bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2005: Optimized range of osmotic pressure and viscosity for a lymphatic drug delivery system
Authors: Kodama Tetsuya , Mori Shiro
Published: August 2019

The lymphatic drug delivery system (LDDS) is a method that allows the injection of drugs into sentinel lymph nodes (SLNs) to facilitate their delivery to downstream lymph nodes (LNs) via the lymphatic network to treat or prevent cancer cell development in both LNs. A high concentration of drug is effectively maintained in both LNs. Here we show that therapeutic effects in metastatic LNs depend on the osmotic pressure and viscosity of the administered drug in a metastatic mouse (MXH10/Mo-lpr/lpr) model. We found using an in vivo bioluminescence imaging system that indocyanine (ICG) solutions were retained in the proper axillary lymph node (PALN) at an osmotic pressure > 1,700 kPa and 6 mPa·s viscosity, when ICG solution was injected into the SiLN to deliver it to the (PALN). Edematous swelling was detected near the SiLN at > 3,500 kPa osmotic pressure and 400 mPa·s viscosity. Furthermore, we established that the therapeutic effects of drugs on metastatic PALN were observed in the osmotic pressure range 1000 - 2400 kPa and viscosity range 2 - 30 mPa·s, after tumor cells were injected into the SiLN to induce metastasis in the PALN. Cisplatin solution was injected into the accessory axillary lymph node to deliver cisplatin to the metastatic PALN. Our results demonstrate the optimized values of osmotic pressure and viscosity for LDDS. We anticipate our results to be a starting point for more sophisticated drug administration by a LDDS.

Citation Format: Tetsuya Kodama, Shiro Mori. Optimized range of osmotic pressure and viscosity for a lymphatic drug delivery system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2005.
Abstract 2019A: TLR3 facilitate breast cancer metastasis to lymph node
Authors: Torres-Luquis Odalys J. , Mohammed Sulma
Published: August 2019

Tumor cells can only enter the lymphatic circulation at the interface between the invasive edge of the tumor and the adjacent host stroma that contains the lymphatic circulation. The existence of this distance barrier means that it would be unlikely for tumor cells to passively enter the lymph drainage. Instead, Toll-like receptors (TLRs) are important mediators for active migration and chemotaxis for tumor cells entry into the lymphatic drainage from the primary tumor. Here we use a microsurgical technique to collect lymph circulating tumor cells (LCTC) prior to its entry into sentinel lymph node in rat models and determine if the TLR expression induces the migration and tumor progression. In our results, we observed high levels of TLR3 expression in LCTC when cells were treated with Poly (I:C), a synthetic ligand for TLR3 induction. NFkB activation was also observed in LCTCs, after TLR3 activation this pathway can express genes involved in cancer progression. In addition, when looking at the expression of cytokines and chemokines factors present in the lymph or secreted by the tumor cells, CXCL10 had a high expression on induced TLR3 of LCTC and its ligand CXCR3 was also expressed in the lymph. CXCL10 is involved in chemotaxis, induction of apoptosis, and regulation of cell growth. The CXCL10/CXCR3 signaling mediates paracrine interactions between tumor and stromal cells that govern leukocyte trafficking and angiogenesis. Suppression of CXCR3 or receptors signaling limits metastasis suggesting CXCR3 contributes to the metastatic process. TLR3 Knockdown prevented tumor growth and metastasis in rat. Our data suggest that for breast cancer metastasis, TLR3 might play a role in active migration of tumor cells and tumor progression. Activation of TLRs in tumor cells can dampen the anti-tumor functions of infiltrating immune cells, altering the inflammatory response and promoting cancer progression.

Citation Format: Odalys J. Torres-Luquis, Sulma Mohammed. TLR3 facilitate breast cancer metastasis to lymph node [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2019A.
Abstract 2046: Egr-1 promotes bone marrow fibrosis in primary myelofibrosis
Authors: Rao Rekha M. , Kumar Amar , Vekaria Pratikkumar , Yacoub Abdulraheem , Skikne Barry , McGuirk Joseph
Published: August 2019

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) characterized by JAK-STAT-activated proliferation of leukemia cells and progressive bone marrow fibrosis. However little is known about the bone marrow stromal cell (BMSC) intrinsic factors that work in concert with leukemia stem cells (LSCs) to promote fibrosis of the bone marrow in PMF. In this study we developed an in vitro model to simulate bone marrow fibrosis in PMF by exposing cultured BMSC, HS-5 cells to PMF-patient plasma. We observed that exposure of BMSCs to PMF plasma but not normal serum increased total cellular and mitochondrial reactive oxygen species (ROS), as well as Early growth response 1 (Egr-1) and its target gene expression in BMSCs. Egr-1 is a transcription factor that upregulates fibrosis-associated genes, and is acutely induced by numerous cytokines (e.g. TGF-β, PDGFα) that are elevated in PMF. Utilizing this model, we performed RNA sequencing of HS-5 cells exposed to PMF plasma. We observed that PMF plasma exposure alters pathways responsible for multiple DNA damage responses, G1/s cell cycle checkpoint regulation and protein homeostasis. Since PMF progression is associated with enhanced oxidative stress which induces both DNA damage and Egr-1 expression, we determined the effect of anti-oxidants on the induction Egr-1 and fibrosis associated genes in BMSCs following treatment with PMF plasma. We report that treatment with N-acetyl-cysteine (a ROS scavenger) and mitotempo (a mitochondrial ROS scavenger) abrogated the up-regulation of PMF plasma-induced fibrosis-associated genes in BMSCs. Additionally, knockdown of Egr-1 in BMSCs resulted in the down-regulation of collagen 1A1 and connective tissue growth factor (CTGF) following exposure to PMF plasma. These findings suggest that PMF plasma induces ROS and promotes the upregulation of pro-fibrotic genes in an Egr-1-dependent manner in PMF. Our studies suggest that inhibition of ROS and Egr-1-induced gene transcription could be used as a strategy to alleviate bone marrow fibrosis; a disease complication in PMF for which there are no approved therapies.

