Hypomethylating agents are used as frontline therapy for myelodysplastic neoplasms (MDS), but clinical response is unpredictable. To determine whether response was associated with in vivo dynamics of DNA hypomethylation, we conducted a phase 2 trial for MDS using both injection and oral azacitidine (AZA). We established that global DNA methylation levels in peripheral blood and bone marrow mononuclear cells were comparable in AZA responders and non-responders during their course of treatment. However, there were distinct baseline and early drug induced differences in CpG methylation in haematopoietic stem and progenitor cells (HSPCs) in responders compared to non-responders that overlapped with regulatory regions of genes associated with tissue patterning, cell migration and myeloid differentiation. Following six cycles of therapy when clinical response typically manifests, differential hypomethylation in responder HSPCs pointed to marrow adaptation as a driver of enhanced haematopoiesis. Taken together, CpG methylation differences in HSPCs may explain variable response to AZA. ### Competing Interest Statement F.V. is affiliated with OmniOmics.AI Pty Ltd. C.F. is an advisory board member at Amgen, AbbVie, Adaptive Biotech, BeiGene, Pfizer, Otsuka, and Jazz, a consultant at Novotech, and received speaker fees from Amgen, Pfizer, Servier, BMS, and Astella. D.H. has consultancy agreements with GlaxoSmithKline and Pharming Corp. M.H. is a consultant/advisory board member at Roche, Gilead, Otsuka, Janssen, Beigene, and Takeda. M.N.P. received research funding and/or provision of drug for clinical trials (to institution) from AstraZeneca, BRII Biosciences, Celgene/BMS, CSL Behring, Eli Lilly, Emergent Biosciences, Gilead Pharmaceuticals, GlaxoSmithKline, Grifols, Janssen/Johnson and Johnson, Takeda, ViiV Pharmaceuticals and has advisory roles with Celgene/BMS, Gilead Pharmaceuticals, and ViiV Pharmaceuticals. J.E.P. received research funding and/or provision of drug for clinical trials (to institution) from Celgene/BMS, Astex, Verastem Oncology and received honoraria from Abbvie as an advisory board member. The remaining authors declare no competing financial interests. ### Clinical Trial NCT03493646 ### Funding Statement The investigator initiated clinical trial was funded in part by Celgene/BMS (RG172029) with research support from the National Health and Medical Research Council (RG170246, RG211412), Anthony Rothe Memorial Trust (RG182042, RG202657, RG213236), Leukaemia Foundation (RG231257). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The trial protocol received ethical approval from the South Eastern Sydney Local Health District Human Research Ethics Committee, and participating sites received Institution approval to conduct the trial prior to commencing recruitment. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

Hypomethylating agents (HMAs) are frontline therapies for Myelodysplastic Neoplasms (MDS) and Acute Myeloid Leukemia (AML). However, acquired resistance and treatment failure are commonplace. To address this, we perform a genome-wide CRISPR-Cas9 screen in a human MDS-derived cell line, MDS-L, and identify TOPORS as a loss-of-function target that synergizes with HMAs, reducing leukemic burden and improving survival in xenograft models. We demonstrate that depletion of TOPORS mediates sensitivity to HMAs by predisposing leukemic blasts to an impaired DNA damage response (DDR) accompanied by an accumulation of SUMOylated DNMT1 in HMA-treated TOPORS-depleted cells. The combination of HMAs with targeting of TOPORS does not impair healthy hematopoiesis. While inhibitors of TOPORS are unavailable, we show that inhibition of protein SUMOylation with TAK-981 partially phenocopies HMA-sensitivity and DDR impairment. Overall, our data suggest that the combination of HMAs with inhibition of SUMOylation or TOPORS is a rational treatment option for High-Risk MDS (HR-MDS) or AML.

