bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3954: Single-cell misregulation of NRF2 in basal-like breast cancer
Authors: Pereira Elizabeth , Janes Kevin
Published: July 2017

Cancer is driven by a complex and heterogeneous combination of genes and pathways, many of which are not explicitly oncogenic on their own. These so-called non-oncogenes are required for maintenance of the tumorigenic state, but they are not mutated themselves. One way to identify “non-oncogene” pathways is to evaluate how proliferating cells adjust to variable and evolving environments. By examining heterogeneous transcriptional regulatory states of single cells in a 3D culture model of breast epithelial organization, we uncovered NRF2 as a candidate transcription factor upstream of an important regulatory state for morphogenesis. NRF2 is a known oncogene in lung cancer, but mutations in the transcription factor are uncommon in breast cancer. To examine the functional impact of NRF2 signaling, we engineered several triple-negative breast cancer cell lines with inducible short hairpin RNA to knock down NRF2. In claudin-low MDA-MB-231 cells, knockdown of NRF2 caused dramatic reduction in stellate invasion compared with shGFP control. In basal-like HCC-1806 cells, knockdown of NRF2 caused a reduction in “ruptured” tumorspheres, in which cells disseminate from their originating site. In a non-transformed breast epithelial cell line, overexpression of a non-degradable form of NRF2 caused greater variation in the size of acini, while knockdown of NRF2 narrowed the size distribution of acini. Therefore, knockdown of NRF2 substantially normalizes the size and shape of both non-transformed and cancerous 3D structures. We also analyzed expression levels of the genes in the provisional NRF2-regulated cluster after NRF2 knockdown or overexpression. Upon NRF2 knockdown, the p53-regulated transcripts CDKN1A (p21) and RPS27L were strongly induced, suggesting that p53 may be activated after NRF2 loss. Our results suggest a functional importance for NRF2 in multiple breast-epithelial and TNBC contexts, and raise the possibility of interplay between NRF2 and p53. A detailed understanding of NRF2 regulation and dynamics could enable new strategies to disrupt this non-oncogene addiction and re-sensitize cancer cells to their environment and to chemotherapy.

Citation Format: Elizabeth Pereira, Kevin Janes. Single-cell misregulation of NRF2 in basal-like 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 3954. doi:10.1158/1538-7445.AM2017-3954
Abstract 3960: Tumor cell drug resistance induced by the programmed death ligand 1 (PD-L1) immune checkpoint is associated with autophagy
Authors: Minassian Lori M. , MacDonald-Goodfellow Shannyn K. , Truesdell Peter , Sanwalka Daniel , Craig Andrew W. , Koti Madhuri , Siemens D Robert , Graham Charles H.
Published: July 2017

The interaction between the Programmed Death Ligand 1 (PD-L1) immune checkpoint on the tumor cell surface with the Programmed Death-1 (PD-1) receptor on cytotoxic T lymphocytes (CTLs) leads to CTL inactivation, thereby promoting tumor cell escape from adaptive immunity. We previously demonstrated that signaling by PD-L1/PD-1 is bidirectional and leads to activation of oncogenic pathways as well as drug resistance in tumor cells. We also have preliminary evidence that Immunity Related GTPase M, an important mediator of autophagy, is up-regulated by PD-1/PD-L1 reverse signaling. Autophagy is a well-established mechanism of drug resistance in cancer cells. This led us to hypothesize that PD-1/PD-L1 signaling induces drug resistance in tumor cells by up-regulating autophagy. The MEK/ERK and the PI3K/Akt signaling pathways are known to increase and decrease autophagy, respectively. Breast cancer cells exposed to rPD-1 showed a time dependent increase in extracellular signal–regulated kinase (ERK) activation and a decrease in protein kinase B (Akt) activation. Conversion of microtubule-associated protein light chain 3 (LC3)-I to LC3-II is a requirement for autophagosome formation and is a robust marker of autophagy. Exposure of human breast cancer cells to recombinant PD-1 (rPD-1) showed a time-dependent increase in LC-3 II. We are currently conducting additional studies to confirm that the activation of PD-L1 signaling in tumor cells up-regulates autophagy. These results provide evidence that PD-1/PD-L1 reverse signaling activates autophagy as a potential mechanism of cancer cell chemoresistance. (Supported by a grant from the Canadian Institutes of Health Research.)

Citation Format: Lori M. Minassian, Shannyn K. MacDonald-Goodfellow, Peter Truesdell, Daniel Sanwalka, Andrew W. Craig, Madhuri Koti, D Robert Siemens, Charles H. Graham. Tumor cell drug resistance induced by the programmed death ligand 1 (PD-L1) immune checkpoint is associated with autophagy [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 3960. doi:10.1158/1538-7445.AM2017-3960
Abstract 3972: Ex vivo profiling of PD-1 blockade using organotypic tumor spheroids
Authors: Barbie David A. , Jenkins Russell , Wong Kwok-Kin
Published: July 2017

