bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1815: Massively parallel identification of conserved drug resistant mutations in kinases
Authors: Persky Nicole S. , Hernandez Desiree , Cordova Jonathon , Walker Amanda , Brenan Lisa , Piccioni Federica , Pantel Sasha , Lee Yenarae , Goodale Amy , Yang Xiaoping , Mitsuishi Yoichiro , Carmo Mariana Do , Zhu Cong , Andreev Aleksandr , Root David E. , Johannessen Cory M.
Published: August 2018

Drug resistant mutations that arise in therapeutic targets often limit clinical responses. However, the discovery of such mutations has historically been performed one gene or mutation at a time, often over decades of experimental and clinical testing, limiting our understanding of conserved mechanisms of drug resistance.

We hypothesized that deep mutational scanning of canonical kinases may expedite this process and identify novel conserved elements that cause drug resistance when mutated (similar to the well-studied “Gatekeeper” residue). To test this, we generated cDNA-expression libraries containing all possible amino acid substitutions in CDK6, CDK4, ERK2, and EGFR. We screened each library against clinically utilized, ATP-competitive small molecule inhibitors. We then mapped the phenotypic data for over 40,000 missense mutations onto the aligned crystal structures of each protein and searched for shared structural attributes associated with drug resistance.

This analysis revealed 4 equivalent amino acid sites whose mutation conferred drug resistance to ATP-competitive inhibitors in all of our screens: the Gatekeeper residue, as well as three uncharacterized residues. One of these sites, which we have termed the “Keymaster”, was additionally found to cause resistance in published data sets of sub-saturation BRAF, HER2, BCR-ABL, and MEK1 mutagenesis screens against their respective inhibitors. We confirmed that drug resistant phenotypes are caused by these alterations utilizing growth assays and protein target phosphorylation detection assays. Mechanistically, we show preliminary evidence that Keymaster-mutant proteins are competent for drug binding, but may display elevated basal activity. Consistent with our findings, we additionally identified mutations at Keymaster residues in reported patient tumors in a number of oncogene kinases, suggesting that Keymaster mutations could be drivers of tumorigenesis, as well as drug resistance. These efforts may prove useful for characterizing somatic kinase mutations of unknown function, designing next-generation therapeutics and deepening our understanding of kinase regulation.

Citation Format: Nicole S. Persky, Desiree Hernandez, Jonathon Cordova, Amanda Walker, Lisa Brenan, Federica Piccioni, Sasha Pantel, Yenarae Lee, Amy Goodale, Xiaoping Yang, Yoichiro Mitsuishi, Mariana Do Carmo, Cong Zhu, Aleksandr Andreev, David E. Root, Cory M. Johannessen. Massively parallel identification of conserved drug resistant mutations in kinases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1815.
Abstract 1822: Emergence of MET D1228N mutation as a resistance mechanism following an exceptional response to crizotinib in a MET-amplified triple-negative breast cancer patient
Authors: Parsons Benjamin M. , Meier David R. , Gurda Grzegorz T. , Lofgren Kristopher A. , Kenny Paraic A.
Published: August 2018

The MET proto-oncogene encodes a receptor tyrosine kinase which is activated by Hepatocyte Growth Factor binding. Recent reports have described single agent activity for crizotinib in MET-amplified or mutant lung adenocarcinoma, squamous cell carcinoma and papillary renal cell carcinoma. Here we describe a stage IV triple-negative breast cancer patient in her early forties, previously treated with doxorubicin/cyclophosphamide, carboplatin/paclitaxel and with capecitabine. PET/CT imaging after progression on capecitabine revealed extensive metastatic disease including a large left pleural effusion, bilateral hypermetabolic nodular pleural implants, innumerable bilateral lung nodules and widespread mildly hyper-metabolic skeletal metastases in the ribs, spine and pelvis. A next-generation sequencing assay detected a 30-fold amplification of MET. MET amplification, overexpression and hyperactivity was confirmed by FISH and by immunohistochemistry for total MET and phospho-MET, respectively. Single agent crizotinib (250 mg, twice daily) was well-tolerated. Immunostaining of a biopsy taken after 9 days of treatment showed elimination of active phosphorylated MET, demonstrating in-tumor crizotinib efficacy at the clinical dose. CT imaging at 10 weeks showed a resolution of her metastatic disease, meeting RECIST 1.1 criteria for a complete response. This response was sustained at 22 weeks. At 37 weeks, the patient progressed in the pleura. An ultrasound guided biopsy revealed a robust resurgence of MET phosphorylation while still taking crizotinib, suggesting an alteration rendering MET recalcitrant to crizotinib. Next generation sequencing revealed a MET D1228N mutation, previously implicated in crizotinib resistance in lung cancer. This mutation was not detected in the pre-crizotinib biopsy (locus sequenced at 6300x) suggesting de novo acquisition in response to crizotinib. Transition from this type I MET inhibitor to a type II inhibitor (cabozantinib, initially 60 mg/day, later 100 mg/day) resulted in clinically stable disease for a period of 7 weeks, at which point the patient again progressed. Although MET-amplification is rare in breast cancer (~0.6% of cases), these data indicate that substantial clinical benefit may be achieved using single agent MET inhibition however the optimal selection of second-line agents to use upon progression will be critical.

