bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A56: Loss of wild-type Kras promotes oncogenic Kras-induced leukemogenesis
Authors: Chang Yuan-I , Damnernsawad Alisa , Kong Guangyao , Liu Yangang , Chang Qiang , Zhang Jing
Published: May 2015

Although the Ras genes have long been established as proto-oncogenes, substantial evidence indicates that wild-type (WT) Ras genes act as tumor suppressors in a few solid tumor models. However, recent study by Shannon group discovers that loss of WT Nras does not further promote oncogenic Nras-induced leukemias (Xu et al., 2013), raising the possibility that the earlier finding of WT Ras genes as tumor suppressors restricts to specific Ras isoform(s) and/or specific cell types. Here, we show that in mouse cell lines established from oncogenic Kras-induced acute T-cell lymphoblastic leukemia/lymphoma (T-ALL), WT Kras expression is absent or significantly downregulated due to deletion or epigenetic silencing of the locus. Moreover, WT Kras allele is deleted in ∼30% of primary KrasG12D T-ALL samples but not in pre-leukemia specimens. Our results are consistent with a recent study that WT KRAS allele is deleted in early-stage progenitor ALL patients with an oncogenic KRAS mutation (Zhang, et al., 2012), suggesting that loss of WT Kras expression is a pathological mechanism contributing to T-ALL progression.

To test our hypothesis, we generated compound mice carrying a conditional KrasG12D allele, a floxed WT Kras allele and the Mx1-Cre allele. In the absence of pIpC induction, the leaky expression of Cre is sufficient to induce both oncogenic KrasG12D activation and WT Kras deletion in ∼50% of HSCs. We find that KrasG12D/- mice have a much shorter life span than KrasG12D/+ mice and display more severe leukemia phenotypes, including splenomegaly, leukocytosis, thrombocytopenia, and increase of monocytic cells in peripheral blood and spleen. Bone marrow transplantation experiment confirms that loss of WT Kras promotes oncogenic Kras-induced leukemias in a cell-autonomous manner. Further studies demonstrate that loss-of-WT Kras attenuates KrasG12D HSC depletion and enhances GM-CSF signaling in KrasG12D hematopoietic stem/progenitor cells (HSPCs). We are continuously working on the underlying mechanisms. Taken together, our study elucidates the cross-talk between WT and oncogenic Kras in leukemogenesis and provides mechanistic insights of the WT Kras function as a tumor suppressor.

Citation Format: Yuan-I Chang, Alisa Damnernsawad, Guangyao Kong, Yangang Liu, Qiang Chang, Jing Zhang. Loss of wild-type Kras promotes oncogenic Kras-induced leukemogenesis. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A56. doi: 10.1158/1557-3125.RASONC14-A56
Abstract B18: FOXM1 transcription factor is required for the initiation of lung tumorigenesis by oncogenic KrasG12D
Authors: Wang I-Ching , Ustiyan Vladimir , Zhang Yufang , Cai Yuqi , Kalin Tanya V. , Kalinichenko Vladimir V.
Published: May 2015

Lung cancer is the leading cause of deaths in cancer patients in the United States and world wide. Identification of new molecular targets is clearly needed to improve therapeutic outcomes of this devastating human disease. Activating mutations in K-Ras oncogene and increased expression of FOXM1 protein are associated with poor prognosis in patients with non-small cell lung cancer. Transgenic expression of activated KrasG12D in mouse respiratory epithelium is sufficient to induce lung adenocarcinomas, however, transcriptional mechanisms regulated by K-Ras during the initiation of lung cancer remain poorly understood. FOXM1 transcription factor, a downstream target of K-Ras, stimulates cellular proliferation during embryogenesis, organ repair and tumor growth, but its role in tumor initiation is unknown. In the present study, we used transgenic mice expressing KrasG12D under control of Sftpc promoter to demonstrate that FOXM1 was induced in type II epithelial cells prior to the formation of lung tumors. Conditional deletion of Foxm1 from KrasG12D-expressing respiratory epithelium prevented the initiation of lung tumors in vivo. The loss of Foxm1 inhibited expression of K-Ras target genes critical for the NF-κB and JNK pathways, including Ikbkb, Nfkb1, Nfkb2, Rela, Jnk1, N-Myc, Pttg1 and Cdkn2a. Transgenic overexpression of activated FOXM1 mutant was sufficient to induce expression of these genes in alveolar type II cells. FOXM1 directly bound to promoter regions of Ikbkb, Nfkb2, N-Myc, Pttg1 and Cdkn2a, indicating that these genes are direct FOXM1 targets. FOXM1 is required for K-Ras-mediated lung tumorigenesis by activating genes critical for the NF-κB and JNK pathways.

