bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1967: A novel 40kDa CPE-ΔN isoform promotes proliferation and invasion in pancreatic cancer cells
Authors: Yang XuYu , Ling Cong , Lou Hong , Peng Loh Yoke
Published: July 2017

Carboxypeptidase E (CPE) is a prohormone processing exopeptidase that cleaves C-terminal basic residues from peptide hormones liberated endoproteolytically from prohormones. It is a 476- amino acid protein with a signal peptide in its N-terminus and is mainly expressed in neuropeptide-rich areas of brain and endocrine tissues. As a multifunctional protein, CPE plays many non-enzymatic roles such as prohormone sorting, vesicle transport, and secretion in addition to its carboxypeptidase activity. Moreover, high expression of CPE is found in metastatic tumor tissues, including liver cancer, renal clear cell carcinoma, colorectal cancer, cervical cancer and melanoma, suggesting CPE may be involved in tumor metastasis and progression. In this study, a novel CPE splice isoform mRNA was identified by Northern blotting, which is ~1.83kb in size. RACE assay and sequence analysis confirmed existence of this CPE isoform mRNA, which partially lacks a N-terminus region, and has a shorter 3’-UTR compared to WT-CPE mRNA, by removal of 188 nucleotides within the first exon and 589 nucleotides out of the 3’-UTR, respectively. Bioinformatics analysis revealed that this CPE isoform mRNA has a shortened ORF, which starts coding from the 3rd ATG relative to wild-type (WT) CPE mRNA and encodes a 40kDa CPE-αN protein with the N-terminus truncated. Western blot analysis showed that 40kD CPE-αN is expressed in multiple cancer cell lines and tumor tissues. In Panc-1pancreatic cancer cells, knockdown of endogenous CPE(s) inhibited cell growth and invasion; in contrast, ectopic expression of 40kD CPE isoform significantly promoted proliferation, colony formation, invasion and migration, whereas overexpression of WT-CPE had only a small effect. These results suggest that the 40kD CPE-αN isoform plays a role in cell growth and metastasis.

Note: This abstract was not presented at the meeting.

Citation Format: XuYu Yang, Cong Ling, Hong Lou, Loh Yoke Peng. A novel 40kDa CPE-ΔN isoform promotes proliferation and invasion in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1967. doi:10.1158/1538-7445.AM2017-1967
Abstract 199: Silibinin suppresses bladder cancer through down-regulation of actin cytoskeleton and PI3K/Akt signaling pathways
Authors: Imai-Sumida Mitsuho , Chiyomaru Takeshi , Majid Shahana , Kulkarni Priyanka , Dasgupta Pritha , Saini Sharanjot , Kato Taku , Maekawa Shigekatsu , Hashimoto Yutaka , Shiina Marisa , Deng Guoren , Shahryari Varahram , Nip Hannah , Dahiya Rajvir , Tanaka Yuichiro , Yamamura Soichiro
Published: July 2017

Silibinin is the major active constituent of silymarin, an extract of milk thistle seeds. Silibinin has been shown to have significant anti-cancer effects in a variety of malignancies. However, the molecular mechanisms of silibinin action in bladder cancer have not been studied extensively. In the present study, we found that silibinin (10 μM) significantly suppressed proliferation and invasion of T24 and UM-UC-3 human bladder cancer cells. In this study, we investigated the molecular mechanisms underlying these effects of silibinin. Our results showed that silibinin down-regulates actin cytoskeleton-related Ras-Rac-PAK1 pathways and the phosphatidylinositide 3-kinase (PI3K)/Akt signaling pathway in these bladder cancer cell lines. These pathways were found to promote cellular transformation and cancer development and have crosstalk through Ras cascades. We investigated the role of silibinin on Ras, and found that silibinin suppresses levels of trimethylated histone H3 lysine 4 and acethylated H3 at the K-RAS promoter. We also found that silibinin downregulates long non-coding RNAs: HOTAIR and ZFAS1, which are known to play roles as oncogenic lncRNAs in various cancers. This study shows that silibinin exerts anti-cancer effects through down-regulation of the actin cytoskeleton and PI3K/Akt pathways and thus suppresses the development and progression of bladder cancer.

Citation Format: Mitsuho Imai-Sumida, Takeshi Chiyomaru, Shahana Majid, Priyanka Kulkarni, Pritha Dasgupta, Sharanjot Saini, Taku Kato, Shigekatsu Maekawa, Yutaka Hashimoto, Marisa Shiina, Guoren Deng, Varahram Shahryari, Hannah Nip, Rajvir Dahiya, Yuichiro Tanaka, Soichiro Yamamura. Silibinin suppresses bladder cancer through down-regulation of actin cytoskeleton and PI3K/Akt signaling pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 199. doi:10.1158/1538-7445.AM2017-199
Abstract 2023: Artemisinins enhanced the antileukemic efficacy of BCL2 inhibitors
Authors: Moses Blake S. , Fox Jennifer M. , Chen Xiaochun , Tyner Jeffrey W. , Posner Gary H. , Bailey Patrick , Civin Curt I.
Published: July 2017