Flotillin-1 contributes to invasion and metastasis in triple negative breast cancer (TNBC) and is modified post-translationally through palmitoylation. Palmitoylation, the process of conjugating palmitoyl-CoA to proteins, plays an essential role in protein stability and trafficking. Thus far, there has not been any investigation into the role of flotillin-1 palmitoylation in the context of metastasis in vivo. To address the role of flotillin-1 palmitoylation in metastasis, MDA-MB-231 cells expressing palmitoylation defective flotillin-1 constructs were used as models. Compared to flotillin-1 WT expressing tumors, flotillin-1 palmitoylation defective displayed abrogated tumor progression and lung metastasis in vivo in both spontaneous and experimental models. Further mechanistic investigation led to the identification of zDHHC5 as the main palmitoyl acyltransferase responsible for palmitoylating endogenous flotillin-1. Modulation of flotillin-1 palmitoylation status through mutagenesis, zDHHC5 silencing, and 2-bromopalmitate inhibition all resulted in the proteasomal degradation of flotillin-1 protein. To assess if flotillin-1 palmitoylation can be inhibited for potential clinical relevance, we designed a competitive peptide fused to a cell penetrating peptide sequence, which displayed efficacy in blocking flotillin-1 palmitoylation in vitro without altering palmitoylation of other zDHHC5 substrates, highlighting its specificity. Additionally, TNBC xenograft tumor models expressing a doxycycline inducible flotillin-1 palmitoylation inhibiting peptide displayed attenuated tumor growth and lung metastasis. Collectively, these results reveal a novel palmitoylation dependent mechanism which is essential for the stability of flotillin-1 protein. More specifically, disruption of flotillin-1 palmitoylation through mutagenesis or competitive peptide promoted flotillin-1 protein degradation, subsequently impeding its tumor promoting and metastasis-inducing effects in TNBC tumor models.

Hypomethylating agents (HMAs) are frontline therapies effective at altering the natural course of Myelodysplastic Neoplasms (MDS) and Acute Myeloid Leukemia (AML). However, acquired resistance and treatment failure are hallmarks of HMA therapy. To address this clinical need, we performed a genome-wide CRISPR-Cas9 screen in a human MDS-derived cell line, MDS-L, and identified TOPORS as a highly ranked loss-of-function target that synergizes with HMAs, reducing leukemic burden and improving survival in xenograft models. We demonstrate that the depletion of TOPORS mediates sensitivity to HMAs by predisposing leukemic blasts to an impaired DNA damage response (DDR) accompanied by an accumulation of SUMOylated DNMT1 in HMA-treated TOPORS-depleted cells. Importantly, the combination of HMAs with targeting of TOPORS did not functionally impair healthy hematopoiesis. While inhibitors of TOPORS are currently unavailable, we show that inhibition of protein SUMOylation (upstream of TOPORS functions) with TAK-981 partially phenocopies HMA-sensitivity and DDR impairment. Overall, our data suggest that the combination of HMAs with the inhibition of SUMOylation or TOPORS demonstrates a favourable therapeutic index and is a rational treatment framework for High-Risk MDS (HR-MDS) or AML. ### Competing Interest Statement P.T, J.A.I.T, C.J, and J.P are listed as inventors/contributors in P0054922PCT.

Purpose: The objective of this study is to determine the impact of exposure to obesity-related systemic factors on fatty acid synthase enzyme (FASN) expression in breast cancer cells. Methods: MCF-7 breast cancer cells were exposed to sera from patients having obesity or not having obesity and subjected to quantitative reverse transcription polymerase chain reaction (RT-qPCR). Subsequent MTT and colony-forming assays using both MCF-7 and T-47D cells exposed to sera and treated with or without FASN inhibitor, TVB-3166, were used. MCF-7 cells were then treated with insulin and the sterol regulatory element–binding protein (SREBP) processing inhibitor, betulin, prior to analysis of FASN expression by quantitative RT-qPCR and western blot. Insulin-induced SREBP-FASN promoter binding was analyzed by chromatin immunoprecipitation with an anti-SREBP antibody. Results: In response to sera exposure (body mass index [BMI] >30) there was an increase in FASN expression in breast cancer cells. Furthermore, treatment with the FASN inhibitor, TVB-3166, resulted in a decreased breast cancer cell survival and proliferation while increasing apoptosis upon sera exposure (BMI >30). Insulin-exposed MCF-7 cells exhibited an increased FASN messenger RNA and protein expression, which is abrogated upon SREBP inhibition. In addition, insulin exposure induced enhanced SREBP binding to the FASN promoter. Conclusions: Our results implicate FASN as a potential mediator of obesity-induced breast cancer aggression and a therapeutic target of patients with obesity-induced breast cancer.

