Chemoresistant Population Research Articles

Abstract 3211: Inhibition of autophagy potentiates cytotoxicity of CX-5461 treatment in chemoresistant epithelial ovarian cancer

Abstract Epithelial ovarian cancer remains a deadly diagnosis with poor prognosis and patients succumbing to chemoresistant disease in 80% of cases. Mechanisms of chemoresistance are numerous, but we have identified ribosomal biogenesis basal machinery as being a common upregulated signaling network in response to chemotherapeutic insult. Tumors treated with chemotherapy showed dramatic increases in size and morphology of nucleoli, suggesting global changes in ribosomal synthesis with stress. We next sought to use the RNA Polymerase I inhibitor CX-5461 to identify biologic changes in targeting ribosomal machinery that may allow resensitization to chemotherapy. CX-5461 induced autophagy, senescence and mitotic catastrophe in ovarian cancer cell lines Overall, 13 ovarian cancer lines examined were highly sensitive to Pol I inhibition by MTT, with IC50s ranging from 25nM to 2μM, and chemoresistant lines were generally more sensitive to CX-5461. In p53 mutant cells CX-5461 appears to be primarily cytostatic with mitotic catastrophe being a relatively rare event accompanying cytokinesis failure. This could be indicative of the cells successfully using autophagy to decrease ribosome requirements and evade treatment. When treated in combination with chloroquine as an autophagy inhibitor IC50s decreased to 550nM in HeyA8 (from 2μM), 70nM for HeyA8MDR (from 191nM), 110nM in SKOV3 (from 595nM) and 60nM for SKOV3TR-ip2 (from 109nM) with abundant multinucleated cells, suggesting synergy between ribosomal synthesis and autophagy inhibitors in vitro. Chloroquine administration appears to push cell to mitotic catastrophe after co-administration with CX-5461, thereby enhancing cytotoxicity over cytostatic endpoints after treatment, even in multi-drug resistant cell populations. Inhibition of autophagy appears to enhance efficacy of RNA Polymerase I inhibition in both sensitive and chemoresistant populations, prompting a potential new approach to chemoresistant disease. Citation Format: Robert Cornelison, Danielle C. Llaneza, Yulia Petrova, Zachary C. Dobbin, David A. Schneider, Charles N. Landen. Inhibition of autophagy potentiates cytotoxicity of CX-5461 treatment in chemoresistant epithelial ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3211. doi:10.1158/1538-7445.AM2017-3211

Abstract 5118: A novel polyamine sulfonamide with anti-leukemic activity

Abstract The greatest challenge in treating Acute Myeloid Leukemia (AML) is refractory disease. Although 60-80% of AML patients achieve complete remission after induction chemotherapy, majority of these patients relapse and die of progressive disease. New treatment options targeting the vulnerabilities of AML biology are highly critical for disease regression, in particular the chemo-resistant leukemia-initiating population, to ensure relapse-free survival in patients. Identifying novel druggable targets that are selective to AML despite their location in the vascular niche is thus highly warranted. We developed an in vitro co-culture model consisting of AML cells and Bone Marrow- derived Endothelial Cells (BMECs), mimicking the protective effect of the vascular niche in the bone marrow microenvironment. Using this unique model, and taking advantage of combinatorial chemistry, we performed high-throughput chemical-phenotypic screening of approximately 30 million novel compounds and identified a novel polyamine sulfonamide 2470-51 that can selectively kill AML cells by overcoming the protective effect of the BMECs. In addition to AML blasts, 2470-51 also exhibited highly selective activity against the leukemic stem and progenitor cell population while sparing normal hematopoietic stem and progenitor cells. In vivo studies using patient-derived xenograft models indicated significant regression in AML engraftment post 2470-51 treatment. Target identification experiments involving unbiased label-free shotgun proteomic analysis in combination with targeted Selected Ion Monitoring (SIM) revealed covalent drug binding targets of 2470-51. By performing differential protein expression analysis using ITRAQ analysis, we identified downstream mechanisms that led to mitotic cell cycle arrest and cell death. Collectively, our findings display the in vitro as well as in vivo efficacy of a novel polyamine sulfonamide in eliminating AML, including the leukemia initiating compartment. We also uncover a novel mechanism in AML that can be taken advantage of for selective toxicity. Furthermore, we establish the role of 2470-51 as a potential therapeutic agent in treating AML. Citation Format: Vindhya Vijay, Amy Meacham, Leylah Drusbosky, Elizabeth Wise, Christopher Cogle. A novel polyamine sulfonamide with anti-leukemic activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5118. doi:10.1158/1538-7445.AM2017-5118

Tumor-initiating CD49f cells are a hallmark of chemoresistant triple negative breast cancer

ABSTRACTTaxanes are mainstay treatment of triple negative breast cancer (TNBC) patients but resistance often develops. Using TNBC patient-derived orthoxenografts (PDX) we have recently discovered that a CD49f+ chemoresistant population with tumor-initiating ability is present in sensitive tumors and expands in tumors that have acquired resistance. Importantly, sensitivity to taxanes is recovered after long-term drug interruption. The characterization of this chemoresistant CD49f+ cells provides a unique opportunity to identify novel targets for the treatment of chemoresistant TNBC.

Targeting Lung Cancer Stem Cells: Research and Clinical Impacts.

