Mechanisms Of Therapy Resistance Research Articles

Abstract NG06: CHD1-loss confers AR targeted therapy resistance via promoting cancer heterogeneity and lineage plasticity

Abstract NG06: CHD1-loss confers AR targeted therapy resistance via promoting cancer heterogeneity and lineage plasticity

Abstract 279: Long non-coding RNAs in glioblastoma tumor recurrence and therapy resistance

Abstract Glioblastoma multiforme (GBM) is a devastating disease which invariably recurs and is often resistant to standard therapies. GBM patient-derived xenoline (PDX) models of tumor recurrence and radiation resistance were created using serial in vivo selection against radiation therapy (6 × 2Gy fractions over 2 weeks for 6+ rounds). This produced 8 isogenic pairs of patient matched radiation-sensitive, primary GBM PDX to radiation-resistant, recurrent GBM PDX. Long non-coding RNAs (lncRNA) represent novel regulatory mechanisms for therapy resistance and tumor recurrence in GBM. An in silico informatics pipeline employing differential expression, differential gene correlation, machine learning, and semantic networking was devised to identify coding and non-coding transcripts related to radiation resistance from deep total RNA sequencing. This analysis revealed 269 lncRNA and 947 coding transcripts that are associated with adaptive radiation resistance. LncRNA:RNA and lncRNA:DNA interaction prediction software was employed to further uncover mechanisms of epigenetic regulation by lncRNAs. A subset of the lncRNA transcripts were predicted to interact directly with other coding transcripts while other lncRNAs have predicted interactions with DNA in regulatory regions of the human genome. We are further validating lncRNA:DNA interactions through in silico methods to determine if genes proximal to these interaction sites are related to tumorigenesis or therapy response. Semantic linkages have been predicted from references in the current literature for lncRNAs and key molecular processes such as DNA damage response, extracellular matrix, and chromatin remodeling. Modules of highly correlated genes, including lncRNAs, have been identified that are differentially regulated between radiation sensitive and resistant tumors. Cross-validation between our in silico approaches have confirmed high-confidence associations of a subset of lncRNAs with acquired radiation resistance. Subsequently, we will validate the expression and phenotypic relevance of high-confidence transcripts in our cohort of PDX models derived from recurrent GBM tumors. In conclusion, recurrence of therapy resistant GBM is responsible for patient mortality. Not all patients qualify for surgical resection or for chemotherapy, but radiation is almost a universally tolerated therapy. We have generated a novel model of tumor recurrence and radiation resistance that recapitulates recurrent tumor physiology. We have discovered 269 lncRNAs associated with radiation resistance that suggests potential regulatory mechanisms through nucleic acid binding. Evidence suggests that lncRNAs most likely contribute to acquired resistance through epigenetic regulation of transcription and chromatin state. These transcripts may provide novel, druggable targets for treating therapy resistant, recurrent GBM. Citation Format: Christian Tyler Stackhouse, James R. Rowland, Jelai Wang, Thanh Nguyen, Zongliang Yue, Jake Y. Chen, Lara Ianov, G. Yancey Gillespie, Christopher D. Willey. Long non-coding RNAs in glioblastoma tumor recurrence and therapy resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 279.

Natural and herbal compounds targeting breast cancer, a review based on cancer stem cells.

Cancer stem cells (CSCs) are known as the major reason for therapy resistance. Recently, natural herbal compounds are suggested to have a significant role in inhibiting the breast cancer stem cells (BCSCs). The aim of this study was to explore the effective natural herbal compounds against BCSCs.This review article was designed based on the BCSCs, mechanisms of therapy resistance and natural herbal compounds effective to inhibit their activity. Therefore, Science direct, PubMed and Scopus databases were explored and related original articles were investigated from 2010 to 2019. BCSCs use different mechanisms including special membrane transporters, anti-apoptotic, pro-survival, and self-renewal- related signaling pathways. Natural herbal compounds could disturb these mechanisms, therefore may inhibit or eradicate the BCSCs. Studies show that a broad range of plants, either as a food or medicine, contain anti-cancer agents that phenolic components and their different derivatives share a large quantity. Natural herbal compounds play a pivotal role in the eradication of BCSCs, through the inhibition of biological activities and induction of apoptosis. Although it is necessary to conduct more clinical investigation.

