Growth Of Patient-derived Xenograft Tumors Research Articles

Abstract A16: Combinatorial drug testing using 3D microtumors derived from GBM PDX reveals cytotoxic synergisms in pharmacokinomics-informed pathway interactions

Abstract Introduction: Drug testing of Glioblastoma multiforme (GBM) has revealed that many promising preclinical therapies fail when translated to the clinic. Serious limitations in preclinical models (often monolayer, serum-treated immortalized cell models) likely contribute to this translational failure. Although patient-derived xenografts (PDX), in which patient tumors are serially passaged in immunocompromised mice, are potentially superior model systems of human disease, high throughput drug studies in GBM PDX tumors are not feasible due to time, cost and throughput. To overcome these challenges, we have developed a GBM PDX MicroTumor system in which GBM PDX cells are grown in a novel 3D extracellular matrix material (HuBiogel™, Vivo Biosciences Inc., Birmingham, AL). We hypothesized that this 3D MicroTumor system would better emulate in vivo PDX tumor growth while providing a high throughput assay system for drug combination studies that can be performed in a more rapid timeframe. Methods: Six GBM PDX tumor “xenolines” were selected from the UAB Brain Tumor Animal Model Core to represent the 4 known molecular subtypes of GBM: Classical (JX10, X1016, X1046); Proneural (XD456); Neural (JX10) and Mesenchymal (JX22P). Tumor cells were prepared as single cell suspensions from subcutaneously passaged tumors and embedded in HuBiogel™ beads to form MicroTumors using Neuro-basal media devoid of serum. Single and combinatorial drug cytotoxicity studies were performed using MTT mitochondrial viability assays and Calcein AM imaging. Small molecule inhibitors selected for drug testing were: 1) selumetinib-targeting MEK1/2; 2) crizotinib-targeting c-MET and ALK; 3) cediranib-targeting VEGFR, FLT-1, FLT-4, c-KIT, and PDGFR; and 4) WP1066-targeting JAK2/STAT3. Combinatorial interactions were determined by Chou-Talalay combination index (CI) calculation using Calcusyn software. Global kinase signaling (kinomic profiling) was assessed using paired total cell lysates of GBM-PDX from orthotopic tumors grown in athymic nu/nu mice and as 3D MicroTumors. Testing was performed on the PamStation12 high content peptide microarray platform within the UAB Kinome Core. Results: Following optimization of model system conditions, 4 drugs were tested at multiple concentrations to identify effective dose ranges. While crizotinib, cediranib and WP1066 demonstrated significant cytotoxicity in at least 1 of the GBM-PDX MicroTumors tested, selumetinib was ineffective in producing cytotoxicity as a monotherapy. However, in combinatorial drug testing, selumetinib was effective in promoting synergistic activity (CI<1.0) when combined with each of the other drugs in several of the GBM-PDX tested. Conversely, WP1066 demonstrated some antagonistic interactions with the other drugs in certain tumors. Overall, cediranib and crizotinib demonstrated the most consistent level of efficacy both as monotherapies and combinatorial therapies across the GBM-PDX MicroTumors. Kinomic profiles were compared between GBM-PDX MicroTumors and matched GBM-PDX tumor xenolines grown as orthotopic intracranial tumors to confirm kinase signaling fidelity between the model systems. Paired t-test of kinomic phosphopeptide probes revealed only 20 of 246 peptides significantly differed (FDR<0.05) between the model systems suggesting a high level of signaling preservation within the MicroTumors. Conclusions: The GBM-PDX 3D MicroTumor provides a relatively high throughput drug screening model system that displays similar kinase signaling pathway activation as compared to their in vivo intracranial counterpart. Specific synergistic drug combinations were identified through MicroTumor testing. Ongoing drug combination testing of intracranially implanted GBM-PDX will determine how robustly the MicroTumor system predicts in vivo efficacy. Citation Format: Christopher D. Willey, Ashley N. Gilbert, Rachael Shevin, Catherine P. Langford, Christine W. Duarte, Raj Singh, Joshua C. Anderson, G. Yancey Gillespie. Combinatorial drug testing using 3D microtumors derived from GBM PDX reveals cytotoxic synergisms in pharmacokinomics-informed pathway interactions. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A16.

