Drive Tumor Growth Research Articles

Deciphering cell–cell interactions and communication in the tumor microenvironment and unraveling intratumoral genetic heterogeneity via single-cell genomic sequencing

ABSTRACT A tumor’s heterogeneity has important implications in terms of its clonal origin, progression, stemness, and drug resistance. Therefore, because of its significance in treatment, it is important to understand the gene expression pattern of a single cell, track gene expression or mutation in heterogeneous cells, evaluate the clonal origin of cancer cells, and determine the selective evolution of different subpopulations of cancer cells. Researchers are able to trace a cell’s mutation and identify different types of tumor cells by measuring the whole transcriptome with single-cell sequencing (scRNA-seq). This technology provides a better understanding of the molecular mechanisms driving tumor growth than that offered by traditional RNA sequencing methods. In addition, it has revealed changes in the mutations and functions of somatic cells as a tumor evolves; it has also clarified immune cell infiltration and activation. Research on scRNA-seq technology has recently advanced significantly, suggesting new strategies for the treatment of cancer. In short, cancer researchers have become increasingly dependent on scRNA-seq. This paper reviews the development, detection principles, and processes of scRNA-seq technology and their application in tumor research. It also considers potential clinical applications.

Hsa_circ_0000129 drives tumor growth via sequestering miR-485-3p and upregulating SPIN1 in breast cancer.

Breast cancer (BC) is second cancer frequently occurring worldwide. Circular RNA hsa_circ_0000129 (circ_0000129) exerts a tumor-promoting effect in BC. Nevertheless, the molecular mechanisms mediated by the upregulation of circ_0000129 during BC progression are not well understood. Forty-fiveBC patients were recruited for the research. Changes in circ_0000129 levels were detected with quantitative reverse transcription-polymerase chain reaction. Cell proliferation, apoptosis, migration, invasion, and angiopoiesis were determined by cell counting, 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, transwell, and tube formation assays. Protein levels were detected by western blot analysis. The regulatory mechanism of circ_0000129 was predicted by bioinformatics analysis and validated by dual-luciferase reporter and RNA immunoprecipitation assays. In vivo experiments were carried out to verify the function of circ_0000129. Circ_0000129 was overexpressed in BC samples and cell lines. Functionally, circ_0000129 silencing reduced cell proliferation, migration, invasion, and promoted cell apoptosis, as well as induced HUVEC angiopoiesis in vitro. Furthermore, circ_0000129 knockdown decreased BC cell growth in mouse xenograft models. Mechanically, circ_0000129 interacted with miR-485-3p to mediate the inhibiting effect of miR-485-3p on SPIN1. Silenced miR-485-3p expression weakened the inhibiting effect of circ_0000129 knockdown on BC cell malignant behaviors. Also, forced SPIN1 expression weakened miR-485-3p upregulation mediated effects on BC cell malignant behaviors. Circ_0000129 acted as a miR-485-3p sponge molecular to mediate expression, thus promoting BC progression.

Abstract C060: Adipose-tumor crosstalk alters tumor immune profile by promoting PDAC CXCL5 secretion

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer death in the US, due to late detection and limited therapeutic options. While the main cause of PDAC remains unknown, obesity has been shown to be a major risk factor. However, the molecular mechanisms behind adipose-tumor crosstalk are still being elucidated. Therefore, understanding the crosstalk between adipose and PDAC is critical for improving therapeutic approaches. We hypothesize that factors secreted by the adipose tissue reprogram pancreatic cancer cells to drive tumor growth and anti-tumor immunity. We observed enhanced proliferation in cells treated with conditioned media made from adipose tissue collected from PDAC [MV1] patients. Subsequent analysis determined that PDAC cells also secrete high levels of CXCL5 in response to stimulation with adipose conditioned media. Pathway analysis of RNA-sequencing data from conditioned-media-treated human PDAC cell lines implicated IL-1β and TNF-α as being involved in the resulting transcript changes. Using recombinant IL-1β, we stimulated CXCL5 secretion from multiple PDAC cell lines. Additionally, we found an enhanced secretion of IL-1β from obese adipose tissue compared to that from lean adipose and, using an anti-IL-1β blocking antibody we were able to partially depress the CXCL5 secretion from cells stimulated with adipose conditioned media. Because CXCL5 is a known neutrophil activating and attracting protein, we used CRISPR to engineer CXCL5 deficient murine PDAC cells. To determine the effect of tumor-derived CXCL5 on PDAC growth and immune recruitment, we orthotopically injected non-targeting-control and CXCL5-KO K8484 cells into wild-type, syngeneic, obese mice. While CXCL5-KO tumors displayed a similar size, we observed a significant change in the tumor immune profile. Despite an increase in the pro-tumorigenic monocytic myeloid derived suppressor cells (MDSCs), we found that the CXCL5-KO tumors exhibited a significantly enhanced CD8+ T cell infiltration. However, a high percentage of these CD8+ T cells were PD-1 positive, implicating an exhausted phenotype. Subsequently, we treated wildtype and CXCL5 deficient PDAC bearing obese mice with an anti-PD-1 antibody to promote T-cell re-activation, which resulted in a significantly reduced growth of the CXCL5 deficient tumors. In conclusion, obesity and adipose derived factors directly induce tumor cells to support immune suppression and drive PDAC progression. Citation Format: R. McKinnon Walsh, Joseph Ambrose, Bailey A. Bye, Austin E. Eades, Jarrid L. Jack, Mariana T. Ruckert, Appolinaire A. Olou, Fanuel Messaggio, Prabhakar Chalise, Dong Pei, Michael N. VanSaun. Adipose-tumor crosstalk alters tumor immune profile by promoting PDAC CXCL5 secretion [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 C060.

Efferocytosis drives myeloid NLRP3 dependent inflammasome signaling secretion of IL-1β to promote tumor growth

Caspase-1 signaling in myeloid suppressor cells can promote T-cell independent cancer progression, but the regulation of inflammasome signaling within the highly heterogeneous myeloid population in the tumor milieu remains elusive. To resolve this complexity, single cell transcriptomic profile of Head and Neck Squamous Cell Carcinoma (HNSCC) identified distinct inflammasome-associated genes within specific clusters of tumor-infiltrating myeloid cells. Among these myeloid cells, the sensor protein, NLRP3, and downstream effector IL-1β transcripts were enriched in discreet monocytic and macrophage subtypes in the TME. We showed that deletion of NLRP3, but not AIM2, phenocopied caspase-1/IL-1β dependent tumor progression in vivo. Paradoxically, we found myeloid-intrinsic caspase-1 signaling increased myeloid survival contrary to what would be predicted from the canonical pyroptotic function of caspase-1. This myeloid NLRP3/IL-1β signaling axis promotion of tumor growth was found to be gasdermin D independent. Mechanistically, we found that phagocyte-mediated efferocytosis of dying tumor cells in the TME directly activated NLRP3-dependent inflammasome signaling to drive IL-1β secretion. Subsequently we showed that NLRP3-mediated IL-1β production drives tumor growth in vivo. Dynamic RNA velocity analysis showed a robust directional flow from efferocytosis gene-set high macrophages to an inflammasome gene-set high macrophage population. We provide a novel efferocytosis-dependent inflammasome signaling pathway which mediates homeostatic tumor cell apoptosis that characterizes chronic inflammation-induced malignancy.

Estrogen-induced immune changes within the normal mammary gland

Breast cancer (BCa) incidence increases following aberrant hormone exposure, which has been linked to direct effects on estrogen receptor (ER)+ mammary epithelium. While estrogen exposure during mammary involution has been shown to drive tumour growth via neutrophils, the potential for the ER + immune microenvironment to mediate part (in addition to mammary epithelial cells) of hormonally controlled BCa risk during normal development has not been assessed. We collected mammary tissue, lymph nodes and blood from tumour naïve mice treated with, oophorectomy, estrogen (17β estradiol) or Fulvestrant. Flow cytometry was used to examine the impact on the frequency of innate and adaptive immune cells. Oophorectomy and fulvestrant decreased the proportion of macrophages, particularly pro-tumour polarized M2 macrophages and neutrophils. Conversely, dendritic cells were increased by these therapies, as were eosinophils. Estrogen increased the proportion of M2 macrophages and to a lesser extent CD4-CD8- double negative and FoxP3+ regulatory T cells but decreased CD8 + T cells and B cells. Excluding eosinophils, these changes were restricted to the mammary tissue. This suggests that inhibiting estrogen action lowers the immune suppressive myeloid cells, increases in antigen presentation and eosinophil-mediated direct or indirect cytotoxic effects. In contrast, estrogen exposure, which drives BCa risk, increases the suppressive myeloid cells and reduces anti-tumour cytotoxic T cells. The impact of hormonal exposure on BCa risk, may in part be linked to its immune modulatory activity.

Extracellular vesicles from the infarcted and failing heart drive tumor growth

Abstract Aims Heart disease might be an independent risk factor for cancer (reverse cardio-oncology). The co-occurrence of these diseases worsens patients' prognoses and limits therapeutic options. However, the cellular and molecular mechanisms that link heart disease to cancer remain elusive. Therefore, we hypothesized that cardiac extracellular vesicles (cEVs) secreted by diseased hearts carry and disseminate factors that promote tumor growth. Methods and results We subjected female mice to myocardial infarction (MI) or sham-MI and 28 days of follow-up. Left ventricular remodeling and dysfunction were assessed by echocardiography. To determine the role of cEVs in tumor growth, we focused on cardiac mesenchymal stromal cells (cMSCs), which play a central role in cardiac repair, remodeling, and fibrosis. We isolated cMSCs from mice hearts 10 or 28 days after MI or sham MI and purified cMSC-EVs from the conditioned medium using size exclusion chromatography. cEVs were characterized by nanoparticle tracking analysis (NTA), the classical EV markers: CD81 and Tumor susceptibility gene 101, and electron microscopy. cMSCs after MI secreted more small EVs than cMSCs from sham-MI (Fig. 1A, p<0.0001). Proteomic and biological process analysis revealed a distinctive profile of cEVs after MI with more EV-encapsulated proteins related to inflammation, angiogenesis, and cell cycle (Fig. 1B). Purified cMSC-EVs were labeled with PKH26 dye and found to target both breast and lung cancer cells in vitro. Colorimetric proliferation assay showed that MI-cEVs facilitated cancer cells proliferation compared with sham-MI cEVs (n=7 in each group, p<0.0001). Furthermore, by scratch assay, MI-cEVs facilitated cancer cell migration two times faster than sham-MI cEVs (Fig. 1C, p=0.0002). Finally, we established 2 models of heart disease with cancer. Lung or breast cancer cells (750x103 or 250x103) were inoculated into the hind limb or mammary pad 10 days before or after MI. Serial ultrasound examinations monitored tumor growth. While MI significantly stimulated lung cancer growth, EV inhibition by GW4869 markedly attenuated the tumorigenic effect of MI and left ventricular (LV) dysfunction (Fig. 1D, p for GW4869 <0.0001). Moreover, we found an inverse correlation between LV ejection fraction (LVEF) and the volume of breast cancer tumors. cEV inhibition by GW4869 attenuated this inverse correlation (for vehicle group: n=14, r=−0.54 and p=0.04. for GW4869 group: n=13, r=−0.43, and p=0.14). Conclusions Our results suggest, for the first time, that cMSCs from the infarcted and failing heart secret EVs that target tumor cells and accelerate tumor growth. We propose cEVs as potential mediators and therapeutic targets in patients with concomitant heart disease and cancer. Funding Acknowledgement Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): Seymour Fefer Grant

INTRAPLEURAL THERAPY TO DISRUPT IL-6/IL-8 JUXTACTINE SIGNALLING TO BLOCK TUMOR EMT AND TO DRIVE SYSTEMIC ANTI-TUMOR IMMUNITY

The goal of this study was to determine whether antiIL-6Rα block (tocilizumab) will alter the pleural secretome and will diminish tumor-specific immune responses. Pleural T cells were isolated from freshly drained pleural effusions (n=6). Autologous pleural tumor was expanded in vitro using the Mammary Epithelial Growth Medium (Lonza). Pleural T cells were stimulated using anti-CD3/CD28 Dynal beads and low dose IL-2 (60 Cetus U/ml) for 2,4,7, 14 or 21 days in the presence of tocilizumab for the last 48h (0, 0.35, 0.72, 1.43, 2.86 and 5.72ug/ml). Pleural T cell effectors were counted and plated on tumor targets at 12.5:1 E:T in the presence of tocilizumab. Ex vivo expanded pleural T cells were effector-memory phenotype (CD45RA-CD27-) and were highly cytotoxic against autologous tumor (89-100%). The majority of CD8+ T cells were central memory (CD45RA-/CD27-) or effector memory (CD45RA+/CD27-); the majority co-expressed granzyme B, perforin, 20-60% expressed PD-1. Most CD4+ co-expressed granzyme B and perforin and were PD-1+, suggesting cytotoxic CD4+ T cells. The presence of tocilizumab reversed tumor EMT but did not alter cytotoxicity. We show that the IL-6/IL-6Rα axis is prominent in MPE, drives tumor growth and inhibits anti-tumor immunity. Pleural T cells are neither exhausted nor dysfunctional but are suppressed by the pleural environment. Ex vivo expanded MPE CD8 and CD4 T cells are highly cytotoxic against autologous tumor. Anti-IL-6Rα block reverses tumor EMT but does not inhibit effector responses.

Triple negative breast cancer: approved treatment options and their mechanisms of action

PurposeBreast cancer, the most prevalent cancer worldwide, consists of 4 main subtypes, namely, Luminal A, Luminal B, HER2-positive, and Triple-negative breast cancer (TNBC). Triple-negative breast tumors, which do not express estrogen, progesterone, and HER2 receptors, account for approximately 15-20% of breast cancer cases. The lack of traditional receptor targets contributes to the heterogenous, aggressive, and refractory nature of these tumors, resulting in limited therapeutic strategies.MethodsChemotherapeutics such as taxanes and anthracyclines have been the traditional go to treatment regimens for TNBC patients. Paclitaxel, docetaxel, doxorubicin, and epirubicin have been longstanding, Food and Drug Administration (FDA)-approved therapies against TNBC. Additionally, the FDA approved PARP inhibitors such as olaparib and atezolizumab to be used in combination with chemotherapies, primarily to improve their efficiency and reduce adverse patient outcomes. The immunotherapeutic Keytruda was the latest addition to the FDA-approved list of drugs used to treat TNBC.ResultsThe following review aims to elucidate current FDA-approved therapeutics and their mechanisms of action, shedding a light on the various strategies currently used to circumvent the treatment-resistant nature of TNBC cases.ConclusionThe recent approval and use of therapies such as Trodelvy, olaparib and Keytruda has its roots in the development of an understanding of signaling pathways that drive tumour growth. In the future, the emergence of novel drug delivery methods may help increase the efficiency of these therapies whiel also reducing adverse side effects.

BSCI-23 ANEUPLOIDY PROFILING IN GLIOBLASTOMA IDENTIFIES MECHANISMS OF DISEASE PROGRESSION AND TREATMENT VULNERABILITIES

Glioblastoma (GBM) is the most common and malignant adult brain tumor. Despite years of research, few advancements have been made in its management. One challenging area of glioblastoma research is patient stratification in clinical trials based on genomic features. Although several regions of aneuploidy have been known to drive disease progression in GBM, the degree of aneuploidy across the genome varies widely and the significance of regions of aneuploidy has not been assessed. Using whole genome sequencing profiles for matched tumor and non-tumor samples, we were able to accurately determine the degree of aneuploidy and loss of heterozygosity for a set of primary GBM tumors. Next, using machine learning techniques, distinct patterns of aneuploidy and loss of heterozygosity emerged among a set of GBM tumors, allowing us to define distinct aneuploidy subgroups. Interestingly, these aneuploidy subgroups showed distinctly different rates of patient survival, suggesting that regions of aneuploidy may be driving disease progression. Differing rates of various GBM genomic subtypes including IDH mutation, EGFR mutation, MGMT methylation, and tumor subtypes was also seen among the aneuploidy subgroups. We were able to derive a gene expression signature for each of these aneuploidy subgroups and revealed distinct pathways that were driving tumor growth. Furthermore, using a perturbagen-response dataset we were able to predict compounds to distinctly target each subgroup. Collectively, this suggests that aneuploidy profiling provides important clues to varying mechanisms of disease progression and is a promising approach for targeted therapy in a patient-specific manner.

Impact of lncRNA SOX9-AS1 overexpression on the prognosis and progression of intrahepatic cholangiocarcinoma

BackgroundIntrahepatic cholangiocarcinoma (ICC) is a latent and malignant tumor with a dismal prognosis. This study was to evaluate the clinical relevance and therapeutic potential of SOX9-AS1 expression in ICC. MethodsThe cancerous tissues and adjacent normal tissues were collected from ICC patients. Blood samples from ICC, hepatocellular carcinoma (HCC) group, the extrahepatic cholangiocarcinoma (ECC) group and the healthy controls were collected. SOX9-AS1 levels were evaluated in tissues (versus normal tissues) and plasma samples (versus plasma from HCC and ECC by quantitative real-time RT-PCR. The diagnostic value of SOX9-AS1 for ICC was estimated using receiver operating characteristic (ROC) curves. The relevancy between SOX9-AS1 expression and overall survival or recurrence-free survival was assessed by Kaplan-Meier curves multivariate analyses. The overexpression and knockdown of SOX9-AS1 on cell behavior were assessed by CCK-8 and transwell assay. ResultsSOX9-AS1 levels were increased in ICC, both in the tissues and the cell lines. The upregulation of SOX9-AS1 showed a highly discriminative profile, distinguishing ICC patients from healthy subjects or HCC or ECC patients. Upregulation of SOX9-AS1 was related to shorter overall survival and recurrence-free survival. Muli-variate analysis revealed that SOX9-AS1 expression was an independent prognostic purpose factor of worst overall survival and recurrence-free survival. ConclusionsSOX9-AS1 drives tumor growth and metastasis in ICC. SOX9-AS1 may be applied as a new diagnostic and prognostic purposed marker, in addition to a promising therapeutic target in ICC.

097 Epidermal integrin α3β1 is a regulator of cytokine, CSF-1, and of crosstalk from keratinocytes to dermal macrophages

As the major cell surface receptors for the extracellular matrix, integrins regulate adhesion and migration, and have been shown to drive tumor growth and progression. Our earlier findings have identified integrin α3β1 as a key regulator of the keratinocyte secretome and of the skin tumor microenvironment, indicating its potential efficacy as a therapeutic target. Previous mass spectrometric analysis of conditioned medium from immortalized keratinocytes indicated that α3β1 positively regulates colony-stimulating factor 1 (CSF-1), a secreted cytokine that is a primary regulator of macrophage differentiation, proliferation, and survival.

Extrapolating evidence for molecularly targeted therapies from common to rare cancers: a scoping review of methodological guidance

ObjectivesCancer is increasingly classified according to biomarkers that drive tumour growth and therapies developed to target them. In rare biomarker-defined cancers, randomised controlled trials to adequately assess targeted therapies may...

Abstract 3307: Multiomics analysis to uncover the mechanism of radiosensitization of AR-positive triple negative breast cancers with AR inhibition

Abstract Purpose: Expression of the androgen receptor (AR) has been identified as a driver of tumor growth in triple negative breast cancers (TNBC), and previous work has nominated AR inhibition as a strategy for radiosensitization in AR+ TNBC. Despite its role in radioresistance in AR+ TNBC, the mechanistic role of AR and specifically its role in mediating DNA damage repair in response to radiation therapy (RT) remains unknown. Methods: Nuclear fractionation experiments were performed to assess cellular localization of AR protein in AR+ TNBC cell lines (ACC-422, MDA-MB-453). Cells were cultured in media containing hormones (FBS) with treatment of enzalutamide (ENZA), apalutamide (APA), or darolutamide (DARO). Cells were alternatively cultured in media containing charcoal stripped serum (CSS) without hormones with R1881 stimulation. RNA-sequencing was performed to compare AR+ TNBC cells treated with CSS or R1881 stimulation alone or in combination with ionizing radiation. Reverse phase protein arrays were performed in cells treated with ENZA, RT, or combination treatment. Results: While stimulation with R1881 was sufficient to induce nuclear translocation of AR in MDA-MB-453 cells, AR inhibition with ENZA, APA, or DARO blocked AR nuclear translocation under CSS or FBS growth conditions. When cells were treated with R1881+RT, AR nuclear translocation was induced at similar or greater levels compared to R1881 alone in MDA-MB-453 and ACC-422 cells. Combination treatment of RT with ENZA in the presence of hormones reduced AR nuclear localization (39% reduction in MDA-MB-453 cells and 32% reduction in ACC-422 cells) compared to RT alone. These results suggest that decreased promoter region binding, and gene expression upregulation may be a mechanism of radiosensitization with AR inhibition. In addition, transcriptomic analyses demonstrated at least 979 genes differentially expressed in multiple models. Pathway analyses in these models showed common affected pathways included ECM-receptor interaction, PPAR-gamma activation, PI3K-Akt signaling pathway, and the MAPK/ERK signaling pathway. Proteomic analysis in the same cell lines identified apoptosis, DNA damage, and cell cycle pathway changes after RT when AR-signaling was blocked. Common affected pathways in combined analyses identified PI3K-Akt and MAPK/ERK signaling pathway changes that may be responsible for this radiosensitizing phenotype. Conclusions: Our data suggest that AR inhibition in AR+ TNBC is sufficient to inhibit AR nuclear translocation suggesting that AR may play a nuclear role in response to RT to promote DNA repair and radioresistance. We identify potential pathways, including ECM-receptor interaction, PI3K-Akt signaling pathway, and the MAPK/ERK signaling pathway that may be regulated by AR in response to RT and therefore may be responsible for radioresistance. Citation Format: Anna R. Michmerhuizen, Andrea M. Pesch, Benjamin C. Chandler, Lynn M. Lerner, Connor Ward, Leah Moubadder, Stephanie The, Breanna McBean, Caleb Cheng, Lori J. Pierce, Corey W. Speers. Multiomics analysis to uncover the mechanism of radiosensitization of AR-positive triple negative breast cancers with AR inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3307.

Abstract 2979: ElevatedNRASexpression as a potential driver of DCIS progression to basal-like invasive breast cancer

Abstract Background: Ductal carcinoma in situ (DCIS) is the most common type of in situ premalignant breast cancers. What drives a subset of luminal DCIS to transition to basal invasive breast cancers is unclear. Basal-like invasive breast cancers are aggressive and associated with the shortest overall survival and time to distant metastasis. We have previously shown that N-Ras is highly expressed selectively in the basal-like subtypes of invasive breast cancers and can promote their growth and progression. In this study, we investigated whether NRAS expression at the DCIS stage can control transition from luminal DCIS to basal-like invasive breast cancers. Methods: Wilcoxon rank-sum test was conducted to assess the relative expression of NRAS in DCIS compared to invasive breast cancers in a patient cohort (GSE59248). NRAS levels were also determined by fluorescence in situ hybridization (FISH) in a collection of 21 patient tumor microarrays (TMAs) with concurrent normal, DCIS, and invasive breast cancer, and NRAS’s association with DCIS and invasive breast cancer was assessed by Wilcoxon signed rank test. How NRAS levels correlated with basal biomarkers in the TMAs, as well as in patient datasets GSE59248 and GSE3369 was determined by Pearson correlation. Gene expression changes in cell line were assessed by RNA-seq. Results: Invasive breast cancers showed higher NRAS mRNA levels, as compared to DCIS samples. These NRAShigh lesions were also enriched with basal-like features, such as basal gene expression signature, lower ER, higher p53 protein levels, and higher Ki67 levels. We have shown previously that N-Ras is a driver for tumor growth in SUM102 cells, which is a DCIS-like cell line model displaying basal-like features. Here we found that NRAS-silencing in these cells induced a shift to a luminal gene expression pattern as determined by PAM50. Conclusions: These data suggest that the rise of N-RAS levels in DCIS can not only mark but also control the emergence of basal-like features leading to more aggressive tumor activities. Targeting N-Ras and/or its downstream pathway may prevent the emergence of basal invasive breast cancers. Citation Format: Ze-yi Zheng, Hanan Elsarraj, Yang Hong, Jonathan T. Lei, Meenakshi Anurag, Yichao Shen, Flora Lo, Long Feng, Zifan Zhao, Xiang H. Zhang, Fariba Behbod, Eric C. Chang. ElevatedNRASexpression as a potential driver of DCIS progression to basal-like invasive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2979.

Abstract 3720: TACH101, a first-in-class inhibitor of KDM4 histone lysine demethylase for the treatment of diffuse large B-cell lymphoma

Abstract Background: Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma (NHL) in the US accounting for about 22% of newly diagnosed cases of B-cell NHL. It is considered to be more aggressive and to have worse prognosis due to low response to existing treatments. KDM4 is a family of histone demethylases that can drive tumor growth by regulating transcription, cell cycle, and DNA replication/repair. Overexpression of KDM4 alters post-translational histone modification and is associated with many types of cancer, including DLBCL. TACH101 is a novel, potent small molecule inhibitor of KDM4 that is being developed for treatment of advanced cancers, including DLBCL. Methods: TACH101 was evaluated in vitro and in vivo using cancer cell lines and patient-derived organoid (PDO) and xenograft (PDX) models of DLBCL. Results: TACH101 is a reversible, α-ketoglutarate competitive, selective and potent inhibitor of KDM4 isoforms A-D with IC50 values < 0.100 μM for all four isoforms. An initial screen using a 301-cell line panel showed that DLBCL cell lines are sensitive to TACH101 (IC50 < 10 nM). In an additional panel of DLBCL cell lines, TACH101 inhibited proliferation in a dose-dependent manner in all DLBCL cell lines, independent of molecular subtype, with mean IC50’s of 0.03 ± 0.01 μM in ABC DLBCL cell lines (n=6); 0.02 ± 0.01 μM in GCB DLBCL cell lines (n=7) and 0.02 ± 0.01 μM in PMBL DLBCL cell lines (n=2). In OCI-LY19 DLBCL xenografts in vivo, TACH101 was well tolerated and inhibited tumor growth by 55% to 100%, depending on dosing regimen (either QD or BID following a 3 days on/4 days off schedule). In PK studies, TACH101 exhibited low clearance, moderate volume of distribution, and good oral bioavailability in mouse, rat, and dog. Moreover, treatment with TACH101 resulted in little or no inhibitory effects on CYP enzymes. Toxicity studies in rats and dogs identified potential target tissues and provided safety guidance for the use of TACH101 in human studies. Conclusions: The KDM4 inhibitor, TACH101, had compelling activity in preclinical DLBCL models, suggesting that TACH101 could be an effective therapy for DLBCL. Preparations to advance the drug into clinical trials are underway. Citation Format: Frank Perabo, Chandtip Chandhasin, Sanghee Yoo, Joselyn Del Rosario, Young K. Chen, Ellen Filvaroff, Jeffrey A. Stafford, Stephen Quake, Michael F. Clarke. TACH101, a first-in-class inhibitor of KDM4 histone lysine demethylase for the treatment of diffuse large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3720.

Abstract 6064: ADAR1-dependent RNA editing of GLI1 drives hepatocellular carcinoma stem cell self-renewal by initiating mitophagy

Abstract Background: Hepatocellular carcinoma (HCC) is one of the most common and most difficult to cure malignancies worldwide. Identifying new effective therapeutic targets is of great significance for the management of HCC. Cumulative evidence suggests cancer stem cells (CSCs) are key drivers of tumor growth and heterogeneity. Post-transcriptional RNA editing of adenosine to inosine (A-to-I) catalyzed by ADAR1 dramatically alters cellular transcriptome in cancers. However, ADAR1 editase-dependent mechanisms governing hepatic CSC generation have not been elucidated. Therefore, we have systematically and comprehensively investigated ADAR1’s effect on CSC self-renewal in HCC. Methods: An in vitro human hepatocyte differentiation model along with relevant transcriptomic data form GEO and TCGA were analyzed to characterize the oncofetal role of ADAR1. Using genetic approaches, RNA sequencing was performed to identify putative ADAR1-mediated recoding edited genes in HCC cell lines. Clinical implication of GLI1 editing was studied in a cohort of 88 HCC patients. GLI1 editing-mediated change in its promoter activity and protein stability was investigated by dual reporter assay, CHIP, Co-IP, and PLA assays. Functional difference in stemness properties, including abilities of self-renewal, differentiation, tumorigenesis, chemo-resistance and metastasis between wild-type and edited GLI1 (GLI1wt vs. GLI1R701G) and the exact mechanisms were also studied in cell models and mice. Results: Lentiviral ADAR1 wild-type but not editing-defective ADAR1E912A mutant, editing at nt2101 of GLI1 transcript caused arginine-to-glycine substitution at the residue 701. Importantly, increased editing of GLI1 was implicated in the pathogenesis of HCC. Upon editing, C-terminal half of GLI1 harbored a lower susceptibility to the inhibition of SUFU, thus promoting its nuclear translocation and activation. Moreover, GLI1R701G appeared more stable than GLI1wt due to reduced formation of specific K63-GLI1 substrate and β-TrCP-GLI1 complex. Edited GLI1 was found to strongly enhance targeted activation of NANOG and SOX9, resulting in accumulation of hepatic CSCs population, hepatocarcinogenesis, sorafenib-resistance and metastasis. Additionally, GLI1 editing initiated mitophagy via PINK1/Parkin-dependent pathway. Impaired mitophagy effectively antagonized the functions of GLI1R701G on hepatic CSC self-renewal and overcome chemoresistance. Conclusion: The critical advance of this study is that ADAR1 editase activity drives GLI1-dependent maintenance of the hepatic CSC population. Our discovery of a pivotal ADAR1-GLI1-PINK1 self-renewal axis provides the first mechanistic link between RNA-editing-driven malignant progression and mitochondrial homeostasis. Thus, ADAR1 represents a unique therapeutic vulnerability in liver CSCs with active mitophagy mediated by edited GLI1. Citation Format: Jie Luo, Lanqi Gong, Yuma Yang, Jiao Huang, Xiaona Fang, Baifeng Zhang, Ying Tang, Beilei Liu, Ming Liu, Lu Bai, Victor Ho-Fun Lee, Xin-Yuan Guan. ADAR1-dependent RNA editing of GLI1 drives hepatocellular carcinoma stem cell self-renewal by initiating mitophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6064.

Abstract 6383: Paracrine signaling between differentiated tumor cell-secreted fibromodulin and the stroma promotes tumor growth by enhancing angiogenesis

