Resistant Cell Populations Research Articles

Abstract A084: Towards combining androgen deprivation and immunotherapy to prevent progression to castration-resistant prostate cancer

Abstract Most prostate cancer (PCa) deaths are due to castration-resistant PCa (CRPC), following failure of androgen-deprivation therapy (ADT). ADT is the standard of care for patients with advanced PCa. However, nearly universal progression to castration-resistant prostate cancer (CRPC) occurs 2-3 years after ADT is initiated. Although there have been recent improvements in the treatment of CRPC, even the most promising therapies are still not curative. One approach to this problem is to improve the initial treatment of advanced prostate cancers, by combining complementary therapies with ADT, to prevent progression of such advanced cancers to CRPC. Immunotherapy with checkpoint inhibitors (CPIs) has not been effective in prostate cancers, perhaps because such cancers are “cold” (lacking cytolytic CD8 T-cells). Some cold tumors may be caused by infiltration of myeloid cell populations (tumor associated macrophages and myeloid-derived suppressor cells) into the tumor immune cell microenvironment (TIME). Recently, we found that in a PTEN-deficient mouse PCa model, castration induces an immunosuppressive state within the tumor that is concurrent with tumor recurrence. The response to castration/ADT is tri-phasic: a pro-apoptotic regression phase when tumor shrinks, followed by selection for a residual population of resistant tumor cells and finally recurrent growth as CRPC. Using PCa cell lines to model the first two phases of the response to ADT, we have shown that ADT induces apoptosis, thereby enriching for an ADT-resistant stem/progenitor population that we propose is the in vivo source of TNF. Mechanistically, in our model system the response to ADT is driven by the soluble mediators TNF and CCL2, which facilitate communication within the TIME. Specifically, a TNF-CCL2-CCR2 paracrine loop is induced between prostate cancer cells and non-tumor cells in the microenvironment: TNF produced by tumor cells acts on myofibroblasts to induce CCL2 production, which in turn recruits CCR2+ tumor-associated macrophages (TAMs). To investigate the ADT response within the TIME in an in vivo model of prostate cancer, we employed a prostate-specific PTEN-deficient mouse model (PbCre4 x PTENf/f). Castration caused the tumors to regress, consistent with initial phase of the response that is seen in the human disease. At late times post-castration (5-6 weeks), corresponding to the selection phase, we observed a coordinate increase in the stem/progenitor tumor cell population, as well as TNF and CCL2, within the TIME. Immunohistochemical staining of tumors 5 weeks post-castration revealed an increase in TAMs, and a decrease in CD8 T-cells, consistent with an immunosuppressive or immunoevasive state. This phenotype was reversed by a soluble receptor that binds TNF (etanercept). We also observed increased myeloid-derived suppressor cells (MDSC). Thus, following ADT, TNF derived from an ADT-resistant stem/progenitor epithelial tumor cell population promotes an immunosuppressive state via CCL2 in the TIME. Analysis of public human PCa data sets shows TNF and stem/progenitor marker expression are both increased in CRPC, consistent with our hypothesis that ADT drives the development of an immunosuppressive state via a TNF-CCL2-CCR2 axis. Our results set the stage for the future development of immunotherapies that could improve the efficacy of ADT. Citation Format: John J. Krolewski, Kai Sha, Michalis Mastri, Dean Tang, Kevin Eng, Kent L. Nastiuk. Towards combining androgen deprivation and immunotherapy to prevent progression to castration-resistant prostate cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A084.

Ewing sarcoma resistance to SP-2509 is not mediated through KDM1A/LSD1 mutation.

Ewing sarcoma is the second most common solid bone malignancy diagnosed in pediatric and young adolescent populations. Despite global co-operative efforts, outcomes for patients with relapsed and refractory disease remains obstinately poor. It has become increasingly clear that disruption of the epigenome as a result of alterations in epigenetic regulators, plays a pivotal role in tumorigenesis. As such, this study investigated Ewing sarcoma mechanisms of acquired resistance to the small molecule reversible lysine specific demethylase (LSD1/KDM1A) inhibitor SP-2509. Surprisingly, whole exome sequencing analysis of our generated A673 SP-2509 drug resistant cell line revealed an absence of mutations in KDM1A. Compared to parental counterparts, SP-2509 drug resistant cells demonstrated decreased anchorage independent growth capacity, enhanced sensitivity to the HDAC inhibitors vorinostat/entinostat and a distinct transcriptional profile that was enriched for extracellular matrix proteins. SP-2509 drug resistant cells also exhibited elevated expression levels of the multi-drug resistance genes ABCB1, ABCC3, and ABBC5 and decreased expression of the transcriptional repressor RCOR1/CoREST. Following several months of SP-2509 withdrawal, low level SP-2509 resistance was still apparent (6.3 fold increase in IC50), with drug resistant cell populations maintaining their distinct transcriptional profile. Furthermore, compared to parental cells, washout drug resistant lines displayed equal sensitivity to the standard Ewing sarcoma chemotherapeutic agent's vincristine and doxorubicin. Together these findings indicate that resistance to SP-2509 is not fully reversible or driven by direct mutation in KDM1A.

Prolonged Idasanutlin (RG7388) Treatment Leads to the Generation of p53-Mutated Cells.

