bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2069: Overcoming drug resistance by targeting melanoma dedifferentiation through information-theoretic analysis and single cell proteomics
Authors: SU Yapeng , Wei Wei , Robert Lidia , Tsoi Jennifer , Xue Min , Kim Jungwoo , Graeber Thomas , Levine Raphael , Ribas Antoni , Heath James
Published: July 2017

Despite impressive initial response of BRAF targeted therapy in mestastic melanoma patients, acquired drug resistance, often times derived from tumor heterogeneity, always limit its clinical outcome. A dedifferentiation process named melanocyte to neural crest transition (MNT) has shown to be critical in the early stage resistance development. The dedifferentiation could induce a whole cell state change resulting a drug tolerant state of melanoma cells, which will lead to initial drug resistance. Mathematic modeling indicates the nature of the transition as results of both stochastic phenotypic transition and non-genetic drug selection, which indicates and further proved that the whole cell state transition should be reversible during drug removal. Information theoretic analysis reduced the dimensional of thousands of gene expression level changes as results of only two unbalanced processes onto a cellular steady state. Reversible transition trajectory defined through two processes indicates it to resemble a critical transition in physical systems. Refined single cell proteomic analysis of those proteins that associate with major unbalanced processes identify the critical points of the transition as early as day6, which cannot be resolved from bulk proteomic measurement. By co-targeting the drivers at the critical points in combination with original brafi targeted drug successfully arrested the transition and induced a sustained tumor growth inhibition in multiple patient-derived melanoma cell lines. This study provides us a noval methodology of mining whole transcriptomic transition with information theoretic analysis to reduce the dimensionality and then using single cell proteomic analysis for refining critical points and driver proteins which eventually rationalized a effective combination therapy to stop the drug resistance development early on.

Citation Format: Yapeng SU, Wei Wei, Lidia Robert, Jennifer Tsoi, Min Xue, Jungwoo Kim, Thomas Graeber, Raphael Levine, Antoni Ribas, James Heath. Overcoming drug resistance by targeting melanoma dedifferentiation through information-theoretic analysis and single cell proteomics [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 2069. doi:10.1158/1538-7445.AM2017-2069
Abstract 2096: Bozitinib, a highly selective inhibitor of cMet, demonstrates robust activity in gastric, lung, hepatic and pancreatic in vivo models
Authors: Shih Joe , Zhong Boyu , Shi Hepeng , Xue David , Choy Gavin S. , Redkar Sanjeev
Published: July 2017

Background: cMET is a receptor tyrosine kinase that is located on the cell surface and is activated by the binding of its ligand, hepatocyte growth factor (HGF). In cancer cells, MET can be aberrantly active and cause abnormal signaling, which leads to tumor growth, angiogenesis, and metastasis. In vitro studies have demonstrated that bozitinib (CBT-101, PLB-1001) is a highly selective and specific inhibitor (8 nM) of tumor cell proliferation.

Methods: In-vivo PD studies of gastric (MKN45), lung (LUM858, LU1901, LU2503), hepatic (LIM0612, LIM0801), and pancreatic (KP4) were evaluated. These models covered both the HGF-dependent and HGF-independent mechanisms. Among these models, LUM858, LU1901, LU2503, LIM0612 and LIM0801 are PDX models. In particular, in the LU1901 model, bozitinib (BT) was compared to capmatinib (INC280). Groups included: BT at 1, 3 and 10 mg/kg QD×21 and INC280 at 1, 3, and 10 mg/kg QD×21 and 10 mg/kg BID×21 via IG, CDDP 5 mg/kg, Q7D×3 as a positive control via IP and the vehicle control (QD×21 via IG). Each group (n=8 mice) and the tumor volume was evaluated on D21.

Results: In MKN45, LU2503, LIM0612 and LIM0801, the effect of BT seemed superior than that of crizotinib; in LUM858, its effect was higher than that of erlotinib; in LU1901, its effect was higher than that of crizotinib and INC280. In the LU1901 model, the strongest activity was observed at BT 10 mg/kg with a T/C ratio of 2%, compared to an equi-dose of INC280 (T/C of 22%). All doses of BT and INC280 were well tolerated; no mouse experienced weight loss. In MKN45 model, BT showed a PK/PD correlation and dose-dependence. BT inhibited the phosphorylation of c-Met protein; the rate of target inhibition exceeded 90% at >7 mg/kg. The plasma concentration for BT decreased over time with a significant decrease 16h after its administration, conferring at least 16h of phosphorylation inhibition of the c-Met protein.

Conclusions: In conclusion, BT was well-tolerated, with no animal death nor major weight loss. The in vivo experiments demonstrated that BT is a viable candidate with effective anti-tumor activities. BT is currently under evaluation in cMet dysregulated NSCLC (NCT02896231) with additional trials planned.

