agent via inducing cell cycle arrest, apoptosis and DNA damage in several cancer models. To explore potential use of PAB for prostate cancer chemoprevention, we used human LNCaP prostate cancer cells that harbor wild-type p53, functional androgen receptor (AR), and defective PTEN that are characteristic of early stage prostate adenocarcinomas to study cell fates and signaling mechanisms upon PAB exposure in cell culture. Our results show that PAB inhibited LNCaP cell proliferation in a dose- and time-dependent manner (IC50 ~5.4 µM at 24 h and ~ 0.2 µM at 72 h). PAB induced M phase cell cycle arrest as confirmed by increased phosphorylation of histone H3 (p-H3 Ser10). PAB also induced apoptosis evidenced by Annexin V staining and cleavage of the canonical caspase substrate PARP. PAB at lethal exposure concentration decreased AR protein and its best-known target prostate-specific antigen (PSA). Timewise, PAB treatment increased p-H3 (mitosis marker) and the expression of another mitosis/prosurvival protein survivin several hours ahead of PARP cleavage. PAB induced p-H2A.X, which would mark DNA double-strand breaks, and the best-known DNA damage response protein P53 and its target P21Cip1 preceding cPARP. PAB stimulated phosphorylative activation of c-Jun N-terminal kinase (JNK), and the JNK inhibitor SP600125 decreased PAB-induced p-H2A.X, P53/P21Cip1 and cleavage of PARP as well as M phase arrest. Furthermore, PAB significantly increased 2',7'-dichlorofluorescin diacetate (DCF)-reactive oxygen species (DCF-ROS) in LNCaP cells and addition of antioxidant N-acetyl-L-cysteine (NAC) attenuated P53/P21Cip1 cascade and cPARP/apoptosis, but did not affect M phase arrest. Knocking-down of P53 or P21Cip1 by siRNA attenuated cPARP and apoptosis without affecting p-H2A.X. Taken together, these results suggest a rapid induction of M phase arrest by PAB that was primarily mediated by JNK stress signaling, either parallel to or upstream of ROS-DNA damage-driven P53-P21Cip1 apoptosis cascade in LNCaP cells.

Citation Format: Kartick C. Pramanik, Nehal Gupta, Min Ye, Junxuan Lu, Cheng Jiang. Diterpenoid pseudolaric acid B induces M-phase arrest and apoptosis primarily through JNK stress signaling in LNCaP prostate cancer cells [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 271.
Abstract 2721: Selective FcγR engagement by CTLA-4 antibodies results in increased functional activity
Authors: Waight Jeremy D. , Chand Dhan , Dietrich Sylvia , Gombos Randi , Horn Thomas , Gonzalez Ana M. , Manrique Mariana , Tanne Antoine , Dupont Christopher , Swiech Lukasz , Croker Ben A. , Buell Jennifer S. , Stein Robert , Duncan Alex , Savitsky David A. , Wilson Nicholas S.
Published: August 2018

Therapeutic antibodies targeting T cell co-inhibitory pathways, such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein-1 (PD-1), have emerged as an important class of cancer therapies. Insights into how different IgG isotypes modulate biological activities of antibodies have opened new avenues to enhance their therapeutic effects. For example, Fc-FcγR interactions have been shown to enhance antibody-directed effector cell activities, as well as antibody-dependent forward signaling into target cells via receptor clustering. Here, we describe a novel FcγR-dependent mechanism for antibodies targeting CTLA-4. Our findings suggest that selective Fc-FcγR binding dramatically improves the quality of the immune synapse, which in turn modifies apical T cell receptor signaling events to increase effector T cell activity. Our data also suggest that subsets of antigen-presenting cells (APCs), expressing FcγRIV in mice and FcγRIIIA in humans are important mediators of this effect. Importantly, we find this mechanism to be independent of regulatory T cell (Treg) depletion. Altogether, we describe a novel mechanism of action that provides a foundation for a new class of Fc-engineered antibodies to enhance antitumor immune responses.