Citation Format: Benjamin M. Parsons, David R. Meier, Grzegorz T. Gurda, Kristopher A. Lofgren, Paraic A. Kenny. Emergence of MET D1228N mutation as a resistance mechanism following an exceptional response to crizotinib in a MET-amplified triple-negative breast cancer patient [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1822.
Abstract 1832: Acquired resistance mechanisms to afatinib in lung cancer cells harboring HER2 alterations
Authors: Torigoe Hidejiro , Shien Kazuhiko , Takeda Tatsuaki , Yoshioka Takahiro , Namba Kei , Sato Hiroki , Suzawa Ken , Yamamoto Hiromasa , Soh Junichi , Sakaguchi Masakiyo , Tomida Shuta , Miyoshi Shinichiro , Toyooka Shinichi
Published: August 2018

Human epidermal growth factor receptor 2 (HER2) plays an important role in the pathogenesis of various cancers. HER2 alterations have been suggested to be a therapeutic target in non-small cell lung cancer (NSCLC), just as in breast and gastric cancers. We previously reported that the pan-HER inhibitor afatinib could be a useful therapeutic agent as HER2-targeted therapy for patients with NSCLC harboring HER2 alterations. However, acquired resistance to afatinib was observed in the clinical setting, similar to the case for other HER inhibitors. Thus, elucidation of the mechanisms underlying the development of acquired drug resistance and exploring means to overcome acquired drug resistance are important issues in the treatment of NSCLC. In this study, we experimentally established afatinib-resistant cell lines from NSCLC cell lines harboring HER2 alterations (Calu3, H2170, and H1781), by stepwise-escalation or high-concentration exposure methods, and investigated the mechanisms underlying the acquisition of drug resistance. The established cell lines showed several unique afatinib-resistance mechanisms, including MET amplification, loss of HER2 amplification and gene expression, epithelial-to-mesenchymal transition (EMT) and acquisition of cancer stem cell (CSC)-like features. The afatinib-resistant cell lines showing MET amplification were sensitive to the combination of afatinib plus crizotinib (a MET inhibitor), both in vitro and in vivo. The resistant cell lines with the loss of HER2 amplification obtained MEK inhibitors sensitivity in vitro and in vivo. Other resistant cell lines that showed EMT or had acquired CSC-like features remained sensitive to docetaxel, like the parental cells. These findings may provide clues to countering the resistance to afatinib in NSCLC patients with HER2 alterations.

Citation Format: Hidejiro Torigoe, Kazuhiko Shien, Tatsuaki Takeda, Takahiro Yoshioka, Kei Namba, Hiroki Sato, Ken Suzawa, Hiromasa Yamamoto, Junichi Soh, Masakiyo Sakaguchi, Shuta Tomida, Shinichiro Miyoshi, Shinichi Toyooka. Acquired resistance mechanisms to afatinib in lung cancer cells harboring HER2 alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1832.
Abstract 1838: A pre-existing rare PIK3CAE545K subpopulation confers clinical resistance to MEK plus CDK4/6 inhibition in NRAS melanoma and is dependent on S6K1 signaling
Authors: Romano Gabriele , Chen Pei-Ling , Song Ping , McQuade Jennifer , Liang Roger , Liu Mingguang , Roh Whijae , Duose Dzifa , Carapeto Fernando , Li Jun , Teh Jessica , Aplin Andrew , Chen Merry , Zhang Jianhua , Lazar Alexander , Futreal P Andrew , Amaria Rodabe , Zhang David , Wargo Jennifer , Kwong Lawrence N.
Published: August 2018

Combined MEK and CDK4/6 inhibition (MEKi+CDK4i) has shown promising clinical outcomes in NRAS mutant melanoma patients. Here, we interrogated longitudinal biopsies from a patient who initially responded to MEKi+CDK4i therapy but subsequently developed resistance. Whole exome sequencing and functional validation identified an acquired PIK3CAE545K mutation as conferring drug resistance. We demonstrate that PIK3CAE545K pre-existed in a rare subpopulation that was missed by both clinical and research testing, but was revealed upon multi-region sampling due to PIK3CAE545K being non-uniformly distributed. This resistant population rapidly expanded after the initiation of MEKi+CDK4i therapy and persisted in all successive samples even after immune checkpoint therapy. Functional studies identified activated S6K1 as both a key marker and specific therapeutic vulnerability downstream of PIK3CAE545K-induced resistance. These results demonstrate that comprehensive analysis of pre-treatment samples can reveal rare pre-existing resistant subpopulations and posit S6K1 as a common downstream therapeutic nexus for the MAPK, CDK4/6, and PI3K pathways.

Citation Format: Gabriele Romano, Pei-Ling Chen, Ping Song, Jennifer McQuade, Roger Liang, Mingguang Liu, Whijae Roh, Dzifa Duose, Fernando Carapeto, Jun Li, Jessica Teh, Andrew Aplin, Merry Chen, Jianhua Zhang, Alexander Lazar, P Andrew Futreal, Rodabe Amaria, David Zhang, Jennifer Wargo, Lawrence N. Kwong. A pre-existing rare PIK3CAE545K subpopulation confers clinical resistance to MEK plus CDK4/6 inhibition in NRAS melanoma and is dependent on S6K1 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1838.
Abstract 1846: HPV oncoproteins cause specific types of chromosomal instability in head and neck cancer
Authors: Funk Laura C. , Lee Denis L. , Kimple Randall J. , Lambert Paul F. , Weaver Beth A.
Published: August 2018