Citation Format: Joe Shih, Boyu Zhong, Hepeng Shi, David Xue, Gavin S. Choy, Sanjeev Redkar. Bozitinib, a highly selective inhibitor of cMet, demonstrates robust activity in gastric, lung, hepatic and pancreatic in vivo models [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 2096. doi:10.1158/1538-7445.AM2017-2096
Abstract 2109: Transcriptional analysis of human leukemia cells treated with the experimental anticancer drug laromustine
Authors: Loya Amanda J. , Cincotta Dylan J. , Elkahloun Abdel , Bodine David , Rice Kevin P.
Published: July 2017

Laromustine is an experimental anticancer prodrug that demonstrated significant activity against acute myeloid leukemia in human clinical trials. Upon base-catalyzed activation, laromustine yields two reactive, electrophilic species in situ: 90CE, which can chloroethylate the O6 position of guanine in DNA causing lethal interstrand crosslinks, and methyl isocyanate, which can carbamoylate biochemically relevant nucleophiles, such as cysteine sulfhydryl groups. Previous in vitro and ex vivo investigations into laromustine’s molecular mechanism of action reveal several biochemical consequences of methyl isocyanate, many of which may explain either the acute cytotoxicity of the drug’s carbamoylating activity or the observed synergism with the cytotoxicity of guanine O6 chloroethylation. The combined activities of the electrophilic subspecies of laromustine are believed to induce apoptosis in leukemia cells. Presented herein are extensive flow cytometry experiments using FITC-AnnexinV and propidium iodide suggesting a dose-dependent cytotoxicity that includes apoptotosis. So as to gain a more complete understanding of the drug’s toxicity, an investigation into how laromustine modulates gene transcription in HL60 cells is also carried out and reported here. Total mRNA was harvested from cultured HL60 cells treated with sub-lethal doses of laromustine or analogs lacking either carbamoylating or chloroethylating activity. These samples were subjected to GeneChip analyses against control mRNA. More than 4,000 genes were significantly dysregulated upon the cells’ exposure to laromustine. The analog of laromustine possessing only carbamoylating activity caused the dysregulation of nearly 3,000 genes, the vast majority of which were also dysregulated in response to laromustine. 90CE, which lacks carbamoylating activity but retains chloroethylating activity, understood to be principally responsible for therapeutic cytotoxicity, demonstrated very little effect on gene transcription in HL60 cells. Preliminary analysis of gene pathways affected by laromustine suggests that among the altered pathways, those associated with cancer progression, the G1/S checkpoint, and hematological disorders are particularly stressed. The carbamoylating activity of laromustine is responsible for most of these effects. Identifying genetic pathways disrupted by this potentially useful drug may inform clinical strategies or identify potential targets for co-therapeutic agents.

Citation Format: Amanda J. Loya, Dylan J. Cincotta, Abdel Elkahloun, David Bodine, Kevin P. Rice. Transcriptional analysis of human leukemia cells treated with the experimental anticancer drug laromustine [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 2109. doi:10.1158/1538-7445.AM2017-2109
Abstract 2114: Targeting invasive and drug-resistant PDAC with photodynamic therapy
Authors: Cramer Gwendolyn , Jafari Seyedehrojin , El-Hamidi Hamid , Jones Dustin , Celli Jonathan
Published: July 2017

Pancreatic ductal adenocarcinoma (PDAC) is associated with an extensive desmoplastic reaction and a characteristic extracellular matrix (ECM)-rich stromal microenvironment which is a major determinant of disease progression and therapeutic response. Using 3D cell culture models of PDAC, we recently showed that an invasion-promoting ECM composition leads to differential response to chemotherapy agents and photodynamic therapy (PDT), in which cytotoxic response is triggered by light activation of a photosensitizing agent. In particular, ECM-infiltrating populations with characteristic mesenchymal phenotype display a marked increase in sensitivity to PDT (using the photosensitizer verteporfin), while the same cells exhibit increased chemoresistance. Here, motivated by these findings we systematically disentangle influences associated with the physical and biochemical properties of ECM as regulators of PDAC growth behavior and treatment response. In 3D tumor spheroids transplanted into invasion-promoting type 1 collagen gels, we use riboflavin-mediated photocrosslinking as a tool to regulate ECM stiffness (independent of its concentration), combined with high-content imaging to co-register invasive progression and differential response to chemotherapy and PDT. Increased extent of photocrosslinking correlates with increased collagen stiffness as confirmed by bulk oscillatory rheology, and is associated with a decrease in the invasive velocity (migration speed directed away from the primary transplanted spheroid) of ECM-infiltrating PDAC cells. Preliminary comparisons of PDT and oxaliplatin chemotherapy in low and high collagen crosslinking conditions show that PDT is significantly more effective than oxaliplatin in ECM-invading populations regardless of collagen stiffness. We also compare oxaliplatin chemotherapy and PDT before and after PDAC invasion into Matrigel ECM, and find that PDT efficacy is only enhanced after extensive invasion into the surrounding matrix. These results support the efficacy of PDT in targeting locally invasive PDAC while stressing the importance of elucidating how both biochemical and biophysical ECM interactions regulate therapeutic susceptibilities.

Citation Format: Gwendolyn Cramer, Seyedehrojin Jafari, Hamid El-Hamidi, Dustin Jones, Jonathan Celli. Targeting invasive and drug-resistant PDAC with photodynamic therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2114. doi:10.1158/1538-7445.AM2017-2114
Abstract 2117: TRAIL-induced apoptosis in TRAIL-resistant breast carcinoma
Authors: Manouchehri Jasmine M. , Kalafatis Michael
Published: July 2017

Recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL), the optimized form of the endogenous death ligand TRAIL, shows therapeutic potential for cancer due to its ability to induce apoptosis in cancer cells independent of p53, while exhibiting minimal toxicity to normal cells. Despite this, a majority of breast cancers display resistance to rhTRAIL treatment due to up-regulation of pro-apoptotic proteins, down-regulation of anti-apoptotic proteins, and/or up-regulation of death receptors (DR) 4 and 5. To overcome rhTRAIL resistance, natural compounds have been investigated as sensitizing agents. This study considered the application of the naturally occurring flavonol Quercetin (Q). Q has been shown to have the ability to up-regulate DR5 and down-regulate anti-apoptotic proteins in cancer cells, and thereby, making Q a favorable choice to be employed as a sensitizing agent. The intention of this study was to ascertain the capacity of Q to sensitize rhTRAIL-resistant triple negative breast cancer BT-20 cells and hormone-dependent breast cancer MCF-7 cells to rhTRAIL-induced apoptosis and elucidate the underlying mechanism for Q’s sensitization. Q demonstrated the ability to intensify rhTRAIL’s pro-apoptotic effects in the breast cancer BT-20 and MCF-7 cell lines as detected through Annexin V/PI assays followed by FACS analysis. In comparison to single agent treatments, the cotreatment of Q and rhTRAIL enhanced the induction of the extrinsic pathway of apoptosis as marked by PARP cleavage (a hallmark of apoptosis), activation of caspase 8, and activation of the executioner caspases 3 and 7. The mechanism for Q’s augmentation in breast cancer was determined to be through the down-regulation of c-FLIPL (caspase 8 inhibitor) in a dose-dependent manner. Furthermore, Q promoted the ubiquitination of c-FLIPL facilitating the proteasome-mediated degradation of c-FLIPL in breast cancer. An additional mechanism for Q’s sensitization was displayed in breast cancer BT-20 cells. Q up-regulated DR5 membrane and protein expression in breast cancer BT-20 cells in a dose-dependent manner. RT-PCR analysis revealed that Q’s influence on DR5 expression occurred at the transcriptional level in those breast cancer cells. Thus, these data suggest that the cotreatment of Q and rhTRAIL possesses the therapeutic potential to be an effective anti-breast carcinoma regimen.

Citation Format: Jasmine M. Manouchehri, Michael Kalafatis. TRAIL-induced apoptosis in TRAIL-resistant breast carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2117. doi:10.1158/1538-7445.AM2017-2117
Abstract 2119: Acetylated secretory APE1/Ref-1 induces apoptotic cell death in orthotopic xenografts of triple-negative breast cancer
Authors: Lee Yu Ran , Joo Hee Kyoung , Lee Eun Ok , Park Myoung Soo , Jeon Byeong Hwa , Choi Sunga
Published: July 2017

The anticancer properties of acetylated secretory apurinic/apyrimidinic endonuclease-1 (Ape1/Ref-1) was suggested in triple-negative breast cancer (TNBC) cells; Posttranslational modification, hyperacetylation in MDA-MB-231 cells caused extracellular secretion of acetylated-APE1/Ref-1 (Ac-APE1/Ref-1) and initiated apoptotic cell death by auto-, paracrine binding to the receptor for advanced glycation end products (RAGE). In the present study, we observed potential therapeutic efficacy of Ac-APE1/Ref-1 in preclinical orthotopic models of TNBC in response to hyperacetylation. The extracellular Ac-APE1/Ref-1 was confirmed by proximity ligation assay in hyperacetylated tumor tissue, showing direct binding of Ac-APE1/Ref-1 and RAGE. Treatment of orthotopic TNBC xenografts with acetylating agents induced a strong growth inhibition in the tumor development as observed in computed tomography: it caused an increase of RAGE expression and activation of caspase-3 and PARP. The tumors also exhibited markedly higher count of apoptotic bodies and reduced proliferation index and neovascularization compared with control tumors. However, the Ac-APE1/Ref-1-stimulated apoptotic cell death was remarkably retarded in RAGE-knockout tumor even in hyperacetylation compared with RAGE-overexpressed one. The functional role of secreted Ac-APE1/Ref-1 in hyperacetylated TNBC was confirmed in vivo, demonstrating its relevance to the anticancer agent. Our findings suggest that Ac-APE1/Ref-1 protein possesses potent chemotherapeutic efficacy against TNBC, resistant to standard chemotherapeutic agents, warranting further evaluation as an anticancer agent

Citation Format: Yu Ran Lee, Hee Kyoung Joo, Eun Ok Lee, Myoung Soo Park, Byeong Hwa Jeon, Sunga Choi. Acetylated secretory APE1/Ref-1 induces apoptotic cell death in orthotopic xenografts of triple-negative 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 2119. doi:10.1158/1538-7445.AM2017-2119
Abstract 2121: Deguelin-derivative, L80 suppresses tumor growth and metastasis via inhibition of STAT3 activation in triple-negative breast cancer
Authors: Cho Tae-Min , Oh Eunhye , Sung Daeil , Kim Yoon-Jae , Kim Ji Young , Lee Jeewoo , Seo Jae Hong
Published: July 2017

Triple-negative breast cancer TNBC is associated with aggressive metastasis and poor clinical outcomes due to a lack of effective treatment options. Therefore, new drugs that effectively target both cancer cell proliferation and metastasis are needed to improve clinical outcomes. The 90-kDa molecular chaperone heat-shock protein, HSP90 has been implicated in cancer progression and metastasis by modulating the stabilization and maturation of many oncogenic proteins. Our objective was to investigate the mechanism of action of C-terminal HSP90 inhibitor, deguelin-derivative L80 on TNBC proliferation and metastasis in vitro and in vivo. L80 induced apoptosis and suppression of cell viability in TNBC cell lines via inhibition of Akt activation with concomitant nuclear accumulation of p27. L80 treatment also caused a marked suppression of cell migration and invasion. These responses were associated with inhibition of STAT3 phosphorylation (Tyr705) as evidenced by downregulation of STAT3 downstream target genes including cyclin D1 and survivin. The syngeneic orthotopic mouse model with 4T1 cells, L80 administration resulted in significant reduction in tumor growth together with decreased number of Ki-67-positive cells and downregulation of phospho-STAT3. Double-label immunofluorescence analysis showed that individuals receiving L80 exhibited a marked reduction in co-localization and expression of HIF-1α and HSP90 compared to their control groups in vivo. Finally, we observed that L80 administration significantly suppressed lung metastasis, as determined by in vivo bioluminescent imaging system. Our findings suggest that L80 may be potentially effective for the treatment for metastatic TNBC patients.