(1) Background: MALDI imaging is a technique that still largely depends on time of flight (TOF)-based instrument such as the Bruker UltrafleXtreme. While capable of performing targeted MS/MS, these instruments are unable to perform fragmentation while imaging a tissue section necessitating the reliance of MS1 values for peptide level identifications. With this premise in mind, we have developed a hybrid bioinformatic/image-based method for the identification and validation of viral biomarkers. (2) Methods: Formalin-Fixed Paraffin-Embedded (FFPE) mouse samples were sectioned, mounted and prepared for mass spectrometry imaging using our well-established methods. Peptide identification was achieved by first extracting confident images corresponding to theoretical viral peptides. Next, those masses were used to perform a Peptide Mmass Fingerprint (PMF) searched against known viral FASTA sequences against a background mouse FASTA database. Finally, a correlational analysis was performed with imaging data to confirm pixel-by-pixel colocalization and intensity of viral peptides. (3) Results: 14 viral peptides were successfully identified with significant PMF Scores and a correlational result of >0.79 confirming the presence of the virus and distinguishing it from the background mouse proteins. (4) Conclusions: this novel approach leverages the power of mass spectrometry imaging and provides confident identifications for viral proteins without requiring MS/MS using simple MALDI Time Of Flight/Time Of Flight (TOF/TOF) instrumentation.

Azacitidine (AZA) and Decitabine (DAC) are frontline hypomethylating agents (HMAs) capable of altering the natural course of myelodysplastic syndromes (MDS). Unfortunately, acquired resistance and treatment failure are hallmarks of HMA therapy. Developing therapies that effectively combine with HMAs are challenging as the underlying mechanisms driving HMA response remain uncertain. To address this, we leveraged genome-wide CRISPR-Cas9 dropout screening in a human MDS cell line in the presence of low dose AZA to identify novel synthetic lethal HMA-gene relationships. Here we identified 50 genes that, when Cas9-edited, confer hypersensitivity to AZA. These genes largely clustered into biological processes such as nucleotide excision repair and protein SUMOylation. Gene hits were ranked according to their degree of depletion under AZA selection, revealing UBXN7, UBE2K, and TOPORS as top dropout hits. In agreement with our screen, individual perturbations of UBXN7, UBE2K, or TOPORS in MDS-L significantly reduced cellular fitness under AZA selection in a competitive proliferation assay (p <.0001). Subsequent efforts focused on evaluating TOPORS as a potential target for AZA combinatorial therapy as it resulted in the strongest sensitization phenotype. We determined the dose-response relationship for AZA in TOPORS-edited MDS-L after 4 days of consecutive treatment using a range of doses. Targeting TOPORS using CRISPR-Cas9 or shRNA enhanced sensitivity to AZA by up to 3.4 fold (P<.0001). Concordantly, targeting of TOPORS synergistically reduced the clonogenicity of MDS-L exposed to AZA (P<.001). As HMAs are also used for acute myeloid leukemia (AML), we explored whether targeting TOPORS could be generalized; TOPORS-editing sensitized a panel of AML cell lines to AZA by up to 4.3-fold (P < .0001). Importantly, AZA combined with TOPORS-editing was relatively less toxic in healthy CD34+ hematopoietic cells and their function was not impaired. TOPORS is a dual E3 ubiquitin and SUMO1 ligase implicated in regulating the DNA damage response (DDR) through SUMOylation-dependent mechanisms. Since HMAs are known to induce genome instability, we hypothesized that TOPORS contributes to genome stability in response to AZA. Apoptosis, DNA damage, and cell cycle assays revealed that TOPORS-edited MDS-L cells were primed for apoptosis as they accumulated extensive DNA damage and arrested at a late S- or G2/M-checkpoint in response to low dose AZA. Enrichment analysis of bulk transcriptomes identified activation of DNA replication and spliceosome signatures in AZA-treated TOPORS-edited MDS-L compared to control cells. According to the BioPlex interactome database, TOPORS interacts with splicing factors SRSF4 and SRSF6; therefore, we assessed the consequences of targeting TOPORS on alternative splicing. Clustering of RNA splicing profiles revealed that TOPORS-editing or AZA treatment induced global splicing alterations individually and cooperatively. Compared to AZA-treated control cells, we detected 764 misspliced events in AZA-treated TOPORS-edited MDS-L with the majority being exon skipping events (473 events, FDR<0.05, PSI>0.1). Over-representation analysis associated these misspliced genes with pathways relating to the DDR. In parallel, nuclear proteomics of AZA-treated TOPORS-edited MDS-L identified enrichment of late-stage cell cycle proteins and depletion of global nucleotide excision repair factors including SUMO1 (log2-fold change > |1|, FDR <0.05). As there are currently no pharmacological inhibitors of TOPORS, to enable clinical translation we sought to target its key biological processes. Here we piloted the combination of TAK-981, a novel high-specificity inhibitor of SUMOylation, with AZA or DAC. Combining TAK-981 and AZA was found to be cytotoxically additive in MDS-L (ZIP = 5.596 ± 1.44) and synergistic in MOLM-13 (ZIP= 11.968 ± 1.21), while TAK-981 synergized with DAC in both MDS-L (ZIP= 10.072 ± 1.21) and MOLM-13 (ZIP= 11.31 ± 3.29). In summary, we report that targeting TOPORS confers hypersensitivity to AZA through predisposing leukemia cells to a defective DDR. The known preclinical efficacy of TAK-981 to enhance anti-tumour immune responses, combined with our findings, supports translation of HMA plus TAK-981 combinatorial therapy into early phase clinical trials for the treatment of MDS and AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