Aneuploidy, a karyotype that differs from a multiple of the haploid, is a hallmark of cancer. Aneuploidy can be caused by an ongoing rate of chromosome missegregation during mitosis, known as whole chromosomal instability (CIN). Work from our lab and others has shown that while low rates of CIN can promote tumorigenesis, high levels of CIN are tumor suppressive. Combining two insults that each cause low CIN results in high CIN, cell death, and tumor suppression. We recently showed that the chemotherapy drug paclitaxel causes CIN due to multipolar divisions in breast cancer. Preliminary evidence suggests that tumors which exhibit CIN prior to therapy are sensitized to paclitaxel because it increases their level of CIN over a maximally tolerated threshold. Radiation also induces CIN, and rectal adenocarcinomas which exhibit CIN prior to treatment are sensitized to chemoradiation therapy. Human papillomavirus (HPV), specifically HPV subtype 16, is a growing cause of head and neck cancer worldwide. In cervical cancer, HPV is associated with CIN, which has been attributed largely to the expression of its E6 and E7 oncoproteins. HPV+ head and neck cancer patients show improved responses to radiation therapy than HPV- head and neck patients, potentially due to an increased basal rate of CIN. Here, we tested the ability of HPV E6, E7, or E6+E7 expression to induce CIN in normal oral keratinocytes (NOKs). Expression of E6 alone robustly induced misaligned chromosomes and multipolar spindles with supernumerary centrosomes, while E7 expression alone produced more moderate effects. Combined expression of E6 and E7 exacerbated the phenotypes observed in NOKs expressing E6 alone. Expression of E6, either alone or in combination with E7 caused reduced protein levels of the mitotic kinesin CENP-E, which is sufficient to induce misaligned chromosomes. Interestingly, HPV+ patient-derived xenograft (PDX) models of head and neck squamous cell carcinoma showed less evidence of mitotic defects than NOKs expressing E6 alone. A subset of HPV+ PDX tumors showed an increase in misaligned chromosomes when compared to HPV- tumors, but no increase in lagging chromosomes or multipolar spindles was observed. Together, these data suggest HPV infection causes an incompletely penetrant increase in specific types of CIN in head and neck cancer and that a basal rate of CIN may be predictive of treatment response independent of HPV status.

Citation Format: Laura C. Funk, Denis L. Lee, Randall J. Kimple, Paul F. Lambert, Beth A. Weaver. HPV oncoproteins cause specific types of chromosomal instability in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1846.
Abstract 1848: A comprehensive image cytometry method to identify and functionally characterize a rare subpopulation of platinum-induced aneuploid tumor cells in high-grade serous carcinoma
Authors: Yeung Yu Wing , Fung Oliver , Bashkurov Mikhail , Rottapel Robert , Kapus Andras
Published: August 2018

Background: Although platinum treatment effectively reduces tumor bulk for high grade serous carcinomas of tubal-ovarian origin, there are often scattered tumor cells exhibiting reparative and aneuploid features that remained within the peritoneal cavity. The biological importance of these aneuploid cells has not been well elucidated. In this study, we used HGSC cell line models and developed an improved image cytometry approach to identify these aneuploid cells for functional interrogation.

Methods: We have developed a comprehensive image cytometry approach to assess DNA ploidy and cell cycle phase distribution within a standard flow cytometry software platform and validated this methodology using phase-specific markers (EdU, pRb, Geminin, pH3). We further introduced a novel ‘brightness vs. nuclear size' scatter plot to enhance the cell cycle assessment at the single-nucleus level. The method was applied to correlate the kinetics of the DNA damage and checkpoint response with the cell cycle status and cell viability on HGSC cell lines (OVCAR3, TOV3133G) after in-vitro carboplatin treatment.

Results: Using this improved image cytometry method, we identified the emergence of a rare subpopulation of large aneuploid tumor cells at 1 week after platinum treatment. These large aneuploid cells remained viable (PI- and cleaved-PARP negative) but were uniquely dependent on anti-apoptotic signaling for their continual survival. While the bulk population of treated tumor cells underwent mitotic catastrophe following premature G2-M checkpoint exit, a rare subpopulation escaped cell death likely via mitotic slippage as they exhibit characteristic features of aneuploidy. These large aneuploid cells remained KI-67 positive and clonal dilutional experiments are being performed to assess their regrowth potential.

Conclusions: This study has identified a rare subpopulation of tumor cells that avoided mitotic cell death after platinum treatment and then survived in an aneuploid state. The derivation of aneuploid cells via mitotic slippage subsequent to platinum treatment is an under-recognized phenomenon but may pose as an important drug-resistant mechanism due to enhanced survival strategies co-opted by these aneuploid cells.

Research funding: Ontario Institute for Cancer Research

Citation Format: Yu Wing Yeung, Oliver Fung, Mikhail Bashkurov, Robert Rottapel, Andras Kapus. A comprehensive image cytometry method to identify and functionally characterize a rare subpopulation of platinum-induced aneuploid tumor cells in high-grade serous carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1848.
Abstract 1850: CDK inhibitor PHA-848125 preferentially inhibits proliferation of triple negative breast cancer and synergizes with cisplatin
Authors: Cheung Douglas G. , Buzzetti Marta , Croce Carlo M. , Leva Gianpiero di
Published: August 2018