Citation Format: Rekha M. Rao, Amar Kumar, Pratikkumar Vekaria, Abdulraheem Yacoub, Barry Skikne, Joseph McGuirk. Egr-1 promotes bone marrow fibrosis in primary myelofibrosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2046.
Abstract 2060: Combination of CD20 targeted engineered toxin body, MT-3724, with chemotherapy or IMiDs for the treatment of non Hodgkin's lymphoma
Authors: Higgins Jack P. , Iberg Aimee , Howard Caleigh , Willert Erin
Published: August 2019

MT-3724 is a novel recombinant fusion protein consisting of a CD20 binding variable fragment (scFv) fused to Shiga-like toxin-I A1 (SLTA) ribosome inactivating protein. Engineered Toxin Bodies (ETBs) are unique as they can induce internalization after binding to non- or poorly-internalizing receptors with subsequent enzymatic and permanent ribosome destruction through SLTA, representing a novel mechanism of direct cell-kill in oncology. MT-3724 has demonstrated single agent anti-tumor activity in heavily pre-treated relapsed/refractory (R/R) Non-Hodgkin’s lymphoma NHL patients in a Phase I clinical study. Specifically, in R/R diffuse large B cell lymphoma (DLBCL) patients who had low pre-existing serum levels of rituximab, an objective response rate (ORR) of 30% (3 of 10) and disease control rate of 70% (7 of 10) has been observed thus far (NCT02361346).

Given the unique ribosomal inhibition mechanism of action for MT-3724, we hypothesized that combination of MT-3724 with agents possessing a differentiated mechanism of action could result in additive or synergistic cellular cytotoxicity in CD20 positive NHL cell lines. Preclinical studies were conducted to assess MT-3724 in combination with cytotoxic chemotherapeutic agents (doxorubicin, gemcitabine, bendamustine, and vincristine) or an immunomodulatory (IMiD) agent (lenalidomide) on selected NHL cell lines. The combination of MT-3724 with all agents demonstrated additive or synergistic cytotoxicity of NHL cell lines.

The single agent clinical activity of MT-3724 in heavily pre-treated R/R NHL patients along with this preclinical combinatorial effect of MT-3724 with chemotherapy and IMiDs used in the treatment of R/R NHL have lead the opening of a Phase 2a study of MT-3724 in combination with Gemcitabine and oxaliplatin (GEMOX, NCT03488251) and another planned Phase 2a study of MT-3724 in combination with lenalidomide (NCT03645395).

MT-3724 has demonstrated good tolerability and early signs of clinical activity as monotherapy in patients with R/R NHL along with additive and/or synergistic effects in combination with chemotherapy and IMiDs preclinically. Additional clinical studies to evaluate MT-3724 as single agent and in combination with GEMOX or lenalidomide are underway.

Citation Format: Jack P. Higgins, Aimee Iberg, Caleigh Howard, Erin Willert. Combination of CD20 targeted engineered toxin body, MT-3724, with chemotherapy or IMiDs for the treatment of non Hodgkin's lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2060.
Abstract 208: Transcriptional profiling of the microenvironment in pediatric osteosarcoma
Authors: Sorenson Laurie , McEachron Troy A.
Published: August 2019

Osteosarcoma (OS) is the most common bone tumor in pediatric and adolescent/young adult patients. Over the past three decades, significant improvements in the survival rates or therapeutic approaches for these patients have not been made, especially in the context of metastatic disease. While immune checkpoint blockade has revolutionized the therapeutic landscape in various adult malignancies, its impact in OS has been largely underwhelming. Currently, it is unknown whether the lack of therapeutic benefit of immune checkpoint inhibition observed in patients with OS is truly due to treatment inefficacy rather than a limited understanding of the tumor microenvironment that supports this aggressive disease. To address this knowledge gap, we have performed targeted gene expression profiling of metastatic and non-metastatic osteosarcoma specimens. Our profiling experiments have revealed that metastatic OS specimens are immunologically “colder” than non-metastatic OS specimens. Our data also demonstrates that T cells are largely excluded from the metastatic specimens and that this exclusion significantly correlates with markers of vascular instability. In a pathological setting, such as that of cancer, VEGF and ANG2 signaling promote vascular instability which limits leukocyte extravasation and subsequent tumor infiltration. Our data suggests that vascular destabilization mediated by VEGF/ANG2 signaling impedes T cell infiltration specifically in metastatic OS and identifies these molecules as potential targets for therapeutic intervention.

