Non-proliferative State Research Articles

Abstract SP090: Tumor INF signaling - A double edged sword in cancer

Abstract Our pre-clinical research using breast cancer models has revealed how certain inflammatory pathways can have opposing effects on primary disease and metastasis. We also learned that immunological age can impact response to therapy, leading to age-stratified outcomes. In one study, we developed pre-clinical models of triple-negative breast cancer (TNBC) lung metastasis. We uncovered mechanisms by which certain primary breast cancers can prevent their disseminated tumor cells from generating tumors. These primary tumors elicit a systemic innate immune response involving IL-1β-expressing neutrophils that infiltrate the lungs. Our group and others had previously established that IL-1β promotes primary breast cancer growth. However, we found that in the lungs, IL-1β maintains disseminated tumor cells in an undifferentiated, non-proliferative state. Inhibiting either inflammation or IL-1R signaling enables robust outgrowth of lung metastases. Importantly, we found that among patients with lymph node-positive breast cancer (i.e., those at high risk for metastasis) high IL-1β associates with improved overall survival and distant metastasis-free survival. Our findings reveal a double-edged sword in which IL-1R inhibitors might reduce primary disease but promote metastatic disease. To avoid this dichotomy, we are exploring strategies that prevent disseminated tumor cell differentiation, as was achieved here by IL-1β, to prevent metastatic outgrowth and provide a window of opportunity to target early metastases. As another example, we evaluated immune checkpoint blockade (ICB) therapy using various pre-clinical TNBC models in young and aged mice. We observed reduced tumor growth and improved survival in response to anti-PD-1 and anti-CTLA-4 treatment in young mice compared to aged mice. Immune profiling of peripheral blood and tumor samples demonstrated differences between young and aged mice treated with immune checkpoint blockade. To gain insights into the mechanisms behind those results, we performed whole tumor RNAseq on treated young versus aged mice. Notably, the most significantly enriched gene signatures included genes in interferon response pathways - with impaired responses in tumors of aged mice. Informed by those findings, we interrogated the METABRIC tumor gene expression database, stratifying TNBC patients < 40 and > 65 years of age. Two distinct clusters were identified, including enrichment of interferon-related genes in the "young" cohort. Turning back to our pre-clinical models, we used a drug that stimulates interferon response genes (STING agonists) in combination with ICB and found significantly improved survival in the aged cohorts. Our study demonstrated that age determines the T cell inflamed phenotype and affects response to immune checkpoint blockade in TNBC. Evaluating interferon-related genes from tumor genomic data may aid identification of patients for whom combination therapy including an interferon pathway activator with ICB may be required. These findings highlight the need to understand how inflammatory signatures should be interpreted in order to design more effective clinical phase trials and how pre-clinical models need to better mimic intent-to-treat populations. Citation Format: SS McAllister. Tumor INF signaling - A double edged sword in cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr SP090.

Transcriptome-Wide Analysis of Stationary Phase Small ncRNAs in E. coli.

Almost two-thirds of the microbiome’s biomass has been predicted to be in a non-proliferating, and thus dormant, growth state. It is assumed that dormancy goes hand in hand with global downregulation of gene expression. However, it remains largely unknown how bacteria manage to establish this resting phenotype at the molecular level. Recently small non-protein-coding RNAs (sRNAs or ncRNAs) have been suggested to be involved in establishing the non-proliferating state in bacteria. Here, we have deep sequenced the small transcriptome of Escherichia coli in the exponential and stationary phases and analyzed the resulting reads by a novel biocomputational pipeline STARPA (Stable RNA Processing Product Analyzer). Our analysis reveals over 12,000 small transcripts enriched during both growth stages. Differential expression analysis reveals distinct sRNAs enriched in the stationary phase that originate from various genomic regions, including transfer RNA (tRNA) fragments. Furthermore, expression profiling by Northern blot and RT-qPCR analyses confirms the growth phase-dependent expression of several enriched sRNAs. Our study adds to the existing repertoire of bacterial sRNAs and suggests a role for some of these small molecules in establishing and maintaining stationary phase as well as the bacterial stress response. Functional characterization of these detected sRNAs has the potential of unraveling novel regulatory networks central for stationary phase biology.

Purification and Immunophenotypic Characterization of Murine Plasma Cells.

B cells are primarily known for their capacity to differentiate into antibody-secreting cells (ASCs). ASCs are usually viewed as terminally differentiated cells sharing a unique phenotype. However, it lately became evident that ASCs exist in a variety of subsets differing by their lifespan, anatomic location, and immunological function. Thus, ASCs can exist as long-lived plasma cells (LLPC) that can persist for years in a nonproliferating state within particular niches in the bone marrow (BM), or as short-lived plasma cells (SLPC) that are primarily found in secondary lymphoid organs or inflamed tissues and wane upon the termination of the associated immune response. Another layer of ASC diversity was uncovered with the discovery of their capacity to produce various pro- or anti-inflammatory cytokines. Notably, a subset of natural regulatory plasma cells characterized by the distinctive expression of the inhibitory receptor lymphocyte activation gene (LAG)-3 and a unique capacity to produce interleukin (IL)-10 upon stimulation was recently identified. Here, we describe how to immunophenotypically characterize murine plasma cells as well as how to isolate them using cell sorting, with a special focus on these recently described natural regulatory plasma cells.