Abstract B12: EIF4E deregulation drives simultaneous expression of B-cell lymphoma oncogenes.

Abstract The eukaryotic translation initiation factor 4E (eIF4E) is an oncogene elevated in a large number of cancers. The oncogenic potential of eIF4E arises from its critical roles in the cytoplasm in the mRNA translation and in the nucleus in the mRNA export of specific subset of transcripts. These transcripts can be regulated at the cytoplasmic, nuclear export or at both levels. We therefore analyzed eIF4E expression in 105 cases of DLBCLs by IHC, and found that is expressed in almost all the tumors (88%) and for the majority of cases (72%), eIF4E was expressed in both cellular compartments. Since elevated eIF4E activity leads to increased protein production that favors a subset of transcripts including prominent DLBCL oncogenes such as MYC and BCL2, we analyzed eIF4E expression in “double-hit” and “triple hit” DLBCLs. All the pts samples and DLBCL cell lines harboring two or more oncogenic mutations expressed eIF4E at high levels, suggesting a potential mechanistic association. Cells engineered to overexpress wild-type eIF4E, or mutants active in translation but not export (S53A) or mutants active in export but not translation (W73A), confirmed that eIF4E regulates the nuclear export and translation of MYC and BCL2, and discovered that this mechanism regulates additional DLBCL oncogenes such as BCL6. EIF4E immunoprecipitation from triple-hit DLBCL nuclear fractions followed by mRNA amplification by QPCR (RIP-QPCR) determined that MYC, BCL2 and BCL6 were direct eIF4E mRNA export targets. Polysomal profiling demonstrated that these oncogenes are also preferentially translated by eIF4E cytosolic activity. Accordingly, treatment of triple-hit DLBCL cell lines with the eIF4E competitive inhibitor ribavirin significantly increased the nuclear entrapment of BCL6, MYC and BCL2 transcripts, decreased their proportion of heavy polysomes and lead to a decrease in their protein abundance. In an extended panel of six double- and triple-hit DLBCL cell lines, ribavirin induced cell killing in all of them at clinically achievable concentrations. To assess the anti-lymphoma effect of ribavirin in a more clinically relevant context, we established a patient derived xenograft (PDX) in NSG mice. The specimen was isolated from a naïve treatment patient harboring a triple-hit ABC-type DLBCL featuring BCL6 translocation (3q27), BCL2 translocation and MYC amplification. The patient presented a treatment-refractory disease. We then expanded PDX into 10 NSG mice and when tumors were palpable, mice were randomized to receive vehicle or ribavirin for 10 days. We found a significant reduction in tumor growth in PDX. We also found that ribavirin induced mRNA nuclear entrapment of BCL2 and BCL6 and decrease protein levels of BCL2, BCL6 and MYC by day 10. We demonstrated that Hsp90 activity is required to maintain eIF4E-containing heavy polysomes in these oncogene transcripts. Accordingly, Hsp90 inhibition with PU-H71 rapidly promotes ribosomal disassembly leading to increased 40S, 60S and monosomes and decreased heavy polysomes. EIF4E target transcripts such as MYC, BCL6 and BCL2 were more affected, consequently decreasing the protein levels of these transcripts. The combination of Hsp90 and eIF4E inhibition reduced more efficiently the protein levels of these transcripts. To determine whether this molecular effect translates into an improved anti-lymphoma effect, we xenografted two triple-hit DLBCL cell lines in SCID mice and we treated them with vehicle control, ribavirin, PU-H71 or the combination for 10 days. We found that the combination of ribavirin with PU-H71 suppressed lymphoma growth more profoundly in both models. This effect was associated with a decrease in proliferation rate (measured by Ki67 staining) and reduced BCL6 expression. There were no macroscopic or microscopic evidence of toxicity in these mice. In summary, our data provides a novel and potentially non-toxic mechanistically-based approach to target B-cell lymphomas harboring multiple oncogene activation. Given the prevalence of simultaneous chromosomal aberrations in other hematological malignancies we envision a wider role for this strategy. Citation Format: Biljana Kraljacic-Culkjovic, Tharu Fernando, Rebecca Goldstein, Charles Mctavish, Jayeshkumar Patel, Shaoning Yang, Fabrizio Tabbo, Ari Melnick, Giorgio Inghirami, Katherine LB Borden, Leandro Cerchietti. EIF4E deregulation drives simultaneous expression of B-cell lymphoma oncogenes. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B12.