The protein p53 protects the organism against carcinogenic events by the induction of cell cycle arrest and DNA repair program upon DNA damage. Virtually all cancers inactivate p53 either by mutations/deletions of the TP53 gene or by boosting negative regulation of p53 activity. The overexpression of MDM2 protein is one of the most common mechanisms utilized by p53wt cancers to keep p53 inactive. Inhibition of MDM2 action by its antagonists has proved its anticancer potential in vitro and is now tested in clinical trials. However, the prolonged treatment of p53wt cells with MDM2 antagonists leads to the development of secondary resistance, as shown first for Nutlin-3a, and later for three other small molecules. In the present study, we show that secondary resistance occurs also after treatment of p53wt cells with idasanutlin (RG7388, RO5503781), which is the only MDM2 antagonist that has passed phase II and entered phase III clinical trials, so far. Idasanutlin strongly activates p53, as evidenced by the induction of p21 expression and potent cell cycle arrest in all the three cell lines tested, i.e., MCF-7, U-2 OS, and SJSA-1. Notably, apoptosis was induced only in SJSA-1 cells, while MCF-7 and U-2 OS cells were able to restore the proliferation upon the removal of idasanutlin. Moreover, idasanutlin-treated U-2 OS cells could be cultured for long time periods in the presence of the drug. This prolonged treatment led to the generation of p53-mutated resistant cell populations. This resistance was generated de novo, as evidenced by the utilization of monoclonal U-2 OS subpopulations. Thus, although idasanutlin presents much improved activities compared to its precursor, it displays the similar weaknesses, which are limited elimination of cancer cells and the generation of p53-mutated drug-resistant subpopulations.

Is adaptive therapy natural?

Research suggests that progression-free survival can be prolonged by integrating evolutionary principles into clinical cancer treatment protocols. The goal is to prevent or slow the proliferation of resistant malignant cell populations. The logic behind this therapy relies on ecological and evolutionary processes. These same processes would be available to natural selection in decreasing the probability of an organism’s death due to cancer. We propose that organisms’ anticancer adaptions include not only ones for preventing cancer but also ones for directing and retarding the evolution of life-threatening cancer cells. We term this last strategy natural adaptive therapy (NAT). The body’s NAT might include a lower than otherwise possible immune response. A restrained immune response might forego maximum short-term kill rates. Restraint would forestall immune-resistant cancer cells and produce long-term durable control of the cancer population. Here, we define, develop, and explore the possibility of NAT. The discovery of NAT mechanisms could identify new strategies in tumor prevention and treatments. Furthermore, we discuss the potential risks of immunotherapies that force the immune system to ramp up the short-term kill rates of malignant cancer cells in a manner that undermines the body’s NAT and accelerates the evolution of immune resistance.

Phenotypic Basis for Matrix Stiffness-Dependent Chemoresistance of Breast Cancer Cells to Doxorubicin.

The persistence of drug resistant cell populations following chemotherapeutic treatment is a significant challenge in the clinical management of cancer. Resistant subpopulations arise via both cell intrinsic and extrinsic mechanisms. Extrinsic factors in the microenvironment, including neighboring cells, glycosaminoglycans, and fibrous proteins impact therapy response. Elevated levels of extracellular fibrous proteins are associated with tumor progression and cause the surrounding tissue to stiffen through changes in structure and composition of the extracellular matrix (ECM). We sought to determine how this progressively stiffening microenvironment affects the sensitivity of breast cancer cells to chemotherapeutic treatment. MDA-MB-231 triple negative breast carcinoma cells cultured in a 3D alginate-based hydrogel system displayed a stiffness-dependent response to the chemotherapeutic doxorubicin. MCF7 breast carcinoma cells cultured in the same conditions did not exhibit this stiffness-dependent resistance to the drug. This differential therapeutic response was coordinated with nuclear translocation of YAP, a marker of mesenchymal differentiation. The stiffness-dependent response was lost when cells were transferred from 3D to monolayer cultures, suggesting that endpoint ECM conditions largely govern the response to doxorubicin. To further examine this response, we utilized a platform capable of dynamic ECM stiffness modulation to allow for a change in matrix stiffness over time. We found that MDA-MB-231 cells have a stiffness-dependent resistance to doxorubicin and that duration of exposure to ECM stiffness is sufficient to modulate this response. These results indicate the need for additional tools to integrate mechanical stiffness with therapeutic response and inform decisions for more effective use of chemotherapeutics in the clinic.

Abstract A48: MET and EGFR interaction promotes acquired resistance to kinase inhibition in TNBC