Note: This abstract was not presented at the meeting.

Citation Format: Tae-Min Cho, Eunhye Oh, Daeil Sung, Yoon-Jae Kim, Ji Young Kim, Jeewoo Lee, Jae Hong Seo. Deguelin-derivative, L80 suppresses tumor growth and metastasis via inhibition of STAT3 activation in triple-negative 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 2121. doi:10.1158/1538-7445.AM2017-2121
Abstract 2131: Tanshinone IIA can decrease growth factor receptors expression and dural-block both Ras/Raf/MEK/ERK and Ras/PI3K/Akt/mTOR pathways to inhibit human breast cancer BT-20 cells
Authors: Su Chin Cheng
Published: July 2017

Background : Tanshinone IIA (Tan-IIA, C19H18O3) with anti-inflammatory activities and antioxidant properties, is one of the diterpine quinones extracted from Salviae miltiorrhizae radix (Danshen). Tan-IIA can inhibit many human cancer cell lines through different molecular mechanisms. The phosphoinositide-3-kinase (PI3K)/AKT/ mammalian target of rapamycin (mTOR) and RAS/RAF/MEK/ERK pathways are two of the most frequently dysregulated kinase cascades in human cancer. Transmembrane tyrosine kinase has been strongly implicated in the proliferation, survival, and metastasis of human tumors. Both pathways represent important signal transduction mechanisms that facilitate the proliferation and survival of cancers driven by growth factor receptors, such as vascular endothelial growth factor receptor (VEGFR), insulin-like growth factor-I receptor (IGF-IR), or epidermal growth factor receptor (EGFR). Targeting both the Ras/Raf/MEK/ERK and Ras/PI3K/Akt/mTOR Pathways for suppressing inhibitor resistant cells is necessary because the individual downstream components of these signaling cascades either through epigenetic modification or somatic mutation are also known to be frequently altered in cancer, thus contributing to resistance to anticancer therapies and tumorigenesis.

Material and methods: In the present study, the human breast cancer BT-20 cells were treated with Tan-IIA in vitro. The cytotoxicity of Tan-IIA was evaluated by MTT assay. The effects of Tan-IIA on the protein expressions of EGFR, IGF-IR, VEGFR, PI3K, AKT, mTOR, Ras, Raf, MEK and ERK and β-actin in the BT-20 cells were examined by western blot analysis.

Results: The results showed that Tan-IIA can induce the proliferation inhibition with time and dose dependent and inhibit the activity of the Ras/Raf/MEK/ERK and Ras/PI3K/Akt/mTOR pathways. In addition, it was showed that Tan-IIA treatment inhibited the protein expression levels of EGFR, VEGFR and IGF-IR significantly.

Conclusions: These findings indicated that one of the molecular mechanisms for Tan-IIA to inhibit BT-20 cells maybe through inhibiting the protein expression levels of EGFR, VEGFR, IGF-IR and both Ras/Raf/MEK/ERK and Ras/PI3K/Akt/mTOR pathways. The use of Tan-IIA for breast cancer may become a feasible novel therapy option. Further studies are warranted to elucidate its mechanisms fully.

Citation Format: Chin Cheng Su. Tanshinone IIA can decrease growth factor receptors expression and dural-block both Ras/Raf/MEK/ERK and Ras/PI3K/Akt/mTOR pathways to inhibit human breast cancer BT-20 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 2131. doi:10.1158/1538-7445.AM2017-2131
Abstract 2137: JAK2 as a novel therapeutic target in anaplastic thyroid cancer
Authors: Pinto Nicole C. , Ruicci Kara , Prokopec Stephenie , Searle Karlee , Lowerison Matthew , Yoo John , Fung Kevin , MacNeil Danielle , Leong Hon S. , Datti Alessandro , Boutros Paul C. , Barrett John W. , Nichols Anthony C.
Published: July 2017

Introduction: Thyroid carcinoma is the most common endocrine malignancy. Anaplastic thyroid cancer (ATC) is rare (1.3%) and represents arguably the most lethal human malignancy with 1-year survival rates of only 10%. There are currently no effective treatments for the majority of patients, highlighting an urgent need for novel therapeutics to manage this disease.

Objective: To validate the functional importance of JAK2 as a therapeutic target in ATC.

Methods: In this study, we have used siRNA knockdown to interrogate the JAK2 signaling pathway as a therapeutic target in ATC. We investigated the mechanism of action and cell death, and assayed for migration and invasion, in vitro. The chick chorioallantoic membrane (CAM) model was also utilized for drug testing, whereby 1x106 Cal62 ATC cells were on-planted to the chick embryo membrane. Two days post on-plant, CAM models were treated with the vehicle (DMSO) or lestaurtinib to measure outcomes including tumor volume and vascularity.

Results: We identified the JAK2 inhibitor lestaurtinib as an inhibitory agent controlling cell line proliferation at submicromolar mean inhibitory concentrations. Immunoblotting revealed the inhibition of phosphorylation of the downstream signaling molecule STAT5 in a dose-dependent manner. Treatment of Cal62 cells resulted in a decrease in cell migration using the scratch-wound assay. The anti-proliferative effective of lestaurtinib did not cause apoptosis, autophagy or cell senescence. CAM models treated with a 4 uM dose of lestaurtinib showed a significant decrease in both tumor volume and vascularity.