Citation Format: Laurie Sorenson, Troy A. McEachron. Transcriptional profiling of the microenvironment in pediatric osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 208.
Abstract 2080: Novel molecularly targeted combination for renal cell carcinoma
Authors: Jordan Andre R. , Wang Jiaojiao , Lopez Luis E. , Morera Daley S. , Lokeshwar Vinata B.
Published: August 2019

INTRODUCTION: Despite many available treatments, >90% of metastatic renal cell carcinoma (mRCC) patients die within 5 years. Due to its modest efficacy, Sorafenib (SF) is a second line treatment for mRCC. Molecular basis for SF failure is unknown. Hymecromone (HC) is a non−toxic orally bioavailable agent used in Europe for improving liver health. We examined the molecular basis of SF failure in clinical specimens and provided mechanistic evidence for why SF+HC could be a potent therapeutic combination against mRCC.

METHODS: UGT1A9 (A9) levels and SF glucuronidation were measured in kidney specimens (45 normal; 86 tumor) by qPCR and a glucuronidation assay using tumor microsomes. Response to SF+HC combination in vector-only (EV), A9-overexpressing (A9) and A9-knockdown (A9-KO) 786-0 and Caki-1 transfectants, primary RCC spheroids, and endothelial cells was examined by proliferation, apoptosis, motility and invasion assays. Target analysis was performed by immunoblot. Treatment efficacy was evaluated in Caki−1 s.c. and orthotopic xenograft models, with bioluminescence imaging.

RESULTS: A9 has been shown to glucuronidate and inactivate SF. A9 levels were 8-10-fold elevated in mRCC patients’ tumors compared to non-mRCC patients and normal kidney tissues. A9 levels were an independent predictor of metastasis (P=0.022; efficacy: 73%; sensitivity: 93.8%). Tumor microsomes from mRCC patients’ glucuronidated SF 5-fold higher than from non-mRCC patients. In RCC cells, HC treatment downregulated A9 levels, A9 promoter activity and inhibited SF-glucuronidation. SF (5 µM) and HC (20-40 µg/ml) combination inhibited RCC cell and primary tumor spheroid proliferation (>90%), motility/invasion (>80%), and induced apoptosis (5-fold). While A9 transfectants were resistant to SF+HC treatment, A9-KO cells were sensitive to SF alone. Endothelial cells co-cultured with A9 transfectants were resistant to SF+HC treatment. SF+HC upregulated apoptosis effectors, but downregulated CD44, RHAMM, and phospho-Met levels in EV but not A9 transfectants. HC (100 or 200 mg/kg) and SF (30 mg/kg) oral combination abrogated Caki−1 tumor growth (>80%) without serum/tissue toxicity. HC+SF treatment decreased A9 levels, Ki67 index and microvessel density. A9-overexpressing tumors were resistant to treatment.

CONCLUSION: This is the first study to demonstrate that A9 overexpression in mRCC is likely a mechanism of SF failure and A9 downregulation can re-sensitize cells. HC+SF is an orally bioavailable and non-toxic combination that may be an effective therapeutic strategy for mRCC.

Citation Format: Andre R. Jordan, Jiaojiao Wang, Luis E. Lopez, Daley S. Morera, Vinata B. Lokeshwar. Novel molecularly targeted combination for renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2080.
Abstract 2085: Treatment of human melanoma cells with dasatinib requires inactivation of both mTOR and MAPK pathways to achieve high cell eradication efficacy
Authors: Vachtenheim Jiri , Réda Jiri , Vlčková Kateřina , Ondrušová Lubica
Published: August 2019

Advanced stages of melanoma are resilient to therapy and new strategies for the treatment are required. About 60% of melanomas harbor the mutated BRAF oncogene, which activate the MAPK pathway. This BRAF driver mutation can be targeted by a small molecule drug vemurafenib. However, most of patients develop resistance during treatment and even drug addiction is acquired. The nonreceptor SRC tyrosine kinase is highly expressed and deregulated in melanoma and can be efficiently inhibited by the inhibitor dasatinib, an anticancer drug which is already used for the treatment of some tumor types. SRC does not have its own canonical pathway but is capable of activating and augmenting different signaling routes in cancer cells. We found that melanoma tumor cell lines analysed were generally resistant to dasatinib irrespective of BRAF or NRAS mutational status. The exception was the cell line Hbl which was very sensitive even to low dasatinib doses and Hbl cells were completely eradicated by 10 nM of dasatinib in several days. The sensitivity of Hbl to dasatinib was dependent on the drug ability to inhibit completely both the MAPK and AKT/mTOR signaling pathways. Dasatinib was capable of inhibiting SRC activation resulting in the repression of AKT activation in Hbl cells. AKT phosphorylation at Ser473 was fully abolished and Thr308 was severely attenuated through inhibiting SRC by dasatinib in Hbl cells. These two phosphorylations are required for AKT activity. MAPK pathway (ERK1/2 phosphorylation) was also inhibited in Hbl cells. Dasatinib also inhibited the phosphorylation of SRC at Tyr416 (a hallmark of SRC activity) in Hbl cells and, surprisingly, also in other dasatinib-resistant melanoma cells. Importantly, although SRC was dephosphorylated at Tyr416, dasatinib was unable to inactivate AKT and MAPK pathway (ERK1/2) in all other melanoma cells. Molecular changes closely correlated with proliferation assays. Our data indicate that the combination therapy of melanoma with dasatinib would bring benefit to patients only by blocking both MAPK and AKT signaling pathways in melanoma cells. Altogether, more molecular knowledge of the effects of the powerful anticancer drug dasatinib is required in order to be used for the treatment of melanoma. This work was supported by the Institutional research project PROGRESQ25 from Charles University Prague.