Despite the impressive activity of PD-1/PD-L1 immune checkpoint blockade in melanoma, lung, and other cancers, the majority of patients are intrinsically resistant to this therapy. Furthermore, despite years of effort few robust biomarkers of response exist, and tumor PD-L1 expression and mutational load are only partially predictive of activity. Nearly all attempts at biomarker development have relied on fixed tumor tissue or studies in plasma. Meanwhile, emerging functional approaches to study patient derived samples, such as organoid development and circulating tumor cell culture, lack features of the native tumor immune microenvironment and thus are limited in the ability to assess ex vivo anti-PD-1 response. We have developed a novel approach to study short-term ex vivo response to immune checkpoint blockade based upon short term culture of murine- and patient-derived organotypic tumor spheroids (MDOTS/PDOTS) in a 3-dimensional microfluidic system. Spheroids derived from fresh mouse and human tumor samples through partial collagenase digestion and filtration retain autologous tumor infiltrating immune cells, including PD-1 positive CD8 T lymphocytes. Using murine syngeneic mouse cancer models with known responsiveness or resistance to PD-1 blockade, we demonstrate the ability to recapitulate tumor killing ex vivo or lack thereof. Multiplexed cytokine profiling of conditioned media in this system further enables characterization of the cytokine and chemokine response to PD-1 and/or CTLA-4 blockade. We thus analyzed the secreted cytokine/chemokine response to PD-1 inhibition across a large panel of PDOTS, and observed pronounced induction of CCL19/CXCL13, which associated with % PD-1 positive CD8 T cells. This strategy further uncovered production of a class of myeloid attracting chemokines and cytokines in major proportion of samples, which clustered with the presence of plasmacytoid dendritic cells and myeloid derived suppressor cells. Importantly, the presence of this maladaptive cytokine/chemokine signature correlated with lack of response in the subset of patients that had been treated with PD-1 blockade. Together, these data demonstrate the ability to profile the functional response to tumor PD-1 blockade ex vivo and unveil a novel strategy to advance precision immuno-oncology.

Citation Format: David A. Barbie, Russell Jenkins, Kwok-Kin Wong. Ex vivo profiling of PD-1 blockade using organotypic tumor spheroids [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 3972. doi:10.1158/1538-7445.AM2017-3972
Abstract 3977: Neutrophils fertilize the pre-metastatic omental niche in ovarian cancer
Authors: LEE WonJae , Naora Honami
Published: July 2017

More than 60% of ovarian cancer patients present with peritoneal carcinomatosis that almost always involves the omentum, a fat pad that suspends from the stomach. However, the mechanisms that promote the tropism of ovarian cancer cells for the omentum are poorly understood. Because the omentum is the major peritoneal conduit and reservoir for immune cells, we hypothesized that immune cells control the propensity of ovarian cancer cells to implant on the omentum. In this study, we evaluated changes in immune cell populations in the omentum and in other visceral fat pads in the peritoneal cavity in mouse xenograft models of ovarian cancer. Intriguingly, the proportion of neutrophils was observed to dramatically increase in the omentum prior to tumor cell implantation at this site. On the other hand, the proportion of neutrophils did not substantially increase in other visceral fat pads. Furthermore, the proportion of macrophages did not significantly increase in any of the fat tissues prior to tumor cell implantation. Collectively, these findings raise the possibility that early recruitment of neutrophils to the pre-metastatic omental niche promotes a microenvironment that is conducive to ovarian cancer cell implantation at this site.

Citation Format: WonJae LEE, Honami Naora. Neutrophils fertilize the pre-metastatic omental niche in ovarian 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 3977. doi:10.1158/1538-7445.AM2017-3977
Abstract 3981: The Shc1 scaffold protein simultaneously balances Stat1 and Stat3 activity in breast cancer to promote immune suppression and resistance to immunotherapy
Authors: Ahn Ryuhjin , Jacquet Kévin , Fabian Marc , Huang Sidong , Bisson Nicolas , Ursini-Siegel Josie
Published: July 2017

Receptor and cytoplasmic tyrosine kinases are key signal integrators in poor outcome breast cancers that are central to the establishment of an immunosuppressive microenvironment. Immunotherapies represent an emerging approach within the armament of anti-cancer agents. Although the efficacy of tyrosine kinase inhibitors relies, in part, on their ability to augment adaptive immunity, the increased heterogeneity and functional redundancy of the tyrosine kinome in poor outcome breast cancers represents a significant hurdle to achieving durable responses to immunotherapies. We previously identified the Shc1 (ShcA) scaffold protein, a central regulator of tyrosine kinase signaling, as essential for promoting immune suppression. We recently showed that the ShcA pathway simultaneously activates STAT3 immunosuppressive signals and impairs STAT1-driven immune surveillance in breast cancer cells. Impaired phosphorylation of select tyrosine residues on ShcA potently and selectively reduces STAT3 activation in breast tumors, profoundly sensitizing them to immune checkpoint inhibitors and tumor vaccines. Meanwhile, impaired phosphorylation of other select tyrosine residues on ShcA potently increased antigen presentation and sensitivity to tumor vaccines in preclinical mouse models. Based on these results, we have set out to elucidate protein interactors dependent on distinct ShcA phospho-tyrosines to regulate STAT1 and STAT3 signaling axis that aid tumor driven immune suppression. We combined affinity-purification mass spectrometry (AP/MS) and proximity-dependent biotin identification (BioID) followed by mass spectrometry (BioID/MS). Known interactors of ShcA as well novel interactors have been identified. Promising candidates that could be critical in immune suppression downstream of ShcA have been validated to be true interactors by co-IP and BioID assays. They have been screened for immune regulation in vitro for further modulation in vivo. Currently, inhibitors of phospho-tyrosine motifs of ShcA do not exists. Therefore, the development of pharmacological inhibitors to prevent phospho-tyrosine ShcA dependent STAT3 signaling or relieve suppression of STAT1 signaling may be an attractive method to strategically sensitize breast tumors to multiple immunotherapies.