Citation Format: I-Ching Wang, Vladimir Ustiyan, Yufang Zhang, Yuqi Cai, Tanya V. Kalin, Vladimir V. Kalinichenko. FOXM1 transcription factor is required for the initiation of lung tumorigenesis by oncogenic KrasG12D. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B18. doi: 10.1158/1557-3125.RASONC14-B18
Abstract B19: Rapid in vivo screen of pancreatic ductal adenocarcinoma therapeutics
Authors: Ocal Ozhan , Heath Blake , Rivera Lee B. , Pashkov Victor , Swift Galvin H. , Brekken Rolf A. , Wilkie Tom M.
Published: May 2015

Pancreatic ductal adenocarcinoma (PDA) is the fourth leading cause of cancer related deaths. Limited progress has been made in developing effective therapy for PDA. Compounding this challenge has been the lack of a systematic and robust in vivo screen for effective drug combinations. Kras mutations (e.g. KrasG12D) are found in over 90% of human PDA and occur early in tumor progression. Protein kinase and G-Protein Coupled Receptor (GPCR) signaling can initiate Ras activation. Regulators of G-protein Signaling (RGS) proteins are coincidence detectors that can be induced by and integrate multiple inputs to feedback regulate GPCR signaling. We previously described Rgs16 expression during embryonic and postnatal pancreas development in pancreatic progenitors, endocrine cells and ductal-associated cells. Here, we show that the Rgs16::GFP transgene is a KrasG12D dependent marker of all stages of neoplasia in the p48Cre; LSL-KrasG12D; Cdkn2af/f PDA mice. Although KrasG12D is expressed in embryonic pancreas progenitor cells and in all mature acinar cells, Rgs16::GFP expression in tumors first emerges in ductal pancreatic intraepithelial neoplasms as early as 12 days post birth. The distribution and intensity of expression is proportional to and coincident with tumor burden. Rgs16::GFP expression in acinar cells of distal lobes initiates only after tumor occlusion of proximal ducts. The gene expression signature of primary PDA cells in culture obtained via RNA-seq is closely related to embryonic progenitors of pancreatic ducts and endocrine cells. PDA cells in culture are highly sensitive to the chemotherapic drug gemcitabine mediated cell death. In a proof-of-principle for drug screens, we find PDA weanling mice treated with gemcitabine and Nab-paclitaxel for two weeks have significantly lower Rgs16::GFP expression and reduced tumor size and occurrence. Rgs16::GFP is a novel in vivo reporter of PDA progression and sensitivity to new drug regimens that inhibit KrasG12D mediated oncogenesis.

Supported by NCI-CA161624

Citation Format: Ozhan Ocal, Blake Heath, Lee B. Rivera, Victor Pashkov, Galvin H. Swift, Rolf A. Brekken, Tom M. Wilkie. Rapid in vivo screen of pancreatic ductal adenocarcinoma therapeutics. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B19. doi: 10.1158/1557-3125.RASONC14-B19
Abstract B23: CD4+ T lymphocytes promote pancreatic cancer progression by suppressing anti-tumor immune responses
Authors: Zhang Yaqing , Yan Wei , Yan Wei , Mathew Esha , Bednar Filip , Wan Shanshan , Collins Meredith A. , Evans Rebecca A. , Welling Theodore H. , Vonderheide Robert H. , Magliano Marina Pasca di , Magliano Marina Pasca di
Published: May 2015

Pancreatic cancer, one of the deadliest human malignancies, is associated with oncogenic Kras and is most commonly preceded by precursor lesions known as Pancreatic Intraepithelial Neoplasias (PanINs). PanIN formation is accompanied by the establishment of an immune-tolerant microenvironment. However, the immune contribution to the initiation of pancreatic cancer is currently poorly understood. Here, we show that oncogenic Kras-expressing epithelial cells drive the establishment of an immunosuppressive microenvironment through the recruitment and activity of CD4+ T cells. We further show that CD4+ T cells functionally repress the activity of CD8+ T cells. Elimination of CD4+ T cells uncovers CD8+ T cells anti-neoplastic function, and blocks the onset of pancreatic carcinogenesis. Thus, our studies uncover essential and opposing roles of immune cells during PanIN formation, and provide rationale to explore immune-modulatory approaches in pancreatic cancer.

Citation Format: Yaqing Zhang, Wei Yan, Wei Yan, Esha Mathew, Filip Bednar, Shanshan Wan, Meredith A. Collins, Rebecca A. Evans, Theodore H. Welling, Robert H. Vonderheide, Marina Pasca di Magliano, Marina Pasca di Magliano. CD4+ T lymphocytes promote pancreatic cancer progression by suppressing anti-tumor immune responses. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B23. doi: 10.1158/1557-3125.RASONC14-B23
Abstract B28: Multiplexed mass spectrometry analysis of KRAS-associated metabolic reprogramming in colorectal tumors and cell lines
Authors: Hutton Josiah E. , Zimmerman Lisa J. , Slebos Robbert J. , Li Ming , Liebler Daniel C.
Published: May 2015

Mutational activation of KRAS is associated with increased cellular proliferation and tumorigenicity, and metabolic reprogramming. We have used a mass spectrometry analysis by multiple reaction monitoring (MRM) to quantify the expression of 62 proteins encompassing glycolysis (21 proteins), TCA cycle (14 proteins), pentose phosphate pathway (10 proteins), serine biosynthesis (4 proteins), and 13 other proteins involved in metabolism. We applied this MRM panel to the colon tumor cell line DLD-1 (one mutant (G13D) KRAS allele and one WT allele) and the isogenic derivative cell lines DKO-1 (G13D, -) and DKs-8 (+,-). DLD-1 and DKO-1 cells produce lactate at a significantly higher rate than the Dks-8 cells, indicating metabolic reprogramming reflecting a “Warburg” phenotype. MRM panel comparisons of these cell lines revealed that KRAS mutation was associated with the increased expression of glucose and glutamine transporters, glycolytic enzymes, enzymes involved in serine biosynthesis, and a few TCA cycle enzymes. Comparison of the isogenic KRAS mutant/WT pair to each other and to the DLD-1 parental cell line revealed distinct expression changes for the three different KRAS mutant/WT combinations, which suggests that mutant and WT KRAS play distinct roles in metabolic reprogramming. The MRM panel was also used to analyze 16 human formalin-fixed, paraffin-embedded (FFPE) Stage II colorectal tumors. Although the expression of the metabolic proteins varied from patient to patient, a consensus group of 7 metabolic proteins, including enzymes of glycolysis, were significantly increased in KRAS mutant tumors. These results demonstrate that mutational activation of KRAS induces metabolic reprogramming via signaling through both BRAF and PI3K signaling pathways in colorectal cancer cells by the altered expression of metabolic proteins. (Supported by NIH Grant U24CA159988)