Abstract IA06: New strategies for multidimensional molecular characterization and follow-up of high-risk pediatric cancers

Abstract Although 80% of all pediatric patients with cancer can be cured based on modern multimodal therapeutic strategies, at least 20% of these patients will undergo tumor progression or relapse. Understanding the molecular mechanisms underlying tumor progression and resistance to treatment is crucial to the development of new treatment approaches. In high-risk pediatric cancers, somatic DNA alterations such as genomic amplifications, copy number alterations, translocations, or mutations play an important role as molecular diagnostic, prognostic, and predictive biomarkers, which might further define targeted treatment approaches. However, it is now known that in many high-risk pediatric cancers, genetic alterations in a given tumor are not static but undergo modifications, with clonal evolution most likely playing a major role in high-risk tumor progression. Circulating tumor DNA, a fraction of cell-free DNA, can be readily isolated from plasma and now provides an important tool and surrogate for tumor molecular analyses, both at diagnosis and during treatment and follow-up. At diagnosis, a prospective trial, NGSKids (NCT02546453), subsequently enrolled 28 patients with high-risk pediatric cancer (neuroblastoma, rhabdomyosarcoma, Ewing sarcoma, other high-risk cancers) at diagnosis. Whole-exome sequencing (WES) was performed on tumor and germline DNA as well as on cfDNA extracted from plasma at diagnosis, during treatment, and follow-up (2-9 sequential cfDNA samples per patient). Whereas all cfDNA samples obtained at follow-up in patients without evidence of disease revealed no or few tumor cell-specific SNVs, interestingly, cfDNA samples obtained at relapse harbored additional, new relapse-specific SNVs (mean: 10; range 2-42) in all cases, targeting genes such as MAPK and MLL4. Deep sequencing capture techniques enable to develop models of clonal evolution. In nonmetastatic brain tumors, ctDNA isolated from CSF resulted in a higher sensitivity and specificity of detection to tumor cell-specific genetic alterations. A French national program, MICCHADO (NCT03496402), now aims to study clonal evolution based on sequential ctDNA analysis from the time of diagnosis in all high-risk pediatric cancers. At relapse, ctDNA studies can provide complementary information to molecular analyses of tumor samples performed within programs such as MAPPYACTS (NCT02613962). ctDNA also enables to infer expression profiles. Indeed, gene expression levels are reflected by nucleosome positioning, and differences in nucleosome organization at transcription start sites (TSS) lead to differential clipping of fragments upon ctDNA release and distinct nucleosome footprints depending on the expression of a given gene in the originating cells. New technologies are also being explored to analyze epigenetic features. Altogether, the presence of tumor genetic and epigenetic abnormalities in ctDNA can be documented in most patients with high-risk pediatric cancer and frequently suggests spatial and temporal heterogeneity. Sequential studies will further elucidate mechanisms of clonal evolution, tumor progression, and therapy resistance. Thus, sequential studies of ctDNA should now be integrated into the development and optimization of targeted treatment strategies. Citation Format: Gudrun Schleiermacher. New strategies for multidimensional molecular characterization and follow-up of high-risk pediatric cancers [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr IA06.

Linking Cancer Stem Cell Plasticity to Therapeutic Resistance-Mechanism and Novel Therapeutic Strategies in Esophageal Cancer.

Esophageal cancer (EC) is an aggressive form of cancer, including squamous cell carcinoma (ESCC) and adenocarcinoma (EAC) as two predominant histological subtypes. Accumulating evidence supports the existence of cancer stem cells (CSCs) able to initiate and maintain EAC or ESCC. In this review, we aim to collect the current evidence on CSCs in esophageal cancer, including the biomarkers/characterization strategies of CSCs, heterogeneity of CSCs, and the key signaling pathways (Wnt/β-catenin, Notch, Hedgehog, YAP, JAK/STAT3) in modulating CSCs during esophageal cancer progression. Exploring the molecular mechanisms of therapy resistance in EC highlights DNA damage response (DDR), metabolic reprogramming, epithelial mesenchymal transition (EMT), and the role of the crosstalk of CSCs and their niche in the tumor progression. According to these molecular findings, potential therapeutic implications of targeting esophageal CSCs may provide novel strategies for the clinical management of esophageal cancer.

Abstract IA19: Multiple uses of circulating tumor cells in lung cancer?

Abstract Liquid biopsies are increasingly being used in clinical studies as prognostic, predictive, and pharmacodynamics biomarkers, as surrogates of tumor response, to detect minimal residual disease after treatment with curative intent and to inform on mechanisms of treatment resistance and treatment switching. There is also steady progress in the development of liquid biopsies for early detection of cancers. The advantages of liquid biopsies are that they are minimally invasive and readily repeatable. The liquid biopsy that has been most widely adopted is circulating tumor DNA (ctDNA) as it is readily measured without specialist equipment in most molecular biology laboratories. Circulating tumor cells (CTCs) are technically more challenging and have yet to realize their full potential as biomarkers in clinic. However, CTC technology is constantly improving, and CTCs have several exciting applications beyond ctDNA that, once optimized, will assist personalized cancer medicine, including their use as cultures for real-time therapy testing and their utility, in some cancer types, to derive mouse models. I will discuss the use of CTCs from patients with lung cancer in this presentation. CTCs in both non-small cell lung cancer (NSCLC) and SCLC enumerated by CellSearch (EpCAM and cytokerin positive, CD45 negative) hold prognostic information. However, while CellSearch CTCs are scarce in NSCLC, most likely due to epithelial-to-mesenchymal transition (EMT) that leads to downrgulation of the EpCAM surface marker used to capture them for enumeration, they are prevalent in SCLC. Single CTC copy number analysis has led to the development of classifier that, with further validation studies, could predict response to chemotherapy alongside ctDNA analysis for therapy monitoring. We have exploited SCLC CTC prevalence to derive mouse models in immune-incompetent mice (termed CDX) that allow us to explore biology and test new therapeutics. I will also update on our research using our biobank of 46 CDX models, exploring inter and intratumoral heterogeneity. The most important aspect of this approach is the ability to generate pre- and post-therapy CDX models allowing interrogation of therapy resistance mechanisms and the biology of progressing disease in a tumor type where tumor evolution is rapid and where serial tumor biopsies are rarely obtained. I will also describe our approaches to study mechanisms of tumor dissemination, including vasculogenic mimicry and new models of brain metastasis. More recently, we are developing direct CTC cultures that may allow real-time therapy testing with reporting to the clinic. I will report on our NSCLC CTC studies within the UK TRACERx consortium to study tumor evolution. We have explored the potential of CTCs found in the pulmonary draining vein of stage I-IIIa patients at surgery with curative intent to predict risk of disease recurrence and shown in a case study that a lethal subclone that gives rise to metastasis 10 months later was identified in the pulmonary vein at surgery. In summary, this presentation will exemplify utility of CTCs in lung cancer that are distinct from but complement the implementation of ctDNA. Citation Format: Caroline Dive. Multiple uses of circulating tumor cells in lung cancer? [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr IA19.

Targeting STAT3 signaling using stabilised sulforaphane (SFX-01) inhibits endocrine resistant stem-like cells in ER-positive breast cancer

Estrogen receptor (ER) positive breast cancer is frequently sensitive to endocrine therapy. Multiple mechanisms of endocrine therapy resistance have been identified, including cancer stem-like cell (CSC) activity. Here we investigate SFX-01, a stabilised formulation of sulforaphane (SFN), for its effects on breast CSC activity in ER+ preclinical models. SFX‐01 reduced mammosphere formation efficiency (MFE) of ER+ primary and metastatic patient samples. Both tamoxifen and fulvestrant increased MFE and aldehyde dehydrogenase (ALDH) activity of patient-derived xenograft (PDX) tumors, which was reversed by combination with SFX‐01. SFX-01 significantly reduced tumor-initiating cell frequency in secondary transplants and reduced the formation of spontaneous lung micrometastases by PDX tumors in mice. Mechanistically, we establish that both tamoxifen and fulvestrant induce STAT3 phosphorylation. SFX-01 suppressed phospho‐STAT3 and SFN directly bound STAT3 in patient and PDX samples. Analysis of ALDH+ cells from endocrine-resistant patient samples revealed activation of STAT3 target genes MUC1 and OSMR, which were inhibited by SFX-01 in patient samples. Increased expression of these genes after 3 months’ endocrine treatment of ER+ patients (n = 68) predicted poor prognosis. Our data establish the importance of STAT3 signaling in CSC-mediated resistance to endocrine therapy and the potential of SFX-01 for improving clinical outcomes in ER+ breast cancer.

