Mechanisms Of Therapeutic Resistance Research Articles

Abstract 2795: Integrating multiomics discovery approaches to identify biomarkers of therapeutic resistance in metastatic colorectal cancer through analyses of multiple sequential tumor and liquid biopsies; Qcroc01: NCT00984048

Abstract Colorectal cancer (CRC) is the 2nd leading cause of cancer related-death in Canada. Clinical responses of metastatic (m)CRC to first-line treatment range from 35 to 60%, but even responders inevitably develop therapeutic resistance. Studies aiming at understanding mechanisms of resistance have largely investigated primary tumors. However, selective pressures during therapy can lead to the expansion of resistant clones and tumor heterogeneity. This highlights the need to characterize the molecular changes of metastasis and plasma over time of treatment and response to decipher tumor evolution and therapeutic resistance mechanisms. In this multicenter study, 52 tissue samples from liver metastasis were collected at baseline (pre-biopsies) and at the time of resistance (post-biopsies) in responder and non-responder mCRC patients (n=44) undergoing the same standard first-line treatments. Multiple post-biopsies also have been harvested in 4 patients, to allow the assessment of tumor heterogeneity and as well as the evolution of the genomic complexity after treatment exposure. Analyses were carried out across multiple omic platforms to identify resistant signatures and characterize molecular changes during treatment. Biopsies were profiled using exome and transcriptome sequencing as well as high-density SNP array analysis to capture chromosomal anomalies, loss of heterozygosity (LOH) and copy number variations (CNV). Additionally, serial blood samples were collected for proteomic, ctDNA and cytokine analysis. Our preliminary analysis of transcriptomes performed on serial biopsies from a set of 11 patients identified genes consistently overexpressed at resistance. Cytogenetics analysis showed similar genomic profiles of matched pre- and post-biopsies and allowed the establishment of LOH and CNV catalogues of liver metastasis, while exome sequencing revealed cumulative somatic mutations over time of treatment, which suggests subclonal and acquired “driver” mutations of resistance. Plasma-derived ctDNA analysis was performed to investigate the mutational status during treatment and whether they correlate with their relative levels in biopsies. Immune gene expression analysis of a test set of 27 metastases revealed strong clustering of 7 metastases due to overexpression of transcripts related to active immune response, allowing to define novel subgroups of patients based on immune response status. Our study, using a multi-omic strategy and integration of independent molecular platforms to profile liver metastasis samples of responder and non-responder mCRC patients, constitutes an innovative approach to identify clinical biomarkers and molecular signature of resistance, which may enhance individualization of cancer medicine and customized therapy. Citation Format: Karen Gambaro, Maud Marques, Ryan Morin, Claudia Kleinman, Michael Witcher, Simon Turcotte, Benoit Samson, Bernard Lespérance, Yoo-Joung Ko, Richard Dalfen, Eve St-Hilaire, Lucas Sideris, Felix Couture, Sabine Tejpar, Ronald Burkes, Mohammed Harb, Francine Aubin, Thierry Alcindor, Errol Camlioglu, Adriana Aguilar, Mathilde Couetoux du Tertre1, Suzan McNamara, Adrian Gologan, Petr Kavan, Gerald Batist. Integrating multiomics discovery approaches to identify biomarkers of therapeutic resistance in metastatic colorectal cancer through analyses of multiple sequential tumor and liquid biopsies; Qcroc01: NCT00984048 [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 2795. doi:10.1158/1538-7445.AM2017-2795

Preclinical models for translational sarcoma research.

Sarcoma is a basket term for mesenchymal tumors for which more than 75 genetically and histologically distinct subtypes are recognized. Therapeutic progress has largely been achieved with classical chemotherapeutic drugs that were tested in empirical clinical trials. However, outcome in metastatic patients remains poor and with few exceptions numerous trials have failed or only provided limited improvement in recent years. Given the genomic heterogeneity, preclinical model systems will be indispensable to identify new molecular targets and to prioritize drugs and drug combinations. Cell culture is still widely used in preclinical sarcoma research to identify potential novel therapeutic approaches and resistance mechanisms. New and improved techniques in genome-wide and proteome-wide screens enable a better characterization. In addition to cell line xenograft mouse models, patient-derived xenografts crucially broadened and improved preclinical studies using primary human samples. Finally, novel strategies for genome editing, like CRISPR/Cas and sleeping beauty transposon, lead to development of novel genetically engineered cell lines and mouse models. The present review gives a non-comprehensive overview on current model systems used in sarcoma research and discusses their translational relevance. Those include cell lines, subtype-specific patient-derived cell lines and xenografts as well as developments in genome editing and genetically engineered cell lines and mouse models.