How Autophagy Shapes the Tumor Microenvironment in Ovarian Cancer.

Ovarian cancer (OC) is characterized by a high mortality rate due to the late diagnosis and the elevated metastatic potential. Autophagy, a lysosomal-driven catabolic process, contributes to the macromolecular turnover, cell homeostasis, and survival, and as such, it represents a pathway targetable for anti-cancer therapies. It is now recognized that the vascularization and the cellular composition of the tumor microenvironment influence the development and progression of OC by controlling the availability of nutrients, oxygen, growth factors, and inflammatory and immune-regulatory soluble factors that ultimately impinge on autophagy regulation in cancer cells. An increasing body of evidence indicates that OC carcinogenesis is associated, at least in the early stages, to insufficient autophagy. On the other hand, when the tumor is already established, autophagy activation provides a survival advantage to the cancer cells that face metabolic stress and protects from the macromolecules and organelles damages induced by chemo- and radiotherapy. Additionally, upregulation of autophagy may lead cancer cells to a non-proliferative dormant state that protects the cells from toxic injuries while preserving their stem-like properties. Further to complicate the picture, autophagy is deregulated also in stromal cells. Thus, changes in the tumor microenvironment reflect on the metabolic crosstalk between cancer and stromal cells impacting on their autophagy levels and, consequently, on cancer progression. Here, we present a brief overview of the role of autophagy in OC hallmarks, including tumor dormancy, chemoresistance, metastasis, and cell metabolism, with an emphasis on the bidirectional metabolic crosstalk between cancer cells and stromal cells in shaping the OC microenvironment.

MODL-06. PRECLINICAL EFFICACY OF THE IMIPRIDONE ONC-206 AGAINST MEDULLOBLASTOMA

Treatment for medulloblastoma (MB) is typically ineffective for MYC amplified or metastatic SHH, Group 3 and 4 subgroups. Promising preclinical and clinical results have been obtained in brain cancers treated with ONC-201, a selective antagonist of DRD2, a G-protein coupled receptor that regulates prosurvival pathways. Herein, we report the activity of ONC-201 and ONC-206, which has increased non-competitive antagonism of DRD2, against MB. We treated three different MB cell types representative of SHH- and Group 3-like cells, with varied levels of DRD2 expression, and consistently observed increased cell death in a dose-dependent manner at lower doses of ONC-206 compared to ONC-201. We also evaluated ClpP as an additional drug target in MB. ClpP is a mitochondrial protease that has been shown to directly bind and be activated by ONC 201, and is highly expressed at the protein level across pediatric MB, malignant glioma and ATRT, but not normal brain. We observed that similar to ONC-201, ONC-206 treatment of MB cells induces the restoration of mitochondrial membrane potential to the non-proliferative state, degradation of the mitochondrial substrate SDHB, reduction in survivin and elevation in ATF4 (integrated stress response). Importantly, ONC-206 treatment induced significant cell death of patient-derived SHH, WNT, and Group 3 tumors ex vivo and Group 4 cells in vitro, while having no observable toxicity in normal brain. Efficacy studies of ONC-206 against MB in vivo will be reported in preparation for a planned Phase I study of ONC-206 in children with malignant brain tumors.

Fibroblast Senescence in Idiopathic Pulmonary Fibrosis.

Aging is an inevitable and complex natural phenomenon due to the increase in age. Cellular senescence means a non-proliferative but viable cellular physiological state. It is the basis of aging, and it exists in the body at any time point. Idiopathic pulmonary fibrosis (IPF) is an interstitial fibrous lung disease with unknown etiology, characterized by irreversible destruction of lung structure and function. Aging is one of the most critical risk factors for IPF, and extensive epidemiological data confirms IPF as an aging-related disease. Senescent fibroblasts in IPF show abnormal activation, telomere shortening, metabolic reprogramming, mitochondrial dysfunction, apoptosis resistance, autophagy deficiency, and senescence-associated secretory phenotypes (SASP). These characteristics of senescent fibroblasts establish a close link between cellular senescence and IPF. The treatment of senescence-related molecules and pathways is continually emerging, and using senolytics eliminating senescent fibroblasts is also actively tried as a new therapy for IPF. In this review, we discuss the roles of aging and cellular senescence in IPF. In particular, we summarize the signaling pathways through which senescent fibroblasts influence the occurrence and development of IPF. On this basis, we further talk about the current treatment ideas, hoping this paper can be used as a helpful reference for future researches.