Triple-negative breast cancer (TNBC), defined by the lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), is highly aggressive with a poor prognosis as it lacks targeted therapies. Several inhibitors of the cyclin-dependent kinase 4 and 6 (CDK4/6) have been approved by the FDA for the treatment of ER-positive breast cancer. However, the effectiveness of these inhibitors against TNBC tumors is less than expected. Here, we tested the efficacy of PHA-848125, a potent ATP-competitive CDK2 inhibitor, on a panel of breast cancer cell lines showing a selective impaired proliferation in TNBC cells (IC50 of ER-positive MCF7 cells: 25.9 μM vs. IC50 of TNBC MDA-MB-231 cells: 430.8 nM). TNBC cells treated with PHA-848125 exhibited effects consistent with CDK2 inhibition, including G1 cell cycle arrest and reduced phosphorylation levels of the retinoblastoma protein (pRB) and CDK2. Interestingly, PHA-848125 was also effective against pRB-null TNBC cell lines, unlike other approved CDK inhibitors. Oral administration of PHA-848125 to TNBC xenograft mice and MMTV-PyMT transgenic mice impaired both tumor growth and metastasis at a tolerable dose (40 mg/kg), but not in ER-positive xenograft mice. A combination treatment of PHA-848125 and cisplatin inhibited tumor growth and induced apoptosis synergistically in vitro and in vivo. Thus, our preclinical experiments set the rationale for the clinical evaluation of PHA-848125, either alone or in combination with cisplatin, for the treatment of TNBC.

Citation Format: Douglas G. Cheung, Marta Buzzetti, Carlo M. Croce, Gianpiero di Leva. CDK inhibitor PHA-848125 preferentially inhibits proliferation of triple negative breast cancer and synergizes with cisplatin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1850.
Abstract 1854: Treatment of head and neck squamous cell carcinoma cells with BMS754807 and Dasatinib induce synergistic cytotoxicity through altered focal adhesion signaling
Authors: Lehman Christine E. , MEndez Rolando , Axelrod Mark , Wulfkuhle Julia , Petricoin Emanuel , Gioeli Daniel , Jameson Mark
Published: August 2018

Head and neck cancer (HNC) is the 6th most common cancer worldwide and the 9th most common cancer in the United States. Singular, targeted cancer therapies rarely produce a robust, complete and durable response and often do not substantially prolong patient survival. Adaptive survival responses often blunt the cytotoxic effects of drugs resulting in resistance. Therefore, targeting compensatory signaling pathways may provide improved therapeutic responses. Even with the current best treatment, 5-year survival for all patients with head and neck squamous cell carcinomas (HNSCC) is only approximately 65%, further highlighting the need for improved combinatorial treatments. Previous work in our laboratory has shown that inhibitors of Insulin Like Growth Factor 1 Receptor (IGF1R) and Src family kinases, when used in combination, synergistically inhibit HNSCC growth in vitro. Therefore, to explore the mechanisms which may contribute to this synergistic cytotoxicity, we utilized reverse phase protein arrays to assess the proteome of HNSCC lines treated with BMS754807 and Dasatinib to inhibit IGF1R and Src, respectively. We identified focal adhesion signaling as a potentially vital node in the cell signaling network of synergistic cytotoxicity upon treatment of HNSCC with BMS754807 and Dasatinib. Focal Adhesion Kinase (FAK) and Paxillin phosphorylation was decreased synergistically with the combination of BMS754807 and Dasatinib. Using western blot analysis, we confirmed a decrease in phosphorylation of FAK Y576/577 and Paxillin Y118 upon combined treatment with BMS754807 and Dasatinib. Interestingly, FAK is upregulated in radioresistant HNSCC patients and, metastases have increased phosphorylation of FAK compared to normal tissues. FAK is amplified in up to 26% of HNSCC and overexpression of Paxillin is associated with unfavorable prognosis, highlighting the importance of focal adhesion signaling in head and neck cancers. Our data suggests that interactions between integrins, receptor tyrosine kinase, and non-receptor kinase signaling regulate cell survival in HNSCC. Disruption of IGFR and Src activity leads to convergent inhibition of focal adhesion signaling and synergistic cytotoxicity in a variety of HNSCC cell lines.

Citation Format: Christine E. Lehman, Rolando MEndez, Mark Axelrod, Julia Wulfkuhle, Emanuel Petricoin, Daniel Gioeli, Mark Jameson. Treatment of head and neck squamous cell carcinoma cells with BMS754807 and Dasatinib induce synergistic cytotoxicity through altered focal adhesion signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1854.
Abstract 1859: Inhibition of proteasome deubiquitinase activity prevents cytoprotective aggresome formation in cancer cells
Authors: Hillert Ellin-Kristina , Brjnic Slavica , Mazurkiewicz Magdalena , Larsson Rolf , Fryknäs Mårten , Swanton Lisa , High Stephen , Linder Stig , D'Arcy Padraig B.
Published: August 2018

Proteasome inhibitors kill cancer cells by inducing an acute proteotoxic stress response characterized by the cytosolic accumulation of poly-ubiquitinated proteins, ER stress and the production of reactive oxygen species (ROS). The aggresome pathway has been described as an escape mechanism from proteasome inhibitor-induced cytotoxicity that functions by sequestering the accumulated protein. We show that b-AP15, an established small molecule inhibitor of proteasome deubiquitinase activity, induces the accumulation of cytosolic poly-ubiquitin in the absence of aggresome formation. In contrast to the clinically used proteasome inhibitor bortezomib, the accumulated poly-ubiquitin complexes induced by b-AP15 remain localized in the cytoplasm and fail to form typical aggresome structures. Inhibition of aggresome formation is dependent on ubiquitin but independent of tubulin dynamics, and may result in the accumulation of a range of substrates of the intracellular transport system on microtubules. Finally, b-AP15 induces hyper-ubiquitination of the aggresome chaperone HDAC6, as well as altering HDAC6 association with the proteasome. This suggests a potential mechanism for the present findings, which will require further investigation. Our data suggests that b-AP15 inhibition of aggresome formation may circumvent a potential resistance pathway following cytotoxicity due to proteasome inhibition via the blocking of microtubule transport.