Abstract 504: Quantification of nucleic acid quality in postmortem tissues from a cancer research autopsy program

Abstract The last decade has seen a marked rise in the use of cancer tissues obtained from research autopsies. Such resources have been invaluable for studying cancer evolution or the mechanisms of therapeutic resistance to targeted therapies. Degradation of biomolecules is a potential challenge to usage of cancer tissues obtained in the post-mortem setting and remains incompletely studied. We analysed the nucleic acid quality in 371 different frozen tissue samples collected from 80 patients who underwent a research autopsy, including eight normal tissue types, primary and metastatic tumors. Our results indicate that RNA integrity number (RIN) of normal tissues decline with the elongation of post-mortem interval (PMI) in a tissue-type specific manner. Unlike normal tissues, the RNA quality of cancer tissues is highly variable with respect to post-mortem interval. The kinetics of DNA damage also has tissue type-specific features. Moreover, while DNA degradation is an indicator of low RNA quality, the converse is not true. Finally, we show that despite RIN values as low as 5.0, robust data can be obtained by RNA sequencing that reliably discriminates expression signatures. Based on this promising pilot data, we are currently identifying samples for multiregion RNA sequencing of pancreatic cancers with divergent morphologies within the primary and/or metastatic sites. Thus far we have screened 554 RNA samples from 10 autopsy cases. These samples represent 198 geographically distinct regions of tumor with duplicate or triplicate RNA extractions. The number of samples per patient ranges from 17 to 86 with an average of 55. Our data shows that high quality RNA can be acquired from cases with long PMI (>48h). While extensive screening of samples is required to identify those with optimal quality, the downstream data produced is of high quality and can support novel hypotheses or validations of experimental data. Citation Format: Jun Fan, Raya Khanin, Hitomi Sakamoto, Yi Zhong, Chelsea Michael, Derwin Pena, Breanna Javier, Laura Wood, Christine Iacobuzio-Donahue. Quantification of nucleic acid quality in postmortem tissues from a cancer research autopsy program [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 504. doi:10.1158/1538-7445.AM2017-504

Abstract NG01: Evolution of neoantigen landscape during immune checkpoint blockade in non-small cell lung cancer

Abstract NG01: Evolution of neoantigen landscape during immune checkpoint blockade in non-small cell lung cancer

Abstract 2219: Cancer prevention using a novel fatty acid synthase inhibitor

Abstract Tumor growth and survival is dependent upon de novo fatty acid synthesis regulated via preferential upregulation of fatty acid synthase (FASN) in tumor cells. Here a novel FASN inhibitor that has shown antitumor activity in established tumor models (Alwarawrah Y et al., 2016), now shows promise blocking oncogene-induced cell transformation and significantly delaying time to tumor development in a prevention study using a GEMM of triple negative breast cancer. Non-malignant, immortalized MCF10A human breast epithelial cells were infected with lentivirus encoding for the highly oncogenic 110 kDa truncated form of HER2, herein referred to as p110, under a doxycycline inducible promoter. Lentivirus containing a sham insert served as a control. Forty-eight hours after lentiviral infection, p110 was induced by dox and cells treated with FASN (2 and 4 μM) or vehicle alone for 21 days, and then assessed for cell growth and evidence of senescence. The C3TAg GEMM model of triple negative breast cancer was used to study FASN in the prevention setting. The mice (FVB/N background) express C(3)SV40 T-antigen resulting in the inactivation of p53 and Rb. Eighteen mice were randomized to receive FASN treatment (15mg/kg/ 5 days per week) or control (non-treatment [NT]). The mice were randomized and started treatment at 6 weeks of age, and continued until tumors reached an average size of 87.5mm3. All mice were monitored (5 times/week) for weight loss and time to tumor development. Expression of p110 led to the transformation of MCF10A cells, as evidenced by the formation of plaques in cell culture. Treatment with FASN blocked transformation in p110 expressing MCF10A cells and instead induced growth arrest, expression of senescence associated β-gal, and morphologic appearance consistent with cell senescence. Analysis of viable p110 expressing MCF10A cells that were maintained in the continuous presence of FASN for 60 days led to the identification of a potential mechanism of therapeutic resistance. In the C3TAg in vivo prevention study, FASN resulted in a significant prolongation in time to tumor development (Log-rank (Mantel-Cox); p <0.0056). The median days of tumor onset between the NT and treated groups were 111 and 145 with a range of 84-130 and 85-163, respectively. No gross toxicity or weight loss was observed in FASN treated animals. A novel small molecule FASN inhibitor blocked oncogenic cell transformation and induced a state of senescence. Moreover, FASN significantly delayed the onset of mammary tumors in a GEMM model of aggressive triple negative breast cancer. Identification of a mechanism(s) enabling tumor cells to survive long term FASN inhibition will provide rationale combination strategies to induce permanent senescence or apoptosis, and improve the efficacy of FASN inhibitors as preventative agents. Citation Format: Neil L. Spector, Tim Haystead, Sumin Zhao, Yazan Alwarawrah, David Alcorta, William Kim, Jose R. Roques, Michael Trinkler, David B. Darr. Cancer prevention using a novel fatty acid synthase inhibitor [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 2219. doi:10.1158/1538-7445.AM2017-2219