Epigenetic drivers of Cutaneous T Cell Lymphoma revealed at single cell resolution

Abstract Cutaneous T-cell lymphomas (CTCL) are cancers of mature skin-homing T lymphocytes, but the molecular drivers remain poorly understood. We performed whole genome, exome, epigenome, bulk and single cell (sc) transcriptome analyses of serial peripheral blood, skin, and lymph node biopsies from 20 CTCL patients. Initial studies defined markers of therapy resistance and disease progression in pathways that mediate cell migration, cytokine signaling, and epigenetic regulation. Several were novel to CTCL, including BIRC5 (anti-apoptotic); RRM2 (cell cycle); CXCR4 and LAIR2 (migration). On average 7413 cells from 16 serial samples (>100,000 cells total) passed quality thresholds for scRNA- + TCR-seq. We used Seurat, dittoSeq, and SingleR for quality control, dimensionality reduction (TSNE/UMAP), clustering, marker detection, cell type inference, and visualizations, and confirmed the therapy-resistant expression patterns we previously identified, including LAIR2. TCR clonotypes confirmed patient-specific malignant cell populations and defined clonal changes in serial samples. Pseudotime analysis of serial samples with Slingshot identified significant changes in expression related to changes in therapy, particularly in chemokine, TNF, and NFKB signaling. We identified mutations in STAT3&5 in 3/10 and in epigenetic modifiers (KMT2C/D and DNMT3A) in 6/10 patients. Loss of DNMT3A perturbs T cell differentiation and causes leukemia/lymphoma in mouse models. DNMT3A deficiency and additional epigenetic alterations may be previously unrecognized “drivers” of CTCL. These studies define mechanisms of therapy resistance, map clonal evolution, and reveal novel mechanisms of pathogenesis and progression in CTCL.

Generation and Characterization of Patient-Derived Head and Neck, Oral, and Esophageal Cancer Organoids.

Esophageal cancers comprise adenocarcinoma and squamous cell carcinoma, two distinct histologic subtypes. Both are difficult to treat and among the deadliest human malignancies. We describe protocols to initiate, grow, passage, and characterize patient-derived organoids (PDO) of esophageal cancers, as well as squamous cell carcinomas of oral/head-and-neck and anal origin. Formed rapidly (<14 days) from a single-cell suspension embedded in basement membrane matrix, esophageal cancer PDO recapitulate the histology of the original tumors. Additionally, we provide guidelines for morphological analyses and drug testing coupled with functional assessment of cell response to conventional chemotherapeutics and other pharmacological agents in concert with emerging automated imaging platforms. Predicting drug sensitivity and potential therapy resistance mechanisms in a moderate-to-high throughput manner, esophageal cancer PDO are highly translatable in personalized medicine for customized esophageal cancer treatments. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Generation of esophageal cancer PDO Basic Protocol 2: Propagation and cryopreservation of esophageal cancer PDO Basic Protocol 3: Imaged-based monitoring of organoid size and growth kinetics Basic Protocol 4: Harvesting esophageal cancer PDO for histological analyses Basic Protocol 5: PDO content analysis by flow cytometry Basic Protocol 6: Evaluation of drug response with determination of the half-inhibitory concentration (IC50 ) Support Protocol: Production of RN in HEK293T cell conditioned medium.

A review of glioblastoma immunotherapy.

Glioblastoma is a very aggressive cancer with dismal prognosis despite standard of care including surgical resection, radiation therapy, and chemotherapy. There is interest in applying immunotherapy to glioblastoma as this modality has demonstrated remarkable improvements in the management of several solid tumors including melanoma, renal cell carcinoma, and non-small cell lung cancer. This review aims to provide an overview of the current state of glioblastoma immunotherapy. Literature search was performed on PubMed between 1961 and 2020. Initial clinical trials of checkpoint inhibitors and vaccine therapy for glioblastoma have largely been disappointing for both primary and recurrent glioblastoma. This failure has been attributed to glioblastoma's highly immunosuppressive environment and multiple mechanisms of therapy resistance including high tumor heterogeneity, low mutational burden, systemic immunosuppression, and local immune dysfunction. Current clinical trials are exploring combination therapy and novel treatment strategies beyond immune checkpoint therapies and vaccine therapy such as CAR T cells. There is also an effort to establish synergy between immunotherapy and current standard of care. Furthermore, recent advances in personalized neoantigen vaccines suggest a shift towards personalized, patient-specific GBM treatment.