Abstract Triple-negative breast cancer (TNBC) has the worst prognosis of all breast cancers, and the molecular heterogeneity within TNBC heightens the challenge of developing effective targeted therapies. Receptor tyrosine kinases (RTKs), in particular MET and EGFR, are promising therapeutic targets for TNBC due to their high expression in multiple molecular TNBC subtypes and the tendency for cancers to become “kinase addicted.” We and others have demonstrated that MET is highly expressed in TNBC and its expression correlates with poor prognosis. EGFR expression is also elevated in up to 72% of TNBCs and correlates with poor prognosis in TNBC patients. Recently, we demonstrated that MET and EGFR are coordinately and highly expressed across all TNBC subtypes and the efficacy of combined MET and EGFR inhibition in TNBC patient-derived xenograft (PDX) models. Even though MET and EGFR receptors are actionable targets due to their high activity in TNBC, crosstalk between MET and EGFR has been implicated in therapeutic resistance to kinase inhibitors in several cancer types and needs to be evaluated in TNBC. There is strong evidence demonstrating the critical role of redundant RTK signaling networks in resistance to tyrosine kinase inhibitors (TKIs). Specifically, crosstalk between MET and EGFR has been implicated in therapeutic resistance to EGFR or MET inhibitors in colon, gastric, and lung cancers. Since MET and EGFR are highly expressed in a substantial proportion of TNBCs, we used TNBC PDX models and TNBC cell lines to interrogate mechanisms of resistance to MET and EGFR kinase inhibition. We hypothesized that coexpression, interaction, and activation of MET and EGFR promote acquired TKI resistance and an adaptive kinome response in TNBC. Using TNBC PDX models that highly express both MET and EGFR, we evaluated MET and EGFR activity, localization, and interaction after 3 days or 3 weeks of treatment with MET (glesatinib, crizotinib) and/or EGFR inhibitors (erlotinib). After 3 weeks of dual MET and EGFR inhibition in TNBC PDXs, only a small population of “resistant” cells remained. In these resistant populations, we observed increased MET and EGFR colocalization and increased MET and EGFR activation. To understand how colocalization of MET and EGFR promotes signaling redundancy and crosstalk, we asked whether MET and EGFR directly interact in TNBC PDX using a proximity ligation assay (PLA). PLA uses antibody specificity to detect direct protein-protein interactions at physiologic levels in vitro and in vivo. We observed a significant increase in MET EGFR interactions only in resistant cell populations that had been treated with both MET and EGFR inhibitors. AKT and ERK signaling were also increased in TKI-resistant cells, indicating that these are key survival pathways in resistance. Interestingly, we did not observe an increase in MET-EGFR activity or interaction in PDX tumors treated for only 3 days. Even though TNBC cells (HCC70) showed increased colocalization of MET and EGFR with combined glesatinib and erlotinib treatment, there was not a significant increase in MET-EGFR interactions. Evaluation of MET-EGFR interaction in primary TNBC patient samples revealed significant MET-EGFR interactions in a MET-amplified tumor and lymph node metastases. To our knowledge, this is the first time MET-EGFR interactions have been observed in TNBC in vivo and in human patients. Moreover, MET-EGFR interactions are able to maintain MET, EGFR, ERK, and AKT activity through a yet-unknown mechanism. These results imply that MET EGFR interaction may be a unique mechanism of resistance to kinase inhibition that may be inherent in human TNBCs with genomic amplification of MET or EGFR. Citation Format: Elizabeth Tovar, Curt Essenburg, Anderson Peck, Lisa Turner, Zachary Madaj, Matthew Smith, James Christensen, Marianne Melnik, Eric Haura, Matthew Steensma, Carrie Graveel. MET and EGFR interaction promotes acquired resistance to kinase inhibition in TNBC [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A48.

Stroma-derived IL-6, G-CSF and Activin-A mediated dedifferentiation of lung carcinoma cells into cancer stem cells

Cancer stem cells (CSCs) are a small population of resistant cells inhabiting the tumors. Although comprising only nearly 3% of the tumor mass, these cells were demonstrated to orchestrate tumorigenesis and differentiation, underlie tumors’ heterogeneity and mediate therapy resistance and tumor relapse. Here we show that CSCs may be formed by dedifferentiation of terminally differentiated tumor cells under stress conditions. Using a elegant co-culture cellular system, we were able to prove that nutrients and oxygen deprivation activated non-malignant stromal fibroblasts, which in turn established with tumor cells a paracrine loop mediated by Interleukine-6 (IL-6), Activin-A and Granulocyte colony-stimulating factor (G-CSF), that drove subsequent tumor formation and cellular dedifferentiation. However, by scavenging these cytokines from the media and/or blocking exosomes’ mediated communication it was possible to abrogate dedifferentiation thus turning these mechanisms into potential therapeutic targets against cancer progression.

Pharmacodynamic modelling of resistance to epidermal growth factor receptor inhibition in brain metastasis mouse models

PurposeEpidermal growth factor receptor (EGFR) is thought to play a role in the regulation of cell proliferation; with its activation stimulating tumour growth. EGFR inhibitors have shown promise in the treatment of cancer, particularly in non-small cell lung cancer, however, resistance is observed in the majority of patients. A tumour growth model was developed aiming to explain this resistance.MethodsThe model incorporating populations of both sensitive and resistant cells were fitted to data from a study of EGFR inhibitor AZD3759 in brain metastasis mouse models. The observed regrowth of tumours in higher dose groups suggested the development of resistance to treatment. The bioluminescence observations were highly variable, covering many orders of magnitude, so to assess how reliable the model was, the parameter estimates were compared to those found in less noisy subcutaneous mouse models.ResultsThe fitted model suggested that resistance was mainly due to a proportion of cells being resistant at baseline, and the contribution of mutations occurring during the study leading to resistance was negligible. Estimated growth rate and dose–response was found to be comparable between brain metastasis and subcutaneous mouse models.ConclusionsThe developed model to describe resistance suggests that the resistance to EGFR-inhibition seen in these xenografts is best described by assuming a small percentage of cells are resistant to treatment at baseline. This model suggests changes to dosing and dosing schedule may not prevent resistance to treatment developing, and that additional treatments would need to be used in combination to overcome resistance.