Citation Format: Ryuhjin Ahn, Kévin Jacquet, Marc Fabian, Sidong Huang, Nicolas Bisson, Josie Ursini-Siegel. The Shc1 scaffold protein simultaneously balances Stat1 and Stat3 activity in breast cancer to promote immune suppression and resistance to immunotherapy [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 3981. doi:10.1158/1538-7445.AM2017-3981
Abstract 3995: Identification of receptor tyrosine kinases (RTKs) expression in tumor-infiltrating lymphocytes (TILs) and peripheral blood mononuclear cells (PBMC)
Authors: Khochenkov Dmitry , Volkova Maria , Tsimafeyeu Ilya
Published: July 2017

Aim of this study is to discover expression levels of RTKs (VEGFR1, VEGFR2, PDGFRa, PDGFRb) in CD45+ population of PMBC and TILs isolated from patients with clear cell renal cell cancer (RCC). Here we present preliminary data.

Species of tumors and blood were prospective collected from 9 patients with RCC after radical nephrectomy or tumor resection from July to October 2016. TILs isolation was performed under standard protocol with slight modification. Briefly, tumor was shredded with scissors on small pieces of 2-3 mm and twice washed in RPMI 1640 media by centrifugation for 10 min at 1500 rpm. After centrifugation, pellet was diluted RPMI 1640 with enzyme mix (Collagenase and Dispase II) on Petri dish and placed on shaker-thermostat andincubate sample for 12h at 37C with continuous shaking. After incubation, cell suspension was passed thought a cell strainer, mesh size 70 μm, placed on a 50 mL tube and washed twice washed in RPMI 1640 media by centrifugation.

For TILs isolation cell suspension was placed on Ficoll-Paque Plus and isolated by centrifugation for 25 min at 1500 rpm. Cell pellet was resuspended in RPMI 1640 media. PMBC was isolated from whole blood using the Ficoll-Paque method. Isolated TIL and PMBC were prepared for flow cytometry at typical protocol with or without permeabilization.

Cells were stained with anti-CD45 FITC-conjugated antibodies (Novus), primary anti-VEGFR1, anti-VEGFR2, anti-PDGFRa, anti-PDGFRb (Abcam) mouse antibodies and Alexa-568 conjuated secondary anti-mouse antibodies (Invitrogen) and analyzed on BD Canto II flow cytometer.

Expression of RTKs on CD45+ cell population were evaluated. Among PMBC 86±9% of cells was CD45-positive and expressed RTKs (10±1% cells were positive for VEGFR1 and VEGFR2, and 6±2% cells were positive for PDGRFa and PDGFRb). TILs contained 65±18% of CD45-positive cells and did not express RTKs on the cell surface (less than 1% of cells), but intracellular staining showed presence of RTKs: 8±2% of cells were VEGFR1 or VEGFR2-positive, 20±8% of cells were PDGFRa or PDGFRb-positive.

Preliminary data demonstrate extracellular expression of RTKs on PBMC and intracellular expression of RTKs in TILs. The study will be continued.

Citation Format: Dmitry Khochenkov, Maria Volkova, Ilya Tsimafeyeu. Identification of receptor tyrosine kinases (RTKs) expression in tumor-infiltrating lymphocytes (TILs) and peripheral blood mononuclear cells (PBMC) [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 3995. doi:10.1158/1538-7445.AM2017-3995
Abstract 4003: Acral lentiginous melanoma and infiltrating lymphocytes in a Latin American population
Authors: Castaneda Carlos , Torres-Cabala Carlos , Casavilca Sandro , Castillo Miluska , Villegas Valeria , Sanchez Joselyn , Flores Claudio , Gomez Henry , Vidaurre Tatiana
Published: July 2017

Background: Acral lentiginous melanoma (ALM) is unusual in Caucasian but is the most common subtype in Hispanic populations, and has been associated to poor prognosis. Herein we compare clinicopathological features associated to ALM in a Latin-American population and prospectively evaluate its association with tumor infiltrating lymphocytes (TIL).

Patients and Methods: All patients with melanoma presenting from 2005 to 2012 at Instituto Nacional de Enfermedades Neoplasicas in Lima, Peru, were retrospectively identified. Analysis of association between clinicopathological features and ALM was performed. Survival analysis compared the outcome of ALM to whole group and extremity NALM. We performed prospective evaluation of TIL following Azimi et al recently presented classification.