IGF1 Receptor Targeted Theranostic Nanoparticles for Targeted and Image-Guided Therapy of Pancreatic Cancer.

Overcoming resistance to chemotherapy is a major and unmet medical challenge in the treatment of pancreatic cancer. Poor drug delivery due to stromal barriers in the tumor microenvironment and aggressive tumor biology are additional impediments toward a more successful treatment of pancreatic cancer. In attempts to address these challenges, we developed IGF1 receptor (IGF1R)-directed, multifunctional theranostic nanoparticles for targeted delivery of therapeutic agents into IGF1R-expressing drug-resistant tumor cells and tumor-associated stromal cells. These nanoparticles were prepared by conjugating recombinant human IGF1 to magnetic iron oxide nanoparticles (IONPs) carrying the anthracycline doxorubicin (Dox) as the chemotherapeutic payload. Intravenously administered IGF1-IONPs exhibited excellent tumor targeting and penetration in an orthotopic patient-derived xenograft (PDX) model of pancreatic cancer featuring enriched tumor stroma and heterogeneous cancer cells. IGF1R-targeted therapy using the theranostic IGF1-IONP-Dox significantly inhibited the growth of pancreatic PDX tumors. The effects of the intratumoral nanoparticle delivery and therapeutic responses in the orthotopic pancreatic PDX tumors could be detected by magnetic resonance imaging (MRI) with IONP-induced contrasts. Histological analysis showed that IGF1R-targeted delivery of Dox significantly inhibited cell proliferation and induced apoptotic cell death of pancreatic cancer cells. Therefore, further development of IGF1R-targeted theranostic IONPs and MRI-guided cancer therapy as a precision nanomedicine may provide the basis for more effective treatment of pancreatic cancer.

Abstract 684: Nelfinavir inhibits the growth of small cell lung cancer cells and patient-derived xenograft tumors

Abstract Small cell lung cancer (SCLC) is an aggressive lung cancer with a poor prognosis that is characterized by frequent metastasis at the time of diagnosis. Although SCLC is more responsive to chemotherapy and radiation therapy compared to other types of lung cancer, cure is difficult because of frequent recurrence. Molecular alterations in SCLC include inactivation of TP53 and RB1, high copy number of Myc family, and amplifications of FGFR1 and SOX2. Additionally, achaete-scute homolog 1 (ASCL1), a transcription factor for development of pulmonary neuroendocrine cells, is a lineage-dependent oncogene for SCLC. New drugs and molecular targets need to be identified. Previously, we reported nelfinavir (NFV), a HIV protease inhibitor, inhibited proliferation of non-small cell lung cancer (NSCLC) cells in vitro and human NSCLC xenograft tumors through induction of the unfolded protein response (UPR). Here we demonstrate that the effect of NFV in SCLC cells and preclinical treatment studies using SCLC patient-derived xenograft (PDX) mouse models. NFV inhibited SCLC cell proliferation and induced cell death in vitro. Activating transcription factor 4 (ATF4), a marker of the UPR, was rapidly increased, whereas Myc was decreased after 4 hr treatment with NFV. AMP-activated protein kinase (AMPK), which plays a role in cellular energy homeostasis, was activated in the early phase of NFV treatment, whereas the mammalian target of rapamycin (mTOR) that is a serine/threonine kinase that regulates cell growth, was inactivated. NFV inhibited the expression of SOX2, ASCL1 and Aurora B in a cell line- and time-dependent manner. Taken together, NFV had pleiotropic effects against multiple targets during the inhibition of SCLC cell proliferation. In vivo, NFV inhibited growth of SCLC PDX tumors, which correlated with induction of the UPR and decreased expression of ASCL1 and Aurora B. Collectively, these findings show that NFV is highly effective in SCLC in vitro and in vivo, suggesting possible value of incorporating NFV into clinical trials for patients with SCLC. Citation Format: Shigeru Kawabata, Nick Connis, Chunyu Zhang, Joell J. Gills, Christine L. Hann, Phillip A. Dennis. Nelfinavir inhibits the growth of small cell lung cancer cells and patient-derived xenograft tumors. [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 684. doi:10.1158/1538-7445.AM2015-684