Citation Format: Jeremy D. Waight, Dhan Chand, Sylvia Dietrich, Randi Gombos, Thomas Horn, Ana M. Gonzalez, Mariana Manrique, Antoine Tanne, Christopher Dupont, Lukasz Swiech, Ben A. Croker, Jennifer S. Buell, Robert Stein, Alex Duncan, David A. Savitsky, Nicholas S. Wilson. Selective FcγR engagement by CTLA-4 antibodies results in increased functional activity [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 2721.
Abstract 2726: In vitro functional activity of OMP-336B11, a GITRL-Fc fusion protein, on primary human immune cells
Authors: Chan Ivan H. , Xie Ming-Hong , Lam Andrew , Axelrod Fumiko T. , Elechko Jennifer , Park Angie I. , Gurney Austin
Published: August 2018

Glucocorticoid-induced TNFR-related protein (GITR) is a member of the TNF receptor superfamily. GITR, a co-stimulatory surface receptor, is activated upon binding to GITR Ligand (GITRL) and mediates co-stimulation of T-cell and NK responses and inhibits the suppressive activity of regulatory T-cells (Tregs). As such, GITR is an attractive immuno-oncology target for activation via GITR agonists. OMP-336B11 is a single-gene recombinant fusion protein consisting of two trimeric human GITRLs and a human immunoglobulin (IgG1) Fc domain. Murine preclinical studies using a surrogate GITRL-Fc fusion protein demonstrated robust antitumor efficacy. Functional characterization of OMP-336B11 in various human immune cell assays is presented here. In human peripheral blood mononuclear cells (PBMC), OMP-336B11 stimulated IL-2 cytokine release in a dose-dependent manner. In activated human T-cells, OMP-336B11 enhanced cell proliferation in a dose-responsive fashion. OMP-336B11 also augmented IL-2 induced IFNγ from human NK cells. To elicit NK mediated cytotoxicity of high GITR expressing cells (i.e., Tregs), OMP-336B11 is designed with an IgG1 Fc domain. Co-incubation of primary human NK cells (effector) and GITR expressing cells (target) resulted in an OMP-336B11 dependent dose-titratable increase in target cytotoxicity. Furthermore, OMP-336B11 agonistic activity was compared to anti-GITR agonist antibodies. By measuring cell proliferation and IFNγ from activated T-cells, OMP-336B11 demonstrated superior activity compared to the other agonist anti-GITR antibodies. In conclusion, OMP-336B11 is designed to induce effective GITR activation and also to mediate depletion of GITR-high cells. OMP-336B11 is currently undergoing phase I clinical study.

Citation Format: Ivan H. Chan, Ming-Hong Xie, Andrew Lam, Fumiko T. Axelrod, Jennifer Elechko, Angie I. Park, Austin Gurney. In vitro functional activity of OMP-336B11, a GITRL-Fc fusion protein, on primary human immune cells [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 2726.
Abstract 2728: Circulating levels of Resokine, a soluble modulator of the immune system, are upregulated in both experimental cancer models and in patients across multiple tumor types
Authors: Adams Ryan , Mertsching Elisabeth , Nangle Leslie , Ogilvie Kathy , Crampton Steven , Bruner John , Tyler Samantha , Rosengren Sanna , Cubitt Andrea , King David , Mendlein John
Published: August 2018

Resokine is a newly identified regulator of immune cell activity, and circulating levels of Resokine in normal individuals may represent a soluble set-point control to modulate T cell activity. Resokine activity is a non-canonical function arising from the tRNA synthetase gene family, and the activity is effected by a 60 amino acid N-terminal domain arising from the gene for histidyl-tRNA synthetase which is present in the full-length protein as well as multiple splice variants that have lost their original tRNA synthetase functionality. Resokine is secreted from cells, including tumor cell lines, and in vitro studies have demonstrated that Resokine can inhibit the activation of immune cells. In vitro, for example, Resokine addition during T cell activation induced by antibodies to CD3 and CD28, can result in reduced levels of inflammatory cytokines such as IL-2, interferon gamma, and TNF alpha; inhibition of the up-regulation of cell-surface activation markers, such as CD69, CD40L and 4-1BB; and inhibition of release of the cytotoxic mediator granzyme B.

We have tested levels of circulating Resokine in both mice with syngeneic tumors as well as >300 cancer patients across multiple tumor types. In normal C57Bl6 mice serum levels of Resokine ranged from 70-250pM (n=10) whereas in mice bearing B16F10 tumors, levels were significantly higher (450-3000pM, p<0.001) and correlated with tumor size. Resokine levels in normal human volunteers exhibit a more variable range, from 8pM to >2333pM (n=148), with 18% of individuals having levels <30pM, which was set as the active threshold level based on the concentration required to inhibit T cell activation in vitro. In contrast, samples across >300 cancer patients with different tumor types, exhibited higher circulating levels with only 4% of individuals having levels below the activity threshold of 30pM. This data is consistent with the hypothesis that tumors secrete Resokine as an additional mechanism to down-regulate immune activity, and suggests further investigation of the utility of Resokine levels as a new biomarker of immune activity in patients.

Citation Format: Ryan Adams, Elisabeth Mertsching, Leslie Nangle, Kathy Ogilvie, Steven Crampton, John Bruner, Samantha Tyler, Sanna Rosengren, Andrea Cubitt, David King, John Mendlein. Circulating levels of Resokine, a soluble modulator of the immune system, are upregulated in both experimental cancer models and in patients across multiple tumor types [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 2728.
Abstract 2749: Effects of subtype specific chemotherapeutic immunomodulation in bladder cancer
Authors: Kardos Jordan , Bixby Lisa , Truong Andrew , Krishnan Bhavani , Stewart Kyle , Vincent Benjamin , Kim William
Published: August 2018

In patients with bladder cancer, programmed death-ligand-1 (PD-L1) and programmed death-1 (PD-1) inhibitors have been shown to be effective in around twenty percent of patients, and there is evidence indicating that the level of immune infiltration and immune suppression within the tumor microenvironment correlates with response to these treatments. We have previously shown that there are intrinsic subtypes of bladder cancer, with the basal subtype characterized by high levels of immune infiltration, and the luminal subtype characterized by immune exclusion. Here we show that treatment with current standard of care chemotherapeutic agents has a subtype specific effect on the tumor microenvironment. Cisplatin-based chemotherapeutic treatment of luminal tumors induces a mesenchymal phenotype, immune infiltration, and a transition to a more basal-like tumor, while not altering these characteristics of the tumor microenvironment in basal tumors. Two of the most widely used standard of care chemotherapeutic regiments are Cisplatin-Gemcitabine (GemCis) and Methotrexate-Vinblastine-Doxorubicin-Cisplatin (MVAC), and we show that MVAC treatment induces significant immune infiltration within the luminal subtype while GemCis treatment does not, indicating there are treatment specific effects on the immune microenvironment. Furthermore, using mouse models of bladder cancer previously developed by our lab that accurately reflect the basal and luminal subtypes of bladder cancer, treatment with a representative chemotherapeutic regimen induced immune infiltration in the luminal mouse model while not affecting the tumor immune microenvironment in the basal mouse model. These results indicate chemotherapeutic regimens have subtype specific effects on the tumor microenvironment which could potentially be used to increase the efficacy of immune checkpoint inhibitors.

Citation Format: Jordan Kardos, Lisa Bixby, Andrew Truong, Bhavani Krishnan, Kyle Stewart, Benjamin Vincent, William Kim. Effects of subtype specific chemotherapeutic immunomodulation in bladder cancer [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 2749.
Abstract 2754: NT219, a novel dual inhibitor of STAT3 and IRS1/2, converts immuno-oncology resistant tumors to responders
Authors: Kuperschmidt Lana , Reuveni Hadas , Carmi Shani , Moskovits Neta , Shraga Netta R. , Solomonov Evgeny , Ronen Ohad , Stemmer Salomon , Haviv Izhak
Published: August 2018

Feedback activation of STAT3 and IGF1R/IRS plays a prominent role in mediating drug resistance to a broad spectrum of targeted cancer therapies and chemotherapies. Both the IRS1/2 and STAT3 are major signaling junctions regulated by various oncogenes, and altered during EMT and drug resistance. STAT3 has also been known to play an active role in immune-evasion of tumors and inhibition of STAT3, both in the tumor, as well as in the tumor's microenvironment, may therefore potentiate immune attack on the tumor. NT-219 is a dual inhibitor of STAT3 and IRS1/2 developed by TyrNovo Ltd. to overcome cancer drug resistance. NT-219 inhibits STAT3 phosphorylation and eliminates IRS1/2 in a unique 3-step mechanism: dissociation of IRS1/2 from the IGF1 receptor, induction of IRS1/2 serine phosphorylation, and subsequent degradation by the proteasome. We recently demonstrated that the inhibition of both IRS and STAT3 are required and essential for overcoming drug resistance. NT-219 efficacy was demonstrated in Patient-Derived tumor Xenograft (PDX) models of multiple cancer types: melanoma, sarcoma, pancreatic, colon, lung, and head & neck, when added-on to the approved therapies. In these models, NT-219 overcame acquired resistance to several oncology drug families: inhibitors of EGFR (Tarceva®, Erbitux®, Tagrisso®), MEK (Mekinist®), mutated-BRAF (Zelboraf®), mTOR (Afinitor®) as well as with chemotherapy agents (Gemzar®, 5FU, Oxaliplatin). We recently demonstrated that NT-219 works in synergy also with immune-oncology therapies. By using double autologous PDX models we demonstrated that NT-219 converted non-responding tumors to responders to Keytruda®. It also enhanced the immunotherapeutic potential of Cetuximab. The unique mode of action of NT219 may open a new avenue of combined targeted therapies in a wide range of malignancies, and has the potential to expand response duration and target patient population to the applicable drugs.

Citation Format: Lana Kuperschmidt, Hadas Reuveni, Shani Carmi, Neta Moskovits, Netta R. Shraga, Evgeny Solomonov, Ohad Ronen, Salomon Stemmer, Izhak Haviv. NT219, a novel dual inhibitor of STAT3 and IRS1/2, converts immuno-oncology resistant tumors to responders [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 2754.
Abstract 2769: Targeting malignant mesothelioma with an antibody against the tumor extracellular matrix protein fibulin-3
Authors: Nandhu Mohan Sobhana , Goparaju Chandra , Longo Sharon L. , Pass Harvey I. , Viapiano Mariano S.
Published: August 2018

Malignant mesothelioma (MM) is an aggressive tumor of mesothelial tissues that has poor prognosis and limited therapeutic options. Pass et al. (N Engl J Med 2012) identified the extracellular matrix protein fibulin-3 as an accurate biomarker of MM progression, which can be detected in the tumor parenchyma and in pleural effusions. Here, we report a systematic study of the mechanisms of fibulin-3 in MM and its value as a molecular target. Fibulin-3 activated canonical NFkB signaling and regulated the expression of NFkB-downstream genes, including the proteases MMP9 and MMP13, and the proinvasive proteins TNC and MLCK. Fibulin-3 knockdown downregulated NFkB signaling, reduced MM cell viability and potentiated the cytotoxicity of cisplatin in vitro. We next developed a function-blocking antibody ("mAb428.2") against an epitope of fibulin-3 required for NFkB activation (Nandhu et al., Clin Cancer Res 2017). This antibody blocked fibulin-3 signaling in vitro and reduced the expression of this protein, as well as NFkB-regulated MMP9, in subcutaneous (sc) MM tumors implanted in nude mice. Next, we treated mice carrying sc or intrapleural models of MM with mAb428.2 or control IgG1 (8x 30 mg/kg, q24h) delivered IV. Our anti-fibulin-3 antibody caused a significant reduction of tumor burden and extension of overall survival in both models. Overall, downregulation and inhibition of fibulin-3 proved a successful approach to enhance MM therapy. Given the value of this protein as a MM biomarker, we expect that detection of fibulin-3 may serve to identify patients with the highest chance to respond to combination therapies including anti-fibulin-3 approaches, achieving maximum therapeutic against these aggressive tumors.

Citation Format: Mohan Sobhana Nandhu, Chandra Goparaju, Sharon L. Longo, Harvey I. Pass, Mariano S. Viapiano. Targeting malignant mesothelioma with an antibody against the tumor extracellular matrix protein fibulin-3 [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 2769.
Abstract 2780: Engineering human fusion constructs for targeted delivery of granzyme B to CD33 and CEA antigens
Authors: Cheung Lawrence H. , Mohamedali Khalid A. , Hittelman Walter N. , Rosenblum Michael G.
Published: August 2018

Our laboratory has developed a unique platform for the delivery of the highly pro-apoptotic serine protease granzyme B (GrB). The construct contains enzymatically active GrB and therefore does not require release from the targeting carrier by a fragile linker in contrast to conventional antibody-drug conjugates (ADCs). We employed two scFvs targeting the CD33 antigen found on AML and the juxtamembrane epitope of CEA found on numerous solid tumors. These human scFvs (designated HU33 and CEA respectively) were fused to human GrB through an engineered IgG heavy-chain fragment (hinge to Fc and designated Fc) which contains a dimerization domain. The GrB-Fc-HU33 and GrB-Fc-CEA constructs were cloned into the pSECTag vector containing an upstream 5kDa purification tag with an enterokinase (EK) cleavage site for facile removal. The molecules were expressed in transiently-transfected HEK-293E suspension cells and grown at 37°C for 3 days in 5% CO2. The molecules were isolated from culture media and purified by IMAC followed by EK cleavage and SP chromatography to remove the free tag. The yields were 6-10 mg/L for both molecules. Specific binding of each molecule to the antigen target is undergoing Biacore and/or ELISA assessment. The cytotoxicity of the GrB-Fc-HU33 against HL-60 cells showed a specific cytotoxic effect with an IC50 of 120 nM. Tests of this agent against a panel of cells expressing various levels of CD33 are ongoing. The cytotoxic effects of the GrB-Fc-CEA were assessed against HT29, HT1080-CEA and A431 cells and demonstrated IC50 values of 80, 110 and 95 nM respectively. Based on IC50 values, these data demonstrate that the fusion construct targeting CD33 is efficiently internalized and effectively delivers active serine protease to the cytosol even at relatively low antigen expression levels. In addition, the juxtamembrane epitope of the CEA antibody also allows efficient internalization for delivery of GrB to the cytosol. Immunofluorescence and confocal studies to examine the time course of internalization and the pro-apoptotic events triggered by these agents are under examination and will be presented. Research conducted, in part, by the Clayton Foundation for Research.

Citation Format: Lawrence H. Cheung, Khalid A. Mohamedali, Walter N. Hittelman, Michael G. Rosenblum. Engineering human fusion constructs for targeted delivery of granzyme B to CD33 and CEA antigens [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 2780.
Abstract 2785: Potency-matched dual cytokine antibody fusion proteins in combination with PD-L1 blockade
Authors: Luca Roberto De , Neri Dario
Published: August 2018

Among the different pharmacological approaches for cancer treatment, antibody-cytokine fusion proteins (also called “immunocytokines”) represent an emerging class of biopharmaceutical products, capable of boosting the immune system to attack tumor cells. These products promote the selective accumulation of pro-inflammatory molecules at the site of disease, mediated by the antibody fragment. These fusion proteins have been designed to reduce the systemic toxicity of traditional cytokines, currently approved for certain malignancies.

Immunocytokine products based on interleukin-2 (IL2), interleukin-12 (IL12) or tumor necrosis factor (TNF) have been extensively studied in mouse model of cancer and in patients.

When used as single agents, immunocytokines are often not able to cure mice and patients. However it as been shown by our group that the combination of IL2- and TNF- based products was capable to completely eradicate tumors in mouse models and to induce complete responses in patients.

The development of combination products at the industrial level leads to a duplication of activities and costs. On the other hand, the opportunity to develop products with two payloads fused into a single molecular entity may facilitate development. This approach is limited by the fact that different cytokines often show different maximal tolerated doses (MTDs), and their relative potency need to be tuned. For example, TNF is administered at a dose, which is ten times lower compered to the one of IL2.

We have recently described a novel class of immunocytokine products, termed “potency-matched dual cytokine antibody fusion proteins” consisting of a tumor-targeting antibody (for example F8, specific to the alternatively spliced EDA domain of fibronectin), simultaneously fused to both IL2 and TNF. To match biological activity of the two payloads a single-point mutation was inserted in the TNF sequence and the resulting product, named IL2-F8-TNFmut was able to selectively localize to lesions with an excellent tumor-to-organ ratios and it was found to completely eradicate soft-tissue sarcomas in immunocompetent mice, which did not respond to standard chemotherapy. We now report that complete responses were observed in a variety of mouse models of cancer and that the therapeutic efficacy of IL2-F8-TNFmut was further improved when used in combination with an anti-PD-L1 antibody.

Potency-matched dual cytokine antibody fusion proteins may find clinical applications for various types of malignancies, used either as single agents or in combination with immune-checkpoint inhibitors.

Citation Format: Roberto De Luca, Dario Neri. Potency-matched dual cytokine antibody fusion proteins in combination with PD-L1 blockade [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 2785.
Abstract 2788: VB6-845d tumor cell killing elicits biologic features of immunogenic cell death
Authors: Dillon Rachelle L. , Chooniedass Shilpa , Premsukh Arjune , MacDonald Glen C. , Cizeau Jeannick , Adams Gregory P.
Published: August 2018

VB6-845d is a Targeted Protein Therapeutic, TPT, that comprises a Fab fragment specific for the Epithelial Cell Adhesion Molecule (EpCAM) genetically fused to deBouganin via a furin protease sensitive linker. DeBouganin (deB) is a de-immunized variant of bouganin, a ribosome inactivating protein (RIP), that when internalized blocks protein synthesis thereby leading to cell death. While all cytotoxic molecules induce tumor cell killing, only some are capable of inducing biological manifestations indicative of immunogenic cell death (ICD). ICD is characterized by the collective appearance of distinct cellular changes termed damage associated molecular patterns, or DAMPs, which play a role in immune cell activation by triggering pro-inflammatory processes. Key DAMPs necessary for defining ICD are cell surface translocation of calreticulin, an endoplasmic reticulum chaperone protein, ATP secretion and release of HMGB1 (high mobility group box 1). To evaluate whether VB6-845d cell killing elicits the hallmark features of ICD, VB6-845d treated tumor cell lines were assessed for the presence of these distinct signaling molecules. In vitro studies showed that VB6-845d cytotoxicity induces the translocation of calreticulin to the cell surface as well as the release of ATP and HMGB1. The expression of other potential immune regulators following VB6-845d treatment was also examined. In summary, the data presented suggests that VB6-845d mediates tumor cell killing by an ICD pathway. The potential cross-priming effect initiated by VB6-845d-induced ICD suggests the use of VB6-845d in combination with immune checkpoint inhibitors may enhance their effectiveness in EpCAM-positive epithelial cancers.

Citation Format: Rachelle L. Dillon, Shilpa Chooniedass, Arjune Premsukh, Glen C. MacDonald, Jeannick Cizeau, Gregory P. Adams. VB6-845d tumor cell killing elicits biologic features of immunogenic cell death [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 2788.
Abstract 2803: Novel auristatins with high activity on efflux-positive models and demonstrable bystander activity
Authors: Moquist Philip N. , Bovee Tim D. , Waight Andrew B. , Owen Sarah , Mitchell Jamie A. , Zaval Margo , Quick Marsha , Sandall Sharsti , Emmerton Kim K. , Blesie Nicole , Lyon Robert P. , Senter Peter , Doronina Svetlana
Published: August 2018

Auristatins are a class of clinically validated antimitotic agents utilized as payloads in antibody-drug conjugates (ADCs). Auristatins display many of the desirable properties necessary for ADC cytotoxins, such as low nanomolar potency, cell permeability, and activity across multidrug-resistant (MDR+) cell lines. Herein, we report the development of novel auristatins, which have a unique combination of these favorable properties. The hydrophilic monomethyl auristatin F (MMAF) was chosen as the parent structure, and a medicinal chemistry campaign was undertaken to functionalize various sites of the auristatin with hydrophobic moieties in an effort to improve membrane permeability. The structure-activity relationships (SAR) of the new auristatins demonstrated clear trends correlating hydrophobicity, structure, and polarity with permeability and in vitro cytotoxicity. The highest-performing molecules showed a preference for hydrophobic functionalization at the N-terminal dolavaline. The payloads were linked to the C-terminal position of the auristatin with a lysosomally cleavable maleimido-dipeptide linker. Examination of the ADCs revealed low ng/mL activity in CD30+ and CD19+ cell lines in vitro. Anti-CD70 ADCs demonstrated high in vivo efficacy in a 786-O xenograft and complete remissions (CRs) in the efflux-positive renal cell carcinoma model. Anti-CD30 ADCs were dosed in a CD30+ and CD30- admixed Karpas/Karpas-35R xenograft model to demonstrate proof-of-concept in vivo bystander activity. In summary, these novel auristatins showed potential across multiple indications as ADC payloads.

Citation Format: Philip N. Moquist, Tim D. Bovee, Andrew B. Waight, Sarah Owen, Jamie A. Mitchell, Margo Zaval, Marsha Quick, Sharsti Sandall, Kim K. Emmerton, Nicole Blesie, Robert P. Lyon, Peter Senter, Svetlana Doronina. Novel auristatins with high activity on efflux-positive models and demonstrable bystander activity [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 2803.
Abstract 2806: Inhibition of melanoma development by Isatin analogs
Authors: Desai Dhimant H. , Gowda Raghvendra , Gowda Krishne , Dinavahi Saketh S. , Robertson Gavin P. , Amin Shantu G.
Published: August 2018

Despite major breakthroughs in melanoma drug development, there remains a need for new drugs and novel approaches. Of the three forms of skin cancer, malignant melanoma has the most significant impact on human health carrying the highest risk of mortality from metastasis. An estimated 87,110 new cases of invasive melanoma will be diagnosed and 9,730 people will die of melanoma in the USA in 2017. Melanoma accounts for less than one percent of skin cancer cases, but the vast majority of skin cancer deaths. The vast majority of melanomas are caused by the sun. In fact, one UK study found that about 86 percent of melanomas can be attributed to exposure to ultraviolet (UV) radiation from the sun. The estimated 5-year survival rate for patients whose melanoma is detected early is about 98 percent in the USA, whereas, the survival rate falls to 62 percent when the disease reaches the lymph nodes, and 18 percent when the disease metastasizes to distant organs. Thus, novel effective therapies are urgently needed to treat this disease.

The Isatin (1H-indole-2,3-dione) is found as an endogenous molecule in humans and other mammals and its analogs display diverse types of biological activities including anti-cancer activities. Earlier our group synthesized a novel series of 5,7-dibromoisatin analogs. Various melanoma cells were treated with several Isatin derivatives having functional groups like selenocyanate, thiocyanates, thiourea, and selenourea for 72 hours and the cell survival was estimated by MTS assay. Agents were treated with melanoma cell lines UACC 903, 1205 Lu or normal fibroblast cell line FF2441. Treatment with KS99, a thiocyanate analog and KS101, a selenourea analog of 5,7-dibromoisatin effectively killed melanoma cells after 72 h treatment. Overall, IC50 values of Isatin derivatives on melanoma cell lines were 3.0-5.7μM and 2.1-5.7μM, respectively. IC50 value on normal fibroblast cells with these Isatin derivatives was 5.4-20.7μM. KS101 was toxic to the xenograft mice at 1mg/kg body weight, animals developed vein inflammatic symptoms after 20 days of treatment. However, liposomal formulation of KS101 was safe up to 30mg/kg body weight in 1205 xenografted nude mice when treated alternate days by tail vein injection. The melanoma tumor burden was reduced by 47%. We will discuss structural activity relationship (SAR) of Isatin derivatives, and its in vitro and in vivo inhibitory effects against melanoma.

Citation Format: Dhimant H. Desai, Raghvendra Gowda, Krishne Gowda, Saketh S. Dinavahi, Gavin P. Robertson, Shantu G. Amin. Inhibition of melanoma development by Isatin analogs [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 2806.
Abstract 2807: Cytotoxic activity of novel organotin compounds against different cancer cell lines
Authors: Cherek Paulina , Balogova Michaela , Damodaran Krishna , Ögmundsdottir Helga M.
Published: August 2018

Cisplatin has been a successful drug in oncology for decades. Its use is however limited by toxicity and development of resistance. Efforts for improvement by screening a large number of different platinum compounds have only yielded to new drugs to the clinic, oxaloplatin and carboplatin. An alternative approach is therefore to explore the potential of other metal-containing compounds. The toxic properties of organic tin compounds have been exploited in industry for a long time. More recently such compounds have been studied for potential therapeutic cytotoxic effects yielding promising results. Here we describe the first screening of two novel organotin compounds, based on the same basic structure, containing bromide (SNO7) or chloride (SNO8). The compounds are being screened against the following cancer cell lines: T47-D (breast), A549 (lung), OVCAR-3 (ovarian), Aspc-1 (pancreatic). IC50 was determined from survival analysis by Crystal violet, and induction of apoptosis by Annexin V staining, monitored by IncuCyte. Cisplatin was used as reference. IC50 values (µg/ml) for T47-D and Aspc-1 were, respectively: 44.5 and 13.8 (SNO7), 13.4 and 26.3 (SNO8) and 34.9 and 34.5 (Cisplatin). SNO7 induced apoptosis starting after 24 hours in OVCAR-3 and A549 at 10 µg/ml and also T47-D at 25 µg/ml; SNO8 in T47-D at 25 µg/ml (OVCAR-3 and A549 not tested) and Cisplatin in OVCAR-3 at 10 µg/ml (A549 not tested). These early results indicate differences in sensitivities between compounds and cell lines implying some specificity in mode of action and suggest that tin-containing compounds could be active against cancer cells that are resistant to cisplatin.

Citation Format: Paulina Cherek, Michaela Balogova, Krishna Damodaran, Helga M. Ögmundsdottir. Cytotoxic activity of novel organotin compounds against different cancer cell lines [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 2807.