Gene Networks with Transcriptional Bursting Recapitulate Rare Transient Coordinated High Expression States in Cancer.

Non-genetic transcriptional variability is a potential mechanism for therapy resistance in melanoma. Specifically, rare subpopulations of cells occupy a transient pre-resistant state characterized by coordinated high expression of several genes and survive therapy. How might these rare states arise and disappear within the population? It is unclear whether the canonical models of probabilistic transcriptional pulsing can explain this behavior, or if it requires special, hitherto unidentified mechanisms. We show that a minimal model of transcriptional bursting and gene interactions can give rise to rare coordinated high expression states. These states occur more frequently in networks with low connectivity and depend on three parameters. While entry into these states is initiated by a long transcriptional burst that also triggers entry of other genes, the exit occurs through independent inactivation of individual genes. Together, we demonstrate that established principles of gene regulation are sufficient to describe this behavior and argue for its more general existence. A record of this paper's transparent peer review process is included in the Supplemental Information.

Kinome Profiling of NF1-Related MPNSTs in Response to Kinase Inhibition and Doxorubicin Reveals Therapeutic Vulnerabilities.

Neurofibromatosis Type 1 (NF1)-related Malignant Peripheral Nerve Sheath Tumors (MPNST) are highly resistant sarcomas that account for significant mortality. The mechanisms of therapy resistance are not well-understood in MPNSTs, particularly with respect to kinase inhibition strategies. In this study, we aimed to quantify the impact of both the genomic context and targeted therapy on MPNST resistance using reverse phase phosphoproteome array (RPPA) analysis. We treated tumorgrafts from three genetically engineered mouse models using MET (capmatinib) and MEK (trametinib) inhibitors and doxorubicin, and assessed phosphosignaling at 4 h, 2 days, and 21 days. Baseline kinase signaling in our mouse models recapitulated an MET-addicted state (NF1-MET), P53 mutation (NF1-P53), and HGF overexpression (NF1). Following perturbation with the drug, we observed broad and redundant kinome adaptations that extended well beyond canonical RAS/ERK or PI3K/AKT/mTOR signaling. MET and MEK inhibition were both associated with an initial inflammatory response mediated by kinases in the JAK/STAT pathway and NFkB. Growth signaling predominated at the 2-day and 21-day time points as a result of broad RTK and intracellular kinase activation. Interestingly, AXL and NFkB were strongly activated at the 2-day and 21-day time points, and tightly correlated, regardless of the treatment type or genomic context. The degree of kinome adaptation observed in innately resistant tumors was significantly less than the surviving fractions of responsive tumors that exhibited a latency period before reinitiating growth. Lastly, doxorubicin resistance was associated with kinome adaptations that strongly favored growth and survival signaling. These observations confirm that MPNSTs are capable of profound signaling plasticity in the face of kinase inhibition or DNA damaging agent administration. It is possible that by targeting AXL or NFkB, therapy resistance can be mitigated.

Novel Therapeutic Strategies for Ovarian Cancer Stem Cells.

Ovarian cancer (OC) is one of the most lethal gynecologic malignancies. Due to the lack of specific symptoms and screening methods, this disease is usually diagnosed only at an advanced and metastatic stage. The gold-standard treatment for OC patients consists of debulking surgery followed by taxane combined with platinum-based chemotherapy. Most patients show complete clinical remission after first-line therapy, but the majority of them ultimately relapse, developing radio- and chemoresistant tumors. It is now proposed that the cause of recurrence and reduced therapy efficacy is the presence of small populations of cancer stem cells (CSCs). These cells are usually resistant against conventional cancer therapies and for this reason, effective targeted therapies for the complete eradication of CSCs are urgently needed. In this review article, we highlight the mechanisms of CSC therapy resistance, epithelial-to-mesenchymal transition, stemness, and novel therapeutic strategies for ovarian CSCs.

Landscape of cyclin pathway genomic alterations across 7,207 non-prostate genitourinary tumors.