EXTH-62. PRECLINICAL EFFICACY OF THE IMIPRIDONE ONC-206 AGAINST MEDULLOBLASTOMA

Abstract Treatment for medulloblastoma (MB) is typically ineffective for MYC amplified or metastatic SHH, Group 3 and 4 subgroups. Promising preclinical and clinical results have been obtained for adult and pediatric malignant glioma treated with ONC-201, a selective antagonist of DRD2, a G-protein coupled receptor that regulates prosurvival pathways. Herein, we report the activity of ONC-201 and ONC-206, which has increased non-competitive antagonism of DRD2, against MB. We treated three different MB cell types representative of SHH- and Group 3-like cells, with varied levels of DRD2 expression, and consistently observed increased cell death in a dose-dependent manner at lower doses of ONC-206 compared to ONC-201. We also evaluated ClpP as an additional drug target in MB. ClpP is a mitochondrial protease that has been shown to directly bind and be activated by ONC 201, and is highly expressed at the protein level across pediatric MB, malignant glioma and ATRT, but not normal brain. We observed that similar to ONC-201, ONC-206 treatment of MB cells induces the restoration of mitochondrial membrane potential to the non-proliferative state, degradation of the mitochondrial substrate SDHB, reduction in survivin and elevation in ATF4 (integrated stress response). Importantly, ONC-206 treatment induced significant cell death of patient-derived SHH, WNT, and Group 3 tumors ex vivo and Group 4 cells in vitro, while having no observable toxicity in normal brain. ONC-206 treatment of a transgenic mouse model of Shh MB in vivo significantly reduces tumor growth and doubles survival time in a dose-dependent manner following 2 weeks of therapy. Additional in vivo data will be reported in preparation for a planned Phase I study of ONC-206 in children with malignant brain tumors.

A Novel Engineered Single-Chain Antibody Fragment for Targeting Pediatric Philadelphia Chromosome-like Acute Lymphoblastic Leukemia

Introduction: Philadelphia-like acute lymphoblastic leukaemia (Ph-like ALL) is a high-risk ALL subtype characterized by an inferior survival rate and chemotherapeutic drug resistance (Tasian et al, Blood 130: 2064-2072, 2017). Around 50% of Ph-like ALL cases harbour gene rearrangements leading to the overexpression of cytokine receptor-like factor 2 (CRLF2) (Loh et al, Blood 121: 485-488, 2013). CRLF2 (also known as thymic stromal lymphopoietin receptor, TSLPR) heterodimerizes with the interleukin-7 receptor alpha (IL-7Rα) subunit to form the functional TSLPR. Upon TSLP binding, CRLF2 activates the JAK/STAT signalling pathway, leading to enhanced proliferation and survival of leukemia cells resulting in poor outcomes in patients (Harvey et al, Blood 115: 5312-5321, 2010). The extracellular overexpression of CRLF2 and association with poor prognosis suggest the translational value of designing precision-based therapeutics targeting CRLF2 in Ph-like ALL. Although conventional immunotherapy using full-sized antibodies is a promising treatment strategy that can improve treatment efficiency and minimize off-target toxicity, their clinical translation is challenging due to the high production cost and large size affecting targeting efficiency (Holliger et al, Nat Biotech 23: 1126-1136, 2005). Herein, we validated a novel single-chain variable fragment against CRLF2 (CRLF2-ScFv) for targeting Ph-like ALL cells. Methods: A CRLF2-rearranged Ph-like ALL cell line (MHH-CALL-4) was lentivirally transduced with a CRLF2-targeting shRNA driven by an inducible promoter, enabling the inducible knockdown of CRLF2. CRLF2 expression in MHH-CALL-4 cells after shRNA induction (KD-CALL-4) was evaluated using fluorescence-activated cell sorting (FACS). The cellular association of the CRLF2-ScFv was determined in MHH-CALL-4 and KD-CALL-4 at 4° and 37°C using an indirect immunofluorescence assay. Confocal laser scanning microscopy was used to visualize and compare the cellular association of CRLF2-ScFv in MHH-CALL-4 and KD-CALL-4. The cellular association of CRLF2-ScFv was also investigated ex vivo using a small panel of Ph-like and non-Ph-like ALL patient-derived xenografts (PDXs), representing similar immunophenotype and genetic characteristics to their original disease subtypes (Liem et al, Blood 103: 3905-3914, 2004), and peripheral blood mononuclear cells (PBMCs) to investigate the non-selective association. CRLF2 expression in MHH-CALL-4 and Ph-like ALL PDX cells was quantified using indirect immunofluorescence assay. The downstream impact of CRLF2-ScFv on STAT5 phosphorylation (pSTAT5) was determined by FACS either with or without TSLP stimulation. The statistical significance was tested using Unpaired unequal variances t-test or one-way ANOVA followed by multiple comparisons test. Statistical significance was considered when P ≤ 0.05. All experiments were performed in triplicate. Results: KD-CALL-4 showed a 75% reduction in CRLF2 expression compared with MHH-CALL-4 cells (P = 0.0009). CRLF2-ScFv exhibited a 94% higher association with MHH-CALL-4 compared with KD-CALL-4 cells at 37°C (P = 0.0013). The association of CRLF2-ScFv with MHH-CALL-4 cells was reduced by 75% at the non-proliferating state of cells at 4°C compared to 37°C (P = 0.006). Orthogonally viewed confocal microscopy images showed 82% higher intracellular uptake of CRLF2-ScFv in MHH-CALL-4 compared to KD-CALL-4 cells (P = 0.0003). CRLF2-ScFv showed >80% higher association with a Ph-like PDX sample compared with a control CRLF2low PDX and PBMCs (P < 0.001). Of note, a Ph-like ALL PDX exhibited only one-third of the association with CRLF2-ScFv compared with MHH-CALL-4 cells (P = 0.04), corresponding to the significant difference in CRLF2 surface expression (P = 0.01). CRLF2-ScFv significantly reduced pSTAT5 expression in MHH-CALL-4 cells (P = 0.05) and prevented TSLP-induced STAT5 phosphorylation (P = 0.01), suggesting competition with the TSLP binding site. Conclusion: CRLF2-ScFv is a selective targeting moiety for CRLF2 with a significant internalization potential and receptor antagonistic effect, highlighting the therapeutic implications for targeting Ph-like ALL. Keywords: CRLF2, ScFv, STAT5 phosphorylation, Patient-Derived Xenografts. Disclosures No relevant conflicts of interest to declare.

Iron Regulation in Elderly Asian Elephants (Elephas maximus) Chronically Infected With Mycobacterium tuberculosis.

Restriction of nutrients to pathogens (nutritional immunity) is a critical innate immune response mechanism that operates when pathogens such as Mycobacterium tuberculosis have the potential to evade humoral immunity. Tuberculosis is of growing concern for zoological collections worldwide and is well-illustrated by infections of Asian and African elephants, where tuberculosis is difficult to diagnose. Here, we investigated hematological parameters and iron deposition in liver, lung, and spleen of three Asian elephants (Elephas maximus) infected with Mycobacterium tuberculosis. For reference purposes, we analyzed tissue samples from control M. tuberculosis-negative elephants with and without evidence of inflammation and/or chronic disease. Molecular analyses of bacterial lesions of post mortally collected tissues confirmed M. tuberculosis infection in three elephants. DNA sequencing of the bacterial cultures demonstrated a single source of infection, most likely of human origin. In these elephants, we observed moderate microcytic anemia as well as liver (mild), lung (moderate) and spleen (severe) iron accumulation, the latter mainly occurring in macrophages. Macrophage iron sequestration in response to infection and inflammation is caused by inhibition of iron export via hepcidin-dependent and independent mechanisms. The hepatic mRNA levels of the iron-regulating hormone hepcidin were increased in only one control elephant suffering from chronic inflammation without mycobacterial infection. By contrast, all three tuberculosis-infected elephants showed low hepcidin mRNA levels in the liver and low serum hepcidin concentrations. In addition, hepatic ferroportin mRNA expression was high. This suggests that the hepcidin/ferroportin regulatory system aims to counteract iron restriction in splenic macrophages in M. tuberculosis infected elephants to provide iron for erythropoiesis and to limit iron availability for a pathogen that predominantly proliferates in macrophages. Tuberculosis infections appear to have lingered for more than 30 years in the three infected elephants, and decreased iron availability for mycobacterial proliferation may have forced the bacteria into a persistent, non-proliferative state. As a result, therapeutic iron substitution may not have been beneficial in these elephants, as this therapy may have enhanced progression of the infection.

Peptidoglycan Sensing Prevents Quiescence and Promotes Quorum-Independent Colony Growth of Uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) is the leading cause of human urinary tract infections (UTIs), and many patients experience recurrent infection after successful antibiotic treatment. The source of recurrent infections may be persistent bacterial reservoirs in vivo that are in a quiescent state and thus are not susceptible to antibiotics. Here, we show that multiple UPEC strains require a quorum to proliferate in vitro with glucose as the carbon source. At low cell density, the bacteria remain viable but enter a quiescent, nonproliferative state. Of the clinical UPEC isolates tested to date, 35% (51/145) enter this quiescent state, including isolates from the recently emerged, multidrug-resistant pandemic lineage ST131 (i.e., strain JJ1886) and isolates from the classic endemic lineage ST73 (i.e., strain CFT073). Moreover, quorum-dependent UPEC quiescence is prevented and reversed by small-molecule proliferants that stimulate colony formation. These proliferation cues include d-amino acid-containing peptidoglycan (PG) tetra- and pentapeptides, as well as high local concentrations of l-lysine and l-methionine. Peptidoglycan fragments originate from the peptidoglycan layer that supports the bacterial cell wall but are released as bacteria grow. These fragments are detected by a variety of organisms, including human cells, other diverse bacteria, and, as we show here for the first time, UPEC. Together, these results show that for UPEC, (i) sensing of PG stem peptide and uptake of l-lysine modulate the quorum-regulated decision to proliferate and (ii) quiescence can be prevented by both intra- and interspecies PG peptide signaling.IMPORTANCE Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs). During pathogenesis, UPEC cells adhere to and infiltrate bladder epithelial cells, where they may form intracellular bacterial communities (IBCs) or enter a nongrowing or slowly growing quiescent state. Here, we show in vitro that UPEC strains at low population density enter a reversible, quiescent state by halting division. Quiescent cells resume proliferation in response to sensing a quorum and detecting external signals, or cues, including peptidoglycan tetra- and pentapeptides.

Abstract 6061: NR2F1 limits dissemination of early cancer mammary epithelial cells

Abstract Cancer cells have been found to disseminate during very early and sometimes asymptomatic stages of tumor progression. At secondary organs, early disseminated cancer cells (eDCCs) can enter a non-proliferative state for prolonged periods of time. Intriguingly, eDCCs also carry metastasis- initiating capacity. However, the mechanisms by which these early cancer cells complete all steps of metastasis including metastasis-initiating capacity are unknown. Here we report that the orphan nuclear receptor and lineage commitment regulator NR2F1 serves as an early barrier to early dissemination propelled by HER2 signaling. NR2F1 loss of function in HER2 early cancer cells induced cell motility, loss of E-cadherin junctions, disorganized laminin-V deposition, decreased β-catenin membrane localization and increased micro-invading cells with CK14+/CK18 phenotype. Importantly, upon NR2F1 depletion in HER2 early cancer acini, TWIST, ZEB1 and PRRX1 levels were upregulated. Our findings reveal a previously unrecognized function of NR2F1 as a suppressor of dissemination during early stages of breast tumor progression. Thus, therapies that restore and activate NR2F1 function might limit early dissemination and metastatic outgrowth. Citation Format: Maria Soledad Sosa, Nupura Kale, Nitisha Shrivastava, Carolina Rodriguez-Tirado, Maria Carlini, Jiayi Ji, Javier Bravo-Cordero. NR2F1 limits dissemination of early cancer mammary epithelial cells [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 6061.

The Tomato Guanylate-Binding Protein SlGBP1 Enables Fruit Tissue Differentiation by Maintaining Endopolyploid Cells in a Non-Proliferative State.

Cell fate maintenance is an integral part of plant cell differentiation and the production of functional cells, tissues, and organs. Fleshy fruit development is characterized by the accumulation of water and solutes in the enlarging cells of parenchymatous tissues. In tomato (Solanum lycopersicum), this process is associated with endoreduplication in mesocarp cells. The mechanisms that preserve this developmental program, once initiated, remain unknown. We show here that analysis of a previously identified tomato ethyl methanesulfonate-induced mutant that exhibits abnormal mesocarp cell differentiation could help elucidate determinants of fruit cell fate maintenance. We identified and validated the causal locus through mapping-by-sequencing and gene editing, respectively, and performed metabolic, cellular, and transcriptomic analyses of the mutant phenotype. The data indicate that disruption of the SlGBP1 gene, encoding GUANYLATE BINDING PROTEIN1, induces early termination of endoreduplication followed by late divisions of polyploid mesocarp cells, which consequently acquire the characteristics of young proliferative cells. This study reveals a crucial role of plant GBPs in the control of cell cycle genes, and thus, in cell fate maintenance. We propose that SlGBP1 acts as an inhibitor of cell division, a function conserved with the human hGBP-1 protein.

NFATC4 promotes quiescence and chemotherapy resistance in ovarian cancer.

Development of chemotherapy resistance is a major problem in ovarian cancer. One understudied mechanism of chemoresistance is the induction of quiescence, a reversible nonproliferative state. Unfortunately, little is known about regulators of quiescence. Here, we identify the master transcription factor nuclear factor of activated T cells cytoplasmic 4 (NFATC4) as a regulator of quiescence in ovarian cancer. NFATC4 is enriched in ovarian cancer stem-like cells and correlates with decreased proliferation and poor prognosis. Treatment of cancer cells with cisplatin resulted in NFATC4 nuclear translocation and activation of the NFATC4 pathway, while inhibition of the pathway increased chemotherapy response. Induction of NFATC4 activity resulted in a marked decrease in proliferation, G0 cell cycle arrest, and chemotherapy resistance, both in vitro and in vivo. Finally, NFATC4 drove a quiescent phenotype in part via downregulation of MYC. Together, these data identify NFATC4 as a driver of quiescence and a potential new target to combat chemoresistance in ovarian cancer.

Palmitic acid- and/or palmitoleic acid-containing phosphatidic acids are generated by diacylglycerol kinase α in starved Jurkat T cells

Diacylglycerol kinase (DGK) α enhances the proliferation of melanoma and hepatocellular carcinoma cells whereas, in contrast, DGKα induces a nonproliferative state in T cells. We previously found that DGKα produces palmitic acid (16:0)-containing PA species, such as 16:0/16:0- and 16:0/18:0-PA, in melanoma cells under serum-starved (nonproliferative) conditions. In the present study, we identified the PA species generated by DGKα in T cells under serum-starved (nonproliferative) conditions. We found that serum starvation markedly increased the levels of many PA species, such as 14:1/16:1-, 14:0/16:1-, 14:0/16:0-, 16:1/16:2-, 16:1/16:1-, 16:0/16:1-, 16:0/16:0-, 16:1/18:2-, 16:1/18:1-, 16:0/18:1-, 16:0/18:0-, 18:1/18:2-, 18:1/18:1- and 18:0/18:1-PA, in Jurkat T cells. In lysates from serum-starved Jurkat T cells, DGKα activity, which was Ca2+-dependent and sensitive to a DGKα-specific inhibitor (CU-3), was substantially increased, indicating its activation. Moreover, CU-3 (1–10 μM) significantly reduced the amounts of palmitic acid- and/or palmitoleic acid (16:1)-containing PA species, such as 14:1/16:1-, 14:0/16:1-, 14:0/16:0-, 16:1/16:2-, 16:1/16:1-, 16:0/16:1-, 16:0/16:0-, 16:0/18:1- and 16:0/18:0-PA, which were increased by serum starvation. These results indicate that DGKα generates different PA species in starved melanoma cells (palmitic acid-containing PA species) and T cells (palmitic acid- and/or palmitoleic acid (16:1)-containing PA species). Therefore, the differences in the PA molecular species may account for the opposing functions of DGKα in melanoma and T cells.