Conclusions: Lestaurtinib was found to provide potent control of ATC cell proliferation and migration, and was also found to decrease tumor growth and vascularity in a CAM model. Knockout studies are underway to confirm that the anticancer effect of this drug is indeed mediated through JAK2 signaling. If validated, JAK2 represents a novel therapeutic target for the treatment of aggressive thyroid cancers.

Citation Format: Nicole C. Pinto, Kara Ruicci, Stephenie Prokopec, Karlee Searle, Matthew Lowerison, John Yoo, Kevin Fung, Danielle MacNeil, Hon S. Leong, Alessandro Datti, Paul C. Boutros, John W. Barrett, Anthony C. Nichols. JAK2 as a novel therapeutic target in anaplastic thyroid 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 2137. doi:10.1158/1538-7445.AM2017-2137
Abstract 2140: Understanding molecular mechanisms underlying heme function in non-small cell lung cancer cells
Authors: Sohoni Sagar , Vidal Chantal , Zhang Li
Published: July 2017

Heme is a central molecule for mitochondrial respiration and for all processes involved in oxygen utilization. Heme serves as a prosthetic group or as a cofactor for a number of oxidative phosphorylation enzymes and other oxygen-utilizing hemoproteins. Heme directly regulates the synthesis, translocation and assembly of these enzyme complexes. Most, if not all, human cells can synthesize and uptake heme from the circulation. A number of epidemiological studies have shown that high heme intake is associated with increased risk of cancer, including lung cancer. To assess the status of heme metabolism in lung cancer cells, we performed a series of experiments in non-small-cell lung cancer (NSCLC) cell lines such as HCC4017, H1299, A549, H2009, H1395, H460 and Calu-3. We then compared results with an immortalized normal lung cell line, HBEC30KT and observed significant increase in rates of heme synthesis and heme uptake in NSCLC cells compared to HBEC30KT. Previous studies carried out in our lab showed intensified mitochondrial respiration and increased levels of heme and hemoproteins in NSCLCs. We examined the effect of heme depletion on mitochondrial biogenesis and function. We observed that mitochondrial functions such as ATP production, NAD/NADH ratios and ROS production are also affected by heme depletion. Proteins involved in mitochondrial biogenesis are significantly affected when cells are grown in heme depleted medium. Studies are currently underway to check how heme depletion affects the interactions of various transcription factors involved in mitochondrial biogenesis and function.

Citation Format: Sagar Sohoni, Chantal Vidal, Li Zhang. Understanding molecular mechanisms underlying heme function in non-small cell lung 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 2140. doi:10.1158/1538-7445.AM2017-2140
Abstract 2144: A novel method for high-throughput discovery of neo-antigens and corresponding T-cell receptors
Authors: Peng Songming , Zaretsky Jesse , Bethune Michael T. , Hsu Alice , Baltimore David , Ribas Antoni , Heath James
Published: July 2017

Immunotherapies that boost the ability of endogenous T cells to destroy cancer cells have demonstrated therapeutic efficacy in several human malignancies. Cytotoxic T cells actively recognize peptide antigens, which are displayed on major histocompatibility complexes (MHCs) on the surface of the malignant cells, to play a central role in cancer immunotherapy. However, efficient methods for identifying the tumor antigens and the corresponding T cell receptors are still limited. We report here a DNA-barcode nanoparticle and microfluidics-based method to sort and enumerate neoantigen-specific T cells, at an unprecedented level of sensitivity. The method also permits the matching of a single neoantigen to a specific CD8+ T cell with the TCR a/b gene sequence. The resultant information informs an immunotherapy regimen that is specific for that patient, which can be used to custom-design effective ACT therapies.

Citation Format: Songming Peng, Jesse Zaretsky, Michael T. Bethune, Alice Hsu, David Baltimore, Antoni Ribas, James Heath. A novel method for high-throughput discovery of neo-antigens and corresponding T-cell receptors [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 2144. doi:10.1158/1538-7445.AM2017-2144
Abstract 2145: Regulation of YAP1 during hypoxia and its novel role in vascular mimicry and angiogenesis
Authors: Singhal Namrata Bora , Chellappan Srikumar
Published: July 2017

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality. As in other solid tumors, angiogenesis is necessary for the growth and progression of NSCLC. Vascular mimicry is a phenomenon in which non-endothelial tumor cells form angiogenic vascular structures. We have shown in the past that NSCLC cancer stem-like cells (CSCs) have unique ability to undergo vascular mimicry. The present study shows that transcriptional co-activator YAP1, the oncogenic component of the Hippo pathway, enables the NSCLC CSCs to form angiogenic tubule-like structures in matrigel. Inhibition of YAP1 or depletion of YAP1 suppressed vascular mimicry of CSCs as well as angiogenic tubule formation by HUVECs in matrigel as well as in a fibrin gel bead assay (FIBA) assay. The NSCLC CSCs were found to have higher mRNA expression of VEGF receptor II (KDR) and Angiopoietin-2 (AngPT-2) and depletion of YAP1 reduced the expression of VEGF, KDR and AngPT-2 mRNA; these promoters were induced by YAP1 in transient transfection assays, suggesting a possible molecular mechanism by which YAP1 regulates angiogenesis. Hypoxia is a strong inducer of tumor angiogenesis, cancer progression and metastasis. Our experiments showed that NSCLC cells grown in hypoxic conditions or treated with hypoxia mimetic DMOG have higher YAP1 mRNA and protein expression. However, such a change was not observed in the expression of YAP1 orthologue, TAZ, nor in the canonical Hippo pathway proteins like LATS1&2, Mst1&2, Sav or Mob. We identified a novel regulation of YAP1 by prolyl hydroxylase PHD2, which is mainly known to regulate HIF1α. PHD2 was found to hydroxylate proline residue(s) in YAP1 in a region between aa 284 to aa 289 as seen by mutational analysis. YAP1 was found to directly bind to PHD2 and depletion of PHD2 or treatment with DMOG which is an inhibitor of prolyl hydroxylases, elevated YAP1 protein levels in the nucleus. Further, YAP1 was found to associate with HIF1α as detected by co-immunoprecipitation experiments and could enhance HIF1α-mediated induction of the VEGF promoter. Proximity ligation assays performed on TMA showed enhanced YAP1 and HIF1α interaction in lung tumor tissues compared to normal cells. Our data suggest a novel regulation of YAP1 in hypoxic environment that supports angiogenesis and tumor growth.