Intimate cross-talk between cancer cells and the tumor microenvironment of B-cell lymphomas: The key role of exosomes.

B-cell lymphomas are composed of tumor cells and the tumor microenvironment, which is conventionally thought to be composed of a mixture of stromal cells, blood vessels, immune cells, and non-cell components, such as extracellular matrix, cytokines, and chemokines. Exosomes, small endocytically derived vesicles that have been proved to be present in a variety of tumor niches and involved in mediating cell signaling networks, are increasingly regarded as important components of tumor microenvironment. In this review, we first focus on the biogenesis, biodistribution, transportation, and other general characteristics of exosomes and then highlight the vital roles of exosomes in lymphomagenesis and disease progression, particularly from the perspective of immune dysfunction, virus infection, and therapeutic resistance mechanisms.

Characterization of single circulating tumor cells.

"Liquid biopsy" has been introduced for the analysis of circulating tumor cells (CTCs) in the blood of cancer patients, and this new diagnostic approach has received enormous attention. CTCs are derived from primary tumors and metastatic lesions and, therefore, harbor important information on the molecular characteristics relevant to tumor progression and cancer therapy. Analyses of CTCs have paved new diagnostic avenues with obvious clinical implications for personalized medicine. Key areas of clinical applications of CTCs include detection of cancer, prediction of prognosis in patients with curable disease, monitoring systemic therapies, and stratification of patients based on the detection of therapeutic targets or resistance mechanisms. The present Review will focus on the current knowledge derived from single-cell analysis of CTCs at the DNA, RNA, and protein level and emphasize the unique opportunities for performing functional studies that aim to identify the metastasis-initiator cells in cancer patients and obtain predictive information for steering cancer therapies.

Decellularized human colorectal cancer matrices polarize macrophages towards an anti-inflammatory phenotype promoting cancer cell invasion via CCL18.

Macrophages are frequently identified in solid tumors, playing important roles in cancer progression. Their remarkable plasticity makes them very sensitive to environmental factors, including the extracellular matrix (ECM). In the present work, we investigated the impact of human colorectal tumor matrices on macrophage polarization and on macrophage-mediated cancer cell invasion. Accordingly, we developed an innovative 3D-organotypic model, based on the decellularization of normal and tumor tissues derived from colorectal cancer patients' surgical resections. Extensive characterization of these scaffolds revealed that DNA and other cell constituents were efficiently removed, while native tissue characteristics, namely major ECM components, architecture and mechanical properties, were preserved. Notably, normal and tumor decellularized matrices distinctly promoted macrophage polarization, with macrophages in tumor matrices differentiating towards an anti-inflammatory M2-like phenotype (higher IL-10, TGF-β and CCL18 and lower CCR7 and TNF expression). Matrigel invasion assays revealed that tumor ECM-educated macrophages efficiently stimulated cancer cell invasion through a mechanism involving CCL18. Notably, the high expression of this chemokine at the invasive front of human colorectal tumors correlated with advanced tumor staging. Our approach evidences that normal and tumor decellularized matrices constitute excellent scaffolds when trying to recreate complex microenvironments to understand basic mechanisms of disease or therapeutic resistance.

Mechanisms of Therapeutic Resistance in Prostate Cancer.

Prostate cancer is the second leading cause of cancer deaths in the USA. The challenge in managing castration-resistant prostate cancer (CRPC) stems not from the lack of therapeutic options but from the limited duration of clinical and survival benefit offered by treatments in this setting due to primary and acquired resistance. The remarkable molecular heterogeneity and tumor adaptability in advanced prostate cancer necessitate optimization of such treatment strategies. While the future of CRPC management will involve newer targeted therapies in deliberately biomarker-selected patients, interventions using current approaches may exhibit improved clinical benefit if employed in the context of optimal sequencing and combinations. This review outlines our current understanding of mechanisms of therapeutic resistance in progression to and after the development of castration resistance, highlighting targetable and reversible mechanisms of resistance.

Tumor RAS Gene Expression Levels Are Influenced by the Mutational Status of RAS Genes and Both Upstream and Downstream RAS Pathway Genes.

The 3 human RAS genes play pivotal roles regulating proliferation, differentiation, and survival in normal cells and become mutated in 15% to 20% of all human tumors and amplified in many others. In this report, we examined data from The Cancer Genome Atlas to investigate the relationship between RAS gene mutational status and messenger RNA expression. We show that all 3 RAS genes exhibit increased expression when they are mutated in a context-dependent manner. In the case of KRAS, this increase is manifested by a larger proportional increase in KRAS4A than KRAS4B, although both increase significantly. In addition, the mutational status of RAS genes can be associated with expression changes in other RAS genes, with most of these cases showing decreased expression. The mutational status associations with expression are recapitulated in cancer cell lines. Increases in expression are mediated by both copy number variation and contextual differences, including mutational status of epidermal growth factor receptor (EGFR) and BRAF. These findings potentially reveal an adaptive response during tumor evolution that is dependent on the mutational status of proximal genes in the RAS pathway and cellular context. Cell contextual differences in these adaptations may influence therapeutic responsiveness and alternative resistance mechanisms.

Targeted therapies in breast cancer: New challenges to fight against resistance.

Breast cancer is the most common type of cancer found in women and today represents a significant challenge to public health. With the latest breakthroughs in molecular biology and immunotherapy, very specific targeted therapies have been tailored to the specific pathophysiology of different types of breast cancers. These recent developments have contributed to a more efficient and specific treatment protocol in breast cancer patients. However, the main challenge to be further investigated still remains the emergence of therapeutic resistance mechanisms, which develop soon after the onset of therapy and need urgent attention and further elucidation. What are the recent emerging molecular resistance mechanisms in breast cancer targeted therapy and what are the best strategies to apply in order to circumvent this important obstacle? The main scope of this review is to provide a thorough update of recent developments in the field and discuss future prospects for preventing resistance mechanisms in the quest to increase overall survival of patients suffering from the disease.

The promise of circulating tumor cells for precision cancer therapy.

The rapidly growing array of therapeutic options in cancer requires informative biomarkers to guide the rational selection and precision application of appropriate therapies. Circulating biomarkers such as circulating tumor cells have immense potential as noninvasive, serial 'liquid biopsies' that may be more representative of the complete spectrum of a patient's individual malignancy than spatially and temporally restricted tumor biopsies. In this review, we discuss the current state-of-the-art in the isolation and molecular characterization of circulating tumor cells as well as their utility in a wide range of clinical applications such as prognostics, treatment monitoring and identification of novel therapeutic targets and resistance mechanisms to enable real-time adjustments in the clinical management of cancer.

PLGA nanoparticle encapsulation reduces toxicity while retaining the therapeutic efficacy of EtNBS-PDT in vitro.

Photodynamic therapy regimens, which use light-activated molecules known as photosensitizers, are highly selective against many malignancies and can bypass certain challenging therapeutic resistance mechanisms. Photosensitizers such as the small cationic molecule EtNBS (5-ethylamino-9-diethyl-aminobenzo[a]phenothiazinium chloride) have proven potent against cancer cells that reside within acidic and hypoxic tumour microenvironments. At higher doses, however, these photosensitizers induce “dark toxicity” through light-independent mechanisms. In this study, we evaluated the use of nanoparticle encapsulation to overcome this limitation. Interestingly, encapsulation of the compound within poly(lactic-co-glycolic acid) (PLGA) nanoparticles (PLGA-EtNBS) was found to significantly reduce EtNBS dark toxicity while completely retaining the molecule’s cytotoxicity in both normoxic and hypoxic conditions. This dual effect can be attributed to the mechanism of release: EtNBS remains encapsulated until external light irradiation, which stimulates an oxygen-independent, radical-mediated process that degrades the PLGA nanoparticles and releases the molecule. As these PLGA-encapsulated EtNBS nanoparticles are capable of penetrating deeply into the hypoxic and acidic cores of 3D spheroid cultures, they may enable the safe and efficacious treatment of otherwise unresponsive tumour regions.