BACKGROUND:Fatty acid synthase (FASN) expression is associated with a more aggressive breast cancer phenotype and is regulated downstream of receptor tyrosine kinase (RTK) signaling pathways. Recently, post transcriptional regulation of lipogenic transcripts have been demonstrated as being mediated downstream of serine-arginine rich protein kinase 2 (SRPK2), which acts to phosphorylate serine-arginine rich splicing factors (SRSFs), resulting in RNA binding and various RNA regulatory processes. Though post-transcriptional regulation of FASN has been studied previously, the upstream mediators of these pathways have not been elucidated.METHODS:Western blotting and RT-qPCR were utilized to demonstrate alterations in FASN and mRNA expression upon modulation of the IGF-1-mTORC1-SRPK2 pathway by small molecule inhibitors or RNAi mediated silencing. RNA stability was accessed by using the transcriptional inhibitor actinomycin-D followed by RT-qPCR. Further, we employed RNA-immunoprecipitation to demonstrate the direct binding of SRSF-1 to FASN transcripts.RESULTS:In the current study, we demonstrated an IGF-1 induced increase in FASN mRNA and protein expression that was attenuated by mTORC1 inhibition. This mTORC1 inhibition also resulted in decreases in total and nuclear p-SRPK2 in response to IGF-1 exposure. Upon SRPK2 knockdown and inhibition, we observed a decrease in FASN protein and mRNA stability, respectively, in response to IGF-1 exposure that was specific to triple negative and HER2+ breast cancer cell lines. As we explored further, IGF-1 exposure resulted in an altered localization of eGFP expressed SRSF-1, pEGFP-SRSF-1 that was rescued upon both SRPK2 knockdown and mTORC1 inhibition. Further, we observed an increase binding of SRSF-1 to FASN RNA upon IGF-1 exposure, which was abrogated by SRPK2 knockdown.CONCLUSION:These current findings establish a potential IGF-1-mTORC1-SRPK2-FASN axis in breast cancer, which could be a potential therapeutic target for cancers that overexpress FASN and components of the IGF-1R pathway.