Results: A total of 537 ALM and 287 NALM cases were identified. Older age (p= 0.02), thicker Breslow (p= 0.008), positive lymph node (p= 0.051) and ulceration (p<0.001) were found to be more frequent in ALM. Acral had worse OS compared with both whole group (p= 0.04) and extremity NALM (p <0.001). Stage I-II patients had a median OS of 5.3 (95%CI: 4.3-6.2) for ALM and 9.2 (95% CI: 5.0 - 7.0) for extremity NALM (p=0.016). TIL was prospectively evaluated in 490 cases, and Grade 0-1, II and III of TIL represented 44.7%, 33.6% and 27.1%, respectively. Higher TIL grade was associated to less Breslow thickness (p= 0.021), less mitosis index and NAM. Higher TIL grade was also associated to better OS (p< 0.001)

Conclusion: ALM is highly prevalent in Latin-America and carry poor outcome. Lower TIL levels was associated to poor outcome and ALM.

Citation Format: Carlos Castaneda, Carlos Torres-Cabala, Sandro Casavilca, Miluska Castillo, Valeria Villegas, Joselyn Sanchez, Claudio Flores, Henry Gomez, Tatiana Vidaurre. Acral lentiginous melanoma and infiltrating lymphocytes in a Latin American population [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 4003. doi:10.1158/1538-7445.AM2017-4003
Abstract 4014: Blood-brain-barrier spheroids: Next-generation screening platform for brain-penetrating agents
Authors: Cho Choi-Fong
Published: July 2017

The inability of most systemically delivered therapeutics to cross the blood-brain-barrier (BBB) is considered a major barrier to effective brain cancer treatment. In malignant glioma the tumor neovasculature is known to be leaky, however invasive cells remain protected behind an intact BBB, and continue to thrive within the brain, ultimately leading to tumor regrowth and patient death. This leaves us with an urgent unmet need for the development of next-generation therapeutics with improved brain delivery. For the first time, we describe here the utility of 3D multicellular BBB spheroids made of human brain endothelial cells (ECs), pericytes and astrocytes as a screening tool for brain-penetrating agents. We show that the outer surface of the spheroids, composed primarily of ECs and pericytes, form a tight barrier which is permeabilized in the presence of VEGF. The barrier is characterized by the presence of intact tight junctions and efflux-pump activity (i.e., P-glycoprotein). Furthermore, we have used this model to successfully demonstrate the transport of angiopep-2 (a well-known brain delivery vector) and its conjugates (containing cargoes of various sizes such as peptide, protein and affibody), thereby displaying the versatility of this model to screen and study a wide range of therapeutic agents. We demonstrate that this model is superior to the conventional transwell model in maintaining essential BBB characteristics (i.e., tight/adherens junctions and P-glycoprotein expression) and as a drug-screening tool. We have utilized the spheroid model to screen a panel of cell-penetrating peptides (CPPs) to identify several candidates with high brain-penetration potential. We then verified the ability of the top 4 candidate CPPs to cross the BBB in mice. This high-throughput model can lead to better design and analysis of first-in-class glioma therapeutics, and improve prediction of drug penetration in a living model, paving the way for breakthrough discoveries in brain cancer.

Citation Format: Choi-Fong Cho. Blood-brain-barrier spheroids: Next-generation screening platform for brain-penetrating agents [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 4014. doi:10.1158/1538-7445.AM2017-4014
Abstract 4018: Identifying allosteric modulators of KRas using second harmonic generation
Authors: Vo Elizabeth Donohue , Mercado Gabriel Besserer , Alexander Patrick , Moree Ben , Van Que , Stephen Andrew G. , Salafsky Joshua , McCormick Frank
Published: July 2017

The Ras protein family consists of small GTPases that are critical signaling transducers regulating cellular processes including proliferation, differentiation, and survival. As monomeric GTPases, Ras proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Oncogenic mutations in one of the Ras isoforms, H-, N-, and K-Ras, impair GTP hydrolysis, thus de-regulating Ras signaling. Ras mutations are found in one-third of human cancers, and tumors bearing these mutations are notoriously difficult to treat. Conventional approaches to inhibit Ras with small molecules have been largely unsuccessful leading many to characterize the Ras family as an “undruggable” target. However, there is renewed enthusiasm towards Ras drug discovery in response to a recent surge of reports revealing structural complexities to Ras proteins that provide opportunity for innovative drug discovery efforts.

In light of findings suggesting KRas contains transient and dynamic binding pockets, many of which are undetectable with traditional structural techniques, we used a second harmonic generation (SHG) platform to screen a ~2700 chemically diverse fragment collection for allosteric modulators of KRas. This high throughput SHG platform enables real-time measurement of protein conformational change under physiological conditions. We selected ~50 fragments for validation by SPR, and positive binders will be selected for structural and biochemical characterization. Another advantage of the SHG platform is that we can monitor the conformational dynamics of fully processed, farnesylated KRas bound to a phosphatidylserine-enriched membrane that mimics KRas’ physiological interaction with the plasma membrane. No other biophysical method is suitable to monitor the real-time interactions of small molecules or proteins with membrane-bound KRas. This analysis may reveal surface contacts unavailable in solution-based assays. We have successfully implemented SHG as a drug discovery platform against KRas, and we aim to utilize selected KRas conformational modulators towards the identification of novel binding pockets and towards the development of targeted KRas therapies.

