Mechanisms Of Therapy Resistance Research Articles

PD25-06 COMBINATION TREATMENT WITH INTRAVESICAL INTERFERON GENE THERAPY AND PAN-ERBB RECEPTOR FAMILY BLOCKER IMPROVES SURVIVAL IN MICE WITH BLADDER CANCER

You have accessJournal of UrologyCME1 Apr 2023PD25-06 COMBINATION TREATMENT WITH INTRAVESICAL INTERFERON GENE THERAPY AND PAN-ERBB RECEPTOR FAMILY BLOCKER IMPROVES SURVIVAL IN MICE WITH BLADDER CANCER Akshay Sood, Jan Rudzinski, Alberto Martini, Come Tholomier, Sharada Mokkapati, and Colin Dinney Akshay SoodAkshay Sood More articles by this author , Jan RudzinskiJan Rudzinski More articles by this author , Alberto MartiniAlberto Martini More articles by this author , Come TholomierCome Tholomier More articles by this author , Sharada MokkapatiSharada Mokkapati More articles by this author , and Colin DinneyColin Dinney More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003303.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Recently, intravesical interferon (IFNα) gene therapy has demonstrated success in treatment of BCG-unresponsive NMIBC in a large, randomized, phase-III trial. While the results have been promising, there is room for improvement. One strategy to improve treatment efficacy and durability may be to identify actionable resistance mechanisms and employ targeted combination therapies. METHODS: We performed end-tumor RNA-seq analysis of MB49 mice tumors treated with IFNα gene therapy to identify potential mechanisms of therapy resistance – ErbB pathway was discovered as a potential target (Figure 1a). We therefore tested combination treatment with ErbB pathway blocker and IFNα in MB49 cells using lentiviral IFNα (LV-IFNα) with/without Afatinib in vitro. Next, in vivo studies were conducted in the MB49 orthotopic mice bladder cancer model: mice were randomized in to 5 treatment groups (n=10 each), saline (Ctrl), LV-Ctrl, Afatinib monotherapy, LV-IFNα monotherapy, and the experimental LV-IFNα + Afatinib combination therapy. Overall survival was assessed. RESULTS: Combination therapy significantly reduced the viability of MB49 cells in vitro compared to any of the other treatment conditions (mean relative ATPase activity for combination treatment at 72 h 4%, compared to 100%, 26%, and 28% for Ctrl, LV-IFNα, and Afatinib, p<0.001; Figure 1b). This effect on cell viability appeared to be driven by a combination of additive cytostatic and cytolytic effects (Figure 1c-d). The combination treatment also decreased cell migration (mean migrated cells/10x field on Boyden chamber assay: 92.3 for combination therapy and 631.0, 600.4, and 270.3 for Ctrl, LV-IFNα, and Afatinib, p<0.001; Figure 1e). Finally, in vivo studies demonstrated improved OS with combination therapy (median OS 49 d in the combination group vs 15, 29, and 26 d in the Ctrl, LV-IFNα, and Afatinib groups, Log-rank p<0.001; Figure 1f). No mice in the combination group died of drug toxicity. CONCLUSIONS: Our preliminary investigations suggest that ErbB pathway may be a clinically meaningful resistance mechanism, which when targeted alongside IFNα gene therapy may improve overall treatment efficacy. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e731 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Akshay Sood More articles by this author Jan Rudzinski More articles by this author Alberto Martini More articles by this author Come Tholomier More articles by this author Sharada Mokkapati More articles by this author Colin Dinney More articles by this author Expand All Advertisement PDF downloadLoading ...

Targeting CLDN6 in germ cell tumors by an antibody-drug-conjugate and studying therapy resistance of yolk-sac tumors to identify and screen specific therapeutic options

BackgroundBeing the standard-of-care for four decades, cisplatin-based chemotherapy is highly efficient in treating germ cell tumors (GCT). However, often refractory patients present with a remaining (resistant) yolk-sac tumor (YST(-R)) component, resulting in poor prognosis due to lack of novel treatment options besides chemotherapy and surgery. The aim of this study was to identify novel targets for the treatment of YST by deciphering the molecular mechanisms of therapy resistance. Additionally, we screened the cytotoxic efficacy of a novel antibody-drug-conjugate targeting CLDN6 (CLDN6-ADC), as well as pharmacological inhibitors to target specifically YST.MethodsProtein and mRNA levels of putative targets were measured by flow cytometry, immunohistochemical stainings, mass spectrometry of formalin-fixed paraffin-embedded tissues, phospho-kinase arrays, or qRT-PCR. Cell viability, apoptosis and cell cycle assays of GCT and non-cancerous cells were performed using XTT cell viability assays or Annexin V / propidium iodide flow cytometry, respectively. Druggable genomic alterations of YST(-R) tissues were identified by the TrueSight Oncology 500 assay.ResultsWe demonstrated that treatment with a CLDN6-ADC enhanced apoptosis induction specifically in CLDN6+ GCT cells in comparison with non-cancerous controls. In a cell line-dependent manner, either an accumulation in the G2 / M cell cycle phase or a mitotic catastrophe was observed. Based on mutational and proteome profiling, this study identified drugs targeting the FGF, VGF, PDGF, mTOR, CHEK1, AURKA, or PARP signaling pathways as promising approaches to target YST. Further, we identified factors relevant for MAPK signaling, translational initiation and RNA binding, extracellular matrix-related processes as well as oxidative stress and immune response to be involved in therapy resistance.ConclusionsIn summary, this study offers a novel CLDN6-ADC to target GCT. Additionally, this study presents novel pharmacological inhibitors blocking FGF, VGF, PDGF, mTOR, CHEK1, AURKA, or PARP signaling for the treatment of (refractory) YST patients. Finally, this study shed light on the mechanisms of therapy resistance in YST.

Acquired non-thermal plasma resistance mediates a shift towards aerobic glycolysis and ferroptotic cell death in melanoma.

To gain insights into the underlying mechanisms of NTP therapy sensitivity and resistance, using the first-ever NTP-resistant cell line derived from sensitive melanoma cells (A375). Melanoma cells were exposed to NTP and re-cultured for 12 consecutive weeks before evaluation against the parental control cells. Whole transcriptome sequencing analysis was performed to identify differentially expressed genes and enriched molecular pathways. Glucose uptake, extracellular lactate, media acidification, and mitochondrial respiration was analyzed to determine metabolic changes. Cell death inhibitors were used to assess the NTP-induced cell death mechanisms, and apoptosis and ferroptosis was further validated via Annexin V, Caspase 3/7, and lipid peroxidation analysis. Cells continuously exposed to NTP became 10 times more resistant to NTP compared to the parental cell line of the same passage, based on their half-maximal inhibitory concentration (IC50). Sequencing and metabolic analysis indicated that NTP-resistant cells had a preference towards aerobic glycolysis, while cell death analysis revealed that NTP-resistant cells exhibited less apoptosis but were more vulnerable to lipid peroxidation and ferroptosis. A preference towards aerobic glycolysis and ferroptotic cell death are key physiological changes in NTP-resistance cells, which opens new avenues for further, in-depth research into other cancer types.

Therapy resistance in neuroblastoma: Mechanisms and reversal strategies.

Neuroblastoma is one of the most common pediatric solid tumors that threaten the health of children, accounting for about 15% of childhood cancer-related mortality in the United States. Currently, multiple therapies have been developed and applied in clinic to treat neuroblastoma including chemotherapy, radiotherapy, targeted therapy, and immunotherapy. However, the resistance to therapies is inevitable following long-term treatment, leading to treatment failure and cancer relapse. Hence, to understand the mechanisms of therapy resistance and discover reversal strategies have become an urgent task. Recent studies have demonstrated numerous genetic alterations and dysfunctional pathways related to neuroblastoma resistance. These molecular signatures may be potential targets to combat refractory neuroblastoma. A number of novel interventions for neuroblastoma patients have been developed based on these targets. In this review, we focus on the complicated mechanisms of therapy resistance and the potential targets such as ATP-binding cassette transporters, long non-coding RNAs, microRNAs, autophagy, cancer stem cells, and extracellular vesicles. On this basis, we summarized recent studies on the reversal strategies to overcome therapy resistance of neuroblastoma such as targeting ATP-binding cassette transporters, MYCN gene, cancer stem cells, hypoxia, and autophagy. This review aims to provide novel insight in how to improve the therapy efficacy against resistant neuroblastoma, which may shed light on the future directions that would enhance the treatment outcomes and prolong the survival of patients with neuroblastoma.

Comprehensive liquid biopsy analysis as a tool for the early detection of minimal residual disease in breast cancer

Liquid biopsy (LB) provides a unique minimally invasive tool to follow-up cancer patients over time, to detect minimal residual disease (MRD), to study metastasis-biology and mechanisms of therapy-resistance. Molecular characterization of CTCs offers additionally the potential to understand resistance to therapy and implement individualized targeted treatments which can be modified during the disease evolution and follow-up period of a patient. In this study, we present a long-term follow-up of operable breast cancer patients based on a comprehensive liquid biopsy analysis. We performed a comprehensive liquid biopsy analysis in peripheral blood of 13 patients with early-stage operable breast cancer at several time points for a period of ten years, consisting of: (a) CTC enumeration using the CellSearch system, (b) phenotypic analysis of CTCs using Immunofluorescence, (c) gene expression analysis, in EpCAM(+) CTCs for CK-19, CD24,CD44, ALDH1, and TWIST1, (d) analysis of PIK3CA and ESR1 mutations in EpCAM(+) CTCs and corresponding plasma ctDNA and (e) DNA methylation of ESR1 in CTCs. 10/13 (77%) patients were found negative for LB markers in PB during the whole follow-up period, and these patients did not relapse during the follow-up. However, 3/13(18%) patients that were positive for at least one LB marker relapsed within the follow-up period. The molecular characteristics of CTCs were highly different even for the same patient at different time points, and always increased before the clinical relapse. Our results indicate that liquid biopsy can reveal the presence of MRD at least 4 years before the appearance of clinically detectable metastatic disease demonstrating that a comprehensive liquid biopsy analysis provides highly important information for the therapeutic management of breast cancer patients.

Lysine-specific histone demethylase 1A (KDM1A/LSD1) inhibition attenuates DNA double-strand break repair and augments the efficacy of temozolomide in glioblastoma.

Efficient DNA repair in response to standard chemo and radiation therapies often contributes to glioblastoma (GBM) therapy resistance. Understanding the mechanisms of therapy resistance and identifying the drugs that enhance the therapeutic efficacy of standard therapies may extend the survival of GBM patients. In this study, we investigated the role of KDM1A/LSD1 in DNA double-strand break (DSB) repair and a combination of KDM1A inhibitor and temozolomide (TMZ) in vitro and in vivo using patient-derived glioma stem cells (GSCs). Brain bioavailability of the KDM1A inhibitor (NCD38) was established using LS-MS/MS. The effect of a combination of KDM1A knockdown or inhibition with TMZ was studied using cell viability and self-renewal assays. Mechanistic studies were conducted using CUT&Tag-seq, RNA-seq, RT-qPCR, western blot, homologous recombination (HR) and non-homologous end joining (NHEJ) reporter, immunofluorescence, and comet assays. Orthotopic murine models were used to study efficacy in vivo. TCGA analysis showed KDM1A is highly expressed in TMZ-treated GBM patients. Knockdown or knockout or inhibition of KDM1A enhanced TMZ efficacy in reducing the viability and self-renewal of GSCs. Pharmacokinetic studies established that NCD38 readily crosses the blood-brain barrier. CUT&Tag-seq studies showed that KDM1A is enriched at the promoters of DNA repair genes and RNA-seq studies confirmed that KDM1A inhibition reduced their expression. Knockdown or inhibition of KDM1A attenuated HR and NHEJ-mediated DNA repair capacity and enhanced TMZ-mediated DNA damage. Acombination of KDM1A knockdown or inhibition and TMZ treatment significantly enhanced the survival of tumor-bearing mice. Our results provide evidence that KDM1A inhibition sensitizes GBM to TMZ via attenuation of DNA DSB repair pathways.

Emergent dynamics of underlying regulatory network links EMT and androgen receptor-dependent resistance in prostate cancer

Advanced prostate cancer patients initially respond to hormone therapy, be it in the form of androgen deprivation therapy or second-generation hormone therapies, such as abiraterone acetate or enzalutamide. However, most men with prostate cancer eventually develop hormone therapy resistance. This resistance can arise through multiple mechanisms, such as through genetic mutations, epigenetic mechanisms, or through non-genetic pathways, such as lineage plasticity along epithelial-mesenchymal or neuroendocrine-like axes. These mechanisms of hormone therapy resistance often co-exist within a single patient’s tumor and can overlap within a single cell. There exists a growing need to better understand how phenotypic heterogeneity and plasticity results from emergent dynamics of the regulatory networks governing androgen independence. Here, we investigated the dynamics of a regulatory network connecting the drivers of androgen receptor (AR) splice variant-mediated androgen independence and those of epithelial-mesenchymal transition. Model simulations for this network revealed four possible phenotypes: epithelial-sensitive (ES), epithelial-resistant (ER), mesenchymal-resistant (MR) and mesenchymal-sensitive (MS), with the latter phenotype occurring rarely. We observed that well-coordinated “teams” of regulators working antagonistically within the network enable these phenotypes. These model predictions are supported by multiple transcriptomic datasets both at single-cell and bulk levels, including in vitro EMT induction models and clinical samples. Further, our simulations reveal spontaneous stochastic switching between the ES and MR states. Addition of the immune checkpoint molecule, PD-L1, to the network was able to capture the interactions between AR, PD-L1, and the mesenchymal marker SNAIL, which was also confirmed through quantitative experiments. This systems-level understanding of the driver of androgen independence and EMT could aid in understanding non-genetic transitions and progression of such cancers and help in identifying novel therapeutic strategies or targets.

Mechanisms involved in cancer stem cell resistance in head and neck squamous cell carcinoma.

Despite scientific advances in the Oncology field, cancer remains a leading cause of death worldwide. Molecular and cellular heterogeneity of head and neck squamous cell carcinoma (HNSCC) is a significant contributor to the unpredictability of the clinical response and failure in cancer treatment. Cancer stem cells (CSCs) are recognized as a subpopulation of tumor cells that can drive and maintain tumorigenesis and metastasis, leading to poor prognosis in different types of cancer. CSCs exhibit a high level of plasticity, quickly adapting to the tumor microenvironment changes, and are intrinsically resistant to current chemo and radiotherapies. The mechanisms of CSC-mediated therapy resistance are not fully understood. However, they include different strategies used by CSCs to overcome challenges imposed by treatment, such as activation of DNA repair system, anti-apoptotic mechanisms, acquisition of quiescent state and Epithelial-mesenchymal transition, increased drug efflux capacity, hypoxic environment, protection by the CSC niche, overexpression of stemness related genes, and immune surveillance. Complete elimination of CSCs seems to be the main target for achieving tumor control and improving overall survival for cancer patients. This review will focus on the multi-factorial mechanisms by which CSCs are resistant to radiotherapy and chemotherapy in HNSCC, supporting the use of possible strategies to overcome therapy failure.

Meta-analysis of expression and the targeting of cell adhesion associated genes in nine cancer types – A one research lab re-evaluation

Cancer presents as a highly heterogeneous disease with partly overlapping and partly distinct (epi)genetic characteristics. These characteristics determine inherent and acquired resistance, which need to be overcome for improving patient survival. In line with the global efforts in identifying druggable resistance factors, extensive preclinical research of the Cordes lab and others designated the cancer adhesome as a critical and general therapy resistance mechanism with multiple druggable cancer targets. In our study, we addressed pancancer cell adhesion mechanisms by connecting the preclinical datasets generated in the Cordes lab with publicly available transcriptomic and patient survival data. We identified similarly changed differentially expressed genes (scDEGs) in nine cancers and their corresponding cell models relative to normal tissues. Those scDEGs interconnected with 212 molecular targets from Cordes lab datasets generated during two decades of research on adhesome and radiobiology. Intriguingly, integrative analysis of adhesion associated scDEGs, TCGA patient survival and protein-protein network reconstruction revealed a set of overexpressed genes adversely affecting overall cancer patient survival and specifically the survival in radiotherapy-treated cohorts. This pancancer gene set includes key integrins (e.g. ITGA6, ITGB1, ITGB4) and their interconnectors (e.g. SPP1, TGFBI), affirming their critical role in the cancer adhesion resistome. In summary, this meta-analysis demonstrates the importance of the adhesome in general, and integrins together with their interconnectors in particular, as potentially conserved determinants and therapeutic targets in cancer.

The multifaceted role of MUC1 in tumor therapy resistance.

Tumor therapeutic resistances are frequently linked to the recurrence and poor prognosis of cancers and have been a key bottleneck in clinical tumor treatment. Mucin1 (MUC1), a heterodimeric transmembrane glycoprotein, exhibits abnormally overexpression in a variety of human tumors and has been confirmed to be related to the formation of therapeutic resistance. In this review, the multifaceted roles of MUC1 in tumor therapy resistance are summarized from aspects of pan-cancer principles shared among therapies and individual mechanisms dependent on different therapies. Concretely, the common mechanisms of therapy resistance across cancers include interfering with gene expression, promoting genome instability, modifying tumor microenvironment, enhancing cancer heterogeneity and stemness, and activating evasion and metastasis. Moreover, the individual mechanisms of therapy resistance in chemotherapy, radiotherapy, and biotherapy are introduced. Last but not least, MUC1-involved therapy resistance in different types of cancers and MUC1-related clinical trials are summarized.

Enhancing CAR T-cell therapies against solid tumors: Mechanisms and reversion of resistance.

Chimeric antigen receptor (CAR) T-cell therapy, belonging to adoptive immune cells therapy, utilizes engineered immunoreceptors to enhance tumor-specific killing. By now new generations of CAR T-cell therapies dramatically promote the effectiveness and robustness in leukemia cases. However, only a few CAR T-cell therapies gain FDA approval till now, which are applied to hematologic cancers. Targeting solid tumors through CAR T-cell therapies still faces many problems, such as tumor heterogeneity, antigen loss, infiltration inability and immunosuppressive micro-environment. Recent advances provide new insights about the mechanisms of CAR T-cell therapy resistance and give rise to potential reversal therapies. In this review, we mainly introduce existing barriers when treating solid tumors with CAR T-cells and discuss the methods to overcome these challenges.

Scientists discover key mechanism of cancer therapy resistance

Scientists discover key mechanism of cancer therapy resistance

Abstract B045: Evaluating the role of altered RNA metabolism in pancreatic cancer therapy resistance

Abstract Pancreatic ductal adenocarcinomas (PDACs) are highly lethal, mostly because they quickly develop resistance to current chemotherapies: Gemcitabine (GEM)/paclitaxel or cocktail FOLFIRINOX. Both chemotherapies rely on nucleoside analogues (GEM and 5-fluorouracil [5-FU], and resistance is developed without acquiring mutations, suggesting the role of non-mutational mechanisms for therapy resistance. Our recent findings demonstrated that PDACs depend on RNA splicing proteins to maintain tumors and are exquisitely susceptible to a range of therapies directed at RNA splicing, supporting the role of aberrant RNA splicing in PDAC therapy resistance. Here we evaluated the role of altered RNA splicing in PDAC therapy resistance. Three murine and human isogenic lines with either sensitivity or resistance to GEM were developed. We performed deep RNA sequencing (RNASeq, 80-100 million reads, traditional is 20-40 million reads), differential gene expression, and splicing analyses. GEM resistant PDAC cells demonstrated &amp;gt;40% of non-canonical RNA splice variants and altered expression of 30% of RNA-binding proteins encoded in the human/murine genome, compared to GEM sensitive ones. Specific alternatively spliced exons with common cis-elements were identified in therapy resistant cells, suggesting that these splicing changes are guided by a splicing factor or other RNA-binding protein(s) (RBPs). Top splicing changes were associated to mRNAs encoding ether lipid and pyruvate metabolism. This finding is important as previous studies have linked these metabolic pathways to therapy resistance, however, they have overlooked if protein isoforms, resulting from aberrant splicing, have different impacts in therapy resistance. In addition, GEM and H3B-8800, a novel spliceosome inhibitor showed higher synergistic activity in GEM resistant PDAC cells compared to GEM sensitive cells, suggesting that the development of resistance in PDAC is highly dependent on RNA splicing. Combinedly, these data strongly suggest that resistance to chemotherapeutic agents in PDAC cells requires altered RNA splicing and aberrant expression of associated proteins. Further investigation is ongoing to identify the splicing events and RBPs related to chemotherapy resistance, which will uncover novel mechanisms of therapy resistance and therapeutic modalities by targeting RNA splicing in pancreatic cancer. Citation Format: Md Afjalus Siraj, Xinning Shan, Robert Tseng, Luisa Escobar-Hoyos. Evaluating the role of altered RNA metabolism in pancreatic cancer therapy resistance [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B045.

Translatome Proteomics Identifies Autophagy As a Resistance Mechanism to on-Target FLT3 Inhibitors in Acute Myeloid Leukemia

Internal tandem duplications (ITD) in the receptor tyrosine kinase FLT3 are among the most frequent mutational targets in acute myeloid leukemia (AML), drive leukemia progression and confer a poor prognosis. Primary resistance to FLT3 kinase inhibitors (FLT3i) such as quizartinib, crenolanib and gilteritinib is a frequent clinical challenge and often occurs in the absence of identifiable genetic causes, suggesting that adaptive cellular mechanisms mediate primary resistance to on-target FLT3i therapy. We therefore systematically investigated acute cellular responses to on-target therapy with multiple FLT3i in FLT3-ITD+ AML using recently developed translatome proteomics to measure changes in the nascent proteome in combination with kinase signaling mapping by phosphoproteomics to identify therapy resistance mechanisms to FLT3 inhibitors in vitro. This systems-biology approach showed that the autophagy network escapes global translation repression caused by FLT3i and pinpointed autophagy induction mediated by the AKT-mTORC1-ULK1 axis as the most dominant stress response mechanism to on-target FLT3i therapy. FLT3i induced autophagy in a concentration- and time-dependent manner specifically in FLT3-ITD+ cells in vitro and in primary human AML cells ex vivo. Inhibition of FLT3-ITD signaling led to decreased phosphorylation at AKT S473, decreased phosphorylation at the AKT substrate mTOR S2448, and decreased phosphorylation at the mTORC1 substrate ULK1 S757. CRISPR/Cas9-targeting of TSC2, a negative mTORC1 regulator, ULK1 or ATG3, a core autophagy protein required for autophagosome formation, significantly attenuated or largely abolished autophagy induction by FLT3i. Pharmacological autophagy inhibition (ROC-325, Lys05) or genetic ablation of autophagy (shRNA-mediated knockdown of ATG3, ULK1 or its target Beclin1) increased the sensitivity to FLT3-targeted therapy in cell lines, patient-derived xenografts and primary AML cells ex vivo. In immunodeficient mice xenografted with human FLT3-ITD+ AML cells, oral co-treatment with gilteritinib and autophagy inhibitor ROC-325 synergistically impaired leukemia progression and extended overall survival. Our findings show the utility of functional translatome proteomics to systematically investigate cell-autonomous therapy resistance mechanisms, identify a molecular mechanism responsible for primary on-target FLT3i treatment resistance and demonstrate the pre-clinical efficacy of a rational combination treatment strategy targeting both FLT3 and autophagy induction.

Disease Characterization and Response Prediction in Myeloma Patients Undergoing Conventional and Cellular Therapies from Circulating Tumor DNA

Background: Multiple myeloma (MM) is characterized by dramatic biological and clinical heterogeneity. Prognostic biomarkers including genomic alterations and bone marrow (BM) minimal residual disease can resolve some of this heterogeneity; however, these rely on invasive tumor sampling which limits their utility. Here we develop and apply plasma cell-free DNA liquid biopsies via Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq) to characterize tumors and quantify treatment response in patients with MM and explore its utility during anti-BCMA CAR T-cell therapy. Methods: We developed a 480 kb MM-specific CAPP-Seq panel, which covers 86 recurrently mutated coding genes as well as other recurrently mutated regions, for disease detection and quantification from circulating tumor DNA (ctDNA). Using this hybrid-capture panel, we profiled 214 plasma, germline and tumor DNA samples from 35 patients including 15 patients undergoing idecabtagene vicleucel (ide-cel) therapy. Samples were sequenced to ultra-high unique molecular depth as previously described (Newman et al, Nat Biotechnol 2016) to quantify ctDNA. Copy number variants (CNVs) were identified using our recently developed method, CANARy (Chabon et al, Nature 2020). Tumor variants and ctDNA levels were identified prior to and following therapy to assess ctDNA dynamics, and were correlated with clinical indices and patient outcomes. Results: We applied CAPP-Seq to samples from 35 patients with newly diagnosed (ND, n=10) or relapsed/refractory (RR, n=25) plasma cell disorders. A median of 87 single-nucleotide variants (SNVs, range 2-283) were detected per case. Among these, a median of 5 mutations/patient were in coding genes such as KRAS (25% of patients), TP53 (25%) and NRAS (19%). Most other SNVs occurred in genomic regions targeted by activation induced deaminase (AID), such as IGH, IGK and IGL. To validate tumor genotyping from plasma, we compared SNVs observed in plasma to paired BM samples in 18 cases. Here, we observed 84% (594/703) of all mutations identified in BM were also present in plasma, including 90% (26/29) of all coding alterations. Interestingly, we detected an additional 259 SNVs in the plasma that were not present in the BM, including KRAS, NRAS, and FAM46C mutations, suggesting ctDNA may better capture spatial tumor heterogeneity. We next utilized CAPP-Seq to quantify disease burden prior to treatment. We detected ctDNA in 97% (31/32) of pretreatment plasma with a median allele frequency of 0.91% (range 0.006 to 36%) and 3.82 log haploid genome equivalents per mL of plasma (hGE/mL). Levels of ctDNA were higher in RRMM compared to NDMM patients (4.26 vs 3.08 log hGE/mL, p=0.02) and correlated with clinical risk factors including R-ISS (stage 1: 2.83 vs stage 2/3: 4.18 log hGE/mL, p=0.01) as well as disease burden measurements including M-spike (rho=0.53, p=0.02) and free light chain ratio (rho=0.40, p=0.03). The fold change in ctDNA level at a median of 36 days from treatment initiation correlated with clinical response (median 0.060 for non-progressors vs 7.0 for progressors, p=0.04). Further, we applied CAPP-Seq to serial samples from 15 patients with RRMM receiving ide-cel. Notably, patients with longer response (>90 days) had significantly lower ctDNA levels at day 28 compared to those with early (<90 days) progression (0.6 vs 4.1 log hGE/mL; p=0.009, Fig 1). Lower ctDNA levels at day 28 predicted longer time to progression (proportional hazard model, p=0.02, HR=1.6). Finally, we assessed clonal evolution upon relapse after ide-cel. In multiple patients we observed emergence of new CNVs in ctDNA, including a case with an emergent loss of chr16, where TNFRSF17 (BCMA) resides (Fig 2). This copy number loss was observed 36 days prior to clinical relapse, was validated through WGS of the relapsed tumor, and resulted in loss of BCMA by immunohistochemistry. Conclusions: Assessment of ctDNA has significant potential utility in MM, as it allows for mutational genotyping, quantification of disease burden, and identification of tumor clonal evolution. Lower levels of ctDNA during treatment and its clearance are associated with improved outcomes, while detection of emergent alterations after targeted therapies may inform subsequent treatment strategies. Use of ctDNA may improve response and relapse prediction and reveal mechanisms of therapy resistance. *SS and DMK are co-senior authors Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

OR30-5 Androgen-Regulated Secreted Factors Promote Tumorigenesis and Immune Suppression in Breast Cancer

Abstract Background Breast cancer (BC) is the most diagnosed cancer and the second-leading cause of cancer-associated death in women in the United States. BCs that express the estrogen and/or progesterone receptor (ER/PR) are treated with ER-targeting endocrine therapies that effectively reduce disease burden and extend lifespan. However, up to 50% of patients with ER+BCs will experience recurrence, often at a metastatic site, within 10 years of diagnosis. Novel targeted therapies are needed to treat both metastatic ER+BC resistant to current treatment strategies and chemoresistant ER-BCs.We identified chitinase 3–like-1 (CHI3L1/BRP-39/YKL-40) and prolactin inducible protein (PIP/GCDFP-15) as androgen receptor (AR)-regulated in BC. PIP is reported to be detectable in &amp;gt;90% of BCs, but its function in BC is not well-characterized. CHI3L1 is a glycosyl hydrolase family 18 protein member with reported roles in aging and numerous pathologies. During mammary gland involution, CHI3L1 contributes to a pro-tumorigenic wound-healing-like environment and increases macrophage infiltration. Further, CHI3L1 correlates with tumor-suppressive M2 macrophage infiltration in ER-BC. We previously reported that ER+ cells and patient-derived xenografts with activating mutations in the ESR1 gene encoding ER—a known mechanism of endocrine therapy resistance—had elevated AR and CHI3L1 gene and protein expression. Based on the above and our finding that metastatic BCs biopsies harboring ESR1 mutations exhibited increased AR expression, we hypothesized that AR regulation of CHI3L1 and PIP promote progression to metastatic disease in part through immune suppressive effects. Methods A ConTra(V3) transcription factor binding site analysis was performed on CHI3L1 and PIP promoters. CHI3L1 and PIP were measured via immunohistochemistry in xenograft tumors in mice treated with or without the androgen dihydrotestosterone (DHT). Immunoblotting, immunohistochemistry, and immunofluorescence approaches were also utilized to assess CHI3L1 protein expression in DHT-treated, tamoxifen resistant (TAMR), and long term E2 deprived (LTED) ER+BC cells. CIBERSORT analysis was conducted on the Firehouse Legacy TCGA dataset. Macrophage efferocytosis assays were performed on an IncuCyte Live Cell Imaging system. Results We identified highly conserved AR binding sites in the CHI3L1 and PIP promoters. CHI3L1 protein was elevated in TAMR and LTED BC cells, which was further elevated in BC cells with ESR1 mutations, as well as DHT-treated cells. Further, CHI3L1 and PIP were increased in xenograft tumors after DHT treatment. CIBERSORT analysis revealed that CHI3L1 and PIP high tumors had altered immune profiles, including differential macrophage levels. Finally, CHI3L1 and PIP treatment significantly reduced macrophage efferocytosis of BC cells (p ≤ 0.0001). Conclusions Our work suggests that AR-mediated regulation of CHI3L1 and PIP may alter immune cell infiltration/function in BC and may be important in endocrine therapy resistant ER+BCs. Further characterization and manipulation of these AR-regulated proteins is underway to test the potential to target and reverse their immune suppressive functions. Presentation: Tuesday, June 14, 2022 10:45 a.m. - 11:00 a.m.

Molecular Mechanisms of Anti-Estrogen Therapy Resistance and Novel Targeted Therapies.

Breast cancer (BC) is the most commonly diagnosed cancer in women, constituting one-third of all cancers in women, and it is the second leading cause of cancer-related deaths in the United States. Anti-estrogen therapies, such as selective estrogen receptor modulators, significantly improve survival in estrogen receptor-positive (ER+) BC patients, which represents about 70% of cases. However, about 60% of patients inevitably experience intrinsic or acquired resistance to anti-estrogen therapies, representing a major clinical problem that leads to relapse, metastasis, and patient deaths. The resistance mechanisms involve mutations of the direct targets of anti-estrogen therapies, compensatory survival pathways, as well as alterations in the expression of non-coding RNAs (e.g., microRNA) that regulate the activity of survival and signaling pathways. Although cyclin-dependent kinase 4/6 and phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) inhibitors have significantly improved survival, the efficacy of these therapies alone and in combination with anti-estrogen therapy for advanced ER+ BC, are not curative in advanced and metastatic disease. Therefore, understanding the molecular mechanisms causing treatment resistance is critical for developing highly effective therapies and improving patient survival. This review focuses on the key mechanisms that contribute to anti-estrogen therapy resistance and potential new treatment strategies alone and in combination with anti-estrogen drugs to improve the survival of BC patients.

Therapy Resistance and Disease Progression in CML: Mechanistic Links and Therapeutic Strategies.

Despite the adoption of tyrosine kinasesinhibitors (TKIs) as molecular targeted therapy in chronic myeloid leukemia, some patients do not respond to treatment and even experience disease progression. This review aims to give a broad summary of advances in understanding of the mechanisms of therapy resistance, as well as management strategies that may overcome or prevent the emergence of drug resistance. Ultimately, the goal of therapy is the cure of CML, which will also require an increased understanding of the leukemia stem cell (LSC). Resistance to tyrosine kinase inhibitors stems from a range of possible causes. Mutations of the BCR-ABL1 fusion oncoprotein have been well-studied. Other causes range from cell-intrinsic factors, such as the inherent resistance of primitive stem cells to drug treatment, to mechanisms extrinsic to the leukemic compartment that help CML cells evade apoptosis. There exists heterogeneity in TKI response among different hematopoietic populations in CML. The abundances of these TKI-sensitive and TKI-insensitive populations differ from patient to patient and contribute to response heterogeneity. It is becoming clear that targeting the BCR-ABL1 kinase through TKIs is only one part of the equation, and TKI usage alone may not cure the majority of patients with CML. Considerable effort should be devoted to targeting the BCR-ABL1-independent mechanisms of resistance and persistence of CML LSCs.

Roles of Chromatin Remodelling and Molecular Heterogeneity in Therapy Resistance in Glioblastoma.

Simple SummaryWe review the role of chromatin and epigenetic dysregulation in therapy resistance in glioblastoma. We discuss how epigenetic and genetic forces may cooperate to programme functional cell states that are inherently resistant to therapy. Targeting epigenetic factors that are dysregulated in this malignancy could, therefore, improve clinical outcomes for patients. We highlight some preclinical and clinical compounds that were tested or are currently being explored for glioblastoma. Lastly, we present our thoughts on the requirements for the development of next-generation epigenetic therapies.Cancer stem cells (CSCs) represent a therapy-resistant reservoir in glioblastoma (GBM). It is now becoming clear that epigenetic and chromatin remodelling programs link the stemlike behaviour of CSCs to their treatment resistance. New evidence indicates that the epigenome of GBM cells is shaped by intrinsic and extrinsic factors, including their genetic makeup, their interactions and communication with other neoplastic and non-neoplastic cells, including immune cells, and their metabolic niche. In this review, we explore how all these factors contribute to epigenomic heterogeneity in a tumour and the selection of therapy-resistant cells. Lastly, we discuss current and emerging experimental platforms aimed at precisely understanding the epigenetic mechanisms of therapy resistance that ultimately lead to tumour relapse. Given the growing arsenal of drugs that target epigenetic enzymes, our review addresses promising preclinical and clinical applications of epidrugs to treat GBM, and possible mechanisms of resistance that need to be overcome.

NRF2: A crucial regulator for mitochondrial metabolic shift and prostate cancer progression.

Metabolic alterations are a common survival mechanism for prostate cancer progression and therapy resistance. Oxidative stress in the cellular and tumor microenvironment dictates metabolic switching in the cancer cells to adopt, prosper and escape therapeutic stress. Therefore, regulation of oxidative stress in tumor cells and in the tumor-microenvironment may enhance the action of conventional anticancer therapies. NRF2 is the master regulator for oxidative stress management. However, the overall oxidative stress varies with PCa clinical stage, metabolic state and therapy used for the cancer. In agreement, the blanket use of NRF2 inducers or inhibitors along with anticancer therapies cause adverse effects in some preclinical cancer models. In this review, we have summarized the levels of oxidative stress, metabolic preferences and NRF2 activity in the different stages of prostate cancer. We also propose condition specific ways to use NRF2 inducers or inhibitors along with conventional prostate cancer therapies. The significance of this review is not only to provide a detailed understanding of the mechanism of action of NRF2 to regulate oxidative stress-mediated metabolic switching by prostate cancer cells to escape the radiation, chemo, or hormonal therapies, and to grow aggressively, but also to provide a potential therapeutic method to control aggressive prostate cancer growth by stage specific proper use of NRF2 regulators.