Citation Format: Josiah E. Hutton, III, Lisa J. Zimmerman, Robbert J. Slebos, Ming Li, Daniel C. Liebler. Multiplexed mass spectrometry analysis of KRAS-associated metabolic reprogramming in colorectal tumors and cell lines. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B28. doi: 10.1158/1557-3125.RASONC14-B28
Abstract B31: The RhoGEF GEF-H1 is required for RAS oncogene-driven pancreatic cancer
Authors: Cullis Jane , Meiri Dedi , Jose-Sandi Maria , Kent Oliver , Medrano Mauricio , Mokady Daphna , Tsao Ming , Gingras Anne-Claude , Rottapel Robert
Published: May 2015

Cellular transformation by oncogenic RAS engages the MAPK pathway under strict spatiotemporal regulation by the scaffold protein KSR-1. Here, we find using a whole genome functional shRNA-based screen, that the guanine nucleotide exchange factor GEF-H1 plays a critical role in a positive feedback loop for the RAS/MAPK pathway independently of its RhoGEF activity. GEF-H1 acts as an adaptor protein, linking the PP2A B' subunits to KSR-1, required for the dephosphorylation of KSR-1 S392 and activation of MAPK signaling. GEF-H1 is required for the growth and survival of RASV12-transformed cells and pancreatic xenografts. GEF-H1 expression is induced by oncogenic RAS and is correlated with pancreatic neoplastic progression. Our results therefore identify GEF-H1 as an amplifier of MAPK signaling required for cell transformation and provide mechanistic insight into the progression of RAS-mutant tumors.

Citation Format: Jane Cullis, Dedi Meiri, Maria Jose-Sandi, Oliver Kent, Mauricio Medrano, Daphna Mokady, Ming Tsao, Anne-Claude Gingras, Robert Rottapel. The RhoGEF GEF-H1 is required for RAS oncogene-driven pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B31. doi: 10.1158/1557-3125.RASONC14-B31
Abstract B34: A systems biology approach to elucidate novel drug targets in KRAS mutant tumors
Authors: Wah David A. , Giorgi Federico M. , Petrey Donald , Alvarez Mariano J. , Silkov Tonya , Broyde Josh , Rieckhof Gabrielle E. , Honig Barry , Califano Andrea
Published: May 2015

This abstract is being presented as a short talk in the scientific program. A full abstract is printed in the Proffered Abstracts section (PR09) of the Conference Proceedings.

Citation Format: David A. Wah, Federico M. Giorgi, Donald Petrey, Mariano J. Alvarez, Tonya Silkov, Josh Broyde, Gabrielle E. Rieckhof, Barry Honig, Andrea Califano. A systems biology approach to elucidate novel drug targets in KRAS mutant tumors. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B34. doi: 10.1158/1557-3125.RASONC14-B34
Abstract B37: Prenylation inhibition induces cell death in NRAS mutant melanoma cells
Authors: Garay Tamas , Kenessey Istvan , Laszlo Viktoria , Tovari Jozsef , Berger Walter , Kovacs Laszlo , Timar Jozsef , Hegedus Balazs
Published: May 2015

While targeted therapeutic approaches are now available for the treatment of BRAF mutant melanomas, non-BRAF mutant melanoma still lacks this modality. In order to explore the potential of prenylation inhibition we investigated the response of NRAS or BRAF mutant and double wild-type melanoma to zoledronic acid treatment. Effect of zoledronic acid on proliferation, apoptosis and migration of melanoma cells as well as the activation of downstream elements of the RAS/RAF pathway were characterized in vitro. In vivo subcutaneous xenograft and spleen to liver colonization models were used to evaluate the influence of zoledronic acid treatment on primary tumor growth and metastatic potential of melanoma cells. Zoledronic acid significantly decreased proliferation in NRAS mutant cells as compared to BRAF mutant and double wild-type cells, in vitro. Similarly, TUNEL assay revealed apoptosis induction in an oncogenic mutation specific manner. In line with this finding, activation of ribosomal protein S6 was decreased. Unexpected activation of the downstream effector Erk1/2 and increase in migration was found in BRAF mutant cells. Zoledronic acid failed to show significant synergism with cytotoxic cisplatin or DTIC treatment in vitro. In vivo zoledronic acid did not inhibit the subcutaneous growth of melanoma cells irrespective of oncogenic mutations. In contrast, the lower dose of zoledronic acid resulted in significantly higher liver metastasis in double wild-type cells. Altogether our data demonstrates that prenylation inhibition may be a novel therapeutic approach in NRAS mutant melanoma. However, further investigations are needed to identify drugs that have appropriate pharmacological properties to efficiently target melanoma cells.

Citation Format: Tamas Garay, Istvan Kenessey, Viktoria Laszlo, Jozsef Tovari, Walter Berger, Laszlo Kovacs, Jozsef Timar, Balazs Hegedus. Prenylation inhibition induces cell death in NRAS mutant melanoma cells. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B37. doi: 10.1158/1557-3125.RASONC14-B37
Abstract B46: Stabilized helices targeting the RAS-SOS interaction as inhibitors of RAS-dependent cancer cell growth
Authors: Nickerson Seth , Joy Stephen T. , Arora Paramjit S. , Bar-Sagi Dafna
Published: May 2015

Accumulating evidence supports an essential role for wild-type RAS isoforms in oncogenic RAS-driven tumorigenesis. Recently, we demonstrated that allosteric stimulation of SOS by oncogenic RAS facilitates cross-activation of wild-type RAS and is required for malignancy. To inhibit SOS-mediated activation of RAS we generated stabilized peptide α-helices derived from the catalytic helix of SOS using the hydrogen bond surrogate approach (HBSSOS). Treatment of RAS-mutant cancer cells with HBSSOS inhibits cell proliferation and induces a G2/M phase checkpoint arrest. Importantly, HBSSOS is taken up into cells by macropinocytosis, an endocytic mechanism that is elevated in RAS-mutant cancer cells, and the levels of macropinocytosis for a given cell line correlate inversely with the half maximum effective concentration (EC50) of HBSSOS, indicating preferential uptake of HBSSOS by RAS-mutant cells. Our results identify macropinocytic uptake of stabilized helices as a potential strategy for inhibiting RAS-dependent cancer cell growth.

Citation Format: Seth Nickerson, Stephen T. Joy, Paramjit S. Arora, Dafna Bar-Sagi. Stabilized helices targeting the RAS-SOS interaction as inhibitors of RAS-dependent cancer cell growth. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B46. doi: 10.1158/1557-3125.RASONC14-B46
Abstract B48: Regulation of mutant KRAS protein stability via SMURF2:UBCH5 complex mediated degradation of beta-TrCP1
Authors: Shukla Shirish , Allam Uday Shankar , Ahsan Aarif , Chen Guoan , Beer David G. , Lawrence Theodore S. , Nyati Mukesh K. , Ray Dipankar
Published: May 2015

Attempts to target mutant KRAS have been unsuccessful. Most of the current therapeutic approaches are indirect, mainly via inhibiting KRAS down-stream signaling, which have been marginally successful. Here we report the identification of Smad ubiquitination regulatory factor 2 (SMURF2), a HECT-type ubiquitin ligase (E3) as a critical regulator of mutant KRAS protein stability. We show that the loss of SMURF2 either by si-/sh-RNA mediated gene silencing or by overexpression of a catalytically inactive SMURF2 Cys716Ala (CA) mutant, can cause lysosome-mediated KRAS degradation; whereas, overexpression of wild type SMURF2 enhances KRAS protein stability. Most importantly, we found that mutant KRAS protein is more susceptible to SMURF2 alterations in that mutant protein half-life decreased from >12h in control siRNA-treated cells to <3h with Smurf2 siRNA treatment, whereas only marginal differences were noted for wild-type KRAS protein upon similar treatments. Importantly, this loss of mutant KRAS protein could be rescued by overexpressing a siRNA-resistant (si-R) wild type SMURF2. Our data further show that SMURF2 along with an ubiquitin conjugating enzyme (E2), UBCH5, poly-ubiquitinates and degrades a Ras family E3, beta-Transducing Repeat Containing Protein 1 (beta-TrCP1). Conversely, beta-TrCP1 is accumulated upon the loss of SMURF2, leading to its increased binding and degradation of KRAS. Therefore, as expected, beta-TrCP1 knockdown following Smurf2 siRNA treatment rescues Smurf2 siRNA-mediated mutant KRAS loss. Functionally, we found a positive correlation between SMURF2 and KRAS protein levels among different cancer cell lines and demonstrated that a loss of SMURF2 reduces the clonogenic survival in vitro and prolongs tumor latency of mutant KRAS-dependent cells in vivo. Taken together, we show that SMURF2 catalytic activity is critical in maintaining mutant KRAS protein stability and propose that targeting of SMURF2 may be a unique strategy to degrade mutant KRAS and thus kill mutant KRAS-addicted cancer cells.

Citation Format: Shirish Shukla, Uday Shankar Allam, Aarif Ahsan, Guoan Chen, David G. Beer, Theodore S. Lawrence, Mukesh K. Nyati, Dipankar Ray. Regulation of mutant KRAS protein stability via SMURF2:UBCH5 complex mediated degradation of beta-TrCP1. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B48. doi: 10.1158/1557-3125.RASONC14-B48
Abstract B55: Hypoxanthine wobble base multimutant KRAS2 mRNA PET imaging agent in G12D mice
Authors: Wickstrom Eric , Chen Chang-Po , Wampole Matthew E. , Zhang Kaijun , Paudyal Bishnuhari , Wang Antican , Tripathi Sushil , Kumar Pardeep , Mitchell Edith P. , Lu Bo , Thakur Mathew L. , Gray Brian D. , Mattis Jeffrey A.
Published: May 2015

Activating mutations in KRAS2 oncogene lead to constitutive K-Ras-GTP signals regardless of EGFR signaling, enabling resistance to EGFR-targeted therapies. Some cancers are activated by mutation of the KRAS2 12th codon from the wild type GGU that encodes glycine (G) to a mutant GAU that encodes aspartate (D) or a mutant GUU that encodes valine (V). For this reason, we hypothesize that determining multiple KRAS2 mutations in vivo by external mRNA PET imaging will enable physicians to decide on alternatives to EGFR-directed therapy. We previously pioneered tumor mRNA PET imaging in pancreatic cancer and breast cancer xenografts [Paudyal, et al. (2013) Nuclear Medicine and Biology 40(8):994-9] with peptide nucleic acid (PNA) 12-mers coupled to receptor-targeting peptides and imaging reporter moieties, showing single mismatch specificity. Thus, we designed and synthesized a 12-mer PNA hybridization agent with a hypoxanthine wobble base opposite the middle base of the KRAS2 mRNA 12th codon. At the C-terminus, we included a cyclized tetrapeptide based on insulin-like growth factor 1 (IGF1) to enable IGF1R-mediated cellular uptake. We included an N-terminal N2S2 chelator in order to bind 64Cu. Molecular dynamics and circular dichroism analysis of the wobble base PNA H-bonded to KRAS2 RNA showed preference for mutant A or U over wild type G [Sanders, et al. (2013) Journal of Physical Chemistry B 117(39):11584-95]. We then administered 200 μCi of the [64Cu]PNA wobble base agent, or the complementary G12D agent, or the wild type agent, by tail vein into 3-month-old G12D transgenic mice that developed spontaneous lung tumors. 4 hr and 24 hr later, mice were imaged in a Siemens Inveon microPET/CT scanner. The PET standard uptake value (SUV) with the G12D agent was 1.4, while SUV was 1.3 with the G12DVA wobble base agent, vs. 0.04 with the G12 wild type sequence. The wobble base approach enables detection of three different KRAS2 mutations, G12D, G12V, or G12A, with a single agent. mRNA PET imaging also enables external monitoring of therapeutic efficacy. Supported by NIH CA148565; IP owned by EW/MLT, licensed to MTTI.

Citation Format: Eric Wickstrom, Chang-Po Chen, Matthew E. Wampole, Kaijun Zhang, Bishnuhari Paudyal, Antican Wang, Sushil Tripathi, Pardeep Kumar, Edith P. Mitchell, Bo Lu, Mathew L. Thakur, Brian D. Gray, Jeffrey A. Mattis. Hypoxanthine wobble base multimutant KRAS2 mRNA PET imaging agent in G12D mice. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B55. doi: 10.1158/1557-3125.RASONC14-B55
Abstract IA08: Systematic functional genomics and KRAS
Authors: Hahn William C.
Published: May 2015

Large scale characterization of cancer genomes has led to information regarding the identify, number, and types of alterations found in human tumors. However, the large number of mutations identified to date require complementary approaches to understand the function of these mutated genes and to elucidate pathways involved in cancer initiation and progression. Over the past several years, we have developed genome scale RNAi libraries and open reading frame expression libraries that permit a systematic evaluation of genes involved in cancer initiation and maintenance. We have used these tools to dissect known and novel pathways involved in malignant transformation. In particular, we have dissected the signaling pathways related to one of the most frequently mutated oncogenes KRAS, that has proven to be difficult to target therapeutically. We have identified new components of KRAS effector pathways that are essential for the survival of KRAS driven cancers. We have also developed signatures that allow us to dissect KRAS-driven gene programs that correspond to KRAS-dependence. We have used this information to develop inhibitors of components of KRAS signaling that show promise in KRAS-driven cancer models.

Citation Format: William C. Hahn. Systematic functional genomics and KRAS. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr IA08. doi: 10.1158/1557-3125.RASONC14-IA08
Abstract IA09: Copper is required for oncogenic BRAF signaling and tumorigenesis
Authors: Brady Donita , Crowe Matt , Turski Michelle , Hobbs Aaron , Yao Xiaojie , Chaikuad Apirat , Knapp Stefan , Xiao Kunhong , Campbell Sharon , Thiele Dennis , Counter Chris
Published: May 2015

The BRAF serine/threonine kinase is mutated, typically at V600, to induce an active oncogenic state in a large fraction of melanoma, thyroid, hairy cell leukemia, and to a lesser extent, a wide spectrum of other cancers. BRAFV600E activates the kinases MEK1 and MEK2, which stimulate the MAPK pathway to promote cancer. ATP inhibitors of oncogenic BRAF and MEK1/2 provide a survival advantage in metastatic melanoma, an effect enhanced by co-administering two such inhibitors. Thus, combining multiple approaches to inhibit MAPK signaling is a promising strategy to treat BRAF mutation-positive cancers. We previously found that copper (Cu) influx enhances MEK1 phosphorylation of its substrates ERK1 and ERK2 through a Cu-MEK1 interaction. We now show here that genetic loss of the high affinity Cu transporter Ctr1 or mutations in MEK1 that disrupt Cu binding reduced MAPK signaling and oncogenic BRAFV600E-mediated tumorigenesis. Loss of tumor growth was rescued by expressing a MEK1/5 chimera that phosphorylates ERK1/2 independent of Cu or an activated ERK2. Importantly, Cu chelators used in the treatment of Wilson disease reduced tumor growth of not only BRAFV600E-transformed cells, but also cells resistant to BRAF inhibition. Taken together, these results suggest that Cu-chelation therapy could be repurposed to treat BRAFV600E mutation-positive cancers.

Citation Format: Donita Brady, Matt Crowe, Michelle Turski, Aaron Hobbs, Xiaojie Yao, Apirat Chaikuad, Stefan Knapp, Kunhong Xiao, Sharon Campbell, Dennis Thiele, Chris Counter. Copper is required for oncogenic BRAF signaling and tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr IA09. doi: 10.1158/1557-3125.RASONC14-IA09
Abstract IA13: Discovery of K-Ras inhibitors for the treatment of cancer
Authors: Fesik Stephen W.
Published: May 2015

Aberrant activation of the small GTPase Ras by oncogenic mutation or constitutively active receptor tyrosine kinases (RTKs) results in the deregulation of cellular signals governing growth and survival in cancer. The guanine nucleotide exchange factor Son of Sevenless (SOS) catalyzes the rate-limiting step in the activation of Ras by exchanging GDP for GTP. SOS is therefore a key control point for the propagation of RTK and Ras signaling. Here we report the discovery of small molecules that bind to a unique pocket on the Ras:SOS:Ras complex, increase SOScat-catalyzed nucleotide exchange, and perturb Ras signaling pathways in cells. X-ray crystallographic studies of Ras:SOS:Ras complexed with these small molecules reveal that they bind in a hydrophobic pocket in the CDC25 domain of SOS adjacent to the Switch II region of Ras. The structure-activity relationships exhibited by these compounds can be rationalized on the basis of the x-ray structures of multiple co-complexes. In addition, structure-based mutational analyses indicate that this newly identified pocket is essential for compound activity. As predicted, these molecules increase Ras-GTP levels in cells. However, they unexpectedly inhibit MAPK and PI3K signaling. Our studies suggest a novel way to target K-Ras and offer possible starting points for the discovery of compounds that could be used to treat Ras-driven tumors.

Citation Format: Stephen W. Fesik. Discovery of K-Ras inhibitors for the treatment of cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr IA13. doi: 10.1158/1557-3125.RASONC14-IA13
Abstract IA14: Exploiting context-dependent fragility in oncogenic Ras signaling networks
Authors: White Michael
Published: May 2015

Context-specific molecular vulnerabilities that arise during tumor evolution represent an attractive intervention target class. However, the frequency and diversity of somatic lesions detected among tumors can confound efforts to identify these targets. To help confront this challenge, we have applied parallel screening of chemical and genetic perturbations within a large panel of molecularly annotated non-small cell lung cancer cell lines to identify intervention opportunities tightly linked to molecular response indicators predictive of target sensitivity. This effort revealed actionable targets within onocogenic Ras signaling networks together with collateral vulnerabilities that arise in consequence of oncogenic KRAS expression. Of note, in no case did we find robust synthetic lethal interactions that were attributable solely to the presence or absence of the KRAS oncogene. Rather, detection of significant chemical/genetic interactions required consideration of somatic alterations that co-occur with KRAS mutations at reasonable frequencies in the population. A conceptual extrapolation is that, at least in the setting of lung cancers, KRAS mutation status does not represent a monolithic mechanistic subtype and intervention strategies should account for KRAS-driven vulnerabilities that are specified by discrete genomic contexts.

Citation Format: Michael White. Exploiting context-dependent fragility in oncogenic Ras signaling networks. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr IA14. doi: 10.1158/1557-3125.RASONC14-IA14
Abstract IA16: Identifying KRAS synthetic lethal interactions using systems biology and functional genomics
Authors: Chen Ron , Gwinn Dana , Sweet-Cordero Alejandro
Published: May 2015

Our laboratory uses cross-species genomic approaches to understand the oncogenic effects of point mutations in KRAS. Using cross-species analysis of mouse models of lung cancer and human lung cancer, we have identified “KRAS signatures” of gene expression and used them to identify key drivers of gene regulation downstream of Kras. A key goal of our laboratory is to reverse engineer these oncogenic signatures to identify key transcriptional regulators which could be inhibited for anti-oncogenic therapy. Previously we used this approach to show that Wt1 is required to bypass a senescence response in cells expressing oncogenic KRAS (Vicent et al JCI 2010). We have now carried out a “second generation” synthetic lethal screen using a more comprehensive computational approach to identify “master regulators” of KRAS-driven gene expression. A focused shRNA screen in a large panel of human NSCLC lines confirmed the synthetic lethality of Wt1 in Kras-mutant tumor lines. In addition, we have identified several other likely “hits.” One of these, TCEB1, is a gene involved in regulating both elongation and the hypoxia response. We are currently carrying out further experiments to elucidate the function of TCEB1 and determine why it is required in cells expressing oncogenic Kras.

Citation Format: Ron Chen, Dana Gwinn, Alejandro Sweet-Cordero. Identifying KRAS synthetic lethal interactions using systems biology and functional genomics. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr IA16. doi: 10.1158/1557-3125.RASONC14-IA16
Abstract PR07: Crystal structure of K-Ras G12C bound to an active site inhibitor
Authors: Hunter John , Lim Sang Min , Ficcarro Scott B. , Choi Hwan Geun , Gurbani Deepak , Xie Ting , Marto Jarrod A. , Gray Nathanael S. , Westover Ken
Published: May 2015

Activating K-Ras mutations are common oncogenic events in many cancers but effective direct-acting inhibitors have not been developed. Targeting the nucleotide binding site of GTPases like K-Ras has long been dismissed as a possibility because of the high affinity of these proteins for their natural ligands GTP and GDP. We previously reported the synthesis of a GDP analogue, SML-8-73-1, and a prodrug derivative, SML-10-70-1, which are selective, direct-acting covalent inhibitors of the K-Ras G12C mutant relative to wild-type Ras. Biochemical and biophysical measurements suggested that modification of K-Ras with SML-8-73-1 renders the protein in an inactive state. Here we report a crystal structure of K-Ras G12C labeled with SML-8-73-1. The structure reveals that when bound to SML-8-73-1, K-Ras presents in an inactive conformation. This first-in-class covalent K-Ras inhibitor suggests that irreversible targeting of the K-Ras guanine-nucleotide binding site is a viable therapeutic strategy for inhibition of Ras signaling.

This abstract is also presented as Poster B54.

Citation Format: John Hunter, Sang Min Lim, Scott B. Ficcarro, Hwan Geun Choi, Deepak Gurbani, Ting Xie, Jarrod A. Marto, Nathanael S. Gray, Ken Westover. Crystal structure of K-Ras G12C bound to an active site inhibitor. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr PR07. doi: 10.1158/1557-3125.RASONC14-PR07
Abstract A01: A-803467, a sodium channel blocker, reverses ABCG2-mediated MDR in vitro as well as in vivo
Authors: Anreddy Nagaraju , Kumar Priyank , Patel Atish , Zhang Yun-Kai , Wang Yijun , Kathawala Rishil , Wurpel John D. , Chen Zhe-Sheng
Published: May 2015

The ATP-binding cassette, subfamily G, isoform 2 protein (ABCG2) is a vital member of the ABC transporter superfamily, which has been involved in multidrug resistance (MDR) in cancer. Its diverse range of substrates includes many antineoplastic agents such as doxorubicin and mitoxantrone. ABCG2 expression has been significantly increased in some solid tumors and hematologic malignancies, which is correlated to poorer clinical outcomes. In addition, ABCG2 expression is a distinguishing feature of cancer stem cells, whereby this membranous transporter imparts resistance to the chemotherapeutic drugs. To enhance the chemosensitivity of cancer cells, attention has been focused on MDR modulators. In this study, we investigated the ability of sodium channel blocker, A-803467 to reverse ABCG2-mediated MDR. We found that A-803467 at non-toxic concentration could significantly increase the cellular sensitivity to ABCG2 substrates in drug-resistant cells overexpressing either wild-type or mutant ABCG2. Mechanistic studies indicated that A-803467 (7.5 μM) significantly increased the intracellular accumulation resulted from inhibition of the efflux of mitoxantrone by inhibiting the transport activity without altering expression level of ABCG2 protein. Furthermore, ATPase analysis indicates that A-803467 stimulates the ATPase activity in membranes overexpressing ABCG2. in-vivo results indicating that tumor volume was significantly decreased by combination of A-803467 with topotecan when compared to the topotecan and A-803467 alone group. Our findings suggest that A-803467 has the potential to be used in combination with ABCG2 chemotherapeutic substrates to augment the response in drug resistant cancers.

Citation Format: Nagaraju Anreddy, Priyank Kumar, Atish Patel, Yun-Kai Zhang, Yijun Wang, Rishil Kathawala, John D. Wurpel, Zhe-Sheng Chen. A-803467, a sodium channel blocker, reverses ABCG2-mediated MDR in vitro as well as in vivo. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A01.
Abstract A04: Changes in matrix metalloproteinase expression in SN-38 resistant colorectal cancer cells
Authors: Berg Spencer I T , Cutler Murray J. , Blay Jonathan
Published: May 2015

The matrix metalloproteinases (MMPs) are a complex family of zinc-dependent proteolytic enzymes, which collectively are capable of degrading all components of the extracellular matrix (ECM). The ECM provides structure and support for normal tissues, and acts both as a barrier to and support for cancer cell invasion and metastasis. MMPs are therefore believed to play an important role in regulating the rate of cancer progression. However, the repeated failure of MMP inhibitors in clinical trials, as well as recent discoveries of novel MMP activities and localizations, has led to a re-evaluation of the roles of MMPs to the cancer process.

In colorectal cancer (CRC) the outlook following disease relapse after surgery and initial chemotherapy is poor, due both to drug resistance and further dissemination of disease. We investigated the involvement of MMPs and related molecules in this context.

We have generated a series of derivatives of the human CRC cell line HT29 that are resistant to SN-38, the active metabolite of the chemotherapeutic agent irinotecan. The principal representative cell line, HT29-S, has a slower proliferation rate than parental HT29 cells, and yet forms denser outgrowths in monolayer culture. The abundance and localization of several MMPs and tissue inhibitors of metalloproteinases (TIMPs) have been assessed in both HT29 and HT29-S cells by western blotting of cytosolic and nuclear protein fractions,and by immunofluorescence. HT29-S cells showed a lower expression of MMP1, MMP7, and MMP9, but also TIMP1 and TIMP2, compared to their parental counterparts, with some differences in cellular distribution. In particular, MMP1 gave a strong nuclear signal by both methods in HT29 cells, which was strongly reduced in HT29-S.

A reduction in MMP expression will allow for an increased accumulation of ECM components. This may act through integrin receptors to activate survival pathways and contribute to the chemoresistance observed in HT29-S cells, and may also facilitate the spread of cancer cells by refining the ECM framework in advanced CRC.

Citation Format: Spencer I T Berg, Murray J. Cutler, Jr., Jonathan Blay. Changes in matrix metalloproteinase expression in SN-38 resistant colorectal cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A04.
Abstract A21: Combined treatment increases the efficacy of chemotherapeutics by downregulating of P-glycoprotein
Authors: Samadi Nasser , Mohseni Mahsa , Ganbari Parisa , tabasinezhad Maryam , Yousefi Bahman
Published: May 2015

Introduction: Understanding the mechanisms of chemoresistance is an urgent need for cancer patients. Induction of drug efflux due to overexpression of P-glycoproteins is considered as an important leading cause of multidrug resistance. In this study, we investigated the role of combination treatments of docetaxel and vinblastine in inhibition of proliferation and induction of apoptosis in H1299 cancer cells. Furthermore, the impact of P-glycoprotein activity in changing the effectiveness of the agents was studied.

Methods: Cell proliferation and the percentage of apoptotic cells were assessed using MTT assay and DAPI staining, respectively. The expression of P-glycoprotein was evaluated in gene and protein levels by Real-time RT-PCR and Western blot analysis, respectively.

Results: Combination treatment of the cells decreased the IC50 concentrations for both docetaxel and vinblastine from 30 nM to 15 nM and 30 nM to 5 nM, respectively (p<0.05). P-glycoprotein mRNA level showed a significant up-regulation in the cells incubated with docetaxel or vinblastine (p<0.001). Combination treatment of the cells neutralized P-glycoprotein overexpression noticeably (p<0.05). Adding P-glycoprotein inhibitor to the cells treated with combined docetaxel and vinblastine, enhanced the efficiency of chemotherapeutics considerably.

Conclusion: Combination therapy along with down-regulation of P-glycoproteins can be considered an important strategy to improve the efficacy of therapeutics.

Note: This abstract was not presented at the conference.

Citation Format: Nasser Samadi, Mahsa Mohseni, Parisa Ganbari, Maryam tabasinezhad, Bahman Yousefi. Combined treatment increases the efficacy of chemotherapeutics by downregulating of P-glycoprotein. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A21.
Abstract A26: Analysis of ABCB1, ABCC1 and ABCG2 expression and clinical parameters in 40 primary retinoblastoma cases
Authors: Castro Blanca Elena , Aguilar Eunice , Cabrera Lourdes , Camacho Javier , Hernandez Adriana , Ramón Guillermo , Sadowinski Stanislaw , Orjuela Manuela , Ponce Veronica
Published: May 2015

Multidrug resistance (MDR) is one of the most important mechanisms involved in chemotherapy failure in cancer. ABC transporters constitute a superfamily of pumps that efflux metabolites, xenobiotics and drugs outside the cell. In many types of malignant tumors some of these transporters are overexpressed causing MDR and consequently tumor progression. ABCB1, ABCC1 and ABCG2 are the ABC transporters whose overexpressions are most frequently involved in chemotherapy failure. Eventhough expression of these transporters has been well studied in many types of adult tumors, expression of ABC transporters and correlation with clinical features have been scarcely reported in retinoblastoma, and intraocular malignant tumor occurring in early infancy. Therefore our objective in this work was to determine ABCB1, ABCC1 and ABCG2 expression by qPCR in untreated primary retinoblastoma cases and to explore whether or not correlations with relevant clinical features do exist. Our results on RNA from 40 retinoblastoma cases naive to chemotherapy treatment, show high mRNA expression of ABCC1 followed by ABCB1 and marginal expression of ABCG2, these results are consistent with immunohistochemistry results from others. ABCC1 was found significantly more expressed in bilateral than unilateral cases.

Citation Format: Blanca Elena Castro, Eunice Aguilar, Lourdes Cabrera, Javier Camacho, Adriana Hernandez, Guillermo Ramón, Stanislaw Sadowinski, Manuela Orjuela, Veronica Ponce. Analysis of ABCB1, ABCC1 and ABCG2 expression and clinical parameters in 40 primary retinoblastoma cases. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A26.
Abstract A27: Transposon mutagenesis screen identifies genes conferring resistance to BRAF inhibition in melanoma
Authors: Chen Li , Dastur Anahita , Yin Xunqin , Benes Cyril
Published: May 2015

Drug resistance states can be affected by altered gene expression levels or functions. We sought to systematically identify genes that could confer resistance to BRAF inhibition in BRAF mutant melanoma by applying a novel transposon mutagenesis screening approach. Resistant genes identified in this study include both well-characterized molecules such as BRAF, CRAF, PTEN and many other genes whose functions have not been previously associated with drug resistance, and encompass functional groups such as signaling pathway components, transcription factors, ubiquitination and sumoylation enzymes, and G-protein coupled receptors. Overexpression of these genes also confers resistance to the MEK inhibitor AZD6244 demonstrating a pathway specific activity. The resistance mechanisms are diverse: some demonstrate elevated ERK or PI3K signaling, while others do not appear to be signaling-related. Two genes identified in this study elicit both ERK and AKT signals and respectively increase the EGFR baseline expression and PLX4720-induced phosphorylation. Combined treatment with EGFR and BRAF inhibitors in these two clones demonstrated marked drug synergy. Taken together with recently published studies implicating EGFR in resistance to BRAF our study indicates that multiple mechanisms of resistance to BRAF inhibitors can converge on EGFR signaling. Our study identifies additional genes that could be used to select patients most likely to benefit from treatments combining EGFR inhibitors with BRAF/MEK inhibitors to overcome resistance.

Citation Format: Li Chen, Anahita Dastur, Xunqin Yin, Cyril Benes. Transposon mutagenesis screen identifies genes conferring resistance to BRAF inhibition in melanoma. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A27.