Background: Obese breast cancer patients face a worse prognosis, including an increased risk of recurrence and mortality. While the causative mechanisms have yet to be fully uncovered, emerging evidence implicates palmitate, increased in the obese state, in development of cellular senescence, an inflammatory state associated with proliferation, metastasis, and tumor-associated neutrophil (TAN) polarization, among other measures of carcinogenesis. However, studies have yet to investigate the impact of palmitate on induction of senescence in breast fibroblasts, and no studies have assessed the effect of senescent fibroblasts on neutrophil polarization in the breast tumor microenvironment. This said, we hypothesize that palmitate alters breast cancer cell gene expression and neutrophil phenotype via induction of a senescent-like phenotype in fibroblasts. Methods: HCA2, IMR-90, and human mammary fibroblasts were exposed to palmitate or vehicle in media supplemented with 2% charcoal-stripped fetal bovine serum, after which the cells were measured through qPCR for expression of IL-1a, IL-6 and IL-8, some of the most prominent members of the senescence-associated secretory phenotype. Palmitate-exposed fibroblasts were also subjected to chromogenic staining for senescence-associated beta-galactosidase and immunoenzymatic BrdU analysis, two well-established measures of senescence. Next, in order to study the influence of these fibroblasts on other cells of the breast tumor microenvironment, we assessed their impact on polarization of DMSO-differentiated HL-60 neutrophils by using flow cytometry to measure neutrophil expression of CD54 and CD95, differentially expressed on N1 and N2 neutrophils. Finally, we employed PCR arrays to assess the impact of palmitate-exposed fibroblasts on the expression of 84 genes in MCF-7 and 231 breast cancer cells, measuring activation of pathways related to apoptosis, cell cycle, DNA damage, senescence, telomere maintenance, metabolism, angiogenesis, and the endothelial-to-mesenchymal transition. Results and Conclusions: Palmitate induced pro-inflammatory gene expression and SA-beta-gal activity and decreased BrdU incorporation in fibroblasts. Palmitate also exhibited non-cell-autonomous effects, as palmitate-exposed fibroblasts induced phenotypic changes in both neutrophils and breast cancer cells. These findings are among the first to implicate palmitate-induced fibroblast senescence in the stimulation of non-cell-autonomous changes in the breast tumor microenvironment and will ultimately inform our understanding of the mechanistic connection between the obesity-associated factor palmitate and breast tumorigenesis. Citation Format: Brittany Susanne Harlow, Albert Davalos, Bryan McClellan, Andrew Brenner, Christopher Jolly, Stefano Tiziani, Steve Hursting, Linda deGraffenried. Palmitate induces a senescent-like phenotype in fibroblasts resulting in altered phenotypes in cells of the breast tumor microenvironment [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-06-05.

Urban ecosystems and remnant habitat 'islands' therein, provide important strongholds for many wildlife species including those of conservation significance. However, the persistence of these habitats can be undermined if their structure and function are too severely disrupted. Urban wetlands, specifically, are usually degraded by a monoculture of invasive vegetation, disrupted hydrology, and chronic-contamination from a suite of anthropogenic pollutants. Top predators-as bioindicators-can be used to assess and monitor the health of these ecosystems. We measured eight health parameters (e.g., parasites, wounds and scars, tail loss and body condition) in a wetland top predator, the western tiger snake, Notechis scutatus occidentalis. For three years, snakes were sampled across four wetlands along an urban gradient. For each site, we used GIS software to measure the area of different landscapes and calculate an urbanisation-landscape score. Previously published research on snake contamination informed our calculations of a metal-pollution index for each site. We used generalised linear mixed models to assess the relationship between all health parameters and site variables. We found the metal-pollution index to have the most significant association with poor body condition. Although parasitism, tail loss and wounds differed among sites, none of these parameters influenced body condition. Additionally, the suite of health parameters suggested differing health status among sites; however, our measure of contemporary landscape urbanisation was never a significant predictor variable. Our results suggest that the health of wetland predators surrounding a rapidly growing city may be offset by higher levels of environmental pollution.