549 Background: Cyclin pathway genomic alterations can be a possible therapeutic target as well as a resistance mechanism for therapy. We describe the landscape of cyclin alterations (alt) in non-prostate genitourinary (GU) cancers. Methods: Consecutive samples were analyzed in a CLIA-certified laboratory using comprehensive genomic profiling (CGP) performed by next-generation tissuesequencing (315 genes, &gt;500X coverage). We describe alterations in activating genes (Table) and co-alterations in resistant genes ( RB1 and CCNE1) (related to cyclin inhibition). Results: Alterations in any cyclin pathway genes were found in 37.9% of bladder/urothelial, 33.8% of testicular, 25.2% of penile and 24.6% of kidney cancers. Most alterations were copy number changes and frequencies varied substantially for each tumor type (Table). The high number of cases permitted identification of interesting patterns of outliers for each histology (examples: testis rhabdomyosarcoma and leydig tumor, 50% and 54.5% CDK4 alt; ureter urothelial, 17% CCND1 amp; bladder squamous, 41% CDKN2A del; kidney malignant rhabdoid, 90% SMARCB1 alt; urethra urothelial, 14.7% CCNE1 alt). Alterations in possible resistance genes RB1 and CCNE1 were more frequent in bladder cancers (especially with a neuroendocrine component). Co-occurrence analysis demonstrated a lower likelihood of concomitant vs. isolated alt in cyclin activating and resistance genes (odds ratio for bladder 0.17, p&lt;0.001; OR testis 0.49, p&lt;0.001 and OR kidney 0.45, p&lt;0.001). Conclusions: Cyclin pathway activating and resistance genomic alt are variable in non-prostate GU tumors. Activating alt often occur without simultaneous resistance alt. Our data may inform opportunities for targeted therapy, especially for rare subtypes.[Table: see text]

Abstract P6-04-24: Transcription factor dynamics in endocrine therapy resistance models using a novel chromatin landscape analysis algorithm based on ATAC-Seq

Abstract Endocrine therapy resistance is one of the major challenges in breast cancer therapy today. As the causes are not well understood, therapeutic options are limited. To investigate the molecular mechanisms of endocrine therapy resistance, we established in vitro Long-Term Estrogen Deprivation (LTED) models using multiple human ER-positive breast cancer cell lines. These cells showed differential time-dependent patterns of ESR1 expression with upregulation in some cell lines and downregulation in others. Although all cells continued to grow, none acquired mutations in the ESR1 ligand binding domain, reported to be responsible for endocrine therapy resistance. Current data using gene expression and DNA modification profiles yield limited information for molecular mechanisms of resistance and potential for developing novel therapies. The chromatin landscape is a potential identifier of disease state, and can provide specific information on transcriptional regulation by enhancers. Thus, changes in chromatin open/closed sites could provide important information to predict disease progression and can be used to identify potential therapeutic targets. We analyzed the time-course of chromatin landscape transitions during acquisition of endocrine resistance by the Assay for Transposase Accessible Chromatin (ATAC-seq), and combined these studies with RNA-seq and genomic DNA sequence analysis. ATAC-seq permits the analysis of all sites potentially accessible to the transcriptional machinery. Furthermore, digital footprinting of protected transcription factor motifs within the accessible regions can evaluate the occupancy of hundreds of different transcription factors at one time. We performed bioinformatic analysis of open and closed chromatin sites and selected relevant enhancer signatures. Because transcription factors play a primary role in gene expression and in cell-fate changes, including response to therapies, we derived digital footprints from transcription factor occupancy to correlate this information with phenotypes of LTED cells. Comparing changes in the chromatin landscape with RNA-seq, we created a high throughput algorithm that integrates the information from open/closed chromatin sites, transcription factors occupancy, and gene expression. We found key transcription factors for acquisition of LTED cells; Myc-Max is associated with ESR1 overexpressed LTEDs, whereas Oct1 and Hox genes are associated with LTEDs with absence of ESR1 expression. These methods establish novel mechanisms of endocrine therapy resistance in breast cancer that can also be applied to other tumor types. Our studies provide a rationale for using chromatin landscape and transcription factor networks to expand the range of translational research in the search for novel therapies for breast cancer. These studies will potentially have high impact on development of important novel therapeutic approaches in cancer and many other diseases. Citation Format: Saori Fujiwara. Transcription factor dynamics in endocrine therapy resistance models using a novel chromatin landscape analysis algorithm based on ATAC-Seq [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-24.

The role of the key autophagy kinase ULK1 in hepatocellular carcinoma and its validation as a treatment target

ABSTRACT Although macroautophagy/autophagy is involved in hepatocellular carcinoma (HCC) initiation and development and has been identified as a mechanism of HCC therapy resistance, the role of ULK1 (unc-51 like autophagy activating kinase 1) in HCC remains unclear. Here, we report that both knockdown and knockout of ULK1 inhibited human HCC cell proliferation and invasion, and Ulk1 deletion abrogated tumor growth in a xenograft mouse model. Furthermore, ULK1 ablation in combination with sorafenib significantly inhibited HCC progression compared with sorafenib treatment alone or vehicle control. To identify candidate ULK1 inhibitors, we used a structure-based virtual docking approach to screen 3428 compounds. Among these compounds, XST-14 showed the highest affinity for the ULK1 protein and specifically blocked ULK1 kinase activity. Moreover, the Lys46, Tyr94 and Asp165 amino acid residues of ULK1 were required for its binding to XST-14 according to molecular docking and mutagenesis experiments. Functional assays revealed that XST-14 blocked autophagy and subsequently induced apoptosis and inhibited growth in HCC cells. More importantly, XST-14 acted synergistically with sorafenib to attenuate HCC progression by inhibiting sorafenib-induced autophagy activation both in vitro and in vivo. In addition, XST-14 was well tolerated and exhibited favorable drug metabolism and pharmacokinetic properties and low toxicity in mice. In summary, our study determined that ULK1 may represent a new therapeutic target for HCC and that targeting ULK1 in combination with sorafenib treatment may serve as a promising interventional strategy for treating HCC. Abbreviations: 3MA: 3-methyladenine; ADV: AutoDock Vina; ATP: adenosine triphosphate; EdU: 5-ethynyl-2′-deoxyuridine; ESI: electrospray ionization; HCC: hepatocellular carcinoma; IC50: half maximal inhibitory concentration; KD: kinase domain; q.o.d., every other day; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SPR, surface plasmon resonance.

The role of extracellular vesicles in acquisition of resistance to therapy in glioblastomas.

Glioblastoma (GBM) is the most aggressive primary brain tumor with a median survival of 15 months despite standard care therapy consisting of maximal surgical debulking, followed by radiation therapy with concurrent and adjuvant temozolomide treatment. The natural history of GBM is characterized by inevitable recurrence with patients dying from increasingly resistant tumor regrowth after therapy. Several mechanisms including inter- and intratumoral heterogeneity, the evolution of therapy-resistant clonal subpopulations, reacquisition of stemness in glioblastoma stem cells, multiple drug efflux mechanisms, the tumor-promoting microenvironment, metabolic adaptations, and enhanced repair of drug-induced DNA damage have been implicated in therapy failure. Extracellular vesicles (EVs) have emerged as crucial mediators in the maintenance and establishment of GBM. Multiple seminal studies have uncovered the multi-dynamic role of EVs in the acquisition of drug resistance. Mechanisms include EV-mediated cargo transfer and EVs functioning as drug efflux channels and decoys for antibody-based therapies. In this review, we discuss the various mechanisms of therapy resistance in GBM, highlighting the emerging role of EV-orchestrated drug resistance. Understanding the landscape of GBM resistance is critical in devising novel therapeutic approaches to fight this deadly disease.

Methylome Variation Predicts Exemestane Resistance in Advanced ER+ Breast Cancer.

Background:More than 30% of estrogen receptor-positive breast cancers are resistant to primary hormone therapy, and about 40% that initially respond to hormone therapy eventually acquire resistance. Although the mechanisms of hormone therapy resistance remain unclear, aberrant DNA methylation has been implicated in oncogenesis and drug resistance.Purpose:We investigated the relationship between methylome variations in circulating tumor DNA and exemestane resistance, to track hormone therapy efficacy.Methods:We prospectively recruited 16 patients who were receiving first-line therapy in our center. All patients received exemestane-based hormone therapy after enrollment. We collected blood samples at baseline, first follow-up (after 2 therapeutic cycles) and at detection of disease progression. Disease that progressed within 6 months under exemestane treatment was considered exemestane resistance but was considered relatively exemestane-sensitive otherwise. We obtained circulating tumor DNA-derived methylomes using the whole-genome bisulfide sequencing method. Methylation calling was done by BISMARK software; differentially methylated regions for exemestane resistance were calculated afterward.Results:Median follow-up for the 16 patients was 19.0 months. We found 7 exemestane resistance-related differentially methylated regions, located in different chromosomes, with both significantly different methylation density and methylation ratio. Baseline methylation density and methylation ratio of chromosome 6 [32400000-32599999] were both high in exemestane resistance. High baseline methylation ratios of chromosome 3 [67800000-67999999] (P = .013), chromosome 3 [140200000-140399999] (P = .037), and chromosome 12 [101200000-101399999] (P = .026) could also predict exemestane resistance. During exemestane treatment, synchronized changes in methylation density and methylation ratio in chromosome 6 [32400000-32599999] could accurately stratify patients in terms of progression-free survival (P = .000033). Cutoff values of methylation density and methylation ratio for chromosome 6 [149600000-149799999] were 0.066 and 0.076, respectively.Conclusion:Methylation change in chromosome 6 [149600000-149799999] is an ideal predictor of exemestane resistance with great clinical potential.

Screening of Pleural Mesothelioma Cell Lines for Kinase Activity May Identify New Mechanisms of Therapy Resistance in Patients Receiving Platin-Based Chemotherapy.

Background Malignant pleural mesothelioma (MPM) is a rare, predominantly asbestos-related and biologically highly aggressive tumor associated with a dismal prognosis. Multimodal therapy consisting of platinum-based chemotherapy is the treatment of choice. The reasons underlying the rather poor efficacy of platinum compounds remain largely unknown. Kinase activity might influence cellular response to these regimens. Materials and Methods For this exploratory study, we screened MPM cell lines (NCI-H2452, NCI-H2052, and MSTO-211H) differing in response to cisplatin and benign control fibroblasts (MRC-5) for overall phosphorylation patterns as well as kinase activity with respect to cellular response to cisplatin-based therapeutics. We analysed the cell lines for cellular kinases in a high-throughput manner using the highly innovative technique PamGene. Cell state analysis including apoptosis, necrosis, and cell viability was performed by using enzyme activity and fluorescent-based assays. Results Cisplatin alters cellular phosphorylation patterns affecting cell cycle, migration, adhesion, signal transduction, immune modulation, and apoptosis. In cisplatin-responsive cell lines, phosphorylation of AKT1 and GSK3B was decreased but could not be influenced in cisplatin-resistant NCI-H2452 cells. Cisplatin-responsive cell lines showed increased phosphorylation levels of JNK1/2/3 but decreased phosphorylation in cisplatin-resistant NCI-H2452 cells. Conclusion Kinase phosphorylation and activity might play a crucial role in cellular response to cytostatic agents. Cisplatin influences phosphorylation patterns with distinct features in cisplatin-resistant cells. These alterations exert a significant impact on cell cycle, migration, adhesion, signal transduction, immune modulation, and apoptosis of the respective tumor cells. Based on our results, the induction of p38 or JNK1/3, or inhibition of AKT1 by, for example, BIA-6, might offer a positive synergistic effect by induction of an apoptotic response to cisplatin-based treatment, thus potentially enhancing the clinical outcome of MPM patients.

Gene Expression Alterations during Development of Castration-Resistant Prostate Cancer Are Detected in Circulating Tumor Cells.

Development of castration-resistant prostate cancer (CRPC) is associated with alterations in gene expression involved in steroidogenesis and androgen signaling. This study investigates whether gene expression changes related to CRPC development can be identified in circulating tumor cells (CTCs). Gene expression in paired CTC samples from 29 patients, before androgen deprivation therapy (ADT) and at CRPC relapse, was compared using a panel including 47 genes related to prostate cancer progression on a qPCR platform. Fourteen genes displayed significantly changed gene expression in CTCs at CRPC relapse compared to before start of ADT. The genes with increased expression at CRPC relapse were related to steroidogenesis, AR-signaling, and anti-apoptosis. In contrast, expression of prostate markers was downregulated at CRPC. We also show that midkine (MDK) expression in CTCs from metastatic hormone-sensitive prostate cancer (mHSPC) was associated to short cancer-specific survival (CSS). In conclusion, this study shows that gene expression patterns in CTCs reflect the development of CRPC, and that MDK expression levels in CTCs are prognostic for cancer-specific survival in mHSPC. This study emphasizes the role of CTCs in exploring mechanisms of therapy resistance, as well as a promising biomarker for prognostic and treatment-predictive purposes in advanced mHSPC.