Lung cancer is the most common cancer worldwide, accounting for 1.8 million new cases and 1.6 million deaths in 2012. Non-small cell lung cancer (NSCLC), which is one of two types of lung cancer, accounts for 85–90% of all lung cancers. Despite advances in therapy, lung cancer still remains a leading cause of death. Cancer relapse and dissemination after treatment indicates the existence of a niche of cancer cells that are not fully eradicated by current therapies. These chemoresistant populations of cancer cells are called cancer stem cells (CSCs) because they possess the self-renewal and differentiation capabilities similar to those of normal stem cells. Targeting the niche of CSCs in combination with chemotherapy might provide a promising strategy to eradicate these cells. Thus, understanding the characteristics of CSCs has become a focus of studies of NSCLC therapies.

Resistance to Taxanes in Triple-Negative Breast Cancer Associates with the Dynamics of a CD49f+ Tumor-Initiating Population.

SummaryTaxanes are a mainstay of treatment for breast cancer, but resistance often develops followed by metastatic disease and mortality. Aiming to reveal the mechanisms underlying taxane resistance, we used breast cancer patient-derived orthoxenografts (PDX). Mimicking clinical behavior, triple-negative breast tumors (TNBCs) from PDX models were more sensitive to docetaxel than luminal tumors, but they progressively acquired resistance upon continuous drug administration. Mechanistically, we found that a CD49f+ chemoresistant population with tumor-initiating ability is present in sensitive tumors and expands during the acquisition of drug resistance. In the absence of the drug, the resistant CD49f+ population shrinks and taxane sensitivity is restored. We describe a transcriptional signature of resistance, predictive of recurrent disease after chemotherapy in TNBC. Together, these findings identify a CD49f+ population enriched in tumor-initiating ability and chemoresistance properties and evidence a drug holiday effect on the acquired resistance to docetaxel in triple-negative breast cancer.

Inside the USCAP Journals

Inside the USCAP Journals

Abstract B03: Pancreatic cancer stem cells: can it be restrained?

Abstract Over the past decades world-wide, research towards not only understanding the biology, but also attempting to decrease the mortality rate in pancreatic carcinoma has become the forefront in cancers of solid tumor. Recent evidences suggest that pancreatic carcinomas harbour a distinct subpopulation of putative cancer stem cells defined by their self-renewal capacity, differentiation ability, tumorigenicity and ability to drive towards metastasis. Therefore, there is not only a need to identify this population, and also design newer approaches to target these chemo-resistant cell populations, especially in pancreatic cancer which till date shows 5-year survival rate to be below 5%. Our study identified the potential of CD44hiCD24lo MiaPaCa2/PanC1 cells to possess lower capability to generate spheroids whereas CD44hiCD24hi MiaPaCa2/PanC1 showed higher spheroid forming capacity. Spheroids obtained from both cell-lines showed similar metastatic cancer gene expression pattern. However, a distinctive CXCR4 expression levels were observed between CD44hiCD24lo PanC1-spheroids and CD44hiCD24hi PanC1-spheroids. It was also observed that EMT, CXCR4 and Muc1 were highly expressed in MiaPaCa2-spheroids. Hence, suggesting only a sub-population of tumor cells behave as “cancer initiating cells/stem-like cells” which are associated with cell-cell adhesion and cell matrix interaction. In order to determine the role of cancer stem-like cells (spheroids/CSCs) to migrate and invade to metastasize particularly in pancreatic carcinoma, experiments were designed to evaluate their self-renewal capability and epithelial to mesenchymal transition (EMT) and their association with developmental genes. An exponential spheroid formation was observed along with stem cell transcriptional genes (Oct4 and Nanog). Interestingly, MiaPaCa2-spheroids exhibited higher expression levels of Sox-2, MMP-9 and Vimentin in comparison to PanC1-spheroids suggesting that MiaPaCa2-spheroids may be more aggressive or has higher invasive potential. It appears that pancreatic CSCs undergoing EMT probably express higher levels of Sox-9, Ngn-3, Pdx-1 and c-Myc genes which attributes to its embryonic stemness. The results from the study clearly project the role of CSCs in pancreatic carcinoma and thus further studies to delineate the mechanism which might lead to suppression of pancreatic CSCs is crucial. In this direction, a pilot study has been initiated to explore the potential of Mesenchymal Stem Cells to target the pancreatic CSCs. Citation Format: Neha Chopra, Sangeeta Choudhury, Saima Wajid, Kriti Jain, Rizwana Mirza.{Authors}. Pancreatic cancer stem cells: can it be restrained? [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B03.

Abstract PR10: Exploiting the G2-M cell cycle checkpoint dependency in small cell lung cancer (SCLC) using pharmacological inhibitors of CHK1 and WEE1