Citation Format: Namrata Bora Singhal, Srikumar Chellappan. Regulation of YAP1 during hypoxia and its novel role in vascular mimicry and angiogenesis [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 2145. doi:10.1158/1538-7445.AM2017-2145
Abstract 2157: P73 isoforms regulate cellular survival and response to treatment in diffuse large B-cell lymphoma
Authors: Hassan Hesham M. , Varney Michelle L. , Althof Pamela A. , Caponetti Gabriel C. , Fu Kai , Weisenburger Dennis D. , Singh Rakesh K. , Dave Bhavana J.
Published: July 2017

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin's lymphoma (NHL) and is curable in more than 60% of the cases. Despite long-term remission achieved with current therapies, relapse occurs in almost one-third of the cases. Rearrangements of chromosome locus 1p36 with ensuing deletion or disruption of TP73, one of the most distally located putative tumor suppressor genes, is frequent in many NHL subtypes, including DLBCL, and confers inferior prognosis. Recent reports in normal B-cells and lymphoma animal models suggest an important role of p73 in B-cell lymphomas; however, the biological significance of p73 isoforms in DLBCL is not clear. TP73 is a member of the TP53 family that shares structural homology and hence is capable of transactivating p53 target genes. We investigated the relationship between rearrangements of chromosome locus 1p36 and p73 expression and examined whether differential expression of p73 isoforms (TAp73 and ΔNp73) correlate with proliferation and apoptosis in DLBCL. We utilized molecular techniques in conjunction with fluorescence in situ hybridization (FISH) in untreated histologically confirmed DLBCL patient specimens. In our data set, the 1p36 chromosomal locus was disrupted (mainly heterozygous deletion) in 35% of the cases and was significantly associated with ΔNp73 expression. Subsequent immunohistochemical analysis showed a significant positive correlation between TAp73 and cleaved caspase-3 expression and between ΔNp73 and Ki-67 expression. Additionally, we observed a negative correlation between TAp73 and the anti-apoptotic molecules Bcl-2 and Bcl-6. We then performed in vitro studies in DLBCL cells and modulated the expression of TAp73 and ΔNp73 using expression vectors and siRNA. TAp73-transfected cells were more susceptible to serum deprivation and doxorubicin treatment, conversely the ΔNp73-transfected cells were more resistant to serum deprivation and doxorubicin treatment compared to control cells. Thus, rearrangements of 1p36 consequently deregulates p73 isoform expression in DLBCL. Our data emphasizes the importance of delineating the functional aspect of genetic alterations and underscores the significant role of p73 isoforms in DLBCL pathogenesis and treatment.

Citation Format: Hesham M. Hassan, Michelle L. Varney, Pamela A. Althof, Gabriel C. Caponetti, Kai Fu, Dennis D. Weisenburger, Rakesh K. Singh, Bhavana J. Dave. P73 isoforms regulate cellular survival and response to treatment in diffuse large B-cell lymphoma [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 2157. doi:10.1158/1538-7445.AM2017-2157
Abstract 2158: Targeted intracellular disruption of oncogenic p53/HDM2 and p53/HDMX complexes underlies the therapeutic activity of a stapled peptide drug
Authors: Walensky Loren D. , Wachter Franziska , Morgan Ann , Godes Marina , Bird Gregory
Published: July 2017

The first anti-cancer stapled peptide drug has now been advanced to clinical testing in relapsed human cancers that retain the expression of wild-type p53. A common mechanism for cancer cell suppression of wild-type p53 is overexpression of the negative regulators HDM2 and HDMX, which neutralize p53 through protein interaction. Selective HDM2 inhibitor molecules can effectively reactivate p53 in certain cancers, but the co-expression of HDMX can cause resistance, highlighting the need for dual HDM2/HDMX targeting. We previously developed stapled peptides modeled after the p53 transactivation domain to harness the natural propensity of this alpha-helical motif to engage both targets with high affinity and selectivity. Here, we demonstrate that ALRN-7041, a next-generation, clinical-grade stapled peptide, achieves time-dependent cellular uptake and nuclear localization without membrane perturbation, dose-dependently dissociates p53/HDM2 and p53/HDMX complexes as assessed by real-time protein interaction monitoring in live cells, and impairs the viability of cancer cells bearing wild-type p53 by inducing a surge in p53 protein level. Applying an unbiased statistical approach to determine which biophysical parameters dictate the cellular uptake of stapled peptides, we elucidated the design features of ALRN-7041 that confer intracellular access, providing a roadmap for generating cell-permeable stapled peptides with on mechanism cellular activity for clinical translation. We find that cancer cells exhibiting a signature of HDM2, HDMX, and wild-type p53 co-expression are strikingly susceptible to ALRN-7041, highlighting the therapeutic potential of dual HDM2/HDMX inhibition by a cell-penetrant stapled peptide in human cancer.

Citation Format: Loren D. Walensky, Franziska Wachter, Ann Morgan, Marina Godes, Gregory Bird. Targeted intracellular disruption of oncogenic p53/HDM2 and p53/HDMX complexes underlies the therapeutic activity of a stapled peptide drug [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 2158. doi:10.1158/1538-7445.AM2017-2158
Abstract 2162: MEK signaling marks resistance to synthetic lethality induced by PI3K/Akt and BCL-XL inhibition in PTEN-mutant cancers
Authors: Joshi Raghav , Ren Wenying , Mathew Paul
Published: July 2017

Background: Loss of PTEN represents one of the most common disruptions of tumor suppressor activity in the progression of human neoplasia. There are no established precision medicine approaches defined in PTEN-deficient neoplasms which include prostate, breast, glioblastoma (GBM) and uterine cancers. In PTEN-mutant prostate cancer cells, the PI3K/Akt pathway collaborates with BCL-XL specifically, to regulate the apoptotic threshold (Ren, MCR 2016). Whereas single agent PI3K/Akt inhibitors are ineffective in inducing cell death, combined inhibition of PI3K/Akt and BCL-XL in PTEN-mutant prostate cancer cells results in synergistic apoptosis suggesting a novel therapeutic strategy with translational potential in this important subset of disease.

Hypothesis: We hypothesized that synthetic lethality following combinatorial PI3K/BCL-XL inhibition is conserved across the range of PTEN-mutant cancers.

Methods: PTEN-mutant cell lines (ATCC), validated for PTEN-loss and p-Akt expression, were assessed in apoptosis (cleaved Parp) and cell-viability assays following combined PI3K (Buparlisib, SelleckChem) and BCL-XL (A-1331852, AbbVie) inhibition (1µM each, 24h). Since MEK signaling has been associated with resistance to PI3K pathway inhibition, p-Erk was profiled as a candidate marker of resistance. The contribution of MEK signaling to the apoptotic threshold in p-Erk expressing PTEN-mutant cells was tested with pharmacological MEK inhibition (Trametinib, SelleckChem; 1,3,5 µM 48h) in combination.

Results:

MEK signaling and PI3K/BCL-XL inhibition in PTEN-mutant cancersSourceCell linePTENp-Aktp-ErkSynergistic Apoptosis with PI3K / BCL-XL inhibitionLoss of cell viabilityp-Erk suppression with MEK inhibitionApoptosis with concomitant MEK inhibitionProstatePC3AbsentPresentAbsentYesYesN/AN/AProstateLNCaPAbsentPresentAbsentYesYesN/AN/AProstateC4-2BAbsentPresentAbsentYesYesN/AN/ABreastZR-75-1AbsentPresentWeakYesYesN/AN/AUterineSK-UT-1AbsentPresentPresentNoNoYesYesGBMA-172AbsentPresentPresentNoNoYesNoCervixC33-AAbsentPresentPresentNoNo--

Conclusions: Synthetic lethality in PTEN-mutant tumor cells with PI3K/BCL-XL inhibition was confined to tumor cells without active MEK signaling. MEK inhibition inconsistently overcomes resistance to therapy suggesting heterogeneity of survival pathways in p-erk expressing PTEN-deficient tumors. These data have implications for precision medicine approaches in PTEN-deficient tumors as absence of p-Erk expression may predict for a responsive phenotype.

Citation Format: Raghav Joshi, Wenying Ren, Paul Mathew. MEK signaling marks resistance to synthetic lethality induced by PI3K/Akt and BCL-XL inhibition in PTEN-mutant cancers [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 2162. doi:10.1158/1538-7445.AM2017-2162
Abstract 2163: Combined targeting of MEK and MCL-1 induces apoptosis and tumor regression of KRAS mutant NSCLC
Authors: Hata Aaron N. , Siddiqui Faria M. , Gomez-Caraballo Maria , Bilton Samantha J. , Timonina Daria , Nangia Varuna , Coxon Angela , Caenepeel Sean , Hughes Paul
Published: July 2017

There are currently no effective targeted therapeutic strategies for KRAS mutant non-small cell lung cancer (NSCLC). Single agent MEK inhibitors have demonstrated showed disappointing clinical activity, partly due to inability to induce a robust apoptotic response. Combining MEK inhibitors with BCL-XL/BCL-2 inhibitors may be effective for a subset of KRAS mutant cancers that are dependent on BCL-XL for survival, however this combination is unlikely to be an effective strategy for cancers dependent on MCL-1. We investigated the effect of combining the MEK inhibitor trametinib with a novel MCL-1 inhibitor (compound A), which possesses potent and selective anti-MCL-1 activity in vitro and in vivo, on KRAS mutant cancers. In contrast to colorectal cancer models, which are largely sensitive to combined MEK + BCL-XL inhibition, a subset of cell line and patient-derived mouse xenograft (PDX) KRAS mutant NSCLC models were significantly more sensitive to MEK + MCL-1 inhibition compared to MEK + BCL-XL. To model potential clinical strategies, we tested intermittent dosing regimens and unexpectedly discovered a method strategy for dramatically sensitizing KRAS mutant NSCLC cells to the MEK + MCL-1 combination. These studies provide rationale for the clinical evaluation of combined MEK + MCL-1 inhibitors for KRAS mutant NSCLC.

Citation Format: Aaron N. Hata, Faria M. Siddiqui, Maria Gomez-Caraballo, Samantha J. Bilton, Daria Timonina, Varuna Nangia, Angela Coxon, Sean Caenepeel, Paul Hughes. Combined targeting of MEK and MCL-1 induces apoptosis and tumor regression of KRAS 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 2163. doi:10.1158/1538-7445.AM2017-2163
Abstract 2166: Arsenic trioxide targets BCL6 oncoprotein for degradation in BCL6-dependent diffuse large B-cell lymphoma
Authors: Yue Lok Man , Chau David Hau Wing , Piao Wenying , Tse (Eric) Wai Choi , Kwong Yok Lam
Published: July 2017

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma throughout the world. B-cell lymphoma 6 (BCL6) overexpression is frequently observed in DLBCL. Several recent studies have supported that BCL6 is a critical pathogenic oncoprotein in DLBCL. BCL6 represses various downstream genes, including ATR, TP53 and CDKN1A, thereby impairing DNA repair leading to derangement of cellular proliferation. Most importantly, a specific small molecule inhibitor targeting BCL6 successfully suppressed growth of BCL6-dependent DLBCL cell lines and primary human DLBCL cells, further suggesting that BCL6 plays an important pathogenetic role. Arsenic trioxide (As2O3) has been reported to target various oncogenic proteins, including PML-RARA in acute promyelocytic leukemia, cyclin D1 in mantle cell lymphoma, NPMc+ in acute myeloid leukemia and NPM-ALK in anaplastic large cell lymphoma, for degradation through ubiquitin-proteasome pathway. In this study, we investigated the effects of As2O3 on BCL6 in DLBCL.

As2O3 was found to inhibit cell proliferation and induce cell death via apoptosis in DLBCL cells. More interestingly, BCL6-dependent DLBCL cells were found to show higher sensitivity towards As2O3-induced cytotoxicity. BCL6 was found to be degraded by As2O3 at posttranslational level through the ubiquitin-proteasome pathway. Such degradation led to an upregulation of several downstream targets of BCL6, including PRDM1, CD44 and CD69. Moreover, As2O3 synergized with cisplatin to inhibit cell proliferation and enhance apoptosis in BCL6-dependent DLBCL cell lines. Concomitant treatment with As2O3 and cisplatin further enhanced the phosphorylation of Chk1 and γH2AX in these cells.

In conclusion, our data suggest that As2O3 is a potential therapeutic agent for the treatment of BCL6-dependent DLBCL. On top of that, new combination therapies can be developed to expand the therapeutic spectrum of As2O3 to other neoplasms.

Citation Format: Lok Man Yue, David Hau Wing Chau, Wenying Piao, (Eric) Wai Choi Tse, Yok Lam Kwong. Arsenic trioxide targets BCL6 oncoprotein for degradation in BCL6-dependent diffuse large B-cell lymphoma [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 2166. doi:10.1158/1538-7445.AM2017-2166
Abstract 2174: Inhibition of CDK8 kinase with SEL120-34A allows for a personalized approach in AML
Authors: Rzymski Tomasz , Mikula Michal , Zylkiewicz Eliza , Dreas Agnieszka , Wiklik Katarzyna , Golas Aniela , Wojcik Katarzyna , Masiejczyk Magdalena , Dudzicz Iga , Kucwaj Katarzyna , Statkiewicz Malgorzata , Goryca Krzysztof , Grochowska Aleksandra , Cabaj Aleksandra , Ostrowski Jerzy , Kukliniska Urszula , Brzozka Krzysztof
Published: July 2017

Inhibition of oncogenic transcriptional programs is recognized to be a promising therapeutic strategy. SEL120-34A is a novel inhibitor of Cyclin- dependent kinase 8 (CDK8), which regulates transcription by associating with the Mediator complex. SEL120-34A interacts with the ATP binding site of CDK8 in type I inhibitor fashion and forms several types of interactions, including halogen bonds with the protein’s hinge region and hydrophobic complementarities within its front pocket. Although the compound was only modestly active in solid tumor cell lines, it repressed phosphorylation of STAT5 Ser726 and could differentially inhibit viability of AML and ALL cell lines in vitro and in vivo, along with other type I CDK8 inhibitors. Transcriptomic analysis identified major transcriptional programs altered in responder cell lines, which strongly indicated that apart from repression of survival pathways, CDK8 inhibitors could induce differentiation in cell lines with leukemia stem cells characteristics. Further studies on a large panels of responder and non-responder cell lines identified robust biomarkers which could be used with high confidence for stratification and personalized approach in CDK8-dependent AML cases. Favorable pharmacokinetics, confirmed safety and in vivo efficacy in leukemia models provide the rationale for further clinical development of SEL120-34A.

Citation Format: Tomasz Rzymski, Michal Mikula, Eliza Zylkiewicz, Agnieszka Dreas, Katarzyna Wiklik, Aniela Golas, Katarzyna Wojcik, Magdalena Masiejczyk, Iga Dudzicz, Katarzyna Kucwaj, Malgorzata Statkiewicz, Krzysztof Goryca, Aleksandra Grochowska, Aleksandra Cabaj, Jerzy Ostrowski, Urszula Kukliniska, Krzysztof Brzozka. Inhibition of CDK8 kinase with SEL120-34A allows for a personalized approach in AML [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 2174. doi:10.1158/1538-7445.AM2017-2174