2 Reliance upon ancestral mutations is maintained in colorectal cancers that heterogeneously evolve during targeted therapies

IntroductionClinical effectiveness of targeted therapies is often transitory and virtually all patients develop secondary resistance. The heterogeneous mechanisms of resistance and the sub-clonal evolution pattern of cell populations that emerge upon pharmacological selection limit disease control with subsequent lines of therapy. We used colorectal cancer (CRC) to test the hypothesis that ancestral oncogenic event (such as WNT pathway alterations) shared by every cell sub-clone might be better suited as therapeutic targets than heterogeneous events in the branches, as the former remain present in each drug-resistant cell independently from its genetic drift.Material and methodsExome analysis was performed on a panel of CRC cells that became resistant to commonly used targeted agents to uncover genetic alterations acquired under drug selective pressure. Tumour evolution of resistant cell population was then inferred through EXPANDs software. β-catenin activity, cell growth and death were measured upon functional and pharmacological modulation of WNT pathway in resistant cells and preclinical models established from tissues biopsy obtained from CRC patients who relapsed during treatment with targeted therapies.Results and discussionsExome sequencing and phylogenetic tracking unveiled a complex sub-clonal architecture in CRC resistance cell populations, indicating parallel evolution of multiple independent cellular lineages, each associated with distinct genomic landscapes. Functional and pharmacological inhibition of WNT signalling induces cell death in CRC preclinical models, regardless of drug type or resistance mechanisms. Pharmacological blockade of WNT pathway resulted in translocation of β-catenin to the plasma membrane, decreased of β-catenin dependent Tcf/LEF transcriptional activity, and cell growth impairment despite massive molecular drift of resistant derivatives. Concomitant blockade of WNT and MAPK signalling restrained evolution of drug-resistant clones and prevent onset of secondary resistance.ConclusionIn summary, reliance of CRC on the WNT pathway is preserved in later phases of colorectal carcinogenesis when tumours face genomic bottlenecks and sub-clonal evolution driven by administration of target therapies; thus offering the possibility of a common therapeutic strategy to overcome secondary resistance to treatments.

Abstract 5011: Patient-derived organotypic cancer spheroids (PDOCS) as predictive models for the treatment of cancer in a clinically meaningful time frame

Abstract Background: Standard two-dimensional cell cultures do not retain the key characteristics of the human cancers from which they are derived and treatment effects are not always able to be replicated in vivo, making the development of alternative culturing systems paramount. Specifically, commercially available cell lines do not fully represent the mutation profiles seen in human cancers. Here we investigate the feasibility of three-dimensional PDOCS to more accurately represent the cancers from which they are derived and to predict treatment sensitivity in a clinically meaningful time frame. Methods: Surgical resection, core needle biopsies, paracentesis or thoracentesis samples from patients with various types of cancer were obtained under an approved IRB protocol, digested and spheroid cultures grown suspended in Matrigel. PDOCS were grown for up to two weeks and passaged at least once prior to treatment. PDOCS were imaged using brightfield imaging (4X) prior to treatment with vehicle or 5-fluorouracil (5-FU; 1, 10, or 100 µM) and/or radiation (2 or 5 Gy). After 2 days of treatment, the 5-FU was removed and the cultures were allowed to grow for an additional 2 days. PDOCS were re-imaged and the relative change in diameter was calculated using ImageJ software and compared to untreated controls. Optical metabolic imaging (OMI) was performed with a multiphoton microscope to probe the fluorescence lifetime and optical redox ratio of metabolic co-enzymes NAD(P)H and FAD. Single-cell analysis of each image was completed using Cell Profiler software to parse resistant cell populations in each PDOCS sample tested. DNA for mutation profile analysis was isolated and sequenced using a QIAseq targeted panel. Results: PDOCS were successfully isolated from a variety of cancers including colorectal (CRC), pancreas, lung, neuroendocrine, liver, breast, and ovarian. Key phenotypic characteristics of the tumors were retained in PDOCS cultured including crypt-like structures, mucin production and Ki67 proliferation rates. Cancer hot spot sequencing was performed comparing PDOCS and the tumors from which they were derived. Over 90% of the nonsynonymous mutations were identical, except in the setting of microsatellite instability. All driver mutations were identical (i.e., APC, KRAS, PIK3CA, TP53). Differential sensitivity to chemoradiation was observed among 4 different colorectal PDOCS treated with 5-FU and radiation (Median % PDOCS diameter change vs control: Patient A 17.1, p=0.64; Patient B -3.05, p=0.02; Patient C -19.4, p=2x10-5; Patient D -31.3, p=0.002). Similar response data were determined using OMI; however, single-cell analyses identified potentially resistant cell populations. Conclusions: PDOCS retain key characteristics of the cancers from which they are derived and can be utilized for treatment sensitivity testing in a clinically meaningful time frame. Citation Format: Cheri A. Pasch, Peter F. Favreau, Alex E. Yueh, Kwang P. Nickel, Christopher P. Babiarz, Philip B. Emmerich, Rosabella T. Pitera, Susan N. Payne, Demetra P. Korkos, Joseph T. Sharick, Carley M. Sprackling, Linda Clipson, Kristina A. Matkowskyj, Michael A. Newton, Melissa C. Skala, Michael F. Bassetti, Randall J. Kimple, Dustin A. Deming. Patient-derived organotypic cancer spheroids (PDOCS) as predictive models for the treatment of cancer in a clinically meaningful time frame [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5011.