Abstract B107: Dormant breast tumor cells avoid the adaptive immune system through regulation of the immune and tumor microenvironment

Abstract Many cancer survivors respond to early treatment with complete remission only to succumb to the disease after the tumor returns, sometimes decades later, in a distant metastatic site. Tumor cells can exist in a nonproliferative state of dormancy (quiescence) in specific, perivascular niches in the lungs, brain, and especially bone marrow. In particular, dormant breast tumors can recur after long periods of time, even over 15 years after treatment of the primary tumor. Why the immune system is ineffective at eliminating disseminated/dormant tumor cells before growing into new metastatic lesions is currently unknown. Therefore, in the current study we hypothesized that dormant tumor cells actively elicit a reduced adaptive immune response compared to proliferative tumor cells, ultimately aiding in long-term survival and persistence of dormant cells. We utilized the established syngeneic D2 series of cell lines, which comprises independent clones from a spontaneous mammary tumor in a female Balb/c mouse with varying degrees of dormancy—D2A1 cells grow rapidly upon injection while D2.1 remain dormant. Mammary fat pad (MFP) injections (n=8/group) revealed that proliferative D2A1 tumors rapidly grew in the absence of an adaptive immune system in SCID mice and reached terminal endpoints after 2 to 3 weeks. In comparison, D2A1 tumor growth was significantly reduced in syngeneic Balb/c mice (p<0.0005). Surprisingly, the dormant D2.1 tumors grew at the same rate in Balb/c and SCID mice and persisted post implantation up to 10 weeks and beyond in both, suggesting that these tumors are able to avoid the effects of the adaptive immune system despite very slow growth. To determine potential mechanisms of this differential response, MFP-implanted tumors in Balb/c mice were evaluated via flow cytometry 5 weeks post implantation (n=5/group). Interestingly, the dormant D2.1 tumors contained significantly more Tregs than D2A1 tumors (25.2% vs. 12.6%; p=0.0009), as well as a lower CD4:CD8 ratio (0.45:1 vs. 2.74:1; p=0.032) and enhanced PD-L1 expression on infiltrated myeloid cells (MFI = 1080.75 vs. 1729.25; p = 0.0146). Furthermore, analysis of D2A1 and D2.1 tumor cells revealed that D2.1 cells express significantly more immunomodulatory cytokines and matricellular proteins including IL-10, TGFβ, tenascin-c, and periostin. Taken together, our data illustrate that dormant tumors survive and persist long term despite the immune system, as evidenced by similar growth kinetics of D2.1 cells in the absence or presence of an adaptive immune system. The presence of increased Tregs and higher PD-L1 expression of infiltrated immune cells, along with the altered expression profiles, suggest that dormant tumors elicit an immunosuppressive and protective microenvironment to enable long-term survival. Studies to determine temporal changes in the tumor microenvironment, how dormant cells recruit immunosuppressive cells, and antigenicity of dormant versus proliferative tumor cells are ongoing. Citation Format: Timothy N. Trotter, Cong-Xiao Liu, Tao Wang, H. Kim Lyerly, Zachary C. Hartman. Dormant breast tumor cells avoid the adaptive immune system through regulation of the immune and tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B107.

Establishment of a Temperature-Sensitive Model of Oncogene-Induced Senescence in Angiosarcoma Cells.

Lesions with driver mutations, including atypical nevi and seborrheic keratoses, are very common in dermatology, and are prone to senescence. The molecular events that prevent senescent lesions from becoming malignant are not well understood. We have developed a model of vascular proliferation using a temperature-sensitive, large T antigen and oncogenic HRas. By elevating the temperature to 39 °C, we can turn off large T antigen and study the molecular events in cells with the Ras driver mutation. To assess the signaling events associated with the switch from a proliferative to a nonproliferative state in the constant presence of a driver oncogene, SVR cells were cultivated for 24 and 48 h and compared with SVR cells at 37 °C. Cells were evaluated by Western Blot (WB) gene chip microarray (GC) and quantitative reverse transcription polymerase chain reaction (RT-qPCR). Upon evaluation, a novel phenotype was observed in endothelial cells after switching off the large T antigen. This phenotype was characterized by Notch activation, downregulation of p38 phosphorylation, downregulation of the master immune switch IRF7, and downregulation of hnRNP A0. Switching off proliferative signaling may result in immune privilege and Notch activation, which may account, in part, for the survival of common skin lesions.

Abstract C50: Dissecting vulnerabilities of pancreatic tumors’ silent fraction unravels secrets of an alternative cell state

Abstract Tumors contain a heterogeneous mixture of live cells that are either undergoing proliferation or reside in a nonproliferative state. Cells that do not divide are often associated with increased chemoresistance, challenging therapies that target rapidly proliferating cells. In pancreatic cancer, paucity of blood vessels frequently generates hypoperfused, “arid” regions that constrain tumor cell metabolism and proliferation. Nonetheless, experimental conditions for studying tumor cells in vitro typically mimic well-nourished, “fertile” environments optimized for supporting proliferating cells rather than the more prevalent quiescent state. Characterization of the poorly vascularized KPC model confirmed that the quiescent phenotype is common and is strongly correlated to reduced tissue perfusion. To formulate culture conditions that will emulate the arid microenvironment associated with the nonproliferative state in vitro, we systematically deprived various nutrients from tumor cells and established the necessary components for attaining cell quiescence. Deprivation of amino acids on top of glucose, oxygen, and serum levels was essential for obtaining a complete and reversible cell cycle arrest without loss of viability. Cell cycle arrest was accompanied by a number of physiologic adaptations. Metabolomic analysis indicated reduction in the levels of TCA cycle and mevalonate pathway intermediates indicative of increased reliance on nonglycolytic metabolism. In addition, free amino acid pools were diminished and tumor cells exhibited a significant increase in autophagic flux and micropinocytosis, suggesting that amino acid availability is limited under arid conditions. Concomitant with reduced proliferation and metabolic rewiring, tumor cells under arid conditions exhibited remarkable resistance to gemcitabine. To explore alternative strategies to target these nonproliferating pancreatic tumor cells, we performed a comprehensive genome-wide CRISPR/Cas9-based genetic screen and compared vulnerabilities under ”fertile” and “arid” conditions. Functional annotation of sgRNAs depleted under arid conditions indicated a striking dependence on oxidative phosphorylation for survival. Remarkably, our analysis also revealed that a number of targetable genetic and epigenetic programs driving cell cycle progression were not essential and, in some instances, even hazardous for cells under arid conditions. These results suggest that pancreatic tumor cells that cease proliferation in a nutrient-depleted environment are physiologically rewired and may exhibit a distinct drug response profile from rapidly proliferating cells. The experimental model we developed allows investigation of this dominant yet understudied cell population and may serve as a platform for identification of agents targeting the quiescent fraction of tumors. Citation Format: Yogev Sela, Jinyang Li, Miriam Doepner, Shivahamy Maheswaran, Clementina Mesaros, Ian Blair, Ophir Shalem, Ben Stanger. Dissecting vulnerabilities of pancreatic tumors’ silent fraction unravels secrets of an alternative cell state [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C50.

Abstract A117: The epigenome and gene expression landscape of osimeritib drug tolerant persister cells

Abstract Osimertinib is a potent EGFR inhibitor (EGFRi) that is approved to treat patients with metastatic non-small cell lung cancer (NSCLC) with EGFR gene mutations. Despite the benefit observed for osimertinib many patients still develop resistance and relapse on treatment. One mechanism thought to cause resistance is de novo acquisition of resistant molecular changes in a drug tolerant persister (DTP) cell population. Here we characterized the molecular phenotypes of osimertinib DTPs to identify novel vulnerabilities to eliminate DTPs and prevent the emergence of drug resistance. We used a multiomics approach by combining transcriptomics (RNA-seq), proteomics (reverse phase protein array), and epigenomics assays (ATAC-seq) to profile osimertinib DTPs in four EGFRm NSCLC cell-lines (PC9, H1975, HCC2935, and HCC827). We compared DTPs generated by three weeks exposure to osimertinib to DMSO control, 24 hrs. acute treatment, and DTPs removed from drug. Due to the nature of the time-course we could disentangle effects of acute treatment from long-term DTPs; for example, we observed downregulation of cell-cycle and cell-cycle regulated processes in both acute treatment and DTPs. Phenotypically DTPs were in a non-proliferative state, morphologically larger, and resistant to apoptosis. Unsupervised clustering of multiomics data revealed that DTPs are distinct from acute treated and DMSO control cells at the epigenome, RNA, and protein-level. Interestingly, DTPs removed from drug for up to seven days do not fully return to control state but instead maintain some features in common with DTPs despite regaining proliferative ability. Motif analysis of regions that increased chromatin accessibility in Osimertinib DTPs identified an enrichment for TEAD transcription factor binding sites. TEAD transcription factors are regulated by the transcription co-activators YAP1/TAZ. When the Hippo pathway is on YAP1/TAZ is phosphorylated and localized to the cytoplasm, whereas when the pathway turns off YAP1/TAZ loses phosphorylation, translocate to the nucleus, and acts as a co-factor for TEAD transcription factors. We validated by Western blot that in osimertinib DTPs YAP1 loses phosphorylation indicating that the Hippo pathway switches from on to off. YAP1/TEAD downstream target genes also increase RNA expression in osimertinib DTPs. In addition to the Hippo pathway switch, and potentially related, we observe epithelial to mesenchymal (EMT) transition as a prominent molecular phenotype of DTPs across all four models. The EMT transition was evident at the transcriptome, proteome, and epigenetic level and is a prominent feature of surviving cells but not the 24 hrs. acute treatment. The chromatin accessibility changes at EMT regulated genes suggest the possibility that the EMT transition may be regulated by an epigenetic switch. Consistent with this idea we demonstrate that multiple drugs targeting the epigenome including HDAC inhibitors can effectively block regrowth of DTPs after osimertinib removal. Together our data suggests blocking protective changes to the chromatin landscape may represent a therapeutic strategy to target osimertinib DTPs. Citation Format: Steven W Criscione, Matthew Martin, Yi Yao, Aleksandra Markovets, Jingwen Zhang, Sladjana Gagrica, Oona Delpuech, Zhongwu Lai, Paul Smith, Jonathan Dry. The epigenome and gene expression landscape of osimeritib drug tolerant persister cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A117. doi:10.1158/1535-7163.TARG-19-A117