Citation Format: Ellin-Kristina Hillert, Slavica Brjnic, Magdalena Mazurkiewicz, Rolf Larsson, Mårten Fryknäs, Lisa Swanton, Stephen High, Stig Linder, Padraig B. D'Arcy. Inhibition of proteasome deubiquitinase activity prevents cytoprotective aggresome formation in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1859.
Abstract 186: Small-molecule inhibition of cathepsins L and K as potential therapeutics for macrophage-driven breast cancer
Authors: Dykes Samantha S. , Siemann Dietmar W.
Published: August 2018

Tumor-associated macrophages secrete many factors including proteases and growth factors, which ultimately promote the metastatic phenotype of mammary tumors. Due to their numerous pro-tumor functions, tumor-associated macrophages represent an attractive cell population for stromal-targeted anti-cancer therapies. Interleukin-4 stimulation promotes macrophage differentiation from a baseline M0 state to a more pro-tumorigenic M2 phenotype, which is often associated with increased expression of proteases including the lysosomal cathepsin L. Cathepsin L is important in both macrophage and tumor cell invasion, whereby secreted cathepsin L degrades the extracellular matrix allowing for cell infiltration. M0 to M2 differentiation was not accompanied by an overall increase in the protein expression of cathepsin L, however, secreted cathepsin L was increased in the M2 macrophage population. The present study examined the role of cathepsin L in M0 to M2 differentiation and macrophage-mediated tumor cell invasion using the novel cathepsin L/K inhibitors KGP94 and KGP207 [Dr. Kevin Pinney, Baylor University]. Boyden chamber assays revealed that KGP94 and KGP207 prevented in vitro M2 macrophage invasion and reduced macrophage-stimulated invasion of 4T1 murine breast cancer cells. KGP94 and KGP207 treatment also partially prevented IL-4-stimulated M0 to M2 differentiation of macrophages as determined by a decrease in the IL-4-induced expression of the M2 marker Arginase-1 upon drug treatment. Furthermore, exogenous recombinant cathepsin L partially stimulated the expression of Arginase-1 in M0 macrophages. Together, these data suggest that cathepsin L may play a role in macrophage M0 to M2 differentiation. In summary, the novel cathepsin L/K inhibitors KGP94 and KGP207 altered M0 to M2 differentiation, reduced macrophage invasion, and reduced macrophage-stimulated invasion of breast cancer cells. These data highlight the importance of cathepsin L in macrophage functions and suggest that cathepsin inhibition strategies may be therapeutically beneficial by impairing the progression of tumors, particularly those with high recruitment of M2 macrophages.

Citation Format: Samantha S. Dykes, Dietmar W. Siemann. Small-molecule inhibition of cathepsins L and K as potential therapeutics for macrophage-driven breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 186.
Abstract 1864: Pharmacologic inhibition of NAMPT sensitizes pancreatic cancer cells to the antineoplastic effects of metformin
Authors: Ferbeyre Gerardo , Parisotto Maxime , Rowell Marie-Camille , Bourdeau Véronique , Schmitzer Andreea R.
Published: August 2018

The use of the anti-diabetic drug metformin has been correlated with a reduced cancer incidence, suggesting an unexpected anti-neoplastic activity for this compound. Since metformin treatment is safe and economical, there is considerable interest in exploring its anticancer activity in patients. Pancreatic cancer (pancreatic ductal adenocarcinoma: PDAC) is one of the most aggressive neoplastic diseases, for which there is no treatment significantly increasing patient's survival. Metformin use was associated with reduced pancreatic cancer incidence or better survival in diabetics. In vitro, metformin decreases cell survival and growth of pancreatic cancer cells and appears to target tumor-initiating cells. In vivo, metformin decreases growth of human pancreatic cancer cell lines xenografts in mice. However, clinical trials using metformin failed to decrease pancreatic cancer progression in patients, raising important questions about molecular mechanisms that protect tumor cells from the antineoplastic activities of metformin. We discovered a new mechanism of resistance to the anti-oncogenic properties of metformin in PDAC cells through up-regulation of NAMPT and increase of NAD+ synthesis. Using an inhibitor specific to NAMPT, FK866, we sensitized PDAC cells to the effects of metformin in vitro. In vivo, FK866 increased the efficiency of metformin treatment on KP4 cells xenografts. As both metformin and FK866 clinical trials have failed to efficiently treat cancer, the combination of these two compounds may be a promising strategy to treat pancreatic cancer and maybe other malignancies.