Abstract 3878: A model to assess clonal evolution of metastatic colorectal cancer during chemotherapy utilizing patient derived xenografts

Abstract Introduction Chemotherapy (FOLFOX) with or without surgical resection is the primary treatment for metastatic colorectal cancer (mCRC). However, intrinsic and acquired resistance to chemotherapy poses a major obstacle. The lack of predictors of response to treatment and of understanding of mechanisms of resistance leads to ineffective chemotherapy and, hence, unnecessary morbidities. Patient-derived xenografts (PDXs) represent a model system to evaluate the effectiveness of treatment regimens utilizing human tumors in the context of “precision medicine.” We have developed a heterotopic PDX model of mCRC that emulates acquired chemoresistance. Methods Tumor samples from treatment naïve or chemo-treated mCRC patients were implanted subcutaneously into immunodeficient mice. At an optimal volume, the tumor was either divided into pieces and implanted subcutaneously or processed into a single cell suspension and implanted into mice. Mice were classified into either 5-fluorouracil, leucovorin, plus oxaliplatin (FOLFOX) (n = 10) or vehicle (n = 5) groups. FOLFOX was dosed weekly at the maximum tolerated dosage. Mice were sacrificed when tumors reached 20mm diameter, at which time a single cell suspension was created for implantation into the next generation. Tumors were passaged using this method until FOLFOX resistance was attained. DNA and RNA were isolated from all samples for genetic analysis. Results Liver metastasis from six patients, including chemo-treated (n = 3) and chemo-naïve (n = 3), were consecutively used to generate PDX lines. PDX tumor growth from one of the patients exhibited no difference between treatment and control. Within the remaining treatment cohorts, chemo-treated PDX tumors overall grew significantly slower than matching untreated PDX tumors at the first passage (p<0.05) regardless of whether the tumor originated from single cell suspension or pieces. However, PDX tumors from the first patient entered into the study showed chemo-resistance at passage 3. Further, innate resistance was seen in some PDXs from the same patient displaying rapid growth at initiation of chemotherapy treatment. Utilizing tumor tissue obtained from this model, we are currently assessing the genetic and epigenetic mechanism(s) responsible for resistance. Conclusion We have developed a PDX model that recapitulates acquired chemoresistance in mCRC, which allows for the genomic characterization of FOLFOX resistant liver metastasis. We are currently defining the clonal architecture of chemotherapy resistant and responsive PDXs with the goal of identifying predictors of response and resistance. The ability to treat a patient and their personalized “model” in real time represents an ideal tool to test response to treatment and further investigate treatment resistance. Citation Format: Julie G. Grossman, Matthew Strand, Ha Dang, Brian White, Timothy Fleming, Peter Goedegebuure, Albert Lockhart, Kian Lim, Timothy Ley, Richard Wilson, Elaine Mardis, Christopher Maher, Ryan Fields. A model to assess clonal evolution of metastatic colorectal cancer during chemotherapy utilizing patient derived xenografts. [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 3878. doi:10.1158/1538-7445.AM2015-3878

Abstract 3097: CDKs inhibitor: potential monotherapy for treatment of triple-negative breast cancer