Obesity is associated with low-grade chronic inflammation, and metabolic dysregulation. Evidence shows that chronic inflammation inhibits protective immunity mediated by CD4+ T cells. Additionally, obesity-induced inflammation affects prostate cancer progression. However, the effect of obesity on CD4+ T-cell- response to prostate cancer is not well understood. To investigate whether obesity induces changes in CD4+ T cell cytokine profile, cytokine expression was measured in splenic CD4+ T-cells from 10-week-old male C57Bl/6 mice exposed to conditioned media (CM) from macrophages grown in sera from obese subjects. Additionally, expression levels of key regulators of Epithelial-Mesenchymal Transition (EMT) were measure in prostate cancer epithelial cells exposed to conditioned media from obesity-modified T-cells. Cell migration and invasion was measured in prostate cancer epithelial cells exposed to CM from obesity-modified CD4+ T-cells. Obesity suppressed the expression of IFNγ and IL-2 in CD4+ T-cells but up-regulated the expression of IL-6. Prostate epithelial cancer cells exposed to conditioned media from obesity-modified T cell increased the expression of EMT markers and showed a higher invasive and migratory capacity.

Chronic kidney disease (CKD) is a common cause of morbidity and mortality in domestic cats, but the cause is still largely elusive. While some viruses have been associated with this disease, none have been definitively implicated as causative. Recently, Rodent chaphamaparvovirus 1 was recognized as the cause of murine inclusion body nephropathy, a disease reported for over 40 years in laboratory mice. A novel virus belonging to the same genus, Carnivore chaphamaparvovirus 2, was recently identified in the feces of cats with diarrhea. The goal of this study was to investigate the possible role of chaphamaparvoviruses including members of Rodent chaphamaparvovirus 1 and Carnivore chaphamaparvovirus 2 in the development of feline CKD. The presence of these viruses was retrospectively investigated in formalin-fixed paraffin-embedded feline kidney samples using polymerase chain reaction, in situ hybridization, and immunohistochemistry. Cats were divided into 3 groups: normal (N = 24), CKD (N = 26), and immunocompromised (N = 25). None of the kidney tissues from any of the 75 cats revealed the presence of chaphamaparvovirus DNA, RNA, or antigen. We conclude that viruses belonging to the chaphamaparvovirus genus are unlikely to contribute to the occurrence of feline CKD.