Citation Format: Jiri Vachtenheim, Jiri Réda, Kateřina Vlčková, Lubica Ondrušová. Treatment of human melanoma cells with dasatinib requires inactivation of both mTOR and MAPK pathways to achieve high cell eradication efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2085.
Abstract 2093: Drug induced autophagy and transcriptional gene expression signatures in breast cancer cell lines
Authors: Mascia Francesca , Karagiannis Kostantinos , Wu Wells W. , Shen Rong-Fong , Rao Ashutosh
Published: August 2019

Cancer cells can bypass the desired cell death response to chemotherapy through cytoprotective autophagy. In support of this hypothesis, drug resistant clones display higher basal autophagy than the correspondent drug sensitive cell lines. Autophagy therefore represents a potential target to reduce drug resistance and to enhance drug efficacy. We induced autophagy in several breast cancer cell lines with differing ER/PR/Her2 status by exposing them to tamoxifen (MCF-7/ER positive cells), trastuzumab (SKBR3/Her2 positive cells), bortezomib and rapamycin (MDA-MB-231 triple negative cells) in the presence and absence of an autophagy inhibitor such as hydroxychloroquine. While the ongoing autophagy flux process was confirmed by the amount of LC3II at the lipidated protein level, total RNA was extracted at the same time to perform RNA sequencing analysis. We performed differential expression analysis between all the drug treatments and the relative basal level of each cell type obtaining a cell and drug-specific gene list. We then compared all the gene lists to find a common, cell and drug-independent signature. Bioinformatic analysis was performed using the software IPA (Ingenuity Pathway Analysis) and setting a threshold of 0.01 for the p value. This analysis suggested a set of canonical pathways that are significantly and commonly affected in all the samples tested. The top canonical pathways disrupted in all the gene sets were the one identified as tRNA charging, CHK proteins in cell cycle check point control, hypoxia signaling and unfolded protein response. We plan to further identify the smallest gene list that is associated with the disruption of all these pathways across all the cells. This signature may help to uncover a consistent biomarker of autophagy that can be used during preclinical and clinical drug development aiming to reduce or eliminate drug resistance and improve cancer treatment outcomes.

Citation Format: Francesca Mascia, Kostantinos Karagiannis, Wells W. Wu, Rong-Fong Shen, Ashutosh Rao. Drug induced autophagy and transcriptional gene expression signatures in breast cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2093.
Abstract 2110: WEE1 inhibition could reverse trastuzumab resistance by downregulation of PD-L1 in HER2-positive cancers
Authors: Jin Meihua , Nam Ah-Rong , Park Ji-Eun , Bang Ju-Hee , Oh Kyoung-Seok , Oh Do-Youn , Bang Yung-Jue
Published: August 2019

Background: Trastuzumab in combination with chemotherapy is a standard of care for patients with HER2-positive breast and gastric cancer. Resistance mechanism to trastuzumab, anti-HER2 therapy, includes multiple pathways. Cancer cells could evade immune surveillance through high expression of programmed cell death ligand 1 (PD-L1) on cancer or immune cells. Among resistance mechanisms to trastuzumab, the role of PD-L1 modulation has not yet been discovered in HER2-positive cancers. WEE1 is involved in cell cycle progression and DNA damage response (DDR), and, interestingly, beyond DDR, it could modulate immunes.

We aimed to evaluate the role of PD-L1 in trastuzumab resistance in HER2-positive cancers with/without immune cells and to explore WEE1 inhibitor as a trastuzumab resistance overcoming strategy by modulation of PD-L1.

Methods: Four trastuzumab-resistant (HR) cells (SNU216HR, N87HR, SNU2670HR, SNU2773HR) were established from 2 HER2-amplified gastric cancer cells (SNU216, NCI-N87) and 2 HER2-amplified biliary tract cancer cells (SNU2670, SNU2773). For WEE1 inhibition, AZD1775 was used. MTT assay, colony formation assay, cell cycle analysis by FACS Calibur flow cytometer, and western blot were done.

Results: All four HR cells showed PD-L1 upregulation compared with parental cells. When PD-L1 was knocked-down by transfection with si-PD-L1, anti-growth/proliferation effects were observed in MTT assay and colony forming assay. PD-L1 itself may promote cancer cell growth without interacting with immune cells.

AZD1775 downregulated PD-L1 expression and induced cell cycle arrest at sub-G1 and G2/M phases in all 4 HR cells. The synergistic anti-proliferative effects were found in all 4 HR cells with co-treatment of trastuzumab and AZD1775.

Conclusion: PD-L1 upregulation may contribute to trastuzumab resistance in HER2-positive cancer cells. Targeting WEE1-PD-L1 pathway might be a candidate strategy to overcome trastuzumab resistance in HER2-positive cancers.

Citation Format: Meihua Jin, Ah-Rong Nam, Ji-Eun Park, Ju-Hee Bang, Kyoung-Seok Oh, Do-Youn Oh, Yung-Jue Bang. WEE1 inhibition could reverse trastuzumab resistance by downregulation of PD-L1 in HER2-positive cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2110.
Abstract 2114: Co-delivery nanoparticle to overcome therapeutic resistance in human head and neck cancer promoted by insufficient Src-targeted treatment
Authors: Lang Liwei , Shay Chloe , Wang Xuli , Teng Yong
Published: August 2019

Head and neck cancer (HNC) exhibiting resistance to molecular-targeted therapeutics poses a challenge to their effective clinical management and alternative treatment strategies are actively sought to improve results. Src is commonly hyperactivated in HNC cells and has been implicated as an oncogenic driver in the development and progression of HNC. Unfortunately, the clinical benefit is dampened significantly in HNC patients because only a small fraction of patients respond positively to Src-targeted treatment, and many develop drug resistance. Here, we demonstrate that the addition of AKT blockade in Src-targeted treatment may prevent activation of alternative signaling pathways to bypass the Src inhibition in HNC cells. The combination of Src and AKT inhibitors significantly suppressed HNC growth more efficiently than either drug alone. However, the properties and pharmacokinetics of two inhibitors required a novel approach to optimal delivery into the same tumor cells. To address this, we developed novel tumor-targeting nanoparticles to selectively co-deliver Src and AKT inhibitors to the tumor site in an orthotopic mouse model of HNC. This nanotherapeutic modality significantly improved the efficacy of tumor repression, which was mainly attributed to the highly specific tumor-targeted drug delivery system and synergistic anticancer effect by co-inactivation of AKT and Src. The results of this study will enable us to take a major step toward addressing the urgent need for single drug resistance and failure, and strongly impact on the development of clinically acceptable therapeutics to combat HNC.

Citation Format: Liwei Lang, Chloe Shay, Xuli Wang, Yong Teng. Co-delivery nanoparticle to overcome therapeutic resistance in human head and neck cancer promoted by insufficient Src-targeted treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2114.
Abstract 2117: Different sensitivities of four protein tyrosine kinase inhibitors towards drug-resistant RET mutations
Authors: Liu Xuan , Shen Tao , Huang Qingling , Peng Teng , Hilberg Frank , Cai Jianfeng , Mooers Blaine H. , Wu Jie
Published: August 2019

The RET protein tyrosine kinase (PTK) is a clinically validated target of therapy in non-small cell lung cancer (NSCLC) and thyroid cancer. Mutations in the targeted PTKs is a mechanism of drug resistance in cancer therapy with tyrosine kinase inhibitors (TKIs). Mutation-sensitive secondary drugs have been used successfully to overcome acquired drug-resistance to the first line TKIs. Cabozantinib, lenvatinib, vandetanib, and nintedanib are FDA-approved multikinase TKIs with anti-RET activity. Using RET kinase-dependent BaF3/KIF5B-RET cells, we isolated thirteen mutations resistant to one of these TKIs. Cross-analysis of sensitivities of these four TKIs on these drug-resistant RET mutants and the RETM918T mutant, which is found in medullary thyroid carcinoma, revealed different TKI resistance-sensitivity profiles. In particular, the RETM918T mutant was resistant to cabozantinib, lenvatinib, and vandetanib but did not affect the potency of nintedanib. RETL881V was isolated as a vandetanib-resistant mutation. The RETL881V mutation also induced resistance to cabozantinib and lenvatinib but did not affect the nintedanib sensitivity. Examination of the RET-vandetanib co-crystal structure and the chemical structures of vandetanib and nintedanib suggested that a phenyl group in nintedanib, which corresponds to a methoxy group in vandetanib, may form hydrophobic interaction with the shorter side chain of Val881 and thus allows nintedanib to inhibit RETL881V. To test this possibility, we synthesized two nintedanib analogs (Compound 1 and Compound 2) without the phenyl group and tested their activities. The data showed that RETL881V was resistant to Compound 1 and Compound 2, supporting a role of the phenyl group of nintedanib in mediating inhibition of RETL881V. Taken together, we have identified 13 RET mutations that display different resistance-sensitivity profiles against four anti-RET TKIs. Moreover, nintedanib is effective in inhibiting RETM918T and RETL881V mutants that are resistant to the other three TKIs.

Citation Format: Xuan Liu, Tao Shen, Qingling Huang, Teng Peng, Frank Hilberg, Jianfeng Cai, Blaine H. Mooers, Jie Wu. Different sensitivities of four protein tyrosine kinase inhibitors towards drug-resistant RET mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2117.
Abstract 2141: Mechanistic studies of altertoxin II: A fungal metabolite with selective activity against Ewing sarcoma
Authors: Robles Andrew J. , Dai Wentao , Cichewicz Robert H. , Mooberry Susan L. , Houghton Peter J.
Published: August 2019

Ewing sarcomas (ES) are bone and soft tissue tumors that affect children and adolescents. These cancers are driven by chromosomal translocations that most frequently result in expression of the abnormal EWSR1-FLI1 fusion protein, a transcriptional activator and modulator of RNA splicing, or similar fusion proteins. Despite significant advances in therapies for pediatric hematological malignancies over the past 30 years, there has been less progress in the development of curative therapies for advanced or metastatic pediatric solid tumors. Approximately 35% of ES are resistant to standard-of-care chemotherapeutic agents, and nearly 70% of ES patients with metastatic disease at the time of diagnosis die within 5 years. Therefore, there is a need to develop new, targeted therapies for ES that improve patient survival and decrease toxicity. We performed high-content phenotypic screens of natural product libraries to identify compounds with selective actions against pediatric solid tumor types and found that the fungal metabolite altertoxin II (ATXII) has highly selective cytotoxic activity against 6 ES cells lines compared to a panel of other pediatric and adult cancer cell lines. On average, the IC50 of ATXII is 94-fold higher (range 16- to 400-fold) in rhabdomyosarcoma (RMS) cells than in ES cells, indicating high selectivity for ES. Mechanistic studies were conducted to determine this compound’s mechanisms of actions in ES cells, with the goal of identifying new molecular targets for the treatment of ES. We found that 100 nM ATXII, a concentration that has no effect on RMS cell viability, induces cell cycle accumulation in ES cells in the S and G2/M phases, and rapid phosphorylation of histone H2A.X (gamma-H2A.X) and checkpoint kinase 1, suggesting ATXII selectively induces cell cycle arrest and DNA damage in ES cells. To determine if ES sensitivity to ATXII is a dominant or recessive phenotype, we hybridized sensitive EW8 cells (ES) with resistant Rh30 cells (RMS) and determined the sensitivity of these hybrids to ATXII. Five single-cell subclones derived from these hybrids were resistant to ATXII, suggesting that sensitivity to ATXII is a recessive phenotype in ES cells. Ongoing studies are aimed at determining the cellular uptake and binding kinetics of radiolabeled ATXII and identifying genes that mediate sensitivity and resistance to this compound by performing a genome-wide CRISPR/Cas9 knockout screen.

Citation Format: Andrew J. Robles, Wentao Dai, Robert H. Cichewicz, Susan L. Mooberry, Peter J. Houghton. Mechanistic studies of altertoxin II: A fungal metabolite with selective activity against Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2141.
Abstract 2157: Eliminating Edge Effect in 96-Well Plates by Controlling Thermal Conditions during Cell Plating
Authors: Darou Shannon , Henn Alicia , Alm Kersti , Frank Allayna M. , Yerden Randy
Published: August 2019

The traditional approach to avoiding the increased variability in 96-well plate edge wells has been to avoid using them. This results in a 37% loss in usable assay space, increasing time and materials needed for cell-based assays. Approaches to reducing this “edge effect” have included reducing plate evaporation during long-term incubation. However, we have preliminary data that plating cells at a constant 37 degrees C dramatically reduces edge effect. We have previously showed that in plates filled at room temp (RT), the cells in edge and corner wells experienced the most directional cell rolling during cell settling, preventing random cell deposition. In contrast, cells plated at 37 degrees C settled randomly in edge wells with less or no directional rolling. Here, we extend these findings with studies of well-specific cell settling patterns associated with plate-wide thermal changes after cell plating. Our null hypothesis was that intra-well thermal currents as plates warm to 37 degrees C from RT do not disrupt random cell deposition during settling. A549 human lung carcinoma cells were plated in warmed medium in either traditional uncontrolled RT conditions or in an Xvivo System under completely controllable conditions. In this closed chamber, everything (chamber floor, atmosphere, the pipettor, tips, fluids, gloves, and plate) was at the same temp as the incubator, where cells typically settle in the wells after plating. We used the PHI HoloMonitor M4 microscope to record holographic time-lapse images of cells settling and adhering to the well floor. Crystal violet staining was used to assess cell deposition patterns. We found that not only did constant temp conditions change well-specific cell settling patterns, but when plate-wide thermal changes were inverted, by plating cells at 37 degrees C and letting them settle at RT, we were able to alter well-specific cell rolling and cell settling patterns. We concluded that it is thermal changes that drive edge well variability and that constant temperature control during cell plating can reduce or eliminate edge effect. Plating cells under full-time control of conditions could have a tremendous impact on cell-based drug discovery and pre-clinical testing by allowing full use of 96-well plates. This could reduce assay time and materials as well as improve assay reproducibility.

Citation Format: Shannon Darou, Alicia Henn, Kersti Alm, Allayna M. Frank, Randy Yerden. Eliminating Edge Effect in 96-Well Plates by Controlling Thermal Conditions during Cell Plating [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2157.
Abstract 2178: Targeted delivery of chemotherapeutic agents employing nanovesicles effectively reduced intrahepatic tumors
Authors: George Joseph , Hayashi Nobuhiko , Saito Takashi , Tsutsumi Mikihiro , Tsuchishima Mutsumi
Published: August 2019

Background and Aims: Currently there is no appropriate method for effective and targeted delivery of chemotherapeutic agents to tumor sites. The conventional chemotherapy is afflicted with lack of effective drug delivery to the tumor site, unwanted injury to actively dividing healthy cells, and numerous side effects due to toxicity. The present study was aimed to develop a biologically derived nanoparticle based drug delivery system for the effective treatment of primary hepatic tumors.

Methods: Intrahepatic tumors were induced in immunosuppressed mice using PLC/PRF/5 hepatocellular carcinoma (HCC) cells stably transfected with a mammalian expression vector carrying luciferase gene for in-vivo imaging. Nanovesicles ranging from 80-150 nm were isolated from bovine skim milk using ultracentrifugation and characterized using nanoparticle tracking analysis (NTA) and electron microscopy. Purified nanovesicles were loaded with chemotherapeutic agents and antisense oligonucleotides for grossly deregulated microRNAs specific to HCC. Animals with uniform intrahepatic tumors were injected with nanovesicle loaded anticancer agents through tail vein. Tumor regression was monitored weekly using in-vivo imaging system and the animals were maintained up to 8 weeks.

Results: Bioluminescence based in-vivo imaging demonstrated significant reduction of tumor size in treated animals compared to untreated controls. There was increased survival rate and complete absence of metastasis in all treated animals. Untreated mice developed large intrahepatic tumors, which also metastasized to other organs. There were no side effects or immunological reactions in animals injected with plain nanovesicles without loading of any anticancer agents.

Conclusions: The present study demonstrated that milk derived nanovesicles can be effectively used for successful delivery of anticancer agents into intrahepatic tumors. Nanovesicle based targeted drug delivery system could be an effecient method for the treatment of primary hepatic tumors.* Presenting author E-mail: georgej@kanazawa-med.ac.jp

Note: This abstract was not presented at the meeting.

Citation Format: Joseph George, Nobuhiko Hayashi, Takashi Saito, Mikihiro Tsutsumi, Mutsumi Tsuchishima. Targeted delivery of chemotherapeutic agents employing nanovesicles effectively reduced intrahepatic tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2178.
Abstract 2204: Novel FAK/ALK/ROS1 inhibitor APG-2449 synergizes with osimertinib in preclinical xenograft models of EGFR-mutant NSCLC
Authors: Wang Guangfeng , Fang Douglas D. , Min Ping , Tao Ran , Tang Chunyang , Gu Shoulai , Rui Li , Li Jiajun , Wang Jingwen , Wu Miaoyi , Li Yingfeng , Chen Dingxiong , Zhang Fei , Lian Kejie , Zhang Feifei , Wang Lvcheng , Xu Rongcheng , Yang Dajun , Zhai Yifan
Published: August 2019

Focal adhesion kinase (FAK) plays an important role in cell migration, growth factor signaling, cell cycle progression and cellular survival. It had been shown that FAK and SRC family kinases were able to sustain downstream AKT and MAPK signaling under continuous EGFR inhibition driven by osimertinib (AZD9291), a mutant-selective third-generation EGFR inhibitor. Incomplete inhibition of AKT and MAPK was consistently observed even under increased concentrations of osimertinib and led to acquired resistance to osimertinib, implicating the need of combination therapy with FAK/SRC inhibitors to enhance antitumor activity of osimertinib and overcome its resistance. APG-2449 is a novel oral active small-molecular inhibitor that targets FAK, ALK and ROS1. In this study, we investigated the effect of combination treatment with APG-2449 and osimertinib using NSCLC NCI-H1975 cells carrying EGFRL858R/T790M mutation and an osimertinib-resistant patient-derived xenograft (PDX) model carrying EGFRT790M/19del/C797S mutation. In vitro, the combination treatment with APG-2449 and osimertinib synergistically inhibited the proliferation of NCI-H1975 cells. In vivo, APG-2449 significantly enhanced antitumor activity of osimertinib in NCI-H1975 xenograft models, leading to complete or partial tumor regression. More profound antitumor activity of this combination was also demonstrated in the osimeritinib-resistant PDX model in comparison with single agents. In term of mechanism, the combination arm significantly suppressed the on-target phosphorylation of EGFR, FAK and SRC, as well as AKT and ERK in NCI-H1975 xenografts. Collectively, these results suggest that addition of APG-2449 to osimeritinib may enhance antitumor activity and suppress the development of resistance in NSCLC.

Citation Format: Guangfeng Wang, Douglas D. Fang, Ping Min, Ran Tao, Chunyang Tang, Shoulai Gu, Li Rui, Jiajun Li, Jingwen Wang, Miaoyi Wu, Yingfeng Li, Dingxiong Chen, Fei Zhang, Kejie Lian, Feifei Zhang, Lvcheng Wang, Rongcheng Xu, Dajun Yang, Yifan Zhai. Novel FAK/ALK/ROS1 inhibitor APG-2449 synergizes with osimertinib in preclinical xenograft models of EGFR-mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2204.
Abstract 2206: Anti-tumor activity of infigratinib, a potent and selective inhibitor of FGFR1, FGFR2 and FGFR3, in FGFR fusion-positive cholangiocarcinoma and other solid tumors
Authors: Li Gary , Krook Melanie , Roychowdhury Sameek , Avogadri Francesca , Ye Yining , Moran Susan
Published: August 2019

Abnormal expression and constitutive activation of receptor tyrosine kinases, such as ALK, ROS1 and TRK, as a result of gene rearrangements have been clinically validated as therapeutic targets for cancer. Recently, fusions involving the FGFR family, especially FGFR1, FGFR2 and FGFR3, have been identified in diverse solid tumors such as cholangiocarcinoma, glioblastoma, bladder, lung, breast, thyroid and prostate cancers. FGFR fusions are oncogenic drivers that ligand-independently activate the receptor kinases and their downstream signaling pathways, leading to uncontrolled cell proliferation and invasion.

Infigratinib (BGJ398) is an oral and selective inhibitor of FGFR. At biochemical and cellular levels, infigratinib selectively inhibits the activity of FGFR1, FGFR2 and FGFR3 with low nM potency, while sparing FGFR4, VEGFR2 and other kinases. In FGFR fusion-positive xenograft models, including cell line-derived and patient-derived models representing diverse tumor histologies, infigratinib treatment leads to significant tumor growth inhibition and regression at clinically achievable exposure levels. The anti-tumor effect of infigratinib is accompanied by inhibition of downstream signaling pathways and induction of tumor apoptosis.

Clinically, in an open-label phase II trial, infigratinib demonstrated a cORR of 39.3% in FGFR2 fusion-positive cholangiocarcinoma patients who had received one or fewer prior chemotherapy regimens. Additionally, RECIST responses were achieved in non-cholangiocarcinoma solid tumors tested positive for FGFR fusions.

In conclusion, the available preclinical and clinical data demonstrate the potential of infigratinib as an effective treatment for patients with FGFR fusion-positive tumors, regardless of the fusion partners or the origin of tissue. On the basis of these data there is a clear rationale for performing tumor-agnostic clinical trials in molecularly defined cancers to maximally benefit patients with serious and life-threatening diseases.

Citation Format: Gary Li, Melanie Krook, Sameek Roychowdhury, Francesca Avogadri, Yining Ye, Susan Moran. Anti-tumor activity of infigratinib, a potent and selective inhibitor of FGFR1, FGFR2 and FGFR3, in FGFR fusion-positive cholangiocarcinoma and other solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2206.
Abstract 2209: Novel therapeutic agents for RET-driven cancers, which is highly potent and selective inhibitor to RET kinase
Authors: Li Hua , Jo SeoHyun , Do Woomi , Kim Hyunkyung , Kim Hwan , Seo Kyung-ah , Oh Ji-hoon , Choi Jieun , Ahn Jaeyoung , Son Jung Beom , Kim Nam Doo
Published: August 2019

The rearranged during transfection (RET) tyrosine kinase is mostly altered by fusion and it makes RET as a attractive therapeutic target for medullary thyroid cancer and non-small cell lung cancer (NSCLC). Multi-kinase inhibitors (mKIs) such as cabozantinib and vandetanib expressed poor tolerability and limited efficacy due to off-target kinase inhibitory activity in academia and clinical cases. As for selective RET inhibitor, compounds such as BLU-667 and LOXO-292 are under development, currently in clinical trials. We are currently developing potent and selective RET inhibitors to address unmet medical needs for RET-driven cancers. Our team has validated its high potency and selectivity via kinase profiling and in-vitro assay results. We also anticipate our compound to overcome certain mutation, such as V804M. Our compounds strongly suppress phosphorylation of RET in tumor cells with various RET fusions and mutations by inhibiting proliferation at below 10 nM. This potency result is higher than those of other mKIs with RET inhibitory activity. The inhibitory concentration of our compounds to VEGFR2 is more than dozens of fold compared with RET. In addition, our compounds reveal low cytotoxicity in Ba/F3 naïve cells in the 1 µM concentration. These findings lead us to conclude that our compounds can be potent and highly selective inhibitors of RET kinase, a potential novel therapeutic agents for RET-driven cancers.

Citation Format: Hua Li, SeoHyun Jo, Woomi Do, Hyunkyung Kim, Hwan Kim, Kyung-ah Seo, Ji-hoon Oh, Jieun Choi, Jaeyoung Ahn, Jung Beom Son, Nam Doo Kim. Novel therapeutic agents for RET-driven cancers, which is highly potent and selective inhibitor to RET kinase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2209.
Abstract 2247: Platinoid-induced MHCI antigen presentation enables checkpoint inhibitor responsiveness
Authors: Bastian Ingmar Niels , Li Weihua , Phung Qui , Fisch Kathleen , Lill Jennie , Karin Michael , Shalapour Shabnam
Published: August 2019

Many cancers are refractory to immune checkpoint therapy (ICT), but the causes of ICT resistance remain unknown. Despite low mutational burden, hepatocellular carcinoma (HCC) responds to PD-1 blockade, but prostate cancer (PCa) does not. RNA-seq analysis revealed that, unlike HCC, PCa cells poorly express MHC-I molecules and components of the MHC-I antigen processing and presentation machinery. We found that treatment of PCa cells and other tumor types with platin-based drugs, especially low dose Oxaliplatin, led to a pronounced upregulation of MHC-I molecules and their antigen generating and loading machinery. Most importantly, Oxaliplatin synergistically potentiated the effect of exogenous IFNγ; a conclusion based on two cumulative observations. First, ATAC-seq analyses revealed that Oxaliplatin activates MHC machinery through different pathways compared to well-known IFNγ signaling. Second, Oxaliplatin upregulates IFNγR2 expression in cancer cells. Induction of IFNγR2, which was needed for Oxaliplatin-potentiation of ICT-induced tumor rejection in vivo, depended on NF-κB signaling. Induction of IFNγR2, which was needed for Oxaliplatin-potentiation of ICT-induced tumor rejection in vivo, depended on NF-κB signaling. Moreover, we found that Oxaliplatin activates histone acetyltransferases (HATs) which alter chromatin structure of genes encoding MHC-I complex components, thereby enabling recruitment of a diverse array of activating transcription factors.

Our data indicates that combining ICT with an appropriate low dose platinoid chemotherapeutic agent is useful in reinvigorating the immune response to tumors that have escaped immune destruction due to MHC-I downregulation and do not respond to single agent therapy.

Citation Format: Ingmar Niels Bastian, Weihua Li, Qui Phung, Kathleen Fisch, Jennie Lill, Michael Karin, Shabnam Shalapour. Platinoid-induced MHCI antigen presentation enables checkpoint inhibitor responsiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2247.