Abstract Background: Limitless replicative potential is one of the hallmarks of cancer which occur due to dysregulated cell cycle. Cyclin dependent kinases (CDKs) are major regulators of mammalian cell cycle, which are often hyperactivated in cancer. Therefore, inhibition of cyclin dependent kinases represents an attractive therapeutic approach in cancer therapy. Dinaciclib is a CDK1, CDK2, CDK5 and CDK9 inhibitor, currently being tested in clinical trials in patients with advanced malignancies. The goal of this project was to determine the efficacy and the mechanisms of action of Dinaciclib in triple negative breast cancer (TNBC) by using patient derived xenografts (PDXs).The study results will provide more insights in identification of predictive biomarkers that are important for its clinical development. Methods: The efficacy of Dinaciclib was determined in 3 TNBC PDXs and 3 established TNBC cell lines by in vitro cytotoxic assays and calculating the IC50. Colony formation assay was also performed in 3 dimensional matrigel matrixes to confirm in vitro cytotoxic assay results. Cell cycle analysis was done using PI staining to determine the percentage of cells in G2/M phase after Dinaciclib treatment. Apoptosis analysis was done by Annexin V and PI double staining using FACS. Effect of CDK1, CDK2, and CDK9 on cell proliferation and Dinaciclib efficacy was determined by knocking down these proteins using CRISPER-CAS plasmid. Efficacy of Dinaciclib on tumor growth inhibition was also determined in vivo by implanting PDX cell lines in nude mice. Results: All the PDXs and established breast cancer cell lines tested were sensitive to Dinaciclib as demonstrated by IC50 value ranging from 1 to 20nM. Dinaciclib effectively inhibited the colony formation in 3D culture in all the PDXs and established breast cancer cell lines at 10nM dose. Dinaciclib significantly caused cell cycle arrest at G2/M phase after 24 hours of treatment. It also caused apoptosis induction after 24 hours of treatment. Dinaciclib reduced the CyclinB1, phospho-CDK1, Survivin and MYC protein levels after 24 hour of treatment as analyzed by western blotting. Knockdown of CDK1 and CDK2 inhibited cell proliferation by 30% and 50% in MDA-MB-231 cells in vitro. Dinaciclib significantly inhibited PDX tumor growth in vivo. Conclusions: These data suggest that Dinaciclib is effective in inhibiting triple negative breast cancer growth in vitro and in vivo. Dinaciclib induced both cell cycle arrests at G2/M phase and apoptosis in breast cancer cells. Dinaciclib targets multiple proteins like CDK1, CDK2, CyclinB1 and cMYC, which can be further validated as predictive biomarkers. Dinaciclib can be used as a potential monotherapy for TNBC patients in clinics. Citation Format: Sandeep Rajput, Zhanfang Guo, Cynthia Ma. CDKs inhibitor: potential monotherapy for treatment of triple-negative breast cancer. [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 3097. doi:10.1158/1538-7445.AM2015-3097

Abstract 2699: Targeting GSK-3: a novel approach to enhance glioblastoma chemosensitivity

Abstract Glioblastoma (GBM) is, in essence, an incurable cancer, with most patients surviving 12-15 months following initial diagnosis. Previously published studies identified Glycogen Synthase Kinase-3 (GSK-3) as a new therapeutic target in GBM. Because GSK-3beta is a positive regulator of NF-kappaB-mediated survival and chemoresistance in cancer cells, we hypothesize that the inhibition of GSK-3 may overcome NF-kappaB-mediated chemoresistance to conventional drugs in human GBM. Using IVIS imaging of live mice, we found that: 1) NF-kappaB is constitutively active in orthotopic GBM patient-derived xenograft (PDX) tumors expressing an NF-kappa luciferase reporter; and 2) a single intravenous injection of our novel GSK-3 inhibitor 9-ING-41 (25 mg/kg) significantly reduced NF-kappaB transcriptional activity in orthotopic GBM PDX. Using 3 different orthotopic GBM PDX- tumor models, enabled for bioluminescence imaging through luciferase modification, we evaluated antitumor effects of 9-ING-41, alone as well as in combination with irinotecan, CCNU and temozolomide. Our in vivo results revealed that treatment with a combination of 9-ING-41 and irinotecan delays GBM PDX tumor growth, relative to either agent as a monotherapy, and, even more promising, 9-ING-41+CCNU leads to a complete tumor regression. Histological evaluation of mouse brain confirmed the absence of cancer cells and a formation of cyst at the location of intracranial tumor in mice treated with 9-ING-41+CCNU. Our results provide a rationale to advance 9-ING-41 for clinical evaluation in treating GBM, especially when combined with CCNU cytotoxic therapy. Citation Format: Andrey Ugolkov, Oleksii Dubrovskyi, Irina Gaisina, Alex Yemelyanov, Gennadiy Bondarenko, Charles James, James Chandler, Thomas O'Halloran, Alan Kozikowski, Jeffry Raizer, Andrew Mazar. Targeting GSK-3: a novel approach to enhance glioblastoma chemosensitivity. [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 2699. doi:10.1158/1538-7445.AM2015-2699

Abstract A102: Combination treatment with hypoxia-activated prodrug TH-302 and radiation reduce pancreatic tumor initiating cells and tumor growth in patient-derived xenografts

Abstract Rationale: Using orthotopically-grown primary pancreatic cancer xenografts we recently identified an association between hypoxia and spontaneous metastasis formation, and we hypothesized that metastatic potential is conferred by the presence of tumor-initiating cells (TIC). We therefore investigated the relationship between hypoxia, tumor growth and TIC, and examined the effect of the hypoxia-activated prodrug TH-302 and ionizing radiation (IR) on pancreatic TIC and tumor growth. Methods: Eleven patient-derived pancreatic cancer xenograft models were used to examine tumor-initiation potential in relation to the tumor phenotype. To examine the effect of TH-302 and IR on TIC, tumor-bearing mice were treated with 50 mg/kg or 150 mg/kg TH-302, 10Gy of IR, or with the combination of TH-302 and IR. To examine the effect of TH-302 and IR on tumor growth, mice were treated with either TH-302 or IR alone, or with the combination treatment according to a clinically relevant schedule. Animals were treated with 50mg/kg TH-302 injected ip on days 1, 5 and 9 and/or 2Gy irradiation per day on days 2, 3 and 4. Tumor hypoxia was measured by EF5 administration and immunostaining. Results: TIC frequency varied across the patient-derived xenograft models and rapid tumor growth was strongly correlated with high TIC frequency but not hypoxia. Treatment with TH-302 in combination with IR led to a significant reduction in TIC frequency compared with either treatment alone in all models tested. Treatment-induced reduction in TIC frequency was independent of the magnitude of tumor hypoxia. Treatment-induced reduction of tumor growth was only observed in fast-growing hypoxic models. Additionally, TH-302 treatment also induced DNA damage in tumor tissue adjacent to the hypoxic zone which is consistent with the previously demonstrated bystander effect of TH-302. Conclusions: These results suggest that TIC can reside in both the oxic and the hypoxic microenvironment and that the combination of TH-302 and IR may present a novel therapeutic strategy to reduce the frequency of TIC and tumor growth rate of pancreatic tumors. Citation Format: Ines Lohse, Joanna Rasowski, Pinjian Cao, Melania Pintilie, Trevor Do, Emin Ibrahimov, Ming-Sound Tsao, Richard P. Hill, David W. Hedley. Combination treatment with hypoxia-activated prodrug TH-302 and radiation reduce pancreatic tumor initiating cells and tumor growth in patient-derived xenografts. [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 A102.

Effect of selinexor (KPT-330), a novel oral selective inhibitor of nuclear export (SINE), on tumor suppressors and cell cycle proteins in prostate cancer cells and regression of castration-resistant patient-derived xenograft tumor growth.