Abstract Background: Small cell lung cancer (SCLC), the most lethal form of lung cancer, is characterized by early metastasis and rapid development of drug resistance. SCLC is characterized by ubiquitous loss of TP53 and RB1 and mutually exclusive amplification of the MYC oncogene (CMYC, MYCN, MYCL). The loss of TP53 is thought to render SCLC cells deficient at the G1-S cell cycle checkpoint, thus making these cells completely dependent on the G2-M checkpoint for DNA repair, mainly governed by CHK1 and WEE1. CHK1 is a vital serine-threonine kinase regulating the G2-M checkpoint upon DNA damage. The WEE1 kinase is a regulator of CDK1/2 and inhibition of WEE1 leads to aberrant DNA replication and premature mitosis. In this study we investigated the effect of CHK1 (LY2606368) and WEE1 (AZD1775) inhibitors as therapeutic targets for SCLC. Methods: Protein and gene expression was determined by immunoblot and real-time-RT-PCR respectively. Effect of the drugs on cell viability was determined by CellTiter-Glo. Efficacy of LY2606368 in H69 (chemosensitive) and H69/CR (chemoresistant) models was further determined by in vivo flank tumor regression analysis. The effect of LY2606368 monotherapy was also tested in a spontaneous SCLC genetically engineered mouse model (GEMM) model. The mechanistic studies were conducted by immunoblot analysis, apoptosis assay and flow cytometry. Pre and post-treatment cell lysates were analyzed by reverse phase protein array (RPPA) for biomarker exploration. Result: Higher expression of CHK1 and WEE1 was observed in SCLC patient tumor samples compared to normal lung (p<0.001) and in SCLC cells as compared to NSCLC cells (p<0.05). LY2606368 showed efficacy in both primary and acquired cisplatin resistant in vitro and in vivo models. LY2606368 treatment showed striking single agent activity in a spontaneous GEMM model of SCLC. Proteomic analysis identified an association between LY2606368 sensitivity and high levels of the CMYC protein. The level of CMYC decreased upon LY2606368 treatment (p<0.001) while pro-apoptotic pathways increased (p<0.05). CHK1 was a top marker of cisplatin sensitivity and treatment of cells with LY2606368 reduced activation of the MEK/MAPK pathway which we show as markers of de novo cisplatin resistance. LY2606368 treatment caused accumulation of cells in the sub-G1 phase and increased cell death. WEE1 inhibitor AZD1775 treatment decreased cell viability (IC50<100nM in 70% SCLC cells) in majority of SCLC cells. We identified AXL (receptor tyrosine kinase) and phospho-S6K as markers of AZD1775 resistance and treatment with AXL inhibitor, TP0903, resensitized the cells to AZD1775. Cell cycle analysis revealed increased apoptosis upon treatment of AZD1775 in combination with TP0903. Western blot analysis of pre and post AZD1775 treated samples revealed sustained activation of mTOR pathway in AZD1775 primary resistant lines. Pre-treatment of the cells with the mTOR inhibitor everolimus sensitized SCLC cells to AZD1775 by causing downregulation of AKT/mTOR pathway. Significance: The study establishes CHK1 and WEE1 as therapeutic targets in SCLC, a disease for which the standard of care has remained unchanged for >30 years. Together, these results strongly indicate the potency of G2-M cell cycle checkpoint inhibitors as effective targeted therapies in both chemosensitive and chemoresistant patient population. The CHK1 inhibitor, LY2606368, and the WEE1 inhibitor, AZD1775, are both in current Phase 1 clinical trials. The correlative biomarker data from this study will not only indicate the subset of the patient population who are most likely to respond to these treatments, but it will also guide the selection of most effective combination strategies with CHK1 and WEE1 inhibition. This abstract is also being presented as Poster A25. Citation Format: Triparna Sen, Pan Tong, Catherine Allison Stewart, Sandra Cristea, You Hong-Fan, Bonnie S. Glisson, Don L. Gibbons, Julien Sage, Jing Wang, Lauren A. Byers. Exploiting the G2-M cell cycle checkpoint dependency in small cell lung cancer (SCLC) using pharmacological inhibitors of CHK1 and WEE1. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr PR10.

Abstract B39: Identification and targeting mediators of chemoresistance using the patient-derived xenograft model of ovarian cancer

Abstract Objectives: The objectives of this study were to use the heterogeneity of the patient-derived xenograft (PDX) model to characterize the surviving population for de novo mediators of chemotherapy resistance, and subsequently target pathways contributing to that resistance. Methods: Tumors removed during primary tumor-reductive surgery were implanted directly into SCID mice, either subcutaneously and intraperitoneally. Mice with PDX tumors were treated with combination carboplatin/paclitaxel for 4 weeks. In addition, chemoresistant models were developed by treatment until complete response was achieved, and recurrent tumors retreated until resistant. Patient tumors, developing PDX tumors, and post-chemo surviving residual tumors were subjected to qPCR arrays, RNA-Seq, Methyl-Seq, and proteomic analysis; and IHC for Ki-67 and cancer stem cell markers CD133, ALDH1, and CD44. The Pol-I inhibitor CX-5461 was used to treat five PDX models. Results: At the mRNA level, the original patient tumors were similar to PDX tumors in mice as assessed by an 84-oncogene expression panel. Subcutaneous and intraperitoneal tumors were also remarkably similar. Patient responses correlated well with responses in mice. Proteomic analysis identified 14,023 unique proteins, of which approximately 30% were murine, and 70% were human in each sample. A total of 1,202 of human proteins were found to be differently expressed (p-value < 0.01) between patient and PDX tumors. Of these, 996 (82.9%) were reduced in the PDX model, and predominantly participants in the immune system. PDX tumors collected after chemotherapy showed that the surviving population was significantly more dormant than the original tumor (Ki-67 67% versus 31%, P<0.01), and enriched in all three cancer stem cell populations (2.1-4.7 fold, p<0.05). Principle component analysis of both RNA-Seq and proteomic data show that sensitive and resistant tumors were much more similar to one another than to tumors from different patients, suggesting a limited number of genes contributing to chemoresistance. IPA analysis of RNA-Seq data from treated and untreated PDX tumor pairs demonstrated that the most commonly enriched pathways are those of Protein Kinase A, GNRH, Sphingosine-1-phosphate, and alpha-adrenergic signaling. Additionally, numerous regulators of ribosomal synthesis were significantly altered by treatment. In vivo, 5 PDX models were treated with CX-5461; two had progression, one had stable disease, one had a 60% reduction in tumor growth, and one had a complete response to single agent therapy, still without recurrence after 3 months. Conclusion: The ovarian-PDX model exhibits consistent molecular and biologic similarity to patient tumors. Post-chemo tumors reveal multiple pathways to be consistently altered. Taking this information back to the PDX model demonstrated that inhibition of ribosomal RNA synthesis was highly effective, offering a novel opportunity to preferentially target the chemoresistant population in ovarian cancer. Citation Format: Zachary Dobbin, Robert Cornelison, Ashwini K. Katre, David A. Schneider, Karen Watters, Fabian Coscia, Ernst Lengyel, Aaron Buechlein, Fang Fang, Kenneth Nephew, Charles N. Landen, Jr.. Identification and targeting mediators of chemoresistance using the patient-derived xenograft model of ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B39.