Abstract 4474: An intricate role of p53 and p21 in cellular alterations and drug penetration in spheroids of colorectal cancer cells

Abstract The tumor protein p53 and cyclin-dependent kinase inhibitor p21Waf1/Cip1 (p21) are two proteins with an intriguing and a paradoxical role in cancer. Through p53-dependent signals, p21 negatively controls cell cycle progression to protect cells against deleterious effects of DNA damage. Although p53 is a tumor suppressor protein, recent evidence shows that some patients with wild-type (wt) p53 develop aggressive and drug-resistant cancers. Similarly, recent studies also suggest the oncogenic role of p21. In a p53 deficient background, p21 results in the development of a subset of highly proliferative and resistant population of cancer cells. In this study, we examined the role of p53 and p21 in inducing cellular alterations using a panel of colorectal carcinoma cell lines with different p53 and p21 background and spheroid and spheroid-derived cell cultures. First, our data show that despite the presence of proliferating cells, spheroid-derived cultures of HCT116/p53wt cells continue to display an elevated p21 level even after 6 days of culture as a monolayer. These p21-overexpressing proliferating cells show increased resistance to doxorubicin and paclitaxel in comparison to their parental monolayer counterpart. In contrast to HCT116/p53wt cells, DLD-1/p53mut cells did not show elevated p21 levels following reversal from spheroid to monolayer. The increased p21 level in spheroids and spheroid-derived cultures of HCT116/p53wt cells led us to examine the role of p53 and p21 in drug penetration. Spheroids of HCT116/p53wt cells were less permeable to doxorubicin and showed increased expression of intracellular junctional (IJ) proteins (adherens and tight junctions). Knocking-out p21 in HCT116/p53wt cells decreased the level of IJ proteins, resulting in an increased doxorubicin penetration. Re-expression of p21 in p21-knocked-out HCT116/p53wt cells restored IJ protein levels, reducing doxorubicin penetration and the subsequent effect of doxorubicin on spheroids. Interestingly, however, knocking-out p21 in DLD-1/p53mut cells neither altered intracellular junctions nor affected doxorubicin penetration. These data indicate that the effect of p21 on IJ proteins and drug penetration is pertinent only in cells with wt p53. Overall, the data from our study suggest an intricate interplay between wt p53 and p21 in inducing cellular alterations that affect the sensitivity of cells to anti-cancer drugs in spheroids. Citation Format: Viswanath Das, Narendran Annadurai, Marián Hajdúch. An intricate role of p53 and p21 in cellular alterations and drug penetration in spheroids of colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4474.

Abstract 5829: CDK8 inhibitor SEL120-34A targets CD34 positive AML cells by regulation of various transcriptional programs involved in maintenance of leukemia stem cells

Abstract Despite heterogeneity deregulated transcription is one of the universal features of leukemia cells. This has paved the way for the development of active compounds targeting epigenetic and transcriptional factors. A substituted tricyclic benzimidazole, SEL120-34A, is a novel inhibitor of Cyclin-dependent kinase 8 (CDK8), which regulates transcription by associating with the Mediator complex. In many cancer cells SEL120-34A regulates expression of STAT1- dependent genes as a major transcriptional effect. In AML cells regulation of NUP98-HOXA9 - dependent transcription and other transcriptional programs involved in maintenance of leukemia stem cells have been observed as a dominant mechanism of action. Treatment with the compound resulted in a differential efficacy on CD34 positive AML cells with elevated STAT5 S726 levels and stem cell characteristics. In contrast, resistant cells were negative for activated STAT5 and revealed lineage commitment. Prolonged exposure of AML cells lines to chemotherapy led to the enrichment in population of resistant CD34 positive cells, which proved to be oversensitive to SEL120-34A treatment, providing rationale for rational combinations with approved drugs. Further clinical development of SEL120-34A is warranted as a novel therapeutic approach in relapsed and refractory AML. Citation Format: Eliza Majewska, Milena Mazan, Michal Mikula, Agnieszka Dreas, Katarzyna Wiklik, Aniela Golas, Katarzyna Wojcik, Magdalena Masiejczyk, Malgorzata Statkiewicz, Urszula Kuklinska, Krzysztof Goryca, Aleksandra Grochowska, Aleksandra Cabaj, Michal Combik, Jerzy Ostrowski, Krzysztof Brzozka, Tomasz Rzymski. CDK8 inhibitor SEL120-34A targets CD34 positive AML cells by regulation of various transcriptional programs involved in maintenance of leukemia stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5829.

Abstract 1803: Dynamic pro-survival signaling mediates resistance to androgen receptor targeted therapy in AR-v7 splice variant expressing prostate cancer models

Abstract Patient survival rates for metastatic prostate cancer are limited by therapeutic failure and disease progression. Treatment of castrate resistant prostate cancer (CRPC) remains a clinical challenge as current treatment options offer modest benefit and are not curative. Second generation androgen axis inhibitors such as enzalutamide and abiraterone acetate provide initial clinical benefit for patients. However, responses are not durable and disease progression is inevitable, resulting in limited patient survival. Several mechanisms of resistance have been identified that relate to persistent AR signaling including AR splice variants (ARVs) which encode an AR protein lacking the ligand binding domain (LBD). Since we have demonstrated that enzalutamide induces an apoptotic response in sensitive prostate tumor cells, we hypothesized that anti-apoptotic signaling is a critical mechanism of resistance to AR-targeted therapy in resistant tumor cell populations. Using 22Rv1 cells, a cell line model that expresses high levels of AR-v7, and cell lines that express low or no ARVs, we demonstrate differential activation of intrinsic/Bcl2-mediated apoptosis signaling in response to enzalutamide. Specifically, in 22Rv1 cells we demonstrate robust induction of anti-apoptotic signaling including increased Bcl-2 and Mcl-1 expression and inactivation of the apoptosis sensitizer Bad in response to enzalutamide, where this signaling is opposed in enzalutamide-sensitive cells. Analysis of potential upstream regulators revealed phosphorylation and activation of Akt through PI3K signaling in the ARV-expressing 22Rv1 cells in response to AR antagonism. Pharmacological inhibition of PI3K/Akt signaling in combination with enzalutamide prevented upregulation of Mcl-1 and inactivation of Bad, thus lowering the apoptotic threshold and sensitizing 22Rv1 cells to enzalutamide. In conclusion, this data demonstrates active engagement of pro-survival kinase signaling that mediates an increased apoptotic threshold in response to AR-directed therapy. Therefore, resistance to AR-directed therapy in ARV-expressing tumors may extend beyond the recognized mechanisms of treatment failure (i.e. lack of LBD for drug binding, constitutive AR transcriptional activity) and involve dynamic anti-apoptosis signaling. We demonstrate that strategies that lower the apoptotic threshold can increase sensitivity to AR antagonist in ARV-expressing cells and represents a novel therapeutic approach with potential to manage or overcome therapy resistance in this lethal form of prostate cancer. Citation Format: Amanda Pilling, Clara Hwang. Dynamic pro-survival signaling mediates resistance to androgen receptor targeted therapy in AR-v7 splice variant expressing prostate cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1803.