Abstract AP32: THE ROLE OF NFAT3 IN OVARIAN CANCER QUIESCENCE AND CHEMOTHERAPY RESISTANCE

Abstract Despite the fact that the majority of patients with ovarian cancer will have a complete clinical remission with combined surgical/chemotherapeutic approaches, Epithelial Ovarian Cancer (EOC) is the fifth most common cause of cancer-related death in American women, with the third highest mortality:incidence ratio. The majority of deaths from ovarian cancer are related to disease recurrence and the subsequent development of ultimately fatal chemotherapy-resistant disease. Elucidating mechanisms of chemoresistance in ovarian cancer cells may identify critical therapeutic targets to prevent or treat relapsed ovarian cancer. One feature that might contribute to the chemotherapy resistance is quiescence. Quiescence describes a reversible non-proliferative cell state. As such, quiescent cells have reduced susceptibility to chemotherapeutics which target rapidly proliferating cells. Indeed, quiescence is a known mechanism of chemotherapy resistance of normal stem cells. In normal stem cells quiescence is mediated by the NFAT family of transcription factors. Unfortunately, very little is known about the factors that mediate ovarian cancer cell quiescence. Using qRT-PCR, we evaluated the expression of NFAT family members in ovarian cancer cells. We found NFAT3 to be enriched in patient ovarian cancer stem-like cells (CSLC) compared to bulk cancer cells. Treatment of EOC cells with cisplatin chemotherapy resulted in a nuclear translocation of NFAT3 and increase in NFAT3 transcriptional activity. To directly investigate the functional role of NFAT3 in ovarian cancer, we created ovarian cancer cell lines expressing constitutively nuclear/active (cNFAT3) or inducible constitutively active (IcNFAT3) NFAT3. We found that while cNFAT3 expression in ovarian cancer cells did not impact cell viability, senescence or apoptosis, cNFAT3 expression profoundly restricted ovarian cancer cell proliferation, with a 3-fold decreased cell division rates, and cellular arrest in the G0 phase of the cell cycle. Consistent with a quiescent cell phenotype, this was associated with a 10% decrease in cell size, and a 25% decrease in total cellular RNA. Induction of cNFAT3 expression in vivo resulted in tumor growth arrest. This growth arrest resulted in chemotherapy resistance such that cNFAT3 tumors treated with high dose of chemotherapy rapidly expanded after cNFAT3 inactivation. Furthermore, NFAT inhibition with the peptide inhibitor VIVIT enhance chemotherapy response in vitro and in vivo. Taken together, our data suggests NFAT3 drives a quiescent state in CSLC and thereby mediates chemoresistance. Thus NFAT3 represents a therapeutic target to both overcome chemotherapy resistance in quiescent cancer cells and to restrict the growth of therapy-resistant disease. Citation Format: Alex J Cole, Mangala Iyengar, Patrick O'Hayer, Kun Yang, Tara Sebastian, Lan Coffman, Ronald J Buckanovich. THE ROLE OF NFAT3 IN OVARIAN CANCER QUIESCENCE AND CHEMOTHERAPY RESISTANCE [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr AP32.

The paradox of metabolism in quiescent stem cells

The shift between a proliferating and a nonproliferating state is associated with significant changes in metabolic needs. Proliferating cells tend to have higher metabolic rates, and their metabolic profiles facilitate biosynthesis, as compared to those of nondividing cells of the same sort. Recent studies have elucidated specific molecules that control metabolic changes while cells shift between proliferation and quiescence. Embryonic stem cells, which are rapidly proliferating, tend to have metabolic patterns that are similar to those of nonstem cells in a proliferative state. Moreover, although adult stem cells tend to be quiescent, their metabolic profiles have been reported in multiple organs to more closely resemble those of proliferating than those of nondividing cells in some respects. The findings raise questions about whether there are metabolic profiles that are required for stemness, and whether these profiles relate to the metabolic properties that may be required for quiescence. Here, we review the literature on how metabolism changes upon commitment to proliferation and compare the proliferating and nonproliferating metabolic states of differentiated cells and embryonic and adult stem cells.