INTRODUCTION: Breast cancer accounts for nearly 40,000 deaths annually with the overall prognosis worsened if the patient is obese. Reprogramming of lipid metabolism in cancer is an established hallmark and contributes to tumorigenesis and drug-resistance. The fatty acid synthase enzyme (FASN) is overexpressed in multiple solid and hematopoietic tumors and its expression is associated with tumor grade as well as resistance to therapy. Targeted therapies against the fatty acid synthase enzyme (FASN) are currently in phase II of clinical trials for the treatment of multiple solid tumors. Previously, our lab has shown enhanced expression of FASN in breast cancer cells exposed to obese sera as well as increased sensitivity to the FASN inhibitor, TVB-3166. The obese phenotype is characterized by increased circulating bioactive growth factors and hormones, such as insulin, estrogen, and insulin-like growth factor 1 (IGF-1), that are ligands for the insulin (IR) and insulin-like growth factor receptor-1 (IGF-1R). Activation of these receptors can lead to downstream signaling through the PI3K-Akt-mTOR pathway that mediates lipogenic gene expression through various transcription factors. Of these transcription factors, sterol regulatory element-binding protein-1 (SREBP-1), drives FASN gene expression and is activated downstream of both Akt and mTORC1. HYPOTHESIS: We hypothesize that obesity-induced breast cancer progression is mediated through an SREBP dependent overexpression of FASN. METHODS: MCF-7 cells were exposed to obese or non-obese sera and subjected to quantitative RT-PCR for FASN expression. MTT and colony-forming assays using both MCF-7 and T-47D breast cancer cells conditioned in 2% obese and 2% non-obese sera as well as treated with and without a FASN inhibitor (TVB-3166) were utilized to determine cell survival and viability in response to FASN inhibition. FASN expression in obesity-induced breast cancer was investigated by treating MCF-7 cells with either insulin or SREBP processing inhibitor (Betulin) and subjected to chIP-qPCR against anti-SREBP or normal rabbit IgG. RESULTS: In response to obese sera exposure, there was a nearly 3-fold increase (p=.010) in FASN expression compared to non -obese control. Obese sera exposure increased sensitivity and decrease cell viability to TVB-3166 treatment compared to non-obese sera. ChIP-qPCR against anti-SREBP showed an increase in FASN expression upon treatment with insulin compared to normal rabbit IgG control. This increase in FASN expression was attenuated upon treatment with the SREBP processing inhibitor, Betulin. CONCLUSION: The overexpression of FASN contributes to obesity-induced breast cancer aggression and is regulated by an insulin-SREBP-FASN signaling axis. Citation Format: Bryan Mcclellan, Tommy Pham, Brittany Harlow, Gabby Lee, Duan Quach, Christopher Jolly, Andrew Brenner, Linda deGraffenried. Fatty acid synthase enzyme as a mediator of obesity-induced breast 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 PS19-22.

Abstract INTRODUCTION: Estrogen receptor-positive (ER+) breast cancer accounts for nearly 70% of all cases. Common targeted anti-estrogen therapies such tamoxifen, fulvestrant and aromatase inhibitors have shown success in the clinic, but unfortunately, often lead to resistance. The fatty acid synthase enzyme (FASN) is responsible for endogenously synthesizing long-chain fatty acids, such as palmitate, which can contribute to protein modification, phospholipid biosynthesis for membranes, and lipid raft signaling that favors tumorigenesis. The depletion of intracellular palmitate leads to an alteration of lipid composition within lipid rafts of the plasma membrane as well as lipids within the endoplasmic reticulum membrane. Endoplasmic reticulum stress elicits an inhibition of protein translation through the phosphorylation of eukaryotic initiating factor 2-α (p-eIF2α) and is activated by various stimuli including altered phospholipid composition within the membrane. Our preliminary findings illustrated a degradation of the ERα upon treatment with the FASN inhibitor, TVB-3166, in tamoxifen-resistant breast cancer both in vivo and in vitro. Moreover, previous studies have illustrated FASN inhibition to induce endoplasmic reticulum stress concomitant with a loss of the androgen receptor (AR) in castration-resistant prostate cancer. Additionally, palmitate treatment rescued AR expression that was accompanied by an attenuation in endoplasmic reticulum stress. HYPOTHESIS: We hypothesize FASN inhibition leads to a degradation of ERα in tamoxifen-resistant breast cancer through the induction of endoplasmic reticulum stress. METHODS: Patient tumor explants were incubated for 72h on gelatin sponges in culture medium in the absence or presence of 200nM TVB-3166. Tissue were fixed in 10% formalin and processed into paraffin blocks and stained for ERα and Ki67. To investigate TVB induced endoplasmic reticulum stress, tamoxifen-Resistant (TamR) MCF-7 and MCF-7 cells were treated with TVB-3166 or vehicle control followed by treatment with either palmitate or ER stress inhibitor, 1,2-Bis(2-Aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Expression of ERα, p-eIF2α, eIF2α were measured by western blot. RESULTS: TVB-3166 treatment of primary tumor explants decreased their proliferation (Ki67) compared to untreated controls (14% vs 36%, p<0.01). Both IHC and Western blotting demonstrated a reduction in ERα upon treatment with TVB-3166. In addition to the decreased expression of ERα, there was an increase in the phosphorylation of eukaryotic initiation factor 2α (p-eIF2α). The expression of ERα was rescued upon palmitate treatment while resulting in decreased p-eIF2α. Inhibition of endoplasmic stress using BAPTA also rescued ERα expression after TVB-3166 treatment. CONCLUSION: FASN is a potentially viable target in tamoxifen-resistant breast cancer. Citation Format: Bryan Mcclellan, Aleksandra Gruslova, Christopher Jolly, Linda deGraffenried, Andrew Brenner. Fatty acid synthase inhibition targets ERα in tamoxifen-resistant breast 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 PS17-26.