Citation Format: Elizabeth Donohue Vo, Gabriel Besserer Mercado, Patrick Alexander, Ben Moree, Que Van, Andrew G. Stephen, Joshua Salafsky, Frank McCormick. Identifying allosteric modulators of KRas using second harmonic generation [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 4018. doi:10.1158/1538-7445.AM2017-4018
Abstract 4030: Celsee™ SingleCell Chip can retrieve rare single cells with 100% efficiency for genetic analysis
Authors: Handique Kalyan , Gogoi Priya , Chow William , Gleason Kyle , Payne Auston , Sharma Vishal Premdev , Wang Yixin
Published: July 2017

Tumors are comprised of heterogeneous cells. Only a specific or rare population of tumor cells is capable of metastasizing through blood. While mapping out the genome of cancer cells, gene expression variations indicative of more aggressive cancer cells might be missed among the noise. Therefore, single cell gene expression and mutation analysis provide a more comprehensive and most precise information about the heterogeneity of cancer progression and metastasis. Although there has been a surge in technologies used for selection and analysis of single cells, single cell analysis of rare cells such as circulating tumor cells (CTCs) is fraught with technical challenges. Among many used techniques to isolate single cells, the most common cell separation method is cell sorting by flow cytometry, which requires cell labeling. The numerous steps involved in cell labelling and centrifugation result in significant cell loss and changes in gene expression. In addition, centrifugation may also cause shear-induced gene expression changes in viable cells. For other single cell analysis platforms, there is a minimum input of cells ranging from 200 - 1000 cells. Rare cells like CTCs are found in the range of 1-10 per mL of blood. Another challenge is that the input volume required for these systems is very low usually in microliters. Here, we introduce a novel microfluidic chip with 250,000 microwells which captures cells at 100% efficiency down to one cell with a dynamic range over 5 logs. The underlying principle of this technology is that the geometric pattern of the microfluidic chip captures a single cell from an initial volume of up to 1 mL of fluid due to cell settling. The unique microwell structure promotes easy fluid exchange without perturbing the cells from their captured locations, thereby eliminating cell loss during multistep downstream processing. Preliminary tests have shown that single or cluster of spiked cancer cells in blood can be immunostained, both live or fixed and can also be retrieved very efficiently from this chip using a micro capillary. The retrieved single cells can further be used for several single cell genetic analysis. We will present data describing cancer gene expression and gene mutation analysis in isolated single cells.

Citation Format: Kalyan Handique, Priya Gogoi, William Chow, Kyle Gleason, Auston Payne, Vishal Premdev Sharma, Yixin Wang. Celsee™ SingleCell Chip can retrieve rare single cells with 100% efficiency for genetic analysis [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 4030. doi:10.1158/1538-7445.AM2017-4030
Abstract 4036: Induction of detrimental aneuploidy in basal breast cancer cells treated by MPS1 inhibitors in combination with paclitaxel
Authors: Anderhub Simon J. , Mak Grace WY , Faisal Amir , Walsh Katie , Gurden Mark , Day Liz , Innocenti Paolo , Woodward Hannah L. , Westwood Isaac M. , Cheung Kwai-Ming J. , Naud Sebastien , Hayes Angela , Box Gary , Brandon Alexis de Haven , Valenti Melanie R. , O’Fee Lisa , Saville Harry , Burke Rosemary , Montfort Rob LM van , Raynaud Florence I. , Eccles Suzanne A. , Blagg Julian , Hoelder Swen , Linardopoulos Spiros
Published: July 2017

Basal-like breast cancer has received considerable attention in recent years, but despite all efforts, conventional chemotherapy remains the main therapeutic option in the clinic with taxane therapy, the most frequently prescribed treatment. A significant reason for the lack of effective therapeutics may be related to the high degree of heterogeneity and aneuploidy which is characteristic for basal-like breast cancer. A new approach in this field is the concept that cancer cells may be targeted by the introduction of a detrimental level of aneuploidy. Inhibition of one of the main spindle assembly checkpoint kinases, MPS1 is a rational choice to achieve this goal. We report here the identification of a synergistic effect between the novel, oral, highly potent and selective MPS1 inhibitor CCT289346 and paclitaxel. We demonstrate robust synergism in multiple basal-like breast cancer cell lines and provide mechanistic insights into how both compounds’ individual properties work together to maximise mitotic defects thereby increasing aneuploidy and cell death in a single round of mitosis. Additionally, in vivo studies in basal-like breast cancer xenograft models, including patient derived xenografts and systemic metastatic models, using clinically relevant doses of paclitaxel with well-tolerated doses of CCT289346, demonstrate significant benefit of combination of the two agents in comparison to paclitaxel alone.