NO• and Pancreatic Cancer: A Complex Interaction with Therapeutic Potential.

Pancreatic tumors express high level of nitric oxide synthases (NOSs) in particular inducible (iNOS/NOS2) and endothelial (eNOS/NOS3) forms. However, the role of nitric oxide (NO•) in the development and progression of pancreatic cancer is not clearly defined. Delineating the NO•-induced signaling in pancreatic cancer and its potential contribution in disease aggressiveness may provide therapeutic targets to improve survival in this lethal malignancy. Recent Advances: An increased expression of NOS2/iNOS in tumors is associated with poorer survival in early stage resected patients with pancreatic ductal adenocarcinoma (PDAC). Furthermore, genetic deletion of NOS2 enhanced survival in mice with autochthonous PDAC. Additionally, targeting NOS3/eNOS reduced the abundance of precursor lesions in mice, which trended toward improved survival. The extremely poor prognosis in pancreatic cancer is due to the late diagnosis and lack of effective therapy in advanced disease. One of the most critical issues is to decipher the underlying mechanism of disease aggressiveness and therapeutic resistance for identifying potential therapeutic target and effective treatment. Given the evidence of a strong association between inflammation and pancreatic cancer and clinical evidence, which suggests an association between NOS2 and disease aggressiveness, it is critical to define the role of NO• signaling in this lethal malignancy. Recent preclinical and clinical evidences indicate a potential therapeutic significance of targeting NO• signaling in pancreatic cancer. With the emergence of new preclinical models, including the patient-derived organoids, further preclinical evaluation using clinically tested NOS inhibitors is needed for designing future clinical investigation. Antioxid. Redox Signal. 26, 1000-1008.

ALK alterations and inhibition in lung cancer.

The advent of precision medicine in non-small cell lung cancer has remarkably altered the direction of research and improved clinical outcomes. The identification of molecular subsets with differential response to targeted therapies began with the identification of epidermal growth factor receptor mutated tumors in subsets of non-small cell lung cancer (NSCLC). Emboldened by unprecedented response rates to kinase inhibitors seen in that subset, the oncologic community searched for other molecular subsets featuring oncogene addiction. An early result of this search was the discovery of NSCLC driven by activating rearrangements of the anaplastic lymphoma kinase (ALK) gene. In an astoundingly brief period following the recognition of ALK-positive NSCLC, details of the biology, clinicopathologic features, development of targeted inhibitors, mechanisms of therapeutic resistance, and new generations of treatment were elucidated. This review summarizes the current understanding of the pathologic features, diagnostic approach, treatment options, resistance mechanisms, and future research areas for ALK-positive NSCLC.

Quantification of nucleic acid quality in postmortem tissues from a cancer research autopsy program.

The last decade has seen a marked rise in the use of cancer tissues obtained from research autopsies. Such resources have been invaluable for studying cancer evolution or the mechanisms of therapeutic resistance to targeted therapies. Degradation of biomolecules is a potential challenge to usage of cancer tissues obtained in the post-mortem setting and remains incompletely studied. We analysed the nucleic acid quality in 371 different frozen tissue samples collected from 80 patients who underwent a research autopsy, including eight normal tissue types, primary and metastatic tumors. Our results indicate that RNA integrity number (RIN) of normal tissues decline with the elongation of post-mortem interval (PMI) in a tissue-type specific manner. Unlike normal tissues, the RNA quality of cancer tissues is highly variable with respect to post-mortem interval. The kinetics of DNA damage also has tissue type-specific features. Moreover, while DNA degradation is an indicator of low RNA quality, the converse is not true. Finally, we show that despite RIN values as low as 5.0, robust data can be obtained by RNA sequencing that reliably discriminates expression signatures.

Tumor-associated stromal cells as key contributors to the tumor microenvironment.

The tumor microenvironment is a heterogeneous population of cells consisting of the tumor bulk plus supporting cells. It is becoming increasingly evident that these supporting cells are recruited by cancer cells from nearby endogenous host stroma and promote events such as tumor angiogenesis, proliferation, invasion, and metastasis, as well as mediate mechanisms of therapeutic resistance. In addition, recruited stromal cells range in type and include vascular endothelial cells, pericytes, adipocytes, fibroblasts, and bone-marrow mesenchymal stromal cells. During normal wound healing and inflammatory processes, local stromal cells change their phenotype to become that of reactive stroma. Under certain conditions, however, tumor cells can co-opt these reactive stromal cells and further transition them into tumor-associated stromal cells (TASCs). These TASCs express higher levels of proteins, including alpha-smooth muscle actin, fibroblast activating protein, and matrix metalloproteinases, compared with their normal, non-reactive counterparts. TASCs are also known to secrete many pro-tumorigenic factors, including IL-6, IL-8, stromal-derived factor-1 alpha, vascular endothelial growth factor, tenascin-C, and matrix metalloproteinases, among others, which recruit additional tumor and pro-tumorigenic cells to the developing microenvironment. Here, we review the current literature pertaining to the origins of recruited host stroma, contributions toward tumor progression, tumor-associated stromal cells, and mechanisms of crosstalk between endogenous host stroma and tumor cells.

Analysis of Immune Signatures in Longitudinal Tumor Samples Yields Insight into Biomarkers of Response and Mechanisms of Resistance to Immune Checkpoint Blockade.

Immune checkpoint blockade represents a major breakthrough in cancer therapy; however, responses are not universal. Genomic and immune features in pretreatment tumor biopsies have been reported to correlate with response in patients with melanoma and other cancers, but robust biomarkers have not been identified. We studied a cohort of patients with metastatic melanoma initially treated with cytotoxic T-lymphocyte-associated antigen-4 (CTLA4) blockade (n = 53) followed by programmed death-1 (PD-1) blockade at progression (n = 46), and analyzed immune signatures in longitudinal tissue samples collected at multiple time points during therapy. In this study, we demonstrate that adaptive immune signatures in tumor biopsy samples obtained early during the course of treatment are highly predictive of response to immune checkpoint blockade and also demonstrate differential effects on the tumor microenvironment induced by CTLA4 and PD-1 blockade. Importantly, potential mechanisms of therapeutic resistance to immune checkpoint blockade were also identified. These studies demonstrate that adaptive immune signatures in early on-treatment tumor biopsies are predictive of response to checkpoint blockade and yield insight into mechanisms of therapeutic resistance. These concepts have far-reaching implications in this age of precision medicine and should be explored in immune checkpoint blockade treatment across cancer types. Cancer Discov; 6(8); 827-37. ©2016 AACR.See related commentary by Teng et al., p. 818This article is highlighted in the In This Issue feature, p. 803.

Abstract 2129: Induction of autophagy as a mechanism of therapeutic resistance in head and neck cancer

Abstract Background and purpose: Despite decades of research into improved treatments for head and neck cancer, therapeutic resistance remains a major challenge for this malignancy, with roughly 40% of patients developing recurrent disease. Among the plethora of intrinsic and acquired mechanisms of resistance, recent evidence has suggested the cellular process of autophagy may be an additional contributor. This stress response, which normally functions during periods of nutrient deprivation to maintain cellular homeostasis, may have the unintended consequence of protecting tumor cells from the cytotoxic effects of chemotherapy and radiotherapy. We show that autophagy mediates resistance to chemotherapies targeting the EGFR and mTORC pathways in addition to radiotherapy in several head and neck cancer models, begin to elucidate the mechanism of this resistance, and demonstrate that the addition of a specific autophagy inhibitor can block cytoprotective autophagy. Methods and results: Building on well established evidence that mTORC inhibition can directly induce autophagy, recent work has established a novel mechanism by which kinase inactive EGFR resulting from either growth factor starvation or anti-EGFR antibody (cetuximab) can upregulate this response via an interaction with LAMPT4B. Treatment of a panel of HNC cell lines with either serum starvation or cetuximab produced increased formation of LC3-B puncta, a marker for autophagasome formation, by IF staining, and was confirmed by Western blot for increased LC3-B flux and decreased p62. SiRNA knockdown of either EGFR or LAMPT4B in HNC cells abrogated the induction of autophagy in response to either serum starvation or cetuximab treatment. We have also shown that radiation induces autophagy in a time and dose dependent manner. The mechanism of radiation-induced autophagy remains a subject of study; preliminary data suggest that both EGFR and LAMPT4B play a role as siRNA knockdown of those mRNAs reduces the autophagic response. To determine whether blockade of cytoprotective autophagy can help overcome therapeutic resistance, we have tested specific autophagy inhibitors in combination with either chemotherapy or radiation. The novel ULK1 inhibitor SBI-0206965 successfully blocks the induction of autophagy in a panel of HNC lines. More compellingly, while neither SBI-0206965 nor the NEDD4 inhibitor Heclin, impacted cell growth in untreated cells, both were effective in reducing cell survival following treatment with radiation, cetuximab, or mTORC inhibitor (AZD8055). Summary: These pre-clinical studies have established the proof of concept for the cytoprotective effect of autophagy in response to anti-cancer treatments including EGFR or mTORC inhibition and radiotherapy. Further we have identified the addition of specific autophagy inhibitors to standard treatments as a potential strategy to overcome this mechanism of resistance. Citation Format: Adam D. Swick, Tan Xiaojun, Tyler Fowler, Kwangok Nickel, Richard A. Anderson, Randall J. Kimple. Induction of autophagy as a mechanism of therapeutic resistance in head and neck 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 2129.

Abstract 2414: ADAR1-dependent RNA editing is a mechanism of therapeutic resistance in human plasma cell malignancies

Abstract Introduction Multiple myeloma (MM) is a plasma cell malignancy that accounts for more than 10% of all blood cancers and may progress to plasma cell leukemia (PCL). Despite treatment, virtually all patients become unresponsive to treatment. RNA editing is a post-transcriptional pre-mRNA processing activity that represents an unexplored potential source of clonal molecular heterogeneity contributing to therapeutic resistance. In particular, adenosine deaminase acting on RNA (ADAR) 1, which exists in two isoforms, one constitutive and one inflammation-responsive, has been associated with disease progression and cancer stem cell (CSC) maintenance. The aim of this study was to investigate whether enhanced ADAR1 expression and activity contributed to therapeutic resistance of MM and PCL. Procedures 1) ADAR Quantification: Whole gene and isoform-specific qRT-PCR was used to detect ADAR1 expression in PCL and MM primary samples and in human MM cell lines (HMCL). 2) RNA Editing Detection: We developed a RNA editing site-specific qPCR (RESS-qPCR) assay to detect RNA editing in cancer stem-cell associated transcripts. 3) Therapeutic Resistance Assay. A MM cell line was exposed to lenalidomide continuously in vitro to establish a model of therapeutic resistance. 4) Development of a humanized PCL mouse model: We established novel in vivo PCL primagrafts by intrahepatic transplantation of primary total mononuclear cells into neonatal RAG2-/-gc-/- mice. Results Approximately, 30% of MM patients in the MM Genomic Initiative dataset harbor copy number amplifications of the ADAR locus on chromosome 1q21, which portends a poor prognosis. We observed significantly increased ADAR1 expression in primary PCL samples and aberrant RNA editing of the stem cell transcription factor GLI1 and the DNA cytidine deaminase APOBEC3D. Notably, high-ADAR1-expressing PCL cells successfully engrafted in RAG2-/-gc-/- mice. As the inflammation-responsive isoform of ADAR1 was upregulated in primary samples, we sought to explore the effects of the anti-MM agent and immunomodulatory drug lenalidomide on ADAR1 expression and activity. Continuous in vitro exposure to lenalidomide led to increased ADAR1 mRNA and protein level and a potent induction of RNA editing activity. Increased RNA editing was detected in several cancer and stem cell-associated transcripts, including GLI1, APOBEC3D, AZIN1 and MDM2. Notably, this aberrant RNA editing activity was associated with increased self-renewal capacity in vitro and a cancer stem cell phenotype. Conclusions ADAR1 overexpression and deregulated RNA editing represents a unique source of RNA and proteomic diversity, and may confer a survival and self-renewal advantage to MM cells. This research identifies ADAR1 as a new diagnostic and therapeutic target in MM, and establishes a robust humanized PCL primagraft model for future pre-clinical testing of ADAR1 modulatory agents. Citation Format: Elisa Lazzari, Leslie A. Crews, Christina Wu, Heather Leu, Shawn Ali, Raffaella Chiaramonte, Mark Minden, Caitlin Costello, Catriona H.M. Jamieson. ADAR1-dependent RNA editing is a mechanism of therapeutic resistance in human plasma cell malignancies. [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 2414.