Abstract Cancer stem-like cells (CSCs) and the differentiated bulk cells mainly contribute to cellular heterogeneity within a tumor. The dichotomous division pattern of the CSCs sitting atop the tumor hierarchy allow them to self-renew, and differentiate to initiate and repopulate aggressive tumors. These CSC-differentiation-derived bulk tumor cells reprogram to stem-like states, accounting for high plasticity within a tumor; a phenomenon governed by the distinct epigenetic profiles of the two cell populations. Reciprocal signaling between the tumor and its complex microenvironment is a vital driver of plasticity and heterogeneity in cancer. The indispensable role of CSCs in cellular cross-talk in the tumor niche as critical drivers of tumorigenesis is well-studied. However, the massive outnumbering of the CSCs by the differentiated cells raises several vital questions regarding the role of the latter in tumor growth; whether they contribute to the complex signaling within the tumor microenvironment (TME) to drive tumor growth remains unexplored. In this study, we aimed to mechanistically delineate the distinct roles of the cancer stem-like and differentiated cells by investigating their interactions with other stromal cells in the TME.In our model system, Glioblastoma (GBM), a highly aggressive brain cancer, we adopted a quantitative, label-free, high-throughput proteomics-based approach to identify the differentially abundant secreted proteins between the glioma stem-like cell (GSC) and differentiated glioma cell (DGC). This analysis showed a significantly higher abundance of the Fibromodulin (FMOD), a collagen-binding proteoglycan, in the conditioned medium of the DGCs compared with that of the GSCs. Our investigation revealed that an activated TGF-β signaling leads to p-SMAD2 occupancy on the FMOD promoter region, leading to increased FMOD secretion by the DGCs. DGCs silenced for FMOD fail to cooperate with co-implanted GSCs to promote tumor growth in a subcutaneous mouse model. We also observed that FMOD downregulation neither affects GSC growth and differentiation nor DGC growth and reprogramming in vitro. Moreover, DGC-secreted FMOD activates integrin-dependent Notch signaling in endothelial cells, promoting angiogenesis. FMOD-silencing also inhibits the capability of the transdifferentiated endothelial cells (derived from the DGCs) to form angiogenic networks. Conditional silencing of FMOD in the de novo formed DGCs in an orthotopic intracranial C57BL/6 mouse model inhibited the growth of GSC-initiated tumors due to poorly developed vasculature, thereby increasing mouse survival. The same observation was recapitulated using patient-derived GSCs in an immunocompromised intracranial mouse model, thereby highlighting the human relevance of our findings. Collectively, our findings demonstrate how paracrine signaling between DGC-secreted FMOD and the stromal cells promotes tumor growth by enhancing angiogenesis, thereby highlighting a previously less studied supportive role of DGCs in tumor growth, albeit the GSCs retaining the tumor-initiating capacity. Thus our study identifies DGC-specific FMOD as a potential therapeutic target and emphasizes that targeting both cancer stem cells and differentiated cancer bulk cells is vital to achieving a durable response in GBM. Citation Format: Shreoshi Sengupta, Kumaravel Somasundaram, Dinorah Friedmann Morvinski, Philippe Marin, Prasasvi Reddy, Arani Mukherjee, Prerna Magod, Mainak Mondal, Serge Urbach. Paracrine signaling between differentiated tumor cell-secreted fibromodulin and the stroma promotes tumor growth by enhancing angiogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6383.

Abstract CT253: A Phase 1/2 trial to evaluate the safety and antitumor activity of tipifarnib and alpelisib for patients with HRAS-overexpressing and/or PIK3CA-mutated/amplified recurrent/metastatic head and neck squamous cell carcinoma (The KURRENT trial)

Abstract Background: Understanding connections between key cellular pathways is particularly important when selecting combinatorial cancer therapies. HRAS preferentially activates PI3K 5-fold more efficiently than KRAS, while KRAS is a more efficient activator of RAF (Yan et al. 1998). Further, mutant HRAS is insufficient for oncogenic transformation if it is unable to recruit PI3K in preclinical models (Gupta et al. 2007). Conversely, mutant PI3K requires RAS to drive tumor growth (Zhao and Vogt 2008). HRAS mutation/overexpression and PIK3CA mutations/amplifications account for up to 50% of head and neck squamous cell carcinoma (HNSCC). Recognition of these interdependencies was the basis for the evaluation of HNSCC PDX models (HRAS-mutant, HRAS wildtype-overexpressed, PIK3CA-amplified, and PIK3CA-mutant) which demonstrated additive and synergistic growth effects that confirmed the codependency of these pathways, thus providing robust rationale for investigating combined pathway inhibition in the clinic. The KURRENT trial is enrolling patients with HRAS and/or PIK3CA-dependent tumors who will receive combination treatment with tipifarnib (a potent and selective inhibitor of farnesyltransferase, a critical enzyme for HRAS activity) and alpelisib (an inhibitor of PI3K with inhibitory activity predominantly against the PI3Kα isoform). Methods: The KURRENT trial (KO-TIP-013, NCT04809233) is an ongoing multicenter, open-label, 2-cohort, phase 1/2 trial designed to evaluate the safety of the combination of tipifarnib and alpelisib, determine the recommended combination dose(s) regimen, and evaluate preliminary anti-tumor activity in patients with recurrent/metastatic (R/M) HNSCC whose tumors are dependent upon HRAS and/or PIK3CA signaling. Dependency is defined as an overexpression of the HRAS protein, and/or a PIK3CA mutation and/or amplification. The trial will enroll 40 HNSCC patients (oral cavity, oropharynx, larynx or hypopharynx, sinonasal, nasopharyngeal, or unknown primary subsites regardless of human papillomavirus status); 20 each into two biomarker defined cohorts (Cohort 1: PIK3CA; Cohort 2: HRAS). Participants must have documented treatment failure from at least one prior therapy in the R/M setting and have measurable disease by RECIST v1.1. At the starting dose level, participants will receive tipifarnib at 300 mg twice daily on days 1-7 and 15-21 and alpelisib 200 mg each morning continuously during a 28-day cycle. The trial will use an adaptive dose escalation design (based on a Bayesian logistic regression model) to characterize safety, tolerability, and clinical activity of the combination to identify the Optimal Biologically Active Dose (OBAD) while maintaining a dose limiting toxicity (DLT) rate <33%. No formal interim analysis is planned as the model-based dose escalation process requires decisions based on real-time evaluation of aggregate toxicity and efficacy data. All observed/available data among each cohort will be evaluated before choosing the combination dose for a subsequent cohort. The trial opened for enrollment in October 2021. Citation Format: Glenn J. Hanna, Cesar Perez Batista, Alan L. Ho, Maura L. Gillison, Douglas Adkins, Ashley Dayoub, Bridget Martell, Mollie Leoni, Stephen Dale, Kun Nie, Alex Dmitrienko. A Phase 1/2 trial to evaluate the safety and antitumor activity of tipifarnib and alpelisib for patients with HRAS-overexpressing and/or PIK3CA-mutated/amplified recurrent/metastatic head and neck squamous cell carcinoma (The KURRENT trial) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT253.