277 Background: Androgen deprivation, anti-androgen and androgen biosynthesis inhibitor treatment can initially control the metastatic prostate cancer (PCa), but treatment-refractory progression frequently follows, with the loss of tumor suppressors (TSPs) and increased expression of cell cycle proteins. Inhibition of the nuclear export protein, Exportin 1 (XPO1) leads to nuclear accumulation of cargo proteins such as TSPs & cell-cycle regulators implicated in castration-resistant PCa (CRPC) progression. XPO1 and specific cargo genes are overexpressed in metastatic CRPC relative to benign & primary prostate tumors, implicating XPO1 activity as playing a role in disease progression. Selinexor (KPT-330), a novel, oral SINE currently in Phase 1/2 for both hematological and solid tumors, has potent activity against CRPC. We hypothesized this activity is due selinexor induced nuclear expression of TSPs. Methods: To test this hypothesis, we treated selected PCa cell lines and patient-derived xenografts (PDXs, two adenocarcinomas and one small cell carcinonoma) with selinexor to determine the effect on survival and cargo protein localization. Results: Treatment with selinexor markedly inhibited PCa cell proliferation in vitro, activated the tumor suppressor TP53 & inhibited cell-cycle regulators. Also, treatment of the PDXs with selinexor for at least 3 weeks significantly inhibited tumor growth & reduced the prostate-specific antigen level in the adenocarcinomas. Selinexor increased cell death in all three PDX tumors and reduced cell proliferation in the adenocarcinomas, but not in the small-cell tumor. Expression analyses demonstrated that selinexor induced nuclear accumulation of different cargo proteins unique to the PCa model, accounting for PDX-specific regression. Conclusions: These results point to an anti-tumorigenic effect of selinexor treatment across a spectrum of hormone-refractory PCa that may provide insight into the drivers of PCa treatment resistance and heterogeneity.

Concurrent Targeting of KRAS and AKT by MiR-4689 Is a Novel Treatment Against Mutant KRAS Colorectal Cancer.

KRAS mutations are a major cause of drug resistance to molecular-targeted therapies. Aberrant epidermal growth factor receptor (EGFR) signaling may cause dysregulation of microRNA (miRNA) and gene regulatory networks, which leads to cancer initiation and progression. To address the functional relevance of miRNAs in mutant KRAS cancers, we transfected exogenous KRASG12V into human embryonic kidney 293 and MRC5 cells with wild-type KRAS and BRAF genes, and we comprehensively profiled the dysregulated miRNAs. The result showed that mature miRNA oligonucleotide (miR)-4689, one of the significantly down-regulated miRNAs in KRASG12V overexpressed cells, was found to exhibit a potent growth-inhibitory and proapoptotic effect both in vitro and in vivo. miR-4689 expression was significantly down-regulated in cancer tissues compared to normal mucosa, and it was particularly decreased in mutant KRAS CRC tissues. miR-4689 directly targets v-ki-ras2 kirsten rat sarcoma viral oncogene homolog (KRAS) and v-akt murine thymoma viral oncogene homolog 1(AKT1), key components of two major branches in EGFR pathway, suggesting KRAS overdrives this signaling pathway through inhibition of miR-4689. Overall, this study provided additional evidence that mutant KRAS functions as a broad regulator of the EGFR signaling cascade by inhibiting miR-4689, which negatively regulates both RAS/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT pathways. These activities indicated that miR-4689 may be a promising therapeutic agent in mutant KRAS CRC.

Abstract 1438: Targeting metabolic enzyme with locked nucleic acids in non-small cell lung cancer

Abstract Despite advances in our knowledge of genetics of cancer and some progress in cancer treatment over the last few decades, lung cancer remains the leading cause of cancer mortality worldwide. Recently cancer metabolomics have been widely applied into cancer research, including of cancer diagnosis and therapy. Our recent work has identified tumor initiating cells (TICs) derived from non-small cell lung cancer (NSCLC) patients and shown that metabolic enzyme glycine decarboxylase (GLDC) is critical for TICs in NSCLC. In the clinic, aberrant activation of GLDC correlates with poorer survival in lung cancer patients. Those findings provide potential diagnostic markers and/or therapeutic target for NSCLC. Locked nucleic acids (LNAs) are novel high-affinity DNA analogs that can be used as genotype-specific drugs. In this study we tested NSCLC patient-derived xenograft tumor growth inhibition by GLDC LNAs antisense oligonucleotides. These LNAs antisense oligonucleotides strongly reduce GLDC protein levels. The osmotic minipumps were filled with GLDC LNAs antisense oligonucleotides at 0.5,1 and 5 mg/kg/day dose and implanted dorsally into tumor-bearing NOD/SCID Il2rγ-/- mice. Tumor growth was inhibited at dosages from 1 mg/kg/day. LNAs antisense oligonucleotides appeared to be non-toxic at dosages of 5 mg/kg/day. Furthermore, we found significant changes of serum metabolomics in lung cancer patients compared to normal donors. Our data show that GLDC LNAs antisense oligonucleotides are potent genotype-specific drugs that can inhibit lung tumor growth in vivo. Note: This abstract was not presented at the meeting. Citation Format: Wen Cai Zhang, Bing Lim. Targeting metabolic enzyme with locked nucleic acids in non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1438. doi:10.1158/1538-7445.AM2014-1438

Abstract 5544: Patient-derived xenograft study reveals the pharmacology and the role of ESR1 gene aberrations in endocrine therapy resistance of ER positive breast cancer

Abstract Background: The study of estrogen-receptor-positive patient-derived xenografts (PDXs) from patients with disease progression despite endocrine therapy has revealed recurrent ESR1 mutation, gene amplification and ESR1 gene translocations as likely drivers of resistance. We addressed this hypothesis further using pharmacological and genetic approaches in the PDX system. Methodology: ER positive PDXs that harbored one of four recurrent ligand binding domain (LBD) mutations (Y537S/N/C and D538G) as well as a ESR1/YAP1 translocation were studied in parallel with T47D and MCF7 cells expressing these mutants through lentivirus constructs. Single cell suspensions generated from PDX models were using for in vitro experiments using with a panel of endocrine inhibitors and ESR1 shRNA constructs. In vitro effects were confirmed using in vivo tumor response. Results: Cell suspensions from ER+ PDX models in short-term culture successfully reproduced the endocrine therapy resistant phenotypes exhibited by the donor patient, thereby facilitating drugs screens designed to identify new endocrine approaches for ESR1 mutant breast cancer using authentic models rather than an exclusive reliance on models derived by transfection of mutant constructs into standard indicator cell lines. We were also able to demonstrate that that lentivirus-driven shRNA mediated knockdown of ESR1-YAP1 caused inhibition of PDX tumor growth in in vivo and reduced cell growth in vitro, confirming that this chimeric protein is a driver of estradiol-independent growth. Conclusion: ER+ PDX can be manipulated in vitro and in vivo to successfully model ESR1 mutation and gene-rearrangement driven endocrine therapy resistance. PDX studies complement standard ectopic expression systems for the study of ESR1 mutation-driven resistance and, we hypothesize, will provide pharmacological data that will predict the activity of pharmacological agents designed to improve outcomes for patients with ESR1 mutant driven endocrine therapy resistance. Citation Format: Triparna Sen, Shunqiang Li, Jieya Shao, Robert Crowder, Robert Kitchens, Matthew J. Ellis. Patient-derived xenograft study reveals the pharmacology and the role of ESR1 gene aberrations in endocrine therapy resistance of ER positive breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5544. doi:10.1158/1538-7445.AM2014-5544

Human cancer growth and therapy in immunodeficient mouse models.

Since the discovery of the "nude" mouse more than 40 years ago, investigators have attempted to model human tumor growth in immunodeficient mice. Here, we summarize how the field has advanced over the ensuing years owing to improvements in the murine recipients of human tumors. These improvements include the discovery of the scid mutation and development of targeted mutations in the recombination-activating genes 1 and 2 (Rag1(null), Rag2(null)) that severely cripple the adaptive immune response of the murine host. More recently, mice deficient in adaptive immunity have been crossed with mice bearing targeted mutations designed to weaken the innate immune system, ultimately leading to the development of immunodeficient mice bearing a targeted mutation in the gene encoding the interleukin 2 (IL2) receptor common γ chain (IL2rg(null), also known in humans as cytokine receptor common subunit γ). The IL2rg(null) mutation has been used to develop several immunodeficient strains of mice, including the NOD-scid IL2rg(null) (NSG) strain. Using NSG mice as human xenograft recipients, it is now possible to grow almost all types of primary human tumors in vivo, including most solid tumors and hematological malignancies that maintain characteristics of the primary tumor in the patient. Programs to optimize patient-specific therapy using patient-derived xenograft tumor growth in NSG mice have been established at several institutions, including The Jackson Laboratory. Moreover, NSG mice can be engrafted with functional human immune systems, permitting for the first time the potential to study primary human tumors in vivo in the presence of a human immune system.