Abstract 5848: Histone deacetylase inhibitor selectively targets CD123 positive chemoresistant AML cells

Abstract Acute Myeloid leukemia (AML) is the most common acute leukemia in adults. Chemoresistance remains a major burden for treatment of relapse patient. Studies suggest that AML relapse related to the survival of drug resistant leukemia stem cell (LSC) population. To better understand the mechanism of drug resistance and whether it is correlated to the expression of certain group of LSC surface markers, we collected various human AML cell lines and examined correlations of their IC50 of Ara-C and percentage of individual LSC surface markers in these cell lines. We found that only CD47 (R square = 0.6660, P value = 0.0476) and CD 123 (R square = 0.7421, P value = 0.0275) are positively correlated to chemoresistance. In order to rule out heterogeneous genetics background contribute to chemoresistance of cell lines, we generated a resistant cell line from a drug sensitive AML cell line, OCI-AML2, by gradually increased the Ara-C concentration in culture media. During the selection, the percentage of cells with CD47 and CD123 gradually increased. When the resistant cell line is established, the percent of CD123 positive cells are over 90 percent. RNA-seq analysis shows that genes related to cell cycle regulator and resistance of apoptosis changed significantly. However, signature genes that related to stemness, such as Oct4, Nanog, KLF4, Myc, FLT3, are no significant changed in resistant cells compare with the sensitive line, although Sox2 increased 50% and ALDH1A3 increased 3 folds. Therefore, chemoresistant cells may represent cells in a slow growing, de-differentiate stage, however, they may not bare all characteristics of LSCs. We further investigate the role of epigenetic factors in regulating the survival of chemoresistant leukemia cells. Our data shows epigenetic drugs that targeting histone deacetylases, bromodomain proteins and PRC1 complex are effective to induce apoptosis and cell death of chemoresistant cells, therefore they may be potential second line of chemotherapy drug for chemoresistant patients. Histone deacetylase inhibitor Romidepsin efficiently cleared chemoresistant AML blast in xenographed NSG mice. Furthermore, we found Romidepsin has synergetic effect when combine with Ara-C. Interestingly, Romedepsin preferentially target CD123 positive, slow growing cells while Ara-C mainly target fast growing, CD123 negative cells. Therefore, combine treatment with Romedepsin and Ara-C may prevent the development of chemoresistance in AML cells. In summary, we identify CD123 and CD47 cell surface markers may potentially serve as biomarker for chemresistance AML cells. Our study also sheds light on a new mechanism of drug resistance in leukemia, and provide a rationale to develop and test epigenetic-targeted therapies in leukemia, especially in drug resistant relapse patients. Citation Format: Bowen Yan, Suming Huang, Yi Qiu. Histone deacetylase inhibitor selectively targets CD123 positive chemoresistant AML cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5848.

21 Defining the cell populations responsible for skin cancer initiation and relapse following therapy

IntroductionThe identification of specific cell type from which cancer arises and the cancer cell population that resists upon therapy leading to tumour relapse constitute the main topics of our research. We use the basal cell carcinoma (BCC), the most frequent cancer in humans, as a cancer model for our studies.Material and methodsWe used lineage tracing approach to elucidate the cell at the origin of BCC and study the cancer cell population responsible for relapse following therapy discontinuation.Results and discussionsTo uncover the cancer cell of origin in BCC and the changes in the cellular dynamics that lead to tumour initiation, we assessed the impact of oncogenic hedgehog signalling activation in distinct cell populations and their capacity to induce BCC. We found that only stem cells, and not progenitors, were competent to initiate tumour formation upon oncogenic hedgehog signalling. Interestingly, this difference was due to the hierarchical organisation of tumour growth in oncogene-targeted stem cells, characterised by an increase of symmetric self-renewing divisions and a higher p53-dependent resistance to apoptosis, leading to rapid clonal expansion and progression into invasive tumours.11 Sánchez-Danés A, Hannezo E, Larsimont JC, Liagre M, Youssef KK, Simons BD, Blanpain C. Defining the clonal dynamics leading to mouse skin tumour initation. Nature. 2016 Jul 8;536(7616):298–303. doi: 10.1038/nature19069To study the cancer cell population that mediates BCC relapse upon therapy, we treated two different genetic BCC mouse models with a Smoothened inhibitor (Smoi), the most commonly drug used to treat locally advanced and metastatic BCC. The mechanism by which Smoi leads BCC regression and emergence of resistant tumour cells are currently unknown. We found that Smoi mediates BCC regression by promoting epidermal differentiation and that during the course of Smoi administration, some BCC become resistant to therapy mimicking the situation found in humans. We identify Lgr5 as a marker expressed in the resistant tumour cell population upon Smoi administration.Finally, we demonstrate that combination of Smoi administration with Lgr5 lineage ablation leads to BCC eradication.ConclusionWe demonstrated that stem cells cells are competent to initiate basal cell carcinoma, whereas, committed progenitors resistant.We demonstrated that Smoi administration in combination with Lgr5 lineage ablation leads to BCC eradication, constituting a clinically promising approach to overcome resistance to therapy and cure BCC.

Abstract LB-013: Propranolol alters the intracellular accumulation of doxorubicin and sensitizes angiosarcoma cells to chemotherapy

Abstract Introduction: We and others have found that propranolol, a beta adrenergic receptor antagonist used to treat heart disease, improved the overall survival of patients with angiosarcoma. While combination treatment with chemotherapies has further improved patient responses, identification of chemotherapy drugs that synergize with propranolol may refine treatment protocols. We previously showed that doxorubicin accumulates within the lysosomes of angiosarcoma cell lines, leading to drug resistance. Because propranolol is also lysosomotropic, we sought to determine whether propranolol could reduce the lysosomal accumulation of doxorubicin, increasing tumor cell chemosensitivity. Methods: We used angiosarcoma cell lines to evaluate the effects of doxorubicin in combination with propranolol on cell viability, doxorubicin retention, and drug efflux. The effects of the adrenergic agonist, epinephrine, on doxorubicin retention and efflux were also evaluated. Transmission electron microscopy and cell viability assays were used to assess the long-term effects of propranolol treatment on the lysosome system and doxorubicin resistance, respectively. Results: While propranolol sensitized cells to doxorubicin by reducing the lysosomal accumulation of the drug, it increased the cytosolic concentration of doxorubicin by inhibiting drug efflux. Epinephrine had the opposite effect, reducing the cytosolic accumulation of doxorubicin and activating drug efflux. Although long-term exposure to propranolol expanded both the size and number of cellular lysosomes, cells remained sensitive to doxorubicin treatment in the presence of propranolol; however, removal of propranolol increased drug resistance above previous levels. Conclusions: Angiosarcoma cells sequestered doxorubicin in cellular lysosomes and enhanced drug efflux to circumvent cytotoxicity. Propranolol countered these processes by altering the intracellular distribution of doxorubicin, allowing higher concentrations of drug to reach its nuclear target. Removal of propranolol allowed doxorubicin to re-accumulate in lysosomes, increasing drug resistance. While our results support the use of lysosomotropic chemotherapy drugs in combination with propranolol to increase cytotoxic effects, they also suggest concurrent administration with chemotherapy to reduce the development of resistant cell populations. These results may aid in the rational design of other lysosomotropic chemotherapies in combination with propranolol to treat angiosarcomas. Citation Format: Jhuma Saha, Jong-Hyuk Kim, Clarissa Amaya, Caleb Witcher, Derek M. Korpela, Ali Khammanivong, Josephine Taylor, Brad A. Bryan, Erin B. Dickerson. Propranolol alters the intracellular accumulation of doxorubicin and sensitizes angiosarcoma cells to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-013.

P-251 - Smad4 loss is associated with decreased oxidative metabolism in pancreatic cancer and affects sensitivity to mitochondrial therapy

Pancreatic cancer is one of the most challenging types of tumors to treat. This is likely caused by a population of resistant cancer cells (CS), which are reported to rely on functional mitochondria. We have shown that mitochondrial targeting is an efficient way to eliminate CS. At the same time, we believe that loss of Smad4, a mediator of transforming growth factor 3 (TGF3) pathway, might be the cause of metabolic reprogramming resulting in mitochondrial dysfunction of pancreatic cancer cells (PCC). We demonstrated that respiration of Smad4+ PCC was substantially decreased when TGF3 was applied. By contrast, Smad4-/- PCC as well as Smad4 knocked out (KO) cells showed lower basal respiration level and they were insensitive to TGF3 treatment. Furthermore, we observed significant changes in mitochondrial fragmentation of Smad4+ PCC after TGF3 treatment, while Smad4 KO PCC remained unaffected. Finally, the Smad4 expression profile correlated with the vulnerability of PCC to mitochondrial-targeted inhibitor of complex I, MitoMet. Our results suggest that loss of Smad4 leads to metabolic reprogramming of PCC, which consequently impacts the sensitivity to mitochondrial-targeted therapy. Based on these findings, we propose a new treatment approach aimed at efficient elimination of PCC.

Abstract PD4-04: Combined inhibition of mTOR and CDK4/6 is required for optimal blockade of E2F function and long term growth inhibition in estrogen receptor positive breast cancer

Abstract The cyclin dependent kinase (CDK) –retinoblastoma (RB) -E2F pathway plays a critical role in the control of cell cycle in estrogen receptor positive (ER+) breast cancer. Small molecule inhibitors of CDK4/6 have shown promise in this tumour type in combination with hormonal therapies, reflecting the particular dependence of this subtype of cancer on cyclin D1 and E2F transcription factors. mTOR inhibitors have also shown potential in clinical trials in this disease setting. Recent data has suggested cooperation between the phosphatidylinositol 3-kinase (PI3K) pathway and CDK4/6 inhibition in preventing early adaptation and eliciting growth arrest, but the mechanisms of the interplay between these pathways have not been fully elucidated. Here we show that profound and durable inhibition of ER+ breast cancer growth is likely to require multiple hits on E2F mediated transcription. We demonstrate that inhibition of mTOR using the mTORC1/2 inhibitor vistusertib at 300nM causes a >50% decrease in cyclin D1 protein levels and RB phosphorylation in three cell lines. At these concentrations, vistusertib treatment also elicits marked effects on E2F mediated transcription, causing changes in the mRNA levels of 28 out of 43 (65%) of a selected set of E2F target genes.Combined inhibition of mTOR, CDK4/6 and ER delivers profound and durable regressions in breast cancer cell lines and xenografts (110.2% tumour growth inhibition at day 48). In vivo data show, that over a period of 58 days, tumours failed to re-grow in the presence of the triplet combination compared to either agent alone, suggesting, that the triplet is necessary to maintain growth inhibition. Furthermore, we show that CDK4/6 inhibitor resistant cell lines re-activate the CDK-RB-E2F pathway, but remain sensitive to mTOR inhibition (EC50 52.7 nM in parental cells vs 39.6-73.3 nM in a number of palbociclib resistant cell populations), suggesting that mTORC1/2 inhibitors may represent an option for patients that have relapsed on CDK4/6 therapy. A Phase I study (PASTOR) combining the dual TOR kinase inhibitor Vistusertib with Palbociclib, and Fulvestrant is underway to explore safety and efficacy of the triplet combination in patients with metastatic breast cancer. Citation Format: Oelmann E, Michaloglou C, Crafter C, Siersbaek R, Delpuech O, Curven J, Carnevalli L, Staniszweska A, Polanska U, Cheraghchi-Bashi A, Lawson M, Chernukhin I, McEwen R, Carroll J, Cosulich S. Combined inhibition of mTOR and CDK4/6 is required for optimal blockade of E2F function and long term growth inhibition in estrogen receptor positive breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD4-04.

3 - BBI608, a small molecule stemness inhibitor, packs a big punch against cisplatin resistant NSCLC

The cancer stem cell (CSC) hypothesis is a widely investigated field within oncology, it hypothesises that there is a robustly resistant stem-like population of cells that survive and thrive in initial chemotherapeutic treatment. These surviving CSCs contribute to the recapitulation of a heterogeneous tumour resulting in relapse and resistance. BBI608 is a small molecule inhibitor of cancer stemness, targeting STAT3, leading to the inhibition of critical genes required for the cancer stemness maintenance. Following the initial in vitro and in vivo preclinical promise of BBI608, phase II and III clinical trials are underway to investigate BBI608 in advanced malignancies and in combination with numerous chemotherapies.

Abstract PR14: Polycomb repressive complex 2 inactivation induces primary chemotherapy resistance in T-ALL by upregulating the TRAP1 mitochondrial chaperone

Abstract The tendency of mitochondria to undergo or resist BCL2-controlled apoptosis (so-called mitochondrial priming) is a powerful predictor of the outcome of cytotoxic chemotherapy for cancer. To fully exploit this finding, it will be important to understand the molecular genetic contexts responsible for the relative mitochondrial priming of chemotherapy-sensitive versus resistant cell populations. Here we report that mitochondrial apoptosis resistance in T-cell acute lymphoblastic leukemia (T-ALL) is mediated by inactivation of polycomb repressive complex 2 (PRC2) and consequent downstream upregulation of the TRAP1 gene, which encodes a mitochondrial chaperone protein of the HSP90 family. In clinical samples from 47 T-ALL patients, we found that loss-of-function mutations in any of three core components of PRC2 (EZH2, EED or SUZ12) were associated with resistance to mitochondrial apoptosis, as assessed by BH3 profiling (P = 0.015). In human T-ALL cells, PRC2 depletion induced resistance to mitochondrial apoptosis induction, as assessed by caspase 3/7 activation or annexin V/PI staining, in response to multiple antileukemic drugs with distinct mechanisms of action, including dexamethasone, doxorubicin, vincristine, and asparaginase (P < 0.01). In mouse immature T-cell progenitors, haploinsufficiency for the PRC2 components Ezh2 or Eed was sufficient to induce resistance to mitochondrial apoptosis, as assessed by BH3 profiling analysis (P ≤ 0.01). PRC2 is a histone-modifying complex whose activity is strongly associated with transcriptional repression. We found that PRC2 represses transcription of TRAP1, a nuclearly encoded, mitochondrially localized chaperone of the HSP90 family. Importantly, TRAP1 overexpression was necessary to induce resistance to chemotherapy-induced apoptosis downstream of PRC2 inactivation (P < 0.001), while pharmacologic inhibition of TRAP1 synergized with antileukemic drugs in PRC2-deficient leukemic cells. These findings demonstrate the importance of relative mitochondrial apoptotic priming as a prognostic factor in T-ALL, and implicate mitochondrial chaperone function as a molecular determinant of response to cancer chemotherapy, suggesting a rationale for targeted therapeutic intervention. This abstract is also being presented as Poster 07. Citation Format: Ingrid Aries, Triona Ni Chonghaile, Salmaan Karim, Sebastian Balbach, Melissa Burns, Gayle Pouliot, Stevenson Kristen, Donna Neuberg, Meenakshi Devidas, Loh Mignon, Stephen Hunger, Stuart Winter, David Teachey, Karen Rabin, Kimberly Dunsmore, Brent Wood, Lewis Silverman, Stephen Sallan, Peter Van Vlierberghe, Stuart H. Orkin, Anthony G. Letai, Alejandro Gutierrez. Polycomb repressive complex 2 inactivation induces primary chemotherapy resistance in T-ALL by upregulating the TRAP1 mitochondrial chaperone [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr PR14.