Citation Format: Simon J. Anderhub, Grace WY Mak, Amir Faisal, Katie Walsh, Mark Gurden, Liz Day, Paolo Innocenti, Hannah L. Woodward, Isaac M. Westwood, Kwai-Ming J. Cheung, Sebastien Naud, Angela Hayes, Gary Box, Alexis de Haven Brandon, Melanie R. Valenti, Lisa O’Fee, Harry Saville, Rosemary Burke, Rob LM van Montfort, Florence I. Raynaud, Suzanne A. Eccles, Julian Blagg, Swen Hoelder, Spiros Linardopoulos. Induction of detrimental aneuploidy in basal breast cancer cells treated by MPS1 inhibitors in combination with paclitaxel [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 4036. doi:10.1158/1538-7445.AM2017-4036
Abstract 404: A bi-clustering based computational approach to identify molecular markers for Neoadjuvant chemotherapy outcome prediction in complement to cancer subtype classifications
Authors: Zhang Chi , Liang Sen
Published: July 2017

Molecular stratification models have been well established for most cancer types to classify tumors into sub-types by using genome level molecular characteristics. Tumors of certain sub-type have distinct response to specific therapies. Hence a reliable molecular classification model can be applied to determine treatment strategy. Recent neoadjuvant chemotherapy, immunotherapies and gene targeted drugs and their possible combinations raise a demand for small subsets of novel molecular markers for outcome prediction in addition to the complete molecular profile based classification. However, it is unknown what genes have predictive power for a certain therapeutic strategy while the highly crossed clinical features increase the difficult to determine sample groups for marker identification that form a bi-clustering problem. In addition, predictive maker may be varied among different cancer sub-types. In this work, we developed a bi-clustering based computational framework to identify transcriptomic markers for predicting the outcome of neoadjuvant chemotherapy and adjuvant chemotherapy. Our computational approach identifies functional related gene expression modules show significant expression patterns in a subset of samples that are highly associated with one or several disease sub-types and with high predictive power for the outcome of certain. By applying the analysis to 8 breast carcinoma, 10 colorectal carcinoma and 3 acute myeloid leukemia data sets in public domain, we have identified sets of gene markers with high predictive power for chemotherapy outcome complementary to the existing molecular classifications, suggesting the bi-clustering based approach can improve the whole genome level molecular classification based outcome prediction.

Citation Format: Chi Zhang, Sen Liang. A bi-clustering based computational approach to identify molecular markers for Neoadjuvant chemotherapy outcome prediction in complement to cancer subtype classifications [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 404. doi:10.1158/1538-7445.AM2017-404
Abstract 4040: Short-term romidepsin treatment combined with clotrimazole or bifonazole leads to decreased mitochondrial hexokinase 2 and apoptosis in cancer cells
Authors: Robey Robert W. , McDonald Andrew J. , Kozlowski Hanna , Gottesman Michael M. , Bates Susan E.
Published: July 2017

In light of disappointing clinical trials in solid tumors, we have sought novel combinations to increase the efficacy of the histone deacetylase inhibitor (HDI) romidepsin. Hexokinase 2 (HK2) expression is increased in cancer cells and is postulated to prevent Bax-mediated cytochrome c release from mitochondria. Since romidepsin treatment induces apoptosis via the mitochondrial pathway, combining romidepsin with agents that detach mitochondrial HK2 might result in synergistic apoptotic effects. HCT-116 colon carcinoma cells were treated with 25 ng/ml romidepsin alone for 6h, or with romidepsin in the presence of 25 µM clotrimazole or bifonazole, compounds known to detach mitochondrial HK2. The medium was removed and cells were treated for an additional 42 h with or without clotrimazole or bifonazole. Cells were also treated with clotrimazole or bifonazole alone for 48 h. Apoptosis was then quantitated by annexin staining. While short-term romidepsin treatment alone induced modest annexin staining and 25 µM clotrimazole or bifonazole had little effect, the combination resulted in significant apoptosis. Similar effects were observed for A549 lung cancer cells, MDA-MB-231 breast cancer cells and 786-0 renal carcinoma cells. We also treated HCT-116 and A549 cells lacking Bak, Bax or both with the clotrimazole/romidepsin combination. Apoptosis was slightly increased in Bak-/- cells, reduced in Bax-/- cells and nearly completely abrogated in cells lacking both proteins compared to wild-type cells suggesting apoptosis occurred through the intrinsic pathway. The clotrimazole analog TRAM-34 was similarly active to clotrimazole. Protein expression was determined in cytoplasmic and mitochondrial fractions of cells treated with romidepsin, clotrimazole or both and we found a >60% decrease in mitochondrial HK2 in cells treated with the combination versus untreated cells. Our results suggest that combining romidepsin with a compound that leads to decreased mitochondrial hexokinase, such as clotrimazole or bifonazole, results in increased apoptosis. We postulate that cell death following combined romidepsin and clotrimazole treatment requires an intact intrinsic apoptotic pathway and that HK2 serves an anti-apoptotic function at the mitochondria.

Citation Format: Robert W. Robey, Andrew J. McDonald, Hanna Kozlowski, Michael M. Gottesman, Susan E. Bates. Short-term romidepsin treatment combined with clotrimazole or bifonazole leads to decreased mitochondrial hexokinase 2 and apoptosis in 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 4040. doi:10.1158/1538-7445.AM2017-4040
Abstract 4042: N-121, a novel polypeptide conjugate of SN-38, demonstrated enhanced intratumoral accumulation and sustained release of free SN-38 with potent antitumor activity
Authors: Wang Haojun , Li Wei , Zhou Xiaojian , Wan Liuyi , Wen Guanghui
Published: July 2017

Introduction: SN-38 is a critical chemotherapeutic agent active against a variety of solid tumors including advanced colorectal cancer. Clinical use of SN-38 is hampered by its poor solubility in pharmaceutically acceptable solvents. Although irinotecan, the only approved prodrug of SN-38, is widely used, its efficacy is limited by low conversion to the active SN-38 as well as hematologic and gastrointestinal dose-limiting toxicities. In an attempt to improve the water solubility, antitumor activity and safety profiles of SN-38, we synthesized a water soluble polypeptide-SN-38 conjugate (N-121) and examined its antitumor activity and pharmacokinetics (PK) in mouse models.

Methods: Antitumor activity and PK of SN-38, administered as N-121 or irinotecan, was evaluated following tail vein injection in male nude mice with subcutaneous orthotopic xenografts of the HCT116 human colon cancer. Mice received N-121 400 mgk (mg/kg) (32 mkg of eq. SN-38) or irinotecan 80 mgk once a week for 2 weeks and were followed for up to 46 days.

Results: N-121 drastically improved by over 1000 fold the water solubility of SN-38. At a SN-38 equivalent dose of less than half of that of irinotecan, N-121 resulted in a much longer tumor growth delay (36 days) than irinotecan (14 days). The more durable tumor growth inhibition with N-121 was consistent with its enhanced intratumoral accumulation: intratumoral trough levels of SN-38 were about 250-fold higher than those observed in plasma, compared with only 15-fold with irinotecan. With N-121, Intratumoral levels of SN-38 remained stable over the PK observation period (48 h), contrasting to a marked decline in SN-38 levels with irinotecan; intratumoral trough levels of SN-38 from N-121 were about 50-fold higher than those achieved with irinotecan. N-121 was well tolerated with a slight weight loss of 7.2%.

Conclusion: N-121, a novel water soluble polypeptide-SN-38 conjugate, exhibited enhanced and sustained intratumoral exposure of SN-38 with high antitumor activity in a mouse xenograft tumor model. These promising results warrant further evaluation of N-121.

Note: This abstract was not presented at the meeting.

Citation Format: Haojun Wang, Wei Li, Xiaojian Zhou, Liuyi Wan, Guanghui Wen. N-121, a novel polypeptide conjugate of SN-38, demonstrated enhanced intratumoral accumulation and sustained release of free SN-38 with potent antitumor activity [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 4042. doi:10.1158/1538-7445.AM2017-4042
Abstract 407: Whole-genome pooled CRISPR/Cas library screens to identify new hepatocellular carcinoma drivers and drug targets in combination with sorafenib
Authors: Kodama Takahiro , Newberg Justin Y. , Jenkins Nancy A. , Takehara Tetsuo , Copeland Neal G.
Published: July 2017

Backgrounds and Aims: Hepatocellular carcinoma (HCC) is a deadly cancer and its mortality rate ranks third among all cancer types worldwide. To comprehensively understand its genetic drivers, over 500 HCC genomes have been sequenced but they reveal the existence of thousands of infrequently mutated genes. This intra- and inter-tumor heterogeneity, combined with large number of passenger mutations, has made it difficult to identify the true drivers of HCC. In addition, sorafenib is the currently only FDA-approved molecular target drug in HCC but its anti-tumor effect is very limited. To overcome these problems, we performed two forward genetic screens using recently innovated whole-genome pooled CRISPR/Cas library. Methods: Two human liver cancer cell lines, Huh-7 and SNU-398, were lentivirally transduced with Geckov2 library and transduced cells were selected with puromycin. First, transduced cells were orthotopically injected into the liver of the Nude mice and monitored for tumor formation. To identify potential tumor suppressor genes (TSGs) in the liver, gRNAs enriched in the orthotopic liver tumors were determined by sequencing of gDNA in these tumors. Next, transduced cells were treated with vehicle or sorafenib in vitro for 14 days. To identify potential targets which inhibition show synergistic anti-tumor effect with sorafenib, we determined gRNAs depleted in cells with sorafenib treatment compared to cells with vehicle treatment. Results: In the first in vivo screening, we identified 62 genes targeted by 2 or more highly enriched gRNAs in Huh-7 cells-derived tumors and 47 genes in SNU-398 cells-derived tumors. Twenty-nine genes were common including 4 miRNAs. TCGA analysis revealed that HCC patients with mutation or copy number alteration in any of these genes showed worse overall survival and disease free survival compared to HCC patients without them. In addition, gene expression levels of 3 candidate TSGs, BPHL, CTSZ and PLCG2 were significantly negatively correlated with poor survival of HCC patients. In the second in vitro screening, CYP7B1, HS3ST5, NLRC4, and Mir-1237 were identified as potential drug targets in combination with sorafenib in HCC. Conclusion: Whole-genome pooled CRISPR/Cas library screens discovered potential new hepatocellular carcinoma drivers and drug targets in combination with sorafenib.

Citation Format: Takahiro Kodama, Justin Y. Newberg, Nancy A. Jenkins, Tetsuo Takehara, Neal G. Copeland. Whole-genome pooled CRISPR/Cas library screens to identify new hepatocellular carcinoma drivers and drug targets in combination with sorafenib [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 407. doi:10.1158/1538-7445.AM2017-407
Abstract 4080: Cytochrome P450 enzyme activity is enhanced in hepatocytes grown using a perfused 3D cell culture drug screening system
Authors: Shoemaker James T. , Vukasinovic Jelena
Published: July 2017

A critical requirement for assessing drug toxicity in vitro is cytochrome P450 (CYP) function that is physiologically representative. Due to low levels of CYP enzyme activity, cell lines such as HepG2 hepatocytes have largely been supplanted with primary cells derived from human donor tissue. While these cells retain CYP activity ex vivo, drug screening is limited to short-term assessments due to de-differentiation. This limits their efficacy in testing slow-clearing, large-molecule compounds such as antibody-based therapies and low-turnover drugs. Cell lines in monolayer cultures have been effective in assessing the safety of small molecule drugs, but the disparity between cell lines and in vivo tissue biology proves too substantial for current potential therapies. Three-dimensional (3D) cultures have been shown to be more physiologically relevant models of in vivo tissue. However, even they fail to fully mimic living tissue and suffer from the lack of active oxygen and nutrient transport. Lena Biosciences has developed a groundbreaking in vitro system that facilitates long-term survival of 3D cultures. Cells seeded in 3D hydrogels supported by a rigid scaffold are maintained in statistically independent wells in their own medium which floats atop a high-density blood-substitute in which medium is immiscible. Infusion and withdrawal of this blood-substitute introduces perfusion into a medium-throughput multiwell system without the need for separate pumps for each culture condition.

Internal validation experiments using hepatocytes cultured in a 12-well system showed a two-fold improvement in cellular respiration (measured by alamarBlue assay) over cultures maintained in a standard multiwell plate when the blood-substitute was introduced, and a three-fold improvement when the cultures were perfused. Cell viability (measured by LDH release) was significantly improved when perfusion was introduced. CYP activity (assessed using both fluorescent and luminescent substrate-based assays) was also significantly higher in the perfused hepatocyte cultures. These data demonstrate the substantial advantages to growing cells in this novel perfused cell culture model. Scaling the system to include multiple cell types (primary or cell lines), would allow for simultaneous testing of target and off-target effects of potential small or large molecule therapies. Thus, this system serves as a major improvement over the current organ-on-a-chip solutions in the area of drug safety testing.

Citation Format: James T. Shoemaker, Jelena Vukasinovic. Cytochrome P450 enzyme activity is enhanced in hepatocytes grown using a perfused 3D cell culture drug screening 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 4080. doi:10.1158/1538-7445.AM2017-4080
Abstract 4096: Transglutaminase 2(TG2) induces the intrinsic EGFR-TKI resistance in EGFR-mutant NSCLC
Authors: Choi Junyoung , Yang Hannah , Kim Sang-We , Jung Kyung Hae , Park Kang-Seo , Lee Dae Ho
Published: July 2017

The non-small cell lung cancer (NSCLC) patients with EGFR-sensitive mutations can be therapeutically targeted by EGFR tyrosine kinase inhibitors (EGFR-TKI), such as erlotinib. Unfortunately, even though the intrinsic EGFR-TKI resistant patients harboring the EGFR-sensitizing mutation, a part of those are still resistant to EGFR-TKI. And, it has been reported that both PTEN loss and NF-kB activation contribute to intrinsic EGFR-TKI resistance in EGFR-mutant lung cancer. Transglutaminse 2 (TG2) is post-translational modification enzyme and known to induce degradation of tumor suppressors including PTEN and IκB-α with peptide cross-linking activity. Because TG2 was known as a regulator of PTEN and IκB-α (NF-κB inhibitor) level in cytosol, we have explored if TG2 can be another key regulator to the intrinsic resistance of EGFR-TKI in the intrinsic EGFR-TKI resistant NSCLC cell. In NSCLC with EGFR-sensitive mutations, we first found that higher TG2 expression level and lower PTEN and IκB-α expression levels in the intrinsic EGFR-TKI resistant NSCLC compare with EGFR-TKI sensitive NSCLC. TG2 stably expressing EGFR-TKI sensitive NSCLC cells harboring EGFR mutations showed reduction of both PTEN and IκB-α and exhibited EGFR-TKI resistance. In reverse, TG2 stably downregulated H1650, intrinsic EGFR-TKI resistant NSCLC cell harboring EGFR sensitive mutation, restored EGFR-TKI sensitivity via PTEN and IκB-α restoration by TG2 loss. Xenograft study using TG2 overexpressed and downregulated NSCLC cells also verified our in vitro results. Therefore, we have demonstrated that TG2 elicits the intrinsic EGFR-TKI resistance via PTEN loss and activation of NF-κB pathway. These results suggest that TG2 may be a useful target for overcoming the intrinsic EGFR-TKI resistance of NSCLC harboring EGFR sensitive mutations.

Citation Format: Junyoung Choi, Hannah Yang, Sang-We Kim, Kyung Hae Jung, Kang-Seo Park, Dae Ho Lee. Transglutaminase 2(TG2) induces the intrinsic EGFR-TKI resistance in EGFR-mutant NSCLC [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 4096. doi:10.1158/1538-7445.AM2017-4096