Citation Format: Gerardo Ferbeyre, Maxime Parisotto, Marie-Camille Rowell, Véronique Bourdeau, Andreea R. Schmitzer. Pharmacologic inhibition of NAMPT sensitizes pancreatic cancer cells to the antineoplastic effects of metformin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1864.
Abstract 1865: The combined treatment of 150 kHz Tumor Treating Fields (TTFields) and sorafenib inhibits hepatocellular carcinoma in vitro
Authors: Gotlib Karnit , Zeevi Einav , Schneiderman Rosa S. , Voloshin Tali , Giladi Moshe , Kinzel Adrian , Kison Eilon D. , Weinberg Uri , Palti Yoram
Published: August 2018

Hepatocellular carcinoma (HCC) remains a major health problem worldwide as the third cause of cancer related mortality and the primary cause of death. Currently, sorafenib, an oral multikinase inhibitor that blocks various signaling pathways, is the only drug that has been approved for patients with advanced hepatocellular carcinoma, yet its survival benefit is still limited. Tumor Treating Fields (TTFields) therapy is an effective anti-neoplastic treatment modality delivered via noninvasive application of low intensity, intermediate frequency, alternating electric fields. TTFields are employed as a local treatment with the intent to target dividing cells by disrupting microtubules leading to mitotic catastrophe, abnormal chromosome segregation and the induction of different forms of cancer cell death. The aim of this work is to explore the potential of the use of TTFields alone and in combination with Sorafenib as a treatment for HCC.

HepG2 and Huh-7D12 cells were treated with various TTFields frequencies for 72 hours using the inovitro system. Efficacy of the combined treatment of TTFields and Sorafenib was tested by applying TTFields at the optimal frequency together with various drug concentrations. Cell counts, induction of apoptosis, cell cycle and clonogenic potential were determined at the end of treatment.

The optimal TTFields frequency leading to the highest reduction in cell counts was found to be 150 kHz for both HepG2 and Huh-7D12 cells. TTFields application (1.0 - 1.7 V/cm, 72 hours) at 150 kHz led to 53-64% reduction in cell counts and to additional reduction of over 70% in the clonogenic potential. The combined treatment of TTFields and Sorafenib led to a significant reduction in the number of HepG2 and Huh-7D12 cells (2-way ANOVA, p<0.001 in both cell lines) as compared to each treatment alone.

The results presented in this work demonstrate that TTFields can be an effective treatment against HCC cells and that the combination with Sorafenib may further enhance treatment efficacy. Based on the above, there is a strong rational to continue exploring the potential of the use of TTFields together with standard of care for the treatment of HCC in the clinical settings.

Citation Format: Karnit Gotlib, Einav Zeevi, Rosa S. Schneiderman, Tali Voloshin, Moshe Giladi, Adrian Kinzel, Eilon D. Kison, Uri Weinberg, Yoram Palti. The combined treatment of 150 kHz Tumor Treating Fields (TTFields) and sorafenib inhibits hepatocellular carcinoma in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1865.
Abstract 1868: Upregulation of p62/Nrf2 and downregulation of p-eIF2a provide more susceptible to docosahexaenoic acid-induced cell death in LMP-1 expressing nasopnaryngeal carcinoma cells
Authors: Jing Kaipeng , Shin Soyeon , Han Seung-Hyeon , Wu Tong , Lim Kyu
Published: August 2018

Docosahexaenoic acid (DHA) induces apoptotic cell death through several mechanisms in cancer cells. We have previously demonstrated that DHA triggers apoptosis by increasing reactive oxygen species (ROS) accumulation and the ROS-mediated apoptosis caused by DHA is associated with Nrf2 signaling activation. Here we report that DHA-induced cell death is more susceptible through upregulation of p62-Nrf2 and downregulation of p-eIF2α in LMP-1-expressing nasopharygeal carcinoma (NPC) cells. Treatment of four human NPC cells (CNE, CNE-LMP1, HONE, HONE-EBV) with DHA for 24 hr resulted in a dose-dependent inhibition of cell growth. The DHA effect was due to the induction of apoptosis, given that DHA increased the cleaved form of PARP as well as the number of TUNEL-positive cells. The inhibition of CNE-LMP1 and HONE-EBV cells after DHA treatment is more susceptible compared with CNE and HONE cells without LMP1 by MTT assay. The level of p62 and Nrf2 of LMP1-NPC cells were increased after DHA pretreatment compared to control NPC cells. On the other hand, the level of p-eIF2α produced reverse result. The activation of Nrf2 signal seems to result from decreased Nrf2 inhibitor, Keap1, because DHA remarkably attenuated Keap1 expression levels. Moreover, silencing Nrf2 by small interfering RNAs inhibited the cytotoxic effect of DHA, indicating that Nrf2 activation plays a positive role in the process of DHA-induced apoptosis. Increased staining for LMP1 and p62 was observed in NPC tissues when compared with the nonneplastic (chronic inflammation) tissues. These results suggest that differential regulation of p62/p-eIF2α/NRF2 contributes to susceptible cell death by DHA in LMP-1-expressing NPC cells. Thus, utilization of DHA may represent a promising therapeutic approach for chemoprevention and treatment of human NPC. [This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (NRF-2015R1D1A1A01056887) and by the framework of international cooperation program managed by National Research Foundation of Korea (2015K2A2A6002008)].

Citation Format: Kaipeng Jing, Soyeon Shin, Seung-Hyeon Han, Tong Wu, Kyu Lim. Upregulation of p62/Nrf2 and downregulation of p-eIF2a provide more susceptible to docosahexaenoic acid-induced cell death in LMP-1 expressing nasopnaryngeal carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1868.
Abstract 1870: The anti-proliferative and pro-apoptotic effect of MDM2-p53 antagonists evaluated in human tumor cells lines and chronic lymphocytic leukemia patient samples
Authors: Willmore Elaine , Zhao Yan , Ciardullo Carmela , Woodhouse Laura , Thomas Huw D. , Ahn Maria , Fazal Lynsey , Noble Martin E. , Hardcastle Ian , Howard Steven , Chessari Gianni , Lunec John , Wedge Steve
Published: August 2018

We investigated the cellular response to two MDM2-p53 antagonists (RG7388 and Cpd 1) in a panel of 19 tumor cell lines and in patient-derived chronic lymphocytic leukemia (CLL) cells. RG7388 (Idasanutlin) is an MDM2-p53 antagonist developed by Roche and Cpd1 a novel potent and selective MDM2-p53 antagonist developed as part of an ongoing Alliance between Newcastle University, Cancer Research UK and Astex Pharmaceuticals. RG7388 and Cpd1 potently inhibited the proliferation of many cell lines, with MDM2-amplified SJSA-1 osteosarcoma cells and cells from haematological malignancies being the most sensitive (e.g. GI50 values in the acute myeloid leukemia cell line MOLM13 were 33 + 16 nM for RG7388 and 8 + 1 nM for Cpd 1). We also examined induction of apoptosis by measuring caspase 3/7 activation (24 h treatment). SJSA-1 cells showed a 20-fold increase in caspase activation and Molm13 cells a 6-fold increase (measured at 300nM with RG7388). However, cell lines from solid tumours such as the colorectal carcinoma HCT116 or the hepatocellular carcinoma HepG2 did not show any induction of apoptosis, even at concentrations up to 1µM RG3788. In primary CLL cells, Cpd 1 reduced cell viability (48 h) with an average LC50 of 131 + 46 nM (n = 4), compared to 400 + 55 nM for RG7388 (n =23). We also quantified mRNA levels in 25 primary CLL samples and found that RG7388 induced a predominantly pro-apoptotic gene signature: following 6h treatment, 1µM RG7388 resulted in an average 8-fold induction of PUMA and 3.5-fold increase in BAX compared to a 1.6-fold induction of CDKN1A (p21). Our data confirm that targeting the MDM2-p53 interaction is an effective strategy to inhibit growth and viability of tumor cells, and that some cells, including those expressing high levels of MDM2 or those derived from hematological malignancies, display a striking apoptotic response.

Citation Format: Elaine Willmore, Yan Zhao, Carmela Ciardullo, Laura Woodhouse, Huw D. Thomas, Maria Ahn, Lynsey Fazal, Martin E. Noble, Ian Hardcastle, Steven Howard, Gianni Chessari, John Lunec, Steve Wedge. The anti-proliferative and pro-apoptotic effect of MDM2-p53 antagonists evaluated in human tumor cells lines and chronic lymphocytic leukemia patient samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1870.
Abstract 188: Regulation of breast cancer progression by stromal DDR1
Authors: Sun Xiujie
Published: August 2018

Adipose stromal cells (ASC) constitute a significant component of breast stroma. Emerging evidence demonstrates a functional interaction between ASC and breast cancer (BC) cells. Here, we report that stromal discoidin domain receptor 1 (DDR1) is important for ASC-BC communication. Using a DDR1 knockout mouse model, we find that syngeneic mammary tumors grow more slowly in KO mice than wild-type littermate controls. In vitro, loss of DDR1 in stromal vascular fraction (SVF) impairs its ability to stimulate migration and invasion of tumor cells. Furthermore, we show that DDR1-dependent IL-6 production in SVF is responsible for the SVF effect on tumor cells. These results uncover a previously unrecognized role of stromal DDR1 in BC progression and a potential therapeutic target for BC treatment.

Citation Format: Xiujie Sun. Regulation of breast cancer progression by stromal DDR1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 188.
Abstract 1886: A novel inhibitor of eIF4F protein translation complex sensitizes DLBCL cells to BCL-2 targeted therapy
Authors: Herzog Lee-or , Nguyen Nancy , Chiu Honyin , Mallya Sharmila , Ronai Ze'ev A. , Fruman David A.
Published: August 2018

The purpose of this project is to investigate the feasibility of using small molecules targeting cap-dependent translation to sensitize B cell lymphoma cells to killing by BCL-2 inhibitors (venetoclax/ ABT-199). We hypothesized that diffuse large B-cell lymphoma (DLBCL) cell survival requires cap-dependent translation, which is facilitated by eukaryotic translation initiation factor 4E (eIF4E)-eIF4G interaction and is promoted by mTOR complex 1 (mTORC1). We used the compound SBI-756, a novel inhibitor targeting the scaffolding protein eIF4G (1). SBI-756 is more potent and selective than previously reported compounds targeting the eIF4E cap-binding protein, or compounds targeting eIF4A helicase. By treating DLBCL cells (germinal center B-cell subtype) with SBI-756 (sub μM concentrations), we discovered profound synergistic induction of apoptosis when combined with venetoclax. Cell viability was reduced even more compared to mTOR kinase inhibitor (TOR-KI) treatment combination. Moreover, SBI-756 was found to reduce the viability (IC50 = 653nM) of VAL lymphoma cells lacking 4E-BP1, while VAL were insensitive to TOR-KI treatment. By using Proximity Ligation Assay (PLA) we showed that SBI-756 treatment prevents eIF4E-eIF4G association in intact lymphoma cells. Dual luciferase assays validated this and indicated a dose-dependent reduction in cap-dependent translation following SBI-756 treatment. Also, Western blot analyses confirmed that SBI-756 treatment did not change mTOR substrates' phosphorylation, indicating that the SBI-756 effect is specific to preventing the eIF4E-eIF4G interaction. Furthermore, SBI-756 treatment reduced polysome formation with a corresponding increase in abundance of monosomes. Lastly, induced expression of 4E-BP1 mutant, which irreversibly binds eIF4E, sensitized DLBCL cells to venetoclax treatment to a similar extent as TOR-KI treatment. Ongoing studies will investigate: (1) The molecular mechanism by which translation inhibition by SBI-756 synergizes with venetoclax to prime DLBCL cells toward apoptosis, and (2) pharmacokinetic and pharmacodynamic effects of venetoclax and SBI-756 treatment on DLBCL progression in vivo. Hence, this project highlights a novel combination for treatment of aggressive lymphomas, and establishes its efficacy using preclinical models.

Reference: 1. Feng Y., et al. SBI-0640756 attenuates the growth of clinically unresponsive melanomas by disrupting the eIF4F translation initiation complex. Cancer Res 2015;75(24):OF1-8.

Citation Format: Lee-or Herzog, Nancy Nguyen, Honyin Chiu, Sharmila Mallya, Ze'ev A. Ronai, David A. Fruman. A novel inhibitor of eIF4F protein translation complex sensitizes DLBCL cells to BCL-2 targeted therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1886.
Abstract 1898: Regulation of cysteine supply for survival in hepatocellular carcinoma an breast cancer cell lines
Authors: Kim Do Hyung , Lee Seung Jin
Published: August 2018

Several types of tumor highly rely on extracellular cystine to supply cysteine for essential biological processes, therefore, cystine transporter, system xc-, has been suggested as a potential target for cancer therapy. However, it has not been known about cellular heterogeneity and regulatory mechanisms for cystine-dependency. Cystine dependency of 9 hepatocellular carcinoma cell lines and 6 breast cancer cell lines was measured using cystine-deprived medium or sulfasalazine, an inhibitor of system xc-. Cultivation with cystine-deficient media significantly reduced the growth in 9 cell lines, moderately in 3 cell lines, but in 2 cell lines including SNU398 cells. Sulfasalazine dose-dependently decreased the growth of cystine-dependent cell lines, which was prevented by the addition of 2-mercaptoethanol which reduces cystine to be shuttled via L-type transporter system. To identify regulatory mechanisms to render cells to be less dependent on extracellular cystine, siRNA kinase library was screened in SNU387 cells. The siRNAs to decrease viability of SNU387 cells in cystine-deficient media were identified. These research suggest cellular heterogeneity for the dependency on extracellular L-cystine and the kinase which may support cysteine supply in the absence of extracellular cystine could be a potential candidate for targeting cysteine metabolism.

Citation Format: Do Hyung Kim, Seung Jin Lee. Regulation of cysteine supply for survival in hepatocellular carcinoma an breast cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1898.
Abstract 1901: Structural basis for selective therapeutic targeting of BFL-1 in cancer by a covalent stapled peptide inhibitor
Authors: Harvey Edward P. , Seo Hyuk-Soo , Guerra Rachel M. , Bird Gregory H. , Dhe-Paganon Sirano , Walensky Loren D.
Published: August 2018

BCL-2 family proteins are master regulators of the mitochondrial apoptotic pathway and dictate whether a cell will live or die in response to stress. Cancer cells hijack the anti-apoptotic arm of this signaling network, overexpressing BCL-2, MCL-1, and BFL-1, to enforce cellular immortality. Whereas BCL-2 has been drugged by the selective small-molecule inhibitor, venetoclax, and new MCL-1 inhibitor molecules have just entered phase I clinical testing, anti-apoptotic BFL-1 remains undrugged. Importantly, the expression of BFL-1 can cause resistance to these molecules and has been independently linked to the pathogenesis and chemoresistance of human cancers, including melanomas and hematologic malignancies. Given the emergence of BCL-2 family proteins as high-priority cancer targets, solving their structures, alone and in complex with their natural BH3 domain ligands, yields important blueprints for drug design. Whereas the structures of numerous “BH3-in-groove” complexes have been solved, the corresponding panel of apo structures remains incomplete. Here, we report the crystal structure of apo BFL-1 at 1.69 Å resolution, revealing similarities and key differences among unliganded anti-apoptotic targets. Unlike all other BCL-2 proteins, apo BFL-1 contains a surface-accessible cysteine within its BH3-binding groove, allowing for selective covalent targeting by BH3-based stapled peptide inhibitors. The crystal structure of a complex between BFL-1 and a cysteine-reactive and stapled NOXA BH3 α-helix demonstrated the sulfhydryl bond and fortuitous interactions between the acrylamide-bearing moiety and a newly formed hydrophobic cavity. Comparison of the apo and BH3-liganded structures further revealed an induced conformational change. The two BFL-1 structures expand our understanding of the surface landscapes available for selective targeting of BFL-1, and inform a new therapeutic strategy for overcoming BFL-1-dependent apoptotic blockades in human cancer.

Citation Format: Edward P. Harvey, Hyuk-Soo Seo, Rachel M. Guerra, Gregory H. Bird, Sirano Dhe-Paganon, Loren D. Walensky. Structural basis for selective therapeutic targeting of BFL-1 in cancer by a covalent stapled peptide inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1901.