Abstract 652: An androgen receptor-associated transcription factor regulates fatty acid metabolism driving tumor growth in androgen receptor-positive breast cancer

Abstract Despite advances in the treatment of breast cancer, it is still the second leading cause of cancer-related death in women worldwide. A large number of patients develop recurrence and die of advanced metastatic disease. More than 70% of metastatic breast cancers (mBC) express androgen receptor (AR) representing a potential target for anti-hormone therapies. AR is suggested to directly interact with an associated transcription factor (ARaTF) in breast cancer, however the functional role of ARaTF and its interaction with AR remains to be elucidated. We find that AR expression highly correlates with ARaTF in patients. To study its functional role driving tumorigenesis and metastatic outgrowth, we utilize patient-derived xenograft cell lines derived from liquid biopsies of mBC patients which reflect the highly heterogenous disease. Using genetically manipulated PDX cell models, we demonstrate that repression of ARaTF significantly reduces tumor growth of AR positive (AR+) BC cells in vivo. Notably, we observe the opposite phenotype in an AR negative cell model suggesting a tumor suppressive function when AR is not present. Mechanistically, we find that ARaTF regulates key metabolic processes: (1) Pharmacological inhibition of AR or silencing of ARaTF restricts mitochondrial respiration activity resulting in decreased energy production. (2) We also find that ARaTF positively regulates genes encoding enzymes involved in de novo fatty acid biosynthesis in AR+ BC. We further observe upregulation of the CD36 fatty acid transporter facilitating fatty acid uptake as compensatory pathway upon downregulation of ARaTF. Finally, abolishing CD36 expression in AR+ BC cells significantly reduces cell viability upon ARaTF downregulation. These findings suggest that lipid and energy metabolism are transcriptionally regulated by ARaTF facilitating cell survival in nutrient-depleted environments and thus facilitating metastatic outgrowth. These findings provide knowledge about the functional role of ARaTF regulating the lipogenic metabolic profile of AR+ breast cancer, and might help to identify patients who benefit from an anti-androgen treatment. Citation Format: Sarah-Jane Neuberth, Corinna Schumacher, Marteinn Thor Snaebjörnsson, Roberto Würth, Franziska Maria Zickgraf, Ornella Kossi, Felix Geist, Andrea Geist, Vera Thiel, Jonas Schwickert, Mattia Falcone, Massimo Saini, Almut Schulze, Andreas Trumpp, Martin R. Sprick. An androgen receptor-associated transcription factor regulates fatty acid metabolism driving tumor growth in androgen receptor-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 652.

Abstract 5728: A druggable FOXA1-glucocorticoid receptor transcriptional axis drives tumor growth in NSCLC

Abstract FOXA1 is a pioneer factor that is essential to steroid receptor function in hormone-dependent adenocarcinomas, including breast and prostate cancer. FOXA1 expression is also observed in other tumors of epithelial lineage such as non-small cell lung cancer (NSCLC), but whether and how it is required for tumor growth in these contexts is not known. Analyzing data from genome-scaled pooled loss-of-function screens, we identified and validated a subset of NSCLC cell lines with reduced cellular fitness following FOXA1 knockout. We performed a rapid immunoprecipitation and mass spectrometry of endogenous proteins (RIME) screen to nominate candidate factors that may cooperate with FOXA1 to mediate oncogenic transcription programs. This screen revealed a chromatin-localized interaction between FOXA1 and the glucocorticoid receptor (GR). Depletion of GR expression using shRNA or CRISPR/Cas9 suppressed the proliferation of FOXA1-dependent, but not FOXA1-independent NSCLC models, suggesting GR specifically contributes to tumor growth in the FOXA1-dependent context. To assess the potential therapeutic utility of targeting the FOXA1-GR signaling axis, we employed two structurally distinct ligand antagonists of GR from ORIC Pharmaceuticals: ORIC-101, a steroidal antagonist, and OP-4954, a non-steroidal antagonist. Both antagonists inhibited dexamethasone stimulated GR-transactivation of a glucocorticoid response element (GRE)-luciferase reporter with equal potency. However, proliferation and colony formation of the FOXA1/GR-dependent NSCLC cells were only inhibited upon treatment with ORIC-101, and not OP-4954. Gene expression analysis uncovered a discrete set of GR target genes, including SFTPB, TRAF4 and SGK1, whose expression was reduced by ORIC-101 but unaffected or even induced by OP-4954. The specific inhibition of GR target gene expression and attendant induction of apoptosis by ORIC-101 suggest a hierarchy of GR targets relevant to the FOXA1/GR-growth program in NSCLC. Finally, we evaluated the in vivo therapeutic potential of ORIC-101 using NSCLC xenografts and found that single-agent ORIC-101 exhibited potent antitumor effects in these models. Taken together, these data establish a model of FOXA1-GR-dependent tumor growth amongst a subset of NSCLCs and demonstrate that effective blockade of this complex is a rational therapeutic avenue to be explored in the clinic. Citation Format: Madeline Dorso. A druggable FOXA1-glucocorticoid receptor transcriptional axis drives tumor growth in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5728.