Abstract 324: Targeting RNA-polymerase I using CX-5461 as a mechanism for treating chemotherapy resistant epithelial ovarian cancer

Abstract BACKGROUND: One of the first identified hallmarks of neoplastic transformation was an increased rate of ribosome biogenesis, and increased activity of RNA polymerase I (RNA pol I) is observed in most human cancers. Ribosome biogenesis is an energy intensive and tightly regulated process, and recent studies have shown that CX-5461, a potent and specific RNA pol I inhibitor, has selective toxicity to cancer cells. Here we demonstrate that CX-5461 is an effective anti-proliferative agent in epithelial ovarian carcinoma cells with an increased efficacy in chemoresistant ovarian cancer cells compared to their sensitive parental lines. METHODS AND RESULTS: Ovarian cancer cells were demonstrated to be highly sensitive to RNA pol I inhibition by MTT assay, with an IC50 range from 25nM to 2uM in 13 different ovarian cancer cell lines, and an IC50 of 3uM in the nontransformed immortalized normal surface epithelial line HIO-180. Notably, chemoresistant cell populations were generally more sensitive to CX-5461, compared to matched chemosensitive lines. IC50 in HeyA8MDR was 80nM compared to 2000nM in parental HeyA8, and was 100nM in SKOV3TRip2 compared to 400nM in SKOV3ip1. CX-5461 induces G2/M arrest (averaging 52.7% in G2 compared to 32.5% in controls), primarily through mitotic catastrophe, inducing an accumulation of multinucleated senescent cells. DNA damage pathways are activated, operating through ATM/ATR, as demonstrated by rescue with caffeine and ATR specific inhibitors. In an effort to identify why chemoresistant cells might be more susceptible, ribosomal synthesis processes were examined. Chemoresistant cells were found to have 2-4-fold increased Pol I occupancy on rDNA by ChIP, and increased rRNA synthesis by isotopic incorporation, while ribosome abundance and overall translation efficiency, measured by sucrose gradient fractionation, were unchanged. Treatment of five separate patient-derived xenograft models (PDX) with CX-5461 showed a complete response in one model, partial 55% reduction in tumor size in a second, stable disease in a 3rd, and progression in two models. CONCLUSIONS: CX-5461 shows high activity in ovarian cancer cell lines and patient-derived xenograft models, with a preferential effect on chemoresistant cells. Enhanced sensitivity to the compound is due at least in part to cancer cells’ increased demand for rRNA synthesis. RNA pol I inhibitors represent an exciting new treatment option that may have preferential effect on the chemoresistant population. Citation Format: Robert Cornelison, Zachary C. Dobbin, Ashwini A. Katre, Dae Hoon Jeong, Yulia Petrova, Danielle C. Llaneza, Adam D. Steg, Yinfeng Zhang, David A. Schneider, Charles N. Landen. Targeting RNA-polymerase I using CX-5461 as a mechanism for treating chemotherapy resistant epithelial ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 324.

Abstract SY01-02: Targeting stem cell pathways in human cancer

Abstract SY01-02: Targeting stem cell pathways in human cancer

Abstract A25: Role of ABCB5 in progression and therapeutic resistance of glioblastoma

Abstract Glioblastoma multiforme (GBM), one of the most lethal human cancers, is the most common and aggressive primary malignant brain tumor. Poor prognosis and high lethality result from tumor recurrence and extreme resistance to chemotherapeutic treatment. Compelling evidence suggests that GBM is a heterogeneous tumor composed of bulk tumor cells as well as of subpopulations of slow-proliferating cancer stem cells with tumor-initiating potential, which evade cell killing and are responsible for tumor recurrence. Current therapeutic strategies are thus aimed at eliminating these refractory tumor subpopulations to alleviate chemoresistance and recurrence of GBM. ATP-binding cassette member B5 (ABCB5) has been identified as a molecular marker for distinct subsets of chemoresistant tumor-initiating cell populations in diverse human malignancies, including malignant melanoma, hepatocellular carcinoma and colorectal cancer. Moreover, ABCB5 has been shown to functionally contribute to tumor progression and therapeutic resistance of human cancers. In the current study, we examined the potential role of ABCB5 in malignant growth and chemoresistance of GBM. We found ABCB5 to be co-expressed with GBM stem cell marker CD133 in all of three human GBM cell lines tested thus far, i.e. U87 MG, LN18 and LN229. Functional blockade of ABCB5 using an anti-ABCB5 monoclonal antibody (mAb) (clone 3C2-1D12) inhibited proliferation and survival of human GBM cells, and sensitized GBM cultures to Temozolomide (TMZ)-induced cell killing and apoptosis. Likewise, in vivo systemic administration of anti-ABCB5 mAb to immunodeficient (NSG) mice bearing human GBM xenografts resulted in significant reduction of tumor growth and sensitization of GBM tumors to TMZ therapy, underscoring the potential of ABCB5-targeting strategies for reversal of GBM chemotherapeutic resistance. Mechanistically, we found that ABCB5 blockade significantly altered molecules involved in cell cycle regulatory checkpoints to revoke TMZ-induced G2/M arrest, explaining how treatment with ABCB5 mAb is augmenting drug-mediated apoptosis. Overall, our data reveal a novel role of ABCB5 as a marker of GBM chemoresistance and show that functional ABCB5 blockade inhibits GBM tumor growth in vivo. These findings underline the potential clinical relevance and therapeutic importance of ABCB5 targeting as a novel therapeutic approach to GBM eradication. Citation Format: Pallavi Banerjee, Gretchen Berg, Brian J. Wilson, Qin Guo, Markus H. Frank, Natasha Y. Frank. Role of ABCB5 in progression and therapeutic resistance of glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A25.

Bortezomib Attenuates Adhesion of B Cell Precursor Acute Lymphoblastic Lleukemia Cells to Bone Marrow Mesenchymal Stromal/Stem Cells Via Regulating SPARC Expression

The prognosis of adults with B cell precursor acute lymphoblastic leukemia (BCP-ALL) is poor. Many cases in complete remission experience relapse of leukemia despite intensive chemotherapy or hematopoietic stem cell transplantation. This clinical observation suggests that minimal residual disease (MRD) still exists after these intensive therapies. Cell adhesion-mediated drug resistance (CAM-DR) is one of the mechanisms to support MRD in the bone marrow microenvironment (Clin Cancer Res 14:9, 2008). Mesenchymal stromal/stem cells (MSCs) are a cellular component of bone marrow (BM) and maintain physiological precursor B lymphopoiesis (Nature Rev Immunol 6:107, 2006). We investigated our hypothesis that survival of BCP-ALL cells is supported by their direct adhesion to BM-MSCs in BM microenvironment. First, we confirmed that BCP-ALL cells exhibited their drug resistant phenotype through adhesion to human BM-MSCs using human BCP-ALL cell line, Nalm6. We isolated human BM-MSCs from normal bone marrow samples (AllCells, Emeryville, CA). When Nalm6 cells were co-cultured with human BM-MSCs, they were prone to adhere to BM-MSCs. Nalm6 adhered to BM-MSCs (Nalm6/ad) were more resistant to the treatment with various anti-cancer drugs including doxorubicin than Nalm6 in suspension (Nalm6/su). Immunoblot analysis showed that expression level of anti-apoptotic protein Bcl-2 and phosphorylation level of pro-survival kinase Akt are higher in Nalm6/ad compared with those in Nalm6/su. In cell cycle analysis using Ki67/PI fluorescence-activated cell sorter (FACS) assay, the percentage of populations in G0 phase and S/G2/M phase was higher in Nalm6/ad compared with that in Nalm6/su, which indicated the increase of MRD and the high proliferation of leukemic cells in adhesive population, respectively. Therefore, we considered that detachment of Nalm6 from BM-MSCs is important to restore chemosensitivity of BCP-ALL cells and to eliminate these cells. Accordingly, we screened drugs that are capable of disrupting the adhesion of Nalm6 to BM-MSCs, and found that several proteasome inhibitors had a such activity. BM-MSCs were treated with bortezomib (10nM for 24h or 100nM for 1h), carfilzomib (3 nM for 24 h or 100 nM for 1 h) or oprozomib (10 nM for 24 h or 300 nM for 4 h), and then washed with PBS and co-cultured with Nalm6 cells. We confirmed that the number of BM-MSCs is not affected with these drugs irrespective of their concentration and incubation time by MTT assay. Co-cultured cells were divided into suspension cells and adherent cells. Each population was stained with monoclonal antibodies against CD19 and CD90 to detect Nalm6 and BM-MSCs, respectively, and analyzed by FACS. When BM-MSCs were pre-treated with bortezomib, carfilzomib or oprozomib, the number of Nalm6/ad was significantly decreased, and inversely, the number of Nalm6/su was increased, as compared to that of co-cultures with untreated BM-MSCs. Intriguingly, BM-MSCs treated with bortezomib showed aberrant expression of secreted protein acidic and rich in cysteine (SPARC). Immunoblot analysis showed that SPARC expression of BM-MSCs was transiently increased after bortezomib treatment. In co-cultures with BM-MSCs transfected with siRNA targeting SPARC, the number of Nalm6/ad was increased. In addition, the number of Nalm6 adhered to BM-MSCs pre-treated with recombinant human SPARC was lower than that adhered to control non-treated BM-MSCs. Therefore, SPARC showed anti-adhesive property and was one of the molecules associated with adhesion of BM-MSCs to Nalm6. Finally, we tested whether bortezomib shows therapeutic effects in vivo. Bortezomib administration supported survival of BCP-ALL xenograft model mice utilizing Nalm6, which was concomitant with the decrease in leukemia cells in the femur and in the size of spleen, as compared to those in non-treated control mice. In summary, Nalm6 cells that adhered to BM-MSCs contributed to construction of chemoresistant population similar to MRD. Here, we found that this population could be detached by treatment of BM-MSCs with bortezomib through transient increase in their SPARC expression. Our findings suggest that bortezomib or other proteasome inhibitors might be promising drugs to treat BCP-ALL through attenuating the adhesion of leukemic cells to BM-MSCs, and shed new insight into a therapeutic strategy in BCP-ALL by targeting BM-MSCs. Disclosures No relevant conflicts of interest to declare.

HMGB1 induction of clusterin creates a chemoresistant niche in human prostate tumor cells.

Development of chemoresistance, especially to docetaxel (DTX), is the primary barrier to the cure of castration-resistant prostate cancer but its mechanism is obscure. Here, we report a seminal crosstalk between dying and residual live tumor cells during treatment with DTX that can result in outgrowth of a chemoresistant population. Survival was due to the induction of secretory/cytoplasmic clusterin (sCLU), which is a potent anti-apoptotic protein known to bind and sequester Bax from mitochondria, to prevent caspase 3 activation. sCLU induction in live cells depended on HMGB1 release from dying cells. Supernatants from DTX-treated DU145 tumor cells, which were shown to contain HMGB1, effectively induced sCLU from newly-plated DU145 tumor cells and protected them from DTX toxicity. Addition of anti-HMBG1 to the supernatant or pretreatment of newly-plated DU145 tumor cells with anti-TLR4 or anti-RAGE markedly abrogated sCLU induction and protective effect of the supernatant. Mechanistically, HMGB1 activated NFκB to promote sCLU gene expression and prevented the translocation of activated Bax to mitochondria to block cell death. Importantly, multiple currently-used chemotherapeutic drugs could release HMGB1 from tumor cells. These results suggest that acquisition of chemoresistance may involve the HMGB1/TLR4-RAGE/sCLU pathway triggered by dying cells to provide survival advantage to remnant live tumor cells.

Pre-surgical window study of metformin in obesity-driven endometrial cancer

population was significantly more dormant than the original tumor. RNA sequencing analysis of treated and untreated PDX tumors (n = 6 pair) demonstrated that numerous factors with roles in ribosomal synthesis were significantly altered by treatment. In vitro, an inhibitor of ribosomal synthesis, CX-5461, caused significant G2/M-phase arrest, and effects were much more profound in chemoresistant cells than corresponding chemosensitive cells. In vivo, of the five PDX models that were treated with CX-5461, two had progression, one had stable disease, one had a 60% reduction in tumor growth, and one had a complete response to single-agent therapy, still without recurrence after 3 months. Conclusions: The ovarian PDX model exhibits similarity to patient tumors. Postchemotherapy tumors reveal severe alteration of ribosomal synthesis. Treatment with an inhibitor of ribosomal RNA synthesis was highly effective in vitro and in vivo, offering a novel opportunity to specifically target the chemoresistant population in ovarian cancer.

Abstract 1924: Discovery and characterization of EMT positive and chemo-resistant novel population of HCC like stem cells

Abstract Increasing evidence suggests that tumor heterogeneity facilitates relapse of liver cancer in patients during the post therapeutic period. This concept of heterogeneity has gained increasing support from the liver cancer stem cells (LCSC), which have the potential of differentiation and genetic and epigenetic alterations to adapt to the tumor microenvironment. With the technological advancement, researchers were able to identify new population of LCSCs besides CD133+ clones by discovering new surface markers-CD90, EpCAM, CD13 and CD24. These studies corroborated that CD133- population is highly heterogeneous and might have some additional markers. Recently, by screening colorectal cancer patient samples we discovered a novel surface marker vimentin (Vim) on metastatic liver nodules. We further initiated screening of primary hepatocellular carcinoma murine liver tumor samples and discovered the surface expression of Vim in both species of HCC. Interestingly, we purified Vim+CD133- from primary liver tumor cell suspensions. Using sphere assays, limited dilution assays and staining of the stem cell markers Sox2, Nanog and Oct4A, we showed that Vim+CD133- cells have stem cell like properties similar to Vim-CD133+ population. Then we performed differentiation assays with Vim+CD133- cells under suitable condition and showed hepatocyte like phenotypes by upregulating protein expression of albumin, periodic acid schiff's staining and cholangiocyte type morphologies- tubular and star shaped structures. Several studies correlated that cells undergo epithelial-mesenchymal transition (EMT)phenotypes when vimentin is expressed on their surface. Our investigation revealed that Vim+CD133- population supported EMT like phenotypes by presence of the twist and β-catenin inside the nucleus and absence of E-cadherin on cell surface. Contrary to this Vim-CD133+ population did not show EMT like phenotypes. In next step of our characterization of Vim+CD133- cells, we investigated its chemoresistance properties as LCSCs are well known to cause relapse in patients. We treated these cells with highly toxic doses of 5 Fluorouracil and cisplatin for 4 days and interestingly observed significantly more viability compared to Vim-CD133+ population. We also validated multidrug resistance properties such as expression of multidrug resistance protein 1 (MDR1) and anti-apoptotic markers in Vim+CD133- cells. Finally, we validated their tumorigenic potential using anchorage independent assays and in vivo inoculation in immunocompromised NOD-SCID mice. Collectively our studies characterized a novel population Vim+CD133- in heterogeneous LCSCs, which showed EMT like phenotypes and higher chemo-resistance compared to Vim-CD133+ cells. In the era of personalized cancer therapy, this population should be considered as superior druggable target compared to our canonical Vim-CD133+ cells to obtain greater therapeutic outcome. Citation Format: Abhisek Mitra, Arun Satelli, Xueqing Xia, Shulin Li. Discovery and characterization of EMT positive and chemo-resistant novel population of HCC like stem cells. [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 1924. doi:10.1158/1538-7445.AM2014-1924

Abstract 2266: Investigating the role of the anti-apoptotic protein ARC in breast cancer cell DNA repair

Abstract According to the American Cancer Society, more than 90% of breast cancer patients who succumb to the condition die as a result of the cancer metastasizing to other areas of the body. Our lab has previously shown that the presence of a chemoresistant invasive subpopulation of breast carcinoma cells is correlated with the prevalence of metastasis. Additionally, we have hypothesized that chemoresistance and survival of these cancer cells which eventually leads to relapse and metastasis following initial treatment with chemotherapeutic agents is achieved by enhanced DNA repair capability in this population. We reported that an anti-apoptotic protein ARC (apoptosis repressor with caspase recruitment domain) is over expressed in the invasive mammary cancer cells that are resistant to standard chemotherapeutic agents. Further, we have shown that ARC over expressing cells are resistant to chemotherapy both in vivo and in vitro. In this study, we examined cellular DNA repair signaling pathways in order to delineate the role of ARC in the pathogenesis of breast cancer. Utilizing cell lines where ARC was either knocked down or overexpressed, we investigated potential correlations of ARC with DNA repair capability by means of the single cell gel electrophoresis assay (comet assay). The results of these experiments indicated that over expression of ARC leads to increased cellular DNA repair while knocking it down leads to decreased cellular DNA repair. In order to determine the molecular basis of the role of ARC in the DNA repair pathways, we have performed qRT-PCR based gene expression analysis on ARC modified cell lines using techniques previously established in our lab. The results of these experiments indicated that increased levels of ARC are correlated with upregulation of key genes in single stranded DNA repair pathways such as the base excision and nucleotide excision repair pathways. We are working to identify the specific domain of ARC that serves as its effector in the DNA repair pathways. In particular, we are looking at the caspase recruitment domain (CARD) of ARC which is known to be key for ARC`s function in the apoptotic pathways. Once this domain is identified, it may serve as a potential drug target, the silencing of which could result in the disruption of key pathways required for invasive breast cancer cell survival. Such therapy may facilitate the ablation of chemoresistant breast cancer cell populations, prevent metastasis and tumor relapse, and increase long term patient survival. Citation Format: Eli Grunblatt, Evanka Madan, Sweta Roy, Sumanta Goswami. Investigating the role of the anti-apoptotic protein ARC in breast cancer cell DNA repair. [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 2266. doi:10.1158/1538-7445.AM2014-2266

Using heterogeneity of the patient-derived xenograft model to identify the chemoresistant population in ovarian cancer.

A cornerstone of preclinical cancer research has been the use of clonal cell lines. However, this resource has underperformed in its ability to effectively identify novel therapeutics and evaluate the heterogeneity in a patient's tumor. The patient-derived xenograft (PDX) model retains the heterogeneity of patient tumors, allowing a means to not only examine efficacy of a therapy, but also basic tenets of cancer biology in response to treatment. Herein we describe the development and characterization of an ovarian-PDX model in order to study the development of chemoresistance. We demonstrate that PDX tumors are not simply composed of tumor-initiating cells, but recapitulate the original tumor's heterogeneity, oncogene expression profiles, and clinical response to chemotherapy. Combined carboplatin/paclitaxel treatment of PDX tumors enriches the cancer stem cell populations, but persistent tumors are not entirely composed of these populations. RNA-Seq analysis of six pair of treated PDX tumors compared to untreated tumors demonstrates a consistently contrasting genetic profile after therapy, suggesting similar, but few, pathways are mediating chemoresistance. Pathways and genes identified by this methodology represent novel approaches to targeting the chemoresistant population in ovarian cancer.

Abstract A58: An ovarian patient-derived xenograft model to identify the chemoresistant population

Abstract Introduction: A cornerstone of preclinical ovarian cancer research over the last 30 years has been the use of cell lines both in vitro and in vivo. This resource has underperformed in its ability to identify effective novel therapeutics and evaluate the heterogeneity of an ovarian neoplasm. Developing a patient-derived xenograft (PDX) allows for a comprehensive study of the heterogeneous tumor and potentially the identification of cellular populations responsible for chemoresistance and recurrence, but has not been fully characterized for ovarian cancer. We have developed an ovarian PDX model to demonstrate many important similarities and differences between these and primary patient tumors, and begun to explore its utility in identifying mediators of chemoresistance and personalizing therapy. Methods: Tumors removed from patients undergoing primary tumor reductive surgery were implanted into the subcutaneous flanks of SCID mice. Once a tumor developed, it was expanded into additional mice to propagate a PDX line. Tumors were analyzed for heterogeneity by examination of the tumor initiating cell populations, its stromal content with HLA immunohistochemistry, and proliferation with Ki-67. Cohorts of PDX lines were treated with maximum-tolerated dose of combined carboplatin and paclitaxel or vehicle and response was compared between the PDX and the patient. Chemoresistant PDX lines were generated by treatment with MTD carboplatin and paclitaxel until no evidence of disease (NED), observation until recurrence, and retreatment until fully resistant. RNAseq was conducted comparing treated PDX lines to the untreated PDX lines (n=6 pair) to identify important mediators of chemotherapy resistance. Finally, PDX lines were characterized for defects in homologous recombination (HR) repair with an ex vivo assay, correlating HR status with response to PARP inhibition in vivo. Results: Refinement of xenografting procedures yields an 85% success rate in establishing a PDX. PDX tumors maintain the patient's histology, but the stromal component is replaced by murine cells in the first generation. The xenografts retain primary tumor heterogeneity, at least in expression of putative tumor initiating cells (TIC) (ALDH: 17% vs 19%, CD44: 2.4% vs 5%, CD133 10% vs 3%). However, treatment with chemotherapy significantly increases these populations (ALDH1 increased to 36.1% from 16.2% (p=0.002); CD133 increased to 33.8% from 16.2% (p=0.026), suggesting a role in chemotherapy resistance. The treated PDX tumors showed a decrease in Ki67 staining (from 64% to 34%, p=0.001), suggesting dormancy is induced in the surviving population. The PDX tumors show similar response to chemotherapy as patient tumors, in that PDX tumors from patients with a partial response respond more slowly (if at all) than those from patients achieving a complete response (mean change in tumor volume -5.11% vs -63.73%, p=0.029). RNAseq comparing treated and untreated PDX lines indicate only 54 common genes with a significant change in mRNA (p<0.05), suggesting great heterogeneity among patients not only in primary tumor content, but also mediators of chemoresistance. Finally, patient tumors predicted to respond to PARP inhibition by an ex vivo assay show regression of tumor or stable disease with single-agent PARP inhibitor therapy. Conclusions: The ovarian PDX model recapitulates patient tumor heterogeneity and mirrors response to chemotherapy. This model may prove superior in predicting response to therapy in patients, and allows a better model to study complex tumor biology that is impacted by tumor heterogeneity, such as survival of small populations with chemotherapy. Citation Format: Zachary C. Dobbin, Ashwini K. Katre, Monjri M. Shah, Britt K. Erickson, Hauping Chen, Ronald D. Alvarez, Michael G. Conner, Dongquan Chen, Charles N. Landen. An ovarian patient-derived xenograft model to identify the chemoresistant population. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A58.

Abstract 3717: Aspirin decreases side population cells by targeting the Wnt pathway in esophageal cancer cells in vitro and enhances the combination chemotherapeutic effect of 5-FU and cisplatin in vivo.

Abstract Introduction. The potential use of non-steroidal anti-inflammatory drugs (NSAIDs) in the prevention of esophageal cancer has been highlighted recently. Taking aspirin was reported to reduce the risk of developing Barrett's esophagus and prevent the development of both esophageal squamous cell cancer and adenocarcinoma. However, the underlying mechanism is still unclear. Some researchers indicated that aspirin could induce tumor cells apoptosis and might modulate cancer stem cells regulation through the Wnt/beta-catenin pathway. Pervious results have shown that side population (SP) cells in esophageal cancer cell lines have stem cell-like properties and may be responsible for therapy resistance. Our objective was to determine whether aspirin by targeting Wnt signaling affects the biological properties of side population cells in esophageal cancer and its therapeutic effect both in vitro and in vivo. Methods. In vitro cytotoxicity assays using aspirin were performed in three different esopahgeal cancer cell lines (OE19, OE21, and OE33). After treatment with aspirin Hoechst 33342 staining was used to define the proportion of SP cells. And the expression of Wnt signaling targets with and without aspirin treatment was further evaluated by PCR array. SP and non-SP cells in OE19 were isolated by flow cytometric sorting and further treated with aspirin. The efficacy of aspirin alone and combined with 5-FU and Cisplatin administration by intraperitoneal injection regarding OE19 tumorigenicity was evaluated in a subcutaneous xenograft model. Results. Aspirin inhibits in a dose and time dependent manner cell proliferation in esophageal cancer cell lines. In OE19, SP cells were dramatically decreased from 19.7±1.0 to 6.9±1.0, 3.4±0.9 and 1.8±0.4 (p 2 fold change). In particular, aspirin can reduce 5-FU or cisplatin induced enhancement of SP and displayed a relative stronger effect on SP proliferation as compared to non-SP cells together with a significant reduction of ABCG2+ subpopulation. 5-FU and Cisplatin combined with daily aspirin severely impaired tumor weight comparing to chemotherapy alone (0.14±0.11g vs 0.75±0.2g, p=0.0004) However, administration of aspirin alone did not have effect on tumor growth significantly as compared to vehicle group (1.58±0.36g vs 1.50±0.28, p=0.728). Conclusion. Aspirin may affect on esophageal cancer cells by targeting the chemoresistant side population cells through wnt signaling pathway. This finding indicates a potential therapeutic strategy considering NSAIDs for esophageal cancer. Citation Format: Yue Zhao, Bettina Schwarz, Lu Zhao, Yan Wang, Anneli Tischmacher, Josef Mysliwietz, Joachim Ellwart, Qi Bao, Hanno Nieß, Dominik Modest, Peter Camaj, Karl-Walter Jauch, Peter Nelson, Christiane Bruns. Aspirin decreases side population cells by targeting the Wnt pathway in esophageal cancer cells in vitro and enhances the combination chemotherapeutic effect of 5-FU and cisplatin in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3717. doi:10.1158/1538-7445.AM2013-3717