Abstract Background: The overexpression of the IGF-1R is correlated with an overall worse prognosis for breast cancer patients. IGF-1R contributes to an aggressive phenotype through its downstream signaling cascades, including the classical PI3K-Akt- mTORC1 pathway. Recent studies suggest another important target of IGF-1R regulation is the fatty acid synthase enzyme (FASN), leading to increased endogenous fatty acid synthesis. The importance of FASN activity to breast cancer progression is illustrated by the success of recent clinical trials investigating the efficacy of FASN inhibitors in refractory breast cancer. Previous studies in our lab and others have identified SREBP and SRPK2 as key IGF-1R targets regulating FASN expression. FASN expression can lead to excess palmitate, which can serve as a substrate for palmitoylation shown to aid in the membrane localization and function of various receptor tyrosine kinases, including IGF-1R. However, the role of FASN induced localization and activation of IGF-1R in breast cancer has not been investigated and is critical for a better understanding of the role of FASN in breast cancer progression. Objective: The goal of this study is to investigate a potential feedforward signaling loop between IGF-1R and FASN and its contribution to breast tumorigenesis. Methods: The impact of suppression of mTOR, SREBP, and SRPK2 on FASN gene expression in response to IGF-1 stimulation in MCF-7 and T47D ER+ breast cancer cells over a course of time was evaluated using qPCR. The role of FASN in IGF-1R stabilization and signaling was determined in MCF-7 cells pretreated with 2-bromopalmitate (2-BP, a palmitoylation inhibitor) or the FASN inhibitor, TVB-3166. Colony formation and migration assays were performed for phenotypic analysis of breast cancer cells in response to IGF-1R and SRPK2 modulation. Results: Inhibition of mTORC1 resulted in an attenuation of FASN expression in both MCF-7 and T47D cells upon IGF-1 exposure. Additionally, inhibition of palmitoylation and/or FASN resulted in a decreased stabilization and activation of IGF-1R in MCF-7 breast cancer cells. Further elucidation of specific mechanisms of post-transcriptional regulation of FASN through SRPK2 as well as effects of SRPK2 and IGF-1R inhibition on breast cancer cell migration and survival are on-going. Conclusion: The results of this study suggest that a potential mechanism of breast cancer progression is through an autoregulatory loop between IGF-1R signaling and FASN activity, which can be effectively limited using the new class of FASN inhibitors. These data support the rationale for continued clinical studies evaluating the efficacy of FASN inhibitors in breast cancers driven, at least in part, by IGF-1 signaling. Citation Format: Bryan McClellan, Brittany Harlow, Christopher Jolly, Linda deGraffenried. A potential FASN -IGF1R signaling loop in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2455.

Abstract Background: Numerous epidemiological studies, including the Nurses' Health Study II, found that shift workers have a higher risk for breast cancer. These studies hypothesize that suppressed melatonin and disrupted clock gene expression likely lead to an immune compromised, proliferative phenotype. However, the majority of studies to date rely on correlative evidence. With the onset of the COVID-19 pandemic, shift work has become increasingly prevalent, making the need to understand the mechanisms promoting disease, including breast cancer, even more important. Objective: The objective of this study is to analyze the impact of staggered light exposure on the immune function and breast tissue gene expression in order to identify how breast cancer risk is mediated. Methods: An 18-week study with 10 C57BL/6 female mice was conducted whereby half of the mice were exposed to a staggered light/dark (LD) cycle, as would be experienced by shift workers, and half were exposed to a regular LD cycