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Mutated KRAS (KRAS*) is a fundamental driver in the majority of pancreatic ductal adenocarcinomas (PDAC). Using an inducible mouse model of KRAS*-driven PDAC, we compared KRAS* genetic extinction with pharmacologic inhibition of MEK1 in tumor spheres and in vivo. KRAS* ablation blocked proliferation and induced apoptosis, whereas MEK1 inhibition exerted cytostatic effects. Proteomic analysis evidenced that MEK1 inhibition was accompanied by a sustained activation of the PI3K–AKT–MTOR pathway and by the activation of AXL, PDGFRa, and HER1–2 receptor tyrosine kinases (RTK) expressed in a large proportion of human PDAC samples analyzed. Although single inhibition of each RTK alone or plus MEK1 inhibitors was ineffective, a combination of inhibitors targeting all three coactivated RTKs and MEK1 was needed to inhibit proliferation and induce apoptosis in both mouse and human low-passage PDAC cultures. Importantly, constitutive AKT activation, which may mimic the fraction of AKT2-amplified PDAC, was able to bypass the induction of apoptosis caused by KRAS* ablation, highlighting a potential inherent resistance mechanism that may inform the clinical application of MEK inhibitor therapy. This study suggests that combinatorial-targeted therapies for pancreatic cancer must be informed by the activation state of each putative driver in a given treatment context. In addition, our work may offer explanative and predictive power in understanding why inhibitors of EGFR signaling fail in PDAC treatment and how drug resistance mechanisms may arise in strategies to directly target KRAS. Cancer Res; 75(6); 1091–101. ©2015 AACR.

supplementary Figures S1-S10. S1 growth and regression of iKRAS tumors. S2 culture comparison. S3 immunohistochemistry of p-erk and p-s6 S4 Phospho rtk array S5 Validation of anti AXL antibodies S6 validationn of the AXL inhibitor in vitro S7 validationn of the AXL inhibitor in vivo S8 efficacy of targeting AXL S9 RTK array of iKRAS cells treated with multiple RTK inhibitors S10 P-ERK levels

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You're viewing a past newsletter from the Planet Texas 2050 grand challenge team at The University of Texas at Austin.

Introduction: Inflammatory breast cancer (IBC) is a distinct and aggressive subtype of locally advance breast cancer associated with increased aldehyde dehydrogenase 1 (ALDH1) positive cancer stem cells (CSCs). IBC is associated with a poor survival rate (40% 5-year survival), with few therapeutic strategies identified that effectively blocked the growth and metastasis of IBC. Our previous in vitro studies revealed that the pan-histone deacetylase inhibitor Suberoylanilide Hydroxamic Acid (SAHA) effectively targets self-renewal of IBC tumor cells. Materials and Methods: The IBC cell line, SUM149 was tagged with the Luciferase gene (SUM149-LUC) and their in vivo growth in immune compromised mice tested in both orthotopic and metastatic settings. Results: SUM149-LUC rapidly develop primary tumors and metastatic lesions at a variety of locations including lung, brain, bone, liver, reproductive organs and adrenal glands. SAHA effectively blocked growth of SUM149 primary tumors and inhibited metastasis, which was synergistic with the microtubule stabilizer, Paclitaxel. The therapeutic efficacy of SAHA was associated with a significant decrease in ALDH1+ CSCs populations. Conclusions: Collectively, these results suggest that strategies of combining agents such as SAHA that target CSCs with Paclitaxel that targets the bulk of proliferating tumor cells warrant further investigation for their potential effectiveness in IBC patients, who have the lowest survival of breast cancer patients and have few therapeutic options. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-12-14.

The widening participation agenda and the growing numbers of international students has led to a growing need for centralised academic language and learning (ALL) support for students in Australian universities. One of the challenges that these units face is providing equitable and sustainable models of academic literacy assistance across the university. Support situated within the disciplines is widely espoused in the literature but can lead to inequities, with some students receiving extensive support and others receiving very little. One relatively small ALL unit has addressed this challenge by working within the generic-to-embedded support continuum. On the generic end are academic skills workshops and generic online access materials, while activities at the embedded-literacy end include collaborations with departmental staff to offer targeted activities to improve students’ academic literacy skills. In between, discipline-specific online materials and individual consultations bridge the gap by addressing the concerns of specific disciplines and specific assignments. This strategy aims to provide support that is both effective and equitable, allowing students to access a range of support throughout their academic journey.

SummaryCurrent treatment regimens for pancreatic ductal adenocarcinoma (PDAC) yield poor 5-year survival, emphasizing the critical need to identify druggable targets essential for PDAC maintenance. We developed an unbiased and in vivo target discovery approach to identify molecular vulnerabilities in low-passage and patient-derived PDAC xenografts or genetically engineered mouse model-derived allografts. Focusing on epigenetic regulators, we identified WDR5, a core member of the COMPASS histone H3 Lys4 (H3K4) MLL (1–4) methyltransferase complex, as a top tumor maintenance hit required across multiple human and mouse tumors. Mechanistically, WDR5 functions to sustain proper execution of DNA replication in PDAC cells, as previously suggested by replication stress studies involving MLL1, and c-Myc, also found to interact with WDR5. We indeed demonstrate that interaction with c-Myc is critical for this function. By showing that ATR inhibition mimicked the effects of WDR5 suppression, these data provide rationale to test ATR and WDR5 inhibitors for activity in this disease.

Endosialin/TEM1/CD248 is a cell surface protein expressed at high levels by the malignant cells of about 50% of sarcomas and neuroblastomas. The antibody–drug conjugate (ADC) anti-endosialin-MC-VC-PABC-MMAE was selectively cytotoxic to endosialin-positive cells in vitro and achieved profound and durable antitumor efficacy in preclinical human tumor xenograft models of endosialin-positive disease. MC-VC-PABC-MMAE was conjugated with anti-endosialin with 3–4 MMAE molecules per ADC. The anti-endosialin-MC-VC-PABC-MMAE conjugate was tested for activity in four human cell lines with varied endosialin levels. The HT-1080 fibrosarcoma cells do not express endosialin, A-673 Ewing sarcoma cells and SK-N-AS neuroblastoma cells are moderate expressers of endosialin, and SJSA-1 osteosarcoma cells express very high levels of endosialin. To determine whether endosialin expression was maintained in vivo, A-673 Ewing sarcoma, SK-N-AS neuroblastoma, and SJSA-1 osteosarcoma cells were grown as xenograft tumors in nude mice. The SK-N-AS neuroblastoma and the A-673 Ewing sarcoma lines were selected for in vivo efficacy testing of the anti-endosialin-MC-VC-PABC-MMAE conjugate. The treatment groups included a vehicle control, unconjugated anti-endosialin, an admix control consisting of anti-endosialin and a dose of free MMAE equivalent to the dose administered as the ADC, and the anti-endosialin-MC-VC-PABC-MMAE conjugate. The unconjugated anti-endosialin had no antitumor activity and resulted in similar tumor growth as the vehicle control. The admix control produced a modest tumor growth delay. Administration of the anti-endosialin-MC-VC-PABC-MMAE conjugate resulted in a marked prolonged tumor response of both xenograts. These proof-of-concept results break new ground and open a promising drug discovery approach to these rare and neglected tumors. Mol Cancer Ther; 14(9); 2081–9. ©2015 AACR.

Mutated KRAS (KRAS*) is a fundamental driver in the majority of pancreatic ductal adenocarcinomas (PDAC). Using an inducible mouse model of KRAS*-driven PDAC, we compared KRAS* genetic extinction with pharmacologic inhibition of MEK1 in tumor spheres and in vivo. KRAS* ablation blocked proliferation and induced apoptosis, whereas MEK1 inhibition exerted cytostatic effects. Proteomic analysis evidenced that MEK1 inhibition was accompanied by a sustained activation of the PI3K–AKT–MTOR pathway and by the activation of AXL, PDGFRa, and HER1–2 receptor tyrosine kinases (RTK) expressed in a large proportion of human PDAC samples analyzed. Although single inhibition of each RTK alone or plus MEK1 inhibitors was ineffective, a combination of inhibitors targeting all three coactivated RTKs and MEK1 was needed to inhibit proliferation and induce apoptosis in both mouse and human low-passage PDAC cultures. Importantly, constitutive AKT activation, which may mimic the fraction of AKT2-amplified PDAC, was able to bypass the induction of apoptosis caused by KRAS* ablation, highlighting a potential inherent resistance mechanism that may inform the clinical application of MEK inhibitor therapy. This study suggests that combinatorial-targeted therapies for pancreatic cancer must be informed by the activation state of each putative driver in a given treatment context. In addition, our work may offer explanative and predictive power in understanding why inhibitors of EGFR signaling fail in PDAC treatment and how drug resistance mechanisms may arise in strategies to directly target KRAS. Cancer Res; 75(6); 1091–101. ©2015 AACR.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers in western countries, with a median survival of 6 months and an extremely low percentage of long-term surviving patients. KRAS mutations are known to be a driver event of PDAC, but targeting mutant KRAS has proved challenging. Targeting oncogene-driven signalling pathways is a clinically validated approach for several devastating diseases. Still, despite marked tumour shrinkage, the frequency of relapse indicates that a fraction of tumour cells survives shut down of oncogenic signalling. Here we explore the role of mutant KRAS in PDAC maintenance using a recently developed inducible mouse model of mutated Kras (Kras(G12D), herein KRas) in a p53(LoxP/WT) background. We demonstrate that a subpopulation of dormant tumour cells surviving oncogene ablation (surviving cells) and responsible for tumour relapse has features of cancer stem cells and relies on oxidative phosphorylation for survival. Transcriptomic and metabolic analyses of surviving cells reveal prominent expression of genes governing mitochondrial function, autophagy and lysosome activity, as well as a strong reliance on mitochondrial respiration and a decreased dependence on glycolysis for cellular energetics. Accordingly, surviving cells show high sensitivity to oxidative phosphorylation inhibitors, which can inhibit tumour recurrence. Our integrated analyses illuminate a therapeutic strategy of combined targeting of the KRAS pathway and mitochondrial respiration to manage pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) continues to have a poor prognosis despite new drugs advancing to the clinic. We recently characterized a population of cells able to survive the genetic and pharmacologic extinction of oncogenic pathways and demonstrated that the surviving cells (SC) are tumor stem cells (CSC) able to remain in a quiescent state for months before relapsing. In-depth transcriptomics and metabolomics analyses revealed SCs to exhibit different metabolic features compared to the bulk of tumor cells. Specifically, SC relied on mitochondrial respiration (OXPHOS) and displayed impaired glycolytic capacity. In accord with their decreased dependence on glycolysis these SC were highly sensitive to OXPHOS inhibitors, which prevented tumor recurrence. Notably, preliminary results in patients suggest that SC resistant to conventional chemoradiation also have similar features. We analyzed specimens after neoadjuvant treatment and observed dormant cells positive for CSC markers and characterized by an increased mitochondrial mass. Evaluation of TMA of hundreds of treated tumors revealed that high mitochondrial content is a common feature of SC. Furthermore, functional metabolic characterization of human cells resistant to gemcitabine revealed that SC have a severely impaired glycolytic reserve, closely resembling mouse SC. Based on these findings, we posit that OXPHOS inhibitors may be an effective adjuvant therapy to eradicate resistant cells in patients. To validate the efficacy of OXPHOS inhibition, we developed a new platform to study tumor evolution in response to treatments based on clonal tracking. Lentivirus-based systems have been extensively used as a tool to investigate the clonal dynamics, but they have been limited by lack of sensitivity and the impossibility of tracking identical clones in different animals. Here, using a new version of the barcoded technology coupled with deep-sequencing, we track hundreds of thousands of clones at the single-cell level. We generated patient-derived xenograft animal cohorts in which tumors were clonal replicas of each other (each tumor is maintained by the same clones of all other tumors), representing a unique tool to evaluate responses to treatments. More importantly, resistant tumor clones generated in vivo can be isolated and fully characterized and compared to pre-treatment clones to identify new mechanisms of resistance. Our integrated analysis paves the way for new therapeutic strategies for patients to eradicate treatment-resistant CSC by specifically targeting their unique metabolism. In addition, our clonal tracking-based platform monitoring the efficacy of different treatments in eradicating resistant clones represents an unprecedented tool for exploring treatment responses at the single-cell level, which will help guide the development of personalized treatments. Citation Format: Denise Corti, Alessandro Carugo, Seth Sahil, Matteo Marchesini, Piergiorgio Pettazzoni, Luigi Nezi, Tessa Green, Joseph R Marszalek, Maria Emilia Di Francesco, Timothy P Heffernan, Giulio F Draetta, Andrea Viale. Metabolic eradication of treatment resistant cancer stem cells in pancreatic tumors: A clonal tracking-based platform for identifying the best personalized treatment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 976. doi:10.1158/1538-7445.AM2015-976

Pancreatic ductal adenocarcinoma (PDAC) continues to have a poor prognosis despite extensive characterization of underlying genetic lesions. Although KRAS mutation is a signature driver event required for tumor development and maintenance, all the attempts made to target oncogene-driven signaling pathways have been met with minimal impact on survival and tumors eventually relapse, suggesting that ablation of oncogenic pathways fails to eradicate tumor cells. We used a genetically engineered mouse model of PDAC in which we could control the expression of mutant KRAS in a time- and tissue-specific manner to study the effects of ablating oncogenic signaling on PDAC cells. As previously described (Ying et al. 2012), this model showed rapid tumor growth when KRAS was expressed, followed by robust tumor regression upon oncogene ablation. However a small population of tumor cells survive genetic and pharmacological inactivation of KRAS. These surviving cells are tumor stem cells able to remain in a quiescent state for months before repopulating the original tumor upon reactivation of KRAS signaling. In depth transcriptomic and metabolomic analyses revealed surviving cells have different metabolic features with respect to the bulk of tumor cells, showing a prominent expression of genes governing mitochondrial function, β-oxidation and autophagy, as well as strong reliance on mitochondrial respiration, yet these cells also displayed impaired glycolytic capacity. In accord with their decreased dependence on glycolysis for cellular energetics and their complete absence of glycolytic reserve, these PDAC surviving tumor stem cells show high sensitivity to oxidative phosphorylation inhibitors, which prevented tumor recurrence. Our integrated analysis paves the way for new therapeutic strategies combining KRAS signaling and oxidative phosphorylation inhibition to completely eradicate pancreatic ductal adenocarcinoma. This abstract is also presented as Poster A87. Citation Format: Andrea Viale, Piergiorgio Pettazzoni, Costas Lyssiotis, Haoqiang Ying, Nora Sánchez, Matteo Marchesini, Alessandro Carugo, Tessa Green, Sahil Seth, Virginia Giuliani, Timothy Heffernan, Alec Kimmelman, Huamin Wang, Jason Fleming, Lewis Cantley, Ronald DePinho, Giulio Draetta. Pancreatic tumor stem cells resistant to inhibition of oncogenic signaling are dependent on mitochondrial function. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr PR13.

This abstract is also being presented as a short talk in Session 7: New Therapies. A full abstract is printed in the Proffered Abstracts section (PR13) of the Conference Proceedings. Citation Format: Andrea Viale, Piergiorgio Pettazzoni, Costas Lyssiotis, Haoqiang Ying, Nora Sánchez, Matteo Marchesini, Alessandro Carugo, Tessa Green, Sahil Seth, Virginia Giuliani, Timothy Heffernan, Alec Kimmelman, Huamin Wang, Jason Fleming, Lewis Cantley, Ronald DePinho, Giulio Draetta. Pancreatic tumor stem cells resistant to inhibition of oncogenic signaling are dependent on mitochondrial function. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A87.

TGFβ plays a key role in metastasis by promoting epithelial to mesenchymal transition, up-regulating proteases that degrade extracellular matrix, enhancing tumor cell motility and suppressing antitumor immunity. Previously we have demonstrated that neutralizing TGFβ with the antibody 1D11 significantly inhibited metastasis in preclinical models of breast cancer and melanoma, with minimal effects on primary tumor growth. Neutralizing the protumorigenic effects that TGFβ has on the immune system and tumor microenvironment could, hypothetically, enhance the direct anti-tumor effects of chemotherapeutic agents. To test this hypothesis, several studies were conducted testing combinatorial therapies with 1D11 and chemotherapeutics against subcutaneous (SQ) 4T1, a murine model of triple negative breast cancer (TNBC), with subsequent metastasis to the lung. Paclitaxel is currently the front-line therapy for TNBC in the clinic. Combining 1D11 with paclitaxel had little effect on enhancing the efficacy of paclitaxel alone against primary tumor growth. However, the combinatorial therapy of 1D11 and paclitaxel inhibited spontaneous metastasis from the primary SQ tumor to the lung to a greater extent than either agent alone. In the clinic TNBC typically becomes resistant to taxane therapy and another treatment that has shown some promise in taxane-refractory TNBC is the combinatorial therapy of capecitabine and ixabepilone. In the 4T1 preclinical model, both capecitabine and ixabepilone inhibited the growth of SQ 4T1 tumors as single agents, and the combination of the two therapeutics resulted in slightly greater efficacy. However, the addition of 1D11 to capecitabine and ixabepilone resulted in even greater efficacy than with the combination alone. Currently, the effects of neutralizing TGFβ with 1D11 during the course of capecitabine/ixabepilone therapy on metastasis in the 4T1 model are being investigated. Additionally, mechanism of action studies are being conducted to determine the mechanisms by which 1D11 is enhancing the efficacy of paclitaxel and the combinatorial therapy. The data from these chemo-combo studies with 1D11 suggests that therapies that target TGFβ may enhance the efficacy of paclitaxel against TNBC metastasis and may potentiate the efficacy of the combinatorial therapy of capecitabine/ixabepilone in taxane-refractory TNBC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3826.

TGFβ plays a key role in metastasis by promoting epithelial to mesenchymal transition, up-regulating proteases that degrade extracellular matrix, enhancing tumor cell motility and suppressing antitumor immunity. Preclinical studies were conducted to determine the effect of neutralizing TGFβ on both primary tumor growth and metastases in in vivo models of experimental and spontaneous metastasis. Treatment with the pan-neutralizing TGFβ antibody, 1D11, as a single agent had limited effect on the growth of established, primary, syngeneic tumors (4T1 mammary carcinomas or B16-F10 melanomas) and did not enhance the efficacy of chemotherapeutic agents against these primary tumors. However, TGFβ antagonism was shown to have a significant effect on inhibiting metastasis to the bone following intracardiac injection of 4T1 cells. In addition, it was demonstrated that neutralization of TGFβ in B16-F10 melanoma models inhibited experimental pulmonary metastases following injection of cells into the tail vein, and inhibited spontaneous metastases from a subcutaneous primary tumor to the draining lymph node. Studies aimed at elucidating the mechanism of action of 1D11 in mice lacking specific immune effector cells suggest that the activity of cytotoxic T lymphocytes is important in the inhibition of metastases. The role of natural killer (NK) cells is less clear; NK cell activity is not required for inhibition of experimental metastasis to the lung, but is critical for the ability of 1D11 to inhibit spontaneous metastasis from a primary tumor to the draining lymph node. Investigation into the effects of neutralizing TGFβ on innate immunity and the tumor microenvironment are currently ongoing. Together these data suggest that neutralization of TGFβ significantly inhibits metastasis and anti-TGFβ strategies may be effective at treating metastatic disease in the clinic. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B244.