More effective, less toxic treatments are needed to improve longevity and quality of life for patients with acute leukemias. Artesunate (AS), the current WHO-recommended drug for severe malaria, is a semi-synthetic derivative of the natural compound Artemisinin. AS has broad antineoplastic activity in vitro, and is well tolerated, inexpensive, and can be parenterally or orally administered in humans. Both AS and a more potent synthetic derivative, artemisinin-derived trioxane diphenylphosphate dimer 838 (ART-838), substantially inhibited acute leukemia growth in vivo and in vitro, at doses where normal CD34+ hematopoietic stem-progenitor cell clonogenicity was essentially unaffected. Moreover, AS and ART-838 synergized with several current antileukemic drugs (Fox et al, Oncotarget, 2015). To identify additional potential antileukemic drug combinations, we screened for the growth inhibitory effects of AS and ART-838 against 3 acute leukemia cell lines co-treated with a panel of 122 antileukemic compounds (mainly emerging antineoplastic drugs). Both AS and ART-838 showed synergistic or additive growth inhibition with several compounds. Two BCL2 protein family inhibitors demonstrated among the highest levels of synergy. One of these, ABT-199 (venetoclax), was recently approved by the FDA for the treatment of chronic lymphocytic leukemia (CLL) and received breakthrough designation for acute myeloid leukemia (AML). Therefore, we decided to further investigate the combination of ABT-199 with AS or ART-838. Synergy between AS (or ART-838) and ABT-199 was confirmed across several acute leukemia cell lines with varying sensitivities to each drug. Both leukemia cell proliferation and survival were affected synergistically by exposure to the combination of AS (or ART-838) plus ABT-199. Utilizing in vivo imaging, we assessed changes in growth kinetics of tail vein-injected acute leukemia cell lines and primary patient-derived samples constitutively expressing luciferase in mice treated with AS, ABT-199, or the combination. The combination of AS plus ABT-199 had the greatest antileukemic effect. Based on these data, we propose that Artemisinins plus BCL2 inhibitors comprise an antileukemic drug pair with high potential for incorporation into acute leukemia therapeutic regimens.

Citation Format: Blake S. Moses, Jennifer M. Fox, Xiaochun Chen, Jeffrey W. Tyner, Gary H. Posner, Patrick Bailey, Curt I. Civin. Artemisinins enhanced the antileukemic efficacy of BCL2 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2023. doi:10.1158/1538-7445.AM2017-2023
Abstract 2088: The activity of the FGFR selective inhibitor Debio 1347 is correlated with high mRNA expression
Authors: Brichory Franck , Pokorska-Bocci Anna , Nuciforo Paolo , Rigotti Stefania , Lembrez Nathalie , Vuagniaux Grégoire , Moulon Corinne , Vaslin Anne
Published: July 2017

Dysregulation of the fibroblast growth factor receptor (FGFR) signaling pathway due to receptor overexpression, gene amplification, point mutations or fusions/chromosomal translocations is associated with cancer development and progression.

Debio 1347 (CH5183284) is an oral selective FGFR inhibitor (FGFR1, 2 and 3) currently in clinical development. The aim of this study was to investigate Debio 1347 activity in patient derived xenograft (PDX) mouse models harboring diverse FGFR alterations in multiple indications.

The trial was conducted in 66 PDX models of diverse histotypes selected according to their FGFR1, 2 and 3 alteration status. Debio 1347 was administered orally once daily at 40 up to 80 mg/kg for 10 to 22 consecutive days (N=3/group). Tumor volume was compared to the vehicle control group and measured by caliper twice weekly. Treatment response was determined by relative treatment-to-control ratios (ΔT/ΔC) of which a responding model was defined as ΔT/ΔC <0. PDX tumors were collected at the end of the treatment period and extensively characterized using FISH, nCounter Gene Expression and immunohistochemistry (IHC). Effect on downstream Dual Specificity Phosphatase 6 (DUSP6) signaling was also investigated by qPCR and RNA in situ hybridization (ISH).

Debio 1347 induced tumor regression in 33% of PDX models that exhibited a gene copy number gain and/or the presence of a FGFR fusion gene and/or an FGFR mutation. In addition, Debio 1347 treatment led to tumor regression in 29% of models that did not harbor any FGFR genetic alteration. In contrast, all models that responded to Debio 1347 were shown to display a high expression level (mRNA) of at least one FGFR gene. These findings suggest that high expression of at least one FGFR might be a better predictor of sensitivity to Debio 1347 than genetic alteration. Furthermore, response to Debio 1347 was associated with a decrease in DUSP6 mRNA levels, suggesting that it could be a reliable pharmacodynamic biomarker in clinical trials.

These results provide new mechanistic insights into the predictive sensitivity to Debio 1347 and will help refine patient selection for FGFR-targeted therapy.

Citation Format: Franck Brichory, Anna Pokorska-Bocci, Paolo Nuciforo, Stefania Rigotti, Nathalie Lembrez, Grégoire Vuagniaux, Corinne Moulon, Anne Vaslin. The activity of the FGFR selective inhibitor Debio 1347 is correlated with high mRNA expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2088. doi:10.1158/1538-7445.AM2017-2088
Abstract 2325: Destruction of circulating tumor cells by fluid shear stresses generated in a microfluidic system
Authors: Regmi Sagar , Fu Afu , Lim Sierin , Luo Kathy Qian
Published: July 2017

Circulating tumor cells (CTCs) are mainly responsible for the cause of cancer metastasis. Although most CTCs can be destroyed by the bloodstream, some of them can still manage to withstand hemodynamic shear stress in blood stream. To study the effects of fluidic micro-environment on CTCs, we designed a microfluidic system that can produce various levels of shear stress which can be generated in human artery under resting or exercise condition. We also generated three human breast cancer cell lines with increased ability to form lung metastases in nude mice (i.e. 213-M1A>231-M1>231-C3). All three cell lines can produce a fluorescence resonance energy transfer (FRET)-based sensor which can reveal apoptosis in real-time by changing its color from green to blue. Besides these, we also investigated the effects of shear stress on multiple types of cancer cells including lung cancer (A549), ovarian cancer (2008), breast cancer (UACC-893) and leukemia (K562). For all the cell lines, except the non-attached K562 cells, cells were cultured on petri-dishes, detached by trypsinization and re-suspended in a normal culture medium to a cell density of 2x105 cells/ml. One milliliter of this cell suspensions was circulated in our microfluidic system under the shear stresses of 15, 30, 45 and 60 dyne/cm2 for 2-18 h. The effects of shear stress on morphology and viability of CTCs were determined by FRET imaging microscopy and MTT assay. And the impact of shear stress on causing necrosis in CTCs were examined by propidium iodide (PI) staining and LDH assay. Our findings are summarized below: 1) High shear stress (SS60, 4 h) is more potent to destroy CTCs than low shear stress (SS15, 4 h). 2) High shear stress can destroy most of the CTCs via necrosis. 3) This high level of shear stress is theoretically achievable via physical exercise. 4) Highly metastatic 231-M1A cells are more resistant to high shear stress compared to less metastatic 231-C3 cells. 5) Leukemia K562 cells representing the white blood cells showed stronger resistance to high shear stress compared to the cancer cells tested in this study. We think these findings can help people to understand how metastatic CTCs resist shear force-induced cell death which are useful for designing more effective therapies against metastatic CTCs. Additionally, the finding that high shear stress is more effective to kill CTCs may inspire cancer patients to consider exercise as a way to increase their hemodynamic shear stress, consequently destroy CTCs and prevent cancer metastasis.

Citation Format: Sagar Regmi, Afu Fu, Sierin Lim, Kathy Qian Luo. Destruction of circulating tumor cells by fluid shear stresses generated in a microfluidic system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2325. doi:10.1158/1538-7445.AM2017-2325
Abstract 2369: Transcription activators that regulate PKC-iota expression and are downstream targets of PKC-iota
Authors: Apostolatos Andre H. , Ratnayake Wishrawana S. , Smalley Tracess , Islam Anisul , Acevedo-Duncan Mildred
Published: July 2017

Protein Kinase C-iota (PKC-iota) is an anti-apoptotic oncogene over-expressed in multiple cancers including prostate, ovarian, and glioma. PKC-iota is part of a cycle that helps cancer cell avoid senescence by releasing the transcription factor NFkB and promoting apoptotic resistance. PKC-iota is activated externally by factors like loss of PTEN (Paget 2012). However, while under the effect PKC-iota specific inhibitors, expression levels decreased, suggesting PKC-iota plays a role in regulating its own expression. A previous study showed the ELK1 transcription factor to be a regulator of PKC-iota (Gustafson 2003). Other transcription factors including Jun, ISGF3, PAX3, EGR1, and FOXO1 bind on or near the promoter sequence of the gene and their role in PKC-iota regulation was analyzed. Each transcription factor was systematically silenced with its own siRNA. Western Blotting revealed expression of PKC-iota in the transcription factor silenced cells determining which transcription factors are key players in regulation of PKC-iota. qPCR and microarray were performed to analyze the transcriptome of treated cells to match protein levels with mRNA levels. Targets both up and downstream of PKC-iota were analyzed to find the pathway that PKC-iota uses to help regulate itself.

Citation Format: Andre H. Apostolatos, Wishrawana S. Ratnayake, Tracess Smalley, Anisul Islam, Mildred Acevedo-Duncan. Transcription activators that regulate PKC-iota expression and are downstream targets of PKC-iota [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2369. doi:10.1158/1538-7445.AM2017-2369
Abstract 2508: Profiling the metabolic effects of AGI-5198 treatment on IDH1-mutated gliomas
Authors: Rodado Victor Ruiz , Lita Adrian , Dowdy Tyrone , Gilbert Mark R. , Larion Mioara
Published: July 2017

Background: AGI-5198 (a specific IDH1mut inhibitor) inhibits the formation of D-2-hydroxyglutarate (D-2HG) typically produced in millimolar quantities by the R132H mutation of isocitrate dehydrogenase I (IDH1). AGI-5198 was found to delay growth, to promote differentiation in cells and to decrease tumor volume in IDH1-mutant (IDH1mut) mice xenographs, and as a result, has been advanced to clinical trials in humans with IDH1mut glioblastomas. However, no metabolic study of this drug has been reported to date. Here, we investigated the metabolic changes induced by AGI-5198 treatment in order to determine if the drug restores the normal metabolic status once D-2HG concentration is decreased. Methods: We treated glioma cells carrying an IDH1 mutation with 10 μM AGI-5198 for 72 h. Metabolic changes were explored using an untargeted metabolomics approach. Cell viability analysis verified that the concentration of drug does not lead to significant cell death. NMR and MS-linked metabolomics together with in vivo cell imaging using RAMAN were used. Principal component analysis was employed to explore the differences in the metabolic profiles comparing treated and untreated glioma cells. Results and Conclusions: We confirmed that AGI5198 treatment reduced D-2HG levels in the cell lines investigated, validating previous reports. Moreover, we observed a decrease in phospholipid levels upon AGI-5198 treatment, which suggests a decreased proliferative potential for these cells; of particular importance for clinical application. Our preliminary analysis also suggests that metabolites involved in one carbon metabolism are altered upon AGI-5198 treatment. This pathway provides the methyl group for the DNA methylation by DNA methylases producing a hypermethylated phenotype. Our findings suggest that combining the AGI-5198 treatment with a DNA methylase inhibitor may improve outcomes in IDH1-mutated gliomas.

Citation Format: Victor Ruiz Rodado, Adrian Lita, Tyrone Dowdy, Mark R. Gilbert, Mioara Larion. Profiling the metabolic effects of AGI-5198 treatment on IDH1-mutated gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2508. doi:10.1158/1538-7445.AM2017-2508
Abstract 2578: Investigating the role of mutant p53 in esophageal squamous cell carcinoma
Authors: Lento Ashley , Long Apple , Giroux Veronique , Tang Qiaosi , Sammons Morgan , Klein-Szanto Andres , Berger Shelly , Rustgi Anil
Published: July 2017

Esophageal squamous cell carcinoma (ESCC) is a highly aggressive cancer characterized by a high rate of metastasis, limited therapeutic options, and a poor prognosis. However, there is limited information regarding the molecular mechanisms underlying the metastatic properties of ESCC. p53 is one of the most commonly mutated genes in ESCC, and our group has shown that esophageal cells lines expressing a mutation in human p53 shows signs of malignancy and increased invasion in 3D organotypic culture. A mouse model of mutant p53 (R172H) in ESCC is lacking in the field. To elucidate the role of mutant p53 in ESCC we developed a novel mouse model utilizing a genetic and carcinogenic approach. L2cre;p53-/- and p53R172H/- mice were generated and treated with 4NQO in their drinking water for 16 weeks, which resulted in the development of ESCC. Compared to wildtype mice, p53R172H/- mice and p53-/- mice exhibited a decreased tumor latency time, increased tumor frequency and a more severe tumor diagnosis. However, p53R172H/- mice and p53-/- mice displayed similar tumorigenic properties. RNA-seq was performed on cell lines established from wildtype and p53 mouse models and reveled different gene expression profiles between wildtype, p53R172H/-, and p53-/- cells. p53R172H/- cells displayed an increase in mesenchymal and decrease in epithelial marker expression, supporting the idea that they are undergoing EMT. In addition, several endocytic recycling related genes, including Rab11-fip1, Rab25, and Myo5b were downregulated in mutant and null p53 compared to wildtype cells. Further examination of the differing genetic profiles in our mouse models can provide novel insight into the role of mutant p53 in ESCC tumorigenesis and lead to the identification of new therapeutic targets.

Citation Format: Ashley Lento, Apple Long, Veronique Giroux, Qiaosi Tang, Morgan Sammons, Andres Klein-Szanto, Shelly Berger, Anil Rustgi. Investigating the role of mutant p53 in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2578. doi:10.1158/1538-7445.AM2017-2578
Abstract 2612: Anti-Tigit induces T cell mediated anti-tumor immune response and combines with immune checkpoint inhibitors to enhance strong and long term anti-tumor immunity
Authors: Srivastava Minu K. , Yun Rui , Mayes Erin , Yu Janice , Jie Hyun-Bae , Axelrod Fumiko , Xie Ming-Hong , Monteon Jorge , Lam Andrew , Ji May , Liu Yuwang , Lewicki John , Hoey Tim , Gurney Austin , Park Angie Inkyung
Published: July 2017

TIGIT (T cell immunoreceptor with Ig and ITIM domains) has been recently described as an inhibitory receptor which blocks CD8 T cell-mediated anti-tumor immune responses. We have generated an anti-mouse TIGIT antibody (313R12) to evaluate drug efficacy and mechanism of action in pre-clinical tumor models. Anti-TIGIT as a single agent promoted an anti-tumor immune response in multiple syngeneic mouse tumor models. Anti-TIGIT enhanced tumor specific T cell responses, particularly of the Th1 type and reduced Th2 type responses and also increased the function of cytotoxic T cells. Furthermore, anti-TIGIT displayed combination activity with immune checkpoint inhibitors anti-PD1 and anti-PDL1 in inhibiting tumor growth, promoting complete tumor rejection and significantly increasing mouse survival in the murine CT26 colon carcinoma model as compared to controls and single agents alone. Mice “cured” with anti-TIGIT/anti-PDL1 or anti-TIGIT/anti-PD1 combination treatments did not form tumors upon subsequent re-challenges with increasing number of CT26 tumor cells, suggesting the existence of immunologic memory. IL2 and tumor-specific IFN-γ production by splenic T cells were increased in mice who responded to combination treatment compared to controls. Additionally, both effector and memory CD8+ T cell frequencies were increased within the total CD8+ T cell population in responding mice. We also demonstrated a systemic increase in tumor-specific CD8 T cells after anti-TIGIT/anti-PDL1 combination treatment compared to controls. Therefore, these results suggest that co-targeting of TIGIT and PD1 or PDL1 may be an effective and durable cancer therapy by increasing T cell-mediated anti-tumor immune responses and promoting long-term immunological memory.

Citation Format: Minu K. Srivastava, Rui Yun, Erin Mayes, Janice Yu, Hyun-Bae Jie, Fumiko Axelrod, Ming-Hong Xie, Jorge Monteon, Andrew Lam, May Ji, Yuwang Liu, John Lewicki, Tim Hoey, Austin Gurney, Angie Inkyung Park. Anti-Tigit induces T cell mediated anti-tumor immune response and combines with immune checkpoint inhibitors to enhance strong and long term anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2612. doi:10.1158/1538-7445.AM2017-2612
Abstract 2743: Comparison of enhanced Tagged-Amplicon Sequencing and digital PCR for circulating tumor DNA analysis in advanced breast cancer
Authors: Garcia-Murillas Isaac , Beanney Matthew , Epstein Michael , Howarth Karen , Lawson Andrew , Hrebien Sarah , Green Emma , Rosenfeld Nitzan , Turner Nick
Published: July 2017

Background Circulating tumor DNA (ctDNA) analysis allows non-invasive detection of tumor mutations and amplifications in advanced breast cancer. Multiple technologies have been developed to analyse ctDNA and here we compared two leading ctDNA detection technologies, enhanced Tagged-Amplicon Sequencing™ (eTAm-Seq™) and digital PCR (dPCR) assays, in advanced breast cancer.

Methods We recruited a cohort of 35 women with advanced breast cancer, of whom 23 had two separate blood samples taken in a standard EDTA tube processed immediately or in preservative Streck tubes processed up to 120 hours after venipuncture. Digital PCR was conducted with assays for hotspot actionable mutations in 3 known drivers in breast cancer: PIK3CA exon 9 and 20, ESR1 ligand binding domain and AKT1 (c.49G>A; p.E17K), and ctDNA sequencing was conducted with eTAm-Seq method using a 35-gene panel including cancer hotspots, entire coding regions and copy number variants (CNVs).

Results Across both assays, 37 mutations were detected in 35 patients, with PIK3CA mutation in 13 patients (37%), ESR1 mutations in 10 patients (29%), and no AKT1 mutations. ESR1 mutations were polyclonal in 8 patients, with ctDNA eTAm-Seq method revealing substantially more diversity in mutations, with up to 8 individual mutations detected in a patient. There was 96.15% agreement for PIK3CA mutation detection between assays (Kappa 0.89, 95% CI 0.743 to 1.000), 100% agreement for ESR1 mutations (Kappa 1.00, 95% CI 1.000 to 1.000). There was very high correlation in mutation allele frequency between eTAm-Seq and dPCR (r=0.93, 95%CI 0.86 to 0.96, p<0.0001). The sensitivity and specificity for HER2 amplification detection by eTAm-Seq was 100% compared to tumor HER2 status. Comparison of immediate processing and Streck tubes revealed 97.92% agreement (Kappa 0.95, 95% CI 0.868 to 1.000) for mutation calling.

Conclusions This study demonstrates that ctDNA analysis using eTAmSeq and dPCR have very high agreement in mutation detection in patients with advanced breast cancer patients. Streck tubes present a robust alternative to immediate processing of samples. eTAm-Seq and digital PCR have high clinical validity in mutation detection.

Citation Format: Isaac Garcia-Murillas, Matthew Beanney, Michael Epstein, Karen Howarth, Andrew Lawson, Sarah Hrebien, Emma Green, Nitzan Rosenfeld, Nick Turner. Comparison of enhanced Tagged-Amplicon Sequencing and digital PCR for circulating tumor DNA analysis in advanced breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2743. doi:10.1158/1538-7445.AM2017-2743
Abstract 283: Establishing the predominant type of leukemia among patients attending the Uganda Cancer Institute
Authors: Nsubuga Gerald
Published: July 2017

Introduction: Leukemia is a cancer of white blood cells. It is characterized by an abnormal accumulation of WBC in the bone marrow which leads to bone marrow failure and is manifested in form of anemia and thrombocytopenia. This arises from neoplastic transformation usually of a single cell. There are four common types of leukemia: 1) Acute lymphoblastic leukemia (ALL); 2) Acute myeloblastic leukemia (AML); 3) Chronic lymphocytic leukemia (CLL); and Chronic myeloid/myclocytic leukemia (CML).

Methods: The study was designed to find out the predominant type of leukemia. 80 leukemia cases which comprised of all bone marrow aspirates and peripheral blood slide results with specific age, sex and ward of the patients was analyzed in the hematology laboratory.

Results: The study was revealed that leukemia occurred in following order of types of predominant that is AML 15 (18.75%), CLL 12(15%) and least type was CML 11(13.75%). The result also showed that the most affected age group by all age. Brackets of (01-20) years. From the study a male preponderance (M: F) of 4:3 was found in cases of ALL. The study emphasizes that special staining techniques be adopted in diagnosing and typing of leukemia.

Conclusions: From the results presented, ALL is the predominant type of leukemia affecting children and young adults. AML was found to affect children, young adults, and adults. CLL and CML were found to occur at the same incidence, the elderly (61-70 and 71-80 years). Other types were rare in these age group similarly CLL and CML were rare in children and young adults.

Note: This abstract was not presented at the meeting.

Citation Format: Gerald Nsubuga. Establishing the predominant type of leukemia among patients attending the Uganda Cancer Institute [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 283. doi:10.1158/1538-7445.AM2017-283
Abstract 3023: The antitumor activity of immunotoxins is enhanced by tofacitinib
Authors: Simon Nathan , Antignani Antonella , Hewitt Stephen , Gadina Massimo , Onda Masanori , Alewine Christine , Fitzgerald David Joseph
Published: July 2017

Treatment outcomes of certain aggressive cancers have not improved significantly in the last decade, necessitating development of new therapies. Recombinant immunotoxins (IT) are fusion proteins consisting of the enzymatic domain of a cytotoxin linked to an antibody fragment targeting a cellular surface protein. While the combination of selective targeting and potent cell-killing make ITs well suited for targeted cancer therapy, clinical experience has indicated that patients can generate anti-toxin neutralizing antibodies after one cycle of treatment. Mice treated with the FDA-approved JAK inhibitor, tofacitinib, produced significantly lower anti-toxin antibody titers, suggesting that tofacitinib could be used in combination with immunotoxins to improve therapeutic efficacy. Accordingly, the combination of tofacitinib and IT was tested both in vitro and in vivo using two models of aggressive human cancers: pancreatic ductal adenocarcinoma and triple-negative breast cancer. In vitro combination studies examined the effects on oncogenic signaling pathways and looked for enhancement of cytotoxic potency. Results demonstrated some tofacitinib-mediated inhibition of key signaling pathways but no enhancement of immunotoxin killing. By contrast, when xenograft tumor models were challenged, immunotoxin activity was enhanced greatly by co-treating with tofacitinib. Tofacinib treatments alone did not demonstrate any antitumor activity. The combination of tofacitinib and immunotoxin decreased tumor growth and extended survival compared to either treatment alone. Examination of post-treatment tumor samples and sera from mice indicated that JAK inhibition resulted in alterations of the tumor microenvironment, including a decrease in cancer-promoting cytokine levels and reductions in recruitment of suppressive immune cells to tumor tissue. This study demonstrates that the inhibition of the JAK-STAT pathway can significantly enhance the ability of cytotoxin-based therapeutic agents to elicit anti-tumor effects through alteration of the tumor microenvironment.

Citation Format: Nathan Simon, Antonella Antignani, Stephen Hewitt, Massimo Gadina, Masanori Onda, Christine Alewine, David Joseph Fitzgerald. The antitumor activity of immunotoxins is enhanced by tofacitinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3023. doi:10.1158/1538-7445.AM2017-3023
Abstract 3095: The development of CPI as a novel, next-generation DNA-targeting payload for ADCs
Authors: Kahler Jennifer , Dougher Maureen , Xu Jane , Doroski Matthew , Maderna Andreas , Dushin Russell , Thibault Stephane , Leal Mauricio , Katragadda Madan , O'Donnell Christopher J. , Sung Matthew , Sapra Puja
Published: July 2017

DNA targeting drugs represent one of cornerstones of anti-cancer therapy for both hematologic and solid tumor indications. Low potency anti-DNA compounds (e.g. platins, anthracyclines) are included in many standard-of-care (SOC) regimens, however their modest activity and overall toxicity profiles limit their therapeutic potential. To increase the therapeutic window for DNA-damaging agents, high potency anti-DNA compounds with enhanced anti-tumor activity have been delivered to tumors as payloads of targeting modalities such as antibody-drug conjugates (ADCs). Herein, we describe the development of a novel DNA-damaging compound comprised of a dimeric structure of cyclopropylpyrrolo[e]indolones (CPIs) that was designed to alkylate DNA and generate toxic interstrand crosslinks (ICLs). In response to the CPI-induced formation of ICLs, CPI treatment of cells primarily activates the Fancomia anemia DNA damage response pathway, whereas other successful DNA-damaging ADC payloads such as calicheamicin activate double-strand break response pathways. CPI shows ~860-fold greater potency than calicheamicin in a panel of cell lines derived from a broad spectrum of tumor indications. Importantly, this new CPI payload retains potent activity in calicheamicin- and SOC-resistant tumor models (including overcoming overexpression of drug efflux pumps). When evaluated as payloads on anti-CD33 targeting ADCs, the CPI conjugate showed dramatically improved efficacy over the corresponding calicheamicin conjugates in MDR+ tumor models. As a site-specific conjugate, the CPI ADC shows enhanced in vivo stability and possesses a wider therapeutic window than the corresponding conventional calicheamicin conjugate and other leading DNA-damaging conjugates on the CD33 platform.

Citation Format: Jennifer Kahler, Maureen Dougher, Jane Xu, Matthew Doroski, Andreas Maderna, Russell Dushin, Stephane Thibault, Mauricio Leal, Madan Katragadda, Christopher J. O'Donnell, Matthew Sung, Puja Sapra. The development of CPI as a novel, next-generation DNA-targeting payload for ADCs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3095. doi:10.1158/1538-7445.AM2017-3095
Abstract 3103: Nanoengineered mesenchymal stem cells for targeted lung cancer therapy
Authors: Prabha Swayam , Sadhukha Tanmoy , Layek Buddhadev
Published: July 2017

Solid tumors are characterized by increased interstitial fluid pressure and enhanced solid stress, contributing to poor intra–tumoral penetration of nano drug carriers. This offsets the benefits of enhanced permeation and retention effect often attributed to these drug carriers. We have developed a novel cell based targeting approach using mesenchymal stem cells (MSCs) as cellular drug carriers. MSCs have been indicated in the tumor-targeted therapy owing to their selective ability to home to tumors. Our work is focused on non–genetic modification of MSCs by incorporating nanoparticles loaded with chemotherapeutics to create cellular drug depots capable of homing to tumors and releasing drugs over a sustained period of time. Nano–engineered MSCs were manufactured by incubating MSCs with 100µg/mL of paclitaxel loaded (18.5% w/w paclitaxel) PLGA nanoparticles for 4 hours at 37°C without affecting their viability and migration potential. Anti-cancer efficacy of nano-engineered MSCs was evaluated in the A549 lung tumor model. Bioluminescence imaging was used to monitor tumor growth. Animals that developed tumors (lung bioluminescence between 0.5 x 106 – 1 x 106 photons/sec) were randomly divided into different treatment groups. Animals received 1 million nano-engineered MSCs (equivalent to 5 mg/kg of paclitaxel) or 106 MSCs that were either unmodified or loaded with equivalent concentration of blank nanoparticles. The animals were then re-dosed with half the initial dose of MSCs every two weeks. Additional groups of animals received three doses of 40 mg/kg of nanoparticle-encapsulated or free paclitaxel once every week. Treatment with nano-engineered MSCs resulted in a significant inhibition of the lung tumor growth (p < 0.01) compared to that with other controls. It was interesting to note that despite being dosed at a significantly lower concentration of paclitaxel (~17.5 mg/kg vs. 120 mg/kg total dose), nano-engineered MSCs were significantly more effective than free paclitaxel or paclitaxel nanoparticles, suggesting better delivery to the tumor. The biodistribution studies and pharmacokinetic analysis indicated a rapid migration and enhanced retention of the nano–engineered MSCs to the lung tumors. Our studies demonstrated that nano-engineered MSCs are efficient carriers for tumor-specific drug delivery. Funding: R01EB022558

Citation Format: Swayam Prabha, Tanmoy Sadhukha, Buddhadev Layek. Nanoengineered mesenchymal stem cells for targeted lung cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3103. doi:10.1158/1538-7445.AM2017-3103
Abstract 3143: FBW7 mutations mediate resistance of colorectal cancer to targeted therapies by blocking Mcl-1 degradation
Authors: Tong Jingshan , Tan Shuai , Zou Fangdong , Yu Jian , Zhang Lin
Published: July 2017

Colorectal cancer (CRC), the second leading cause of cancer-related deaths in the US, has been treated with targeted therapies. However, the mechanisms of differential responses and resistance of CRCs to targeted therapies are not well understood. In this study, we found that genetic alterations of FBW7, an E3 ubiquitin ligase and a tumor suppressor frequently mutated in CRCs, contribute to resistance to targeted therapies. CRC cells containing FBW7 inactivating mutations are insensitive to clinically used multi-kinase inhibitors of RAS/RAF/MEK/ERK signaling, including regorafenib and sorafenib. In contrast, sensitivity to these agents is not affected by oncogenic mutations in KRAS, BRAF, PIK3CA, or p53. These cells are defective in apoptosis due to blocked degradation of Mcl-1, a pro-survival Bcl-2 family protein. Deleting FBW7 in FBW7-wild-type CRC cells abolishes Mcl-1 degradation and recapitulates the in vitro and in vivo drug resistance phenotypes of FBW7-mutant cells. CRC cells selected for regorafenib resistance have progressive enrichment of pre-existing FBW7 hotspot mutations, and are cross-resistant to other targeted drugs that induce Mcl-1 degradation. Furthermore, a selective Mcl-1 inhibitor restores regorafenib sensitivity in CRC cells with intrinsic or acquired resistance. Together, our results demonstrate FBW7 mutational status as a key genetic determinant of CRC response to targeted therapies, and Mcl-1 as an attractive therapeutic target.

Citation Format: Jingshan Tong, Shuai Tan, Fangdong Zou, Jian Yu, Lin Zhang. FBW7 mutations mediate resistance of colorectal cancer to targeted therapies by blocking Mcl-1 degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3143. doi:10.1158/1538-7445.AM2017-3143
Abstract 3144: Prediction of ALK mutations associated with acquired resistance to lorlatinib
Authors: Yoda Satoshi , Dardaei Leila , Singh Manrose , Engelman Jeffrey A. , Shaw Alice T. , Hata Aaron N.
Published: July 2017

Anaplastic lymphoma kinase (ALK) rearrangements are important therapeutic targets in non-small cell lung cancer. They are currently treated with the first-generation ALK inhibitor crizotnib followed by more potent, second-generation ALK inhibitors, such as ceritinib, alectinib, or brigatinib. We reported different spectrums of ALK resistance mutations in the biopsies from patients progressing on these drugs. G1202R mutation was found more frequently after treatment with second generation ALK inhibitors. In addition to these drugs, the third-generation ALK inhibitor lorlatinib is currently being evaluated in phase 2 clinical trial. Ba/F3 models indicated that all single ALK mutants are sensitive to lorlatinib and some compound ALK mutations are resistant to lorlatinib. In this study, we performed accelerated mutagenesis screen on Ba/F3 models to predict the resistance mutations which potentially emerge in the patients treated with lorlatinib. Briefly, Ba/F3 cells expressing wild type EML4-ALK or mutant EML4-ALK containing C1156Y, F1174C, L1196M, G1202R, or G1269A were exposed to N-ethyl-N-nitrosourea (ENU). After a 24-hour incubation in normal media, the cells were seeded in 96-well plates and incubated in lorlatinib for 4 weeks. ALK kinase domain was sequenced in clones growing in lorlatinib to identify possible new mutations. As a result, Ba/F3 cells harboring wild type EML4-ALK did not show any mutation on ALK kinase domain. Crizotinib was used as a control to validate the efficiency of mutagenesis. We identified eight different mutations in clones growing in crizotinib, and those were reflecting the spectrum of mutations in the crizotinib-treated patients. Ba/F3 cells with mutant EML4-ALK showed additional compound mutations after incubation with lorlatinib. Those mutations included L1196M + L1198F and G1202R + L1198F which showed high resistance to lorlatinib in Ba/F3 models. Ba/F3 cells with different mutant EML4-ALK showed a distinct spectrum and different frequency of additional mutations. In conclusion, this study predicted that no single mutation would emerge to confer resistance to lorlatinib. Thus, compound mutations and ALK-independent mechanisms become essential mechanisms for lorlatinib resistance.

Citation Format: Satoshi Yoda, Leila Dardaei, Manrose Singh, Jeffrey A. Engelman, Alice T. Shaw, Aaron N. Hata. Prediction of ALK mutations associated with acquired resistance to lorlatinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3144. doi:10.1158/1538-7445.AM2017-3144
Abstract 3148: Activation of Src signaling mediates acquired resistance to ALK inhibition in lung cancer
Authors: Yoshida Ryohei , Sasaki Takaaki , Okumura Shunsuke , Ohsaki Yoshinobu
Published: July 2017

Anaplastic lymphoma kinase (ALK) fusion oncogenes occur in around 3%-5% non-small cell lung cancer (NSCLC) cases. Various ALK inhibitors are in clinical use for the treatment of ALK-NSCLC, including the first generation ALK inhibitor, Crizotinib, and recently the more highly potent Alectinib and Ceritinib. However, most tumors eventually become resistant to ALK specific inhibitors. To address the mechanisms underlying the development of ALK inhibitor resistance, we used iTRAQ quantitative mass spectrometry and phosphor-receptor tyrosine kinase arrays to investigate intracellular signaling alterations in ALK inhibitor resistant NSCLC cell lines. Src signaling was identified as an Alectinib resistance mechanism, and combination treatment with ALK and Src inhibitors was highly effective for inhibiting the growth of ALK inhibitor resistant cells in vitro and in mouse xenograft models. Furthermore, phospho-receptor tyrosine kinase activation and downstream PI3K/AKT signaling was effectively blocked by inhibiting Src in Alectinib resistant cells. Finally, we showed that the combined use of ALK and Src inhibitors inhibited the growth of other ALK-NSCLC cell lines, including those that were Ceritinib or Lorlatinib resistant. Our data suggest that targeting Src signaling may be an effective approach to the treatment of ALK–NSCLC with acquired resistance to ALK inhibitors.

Citation Format: Ryohei Yoshida, Takaaki Sasaki, Shunsuke Okumura, Yoshinobu Ohsaki. Activation of Src signaling mediates acquired resistance to ALK inhibition in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3148. doi:10.1158/1538-7445.AM2017-3148
Abstract 3168: Overcoming acquired drug resistance by TPX-0005, an ALK, ROS1 and pan-TRK inhibitor
Authors: Deng Wei , Huang John , Zhai Dayong , Rogers Evan , Cui Jean
Published: July 2017

The inevitable development of clinical drug resistance presents a common challenge for targeted cancer therapy. In non-small cell lung cancer (NSCLC) patients with ALK and ROS1 rearrangements, the emergence of mutations in the targeted oncogenes was identified as one of the mechanisms that confer drug resistance. In particular, a group of mutations known as solvent front mutations, such as ALKG1202R and ROS1G2032R, render common resistance to ALK and ROS1 inhibitors. Similar solvent front mutations, such as TRKAG595R and TRKCG623R, have also been identified in cancer patients with rearrangement of NTRK family genes who developed resistance to TRK-targeted therapies. TPX-0005, a compact three-dimensional macrocyclic molecule, was designed to completely locate at the adenine binding site of ATP in order to efficiently target the active kinase conformation and systematically circumvent the steric interference from various clinical resistant mutations, especially the solvent front mutations. TPX-0005 is an orally available and potent ATP-competitive inhibitor against ALK, ROS1, TRKA, TRKB and TRKC recombinant kinases and their corresponding clinical resistant mutants. TPX-0005 demonstrated potent anti-proliferative activity in the range of sub-nanomolar to low nanomolar in a number of human cancer cell lines and engineered stable cell lines expressing the targeted oncogenes or their solvent front mutants, accompanied by inhibition of target phosphorylation and concomitant inactivation of downstream effectors such as ERK, AKT and STAT3. In patient derived xenograft tumor models, TPX-0005 treatment resulted in significant regression of tumors harboring the oncogenic ALK, ROS1 and TRKC fusions. Moreover, in a series of mouse xenograft tumor models, TPX-0005 exhibited marked anti-tumor activity not only in tumors harboring the wildtype oncogenic targets but also in tumors harboring the oncogenes with the solvent front mutations via inhibition of the target phosphorylation. Taken together, these pre-clinical studies have demonstrated the potent activities of TPX-0005 against not only wild type oncogenic ALK, ROS1, TRK fusions but also their corresponding solvent front mutations, which will bring in a new therapy for cancer patients resistant to currently available ALK, ROS1, and TRK inhibitors in clinic.

Citation Format: Wei Deng, John Huang, Dayong Zhai, Evan Rogers, Jean Cui. Overcoming acquired drug resistance by TPX-0005, an ALK, ROS1 and pan-TRK inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3168. doi:10.1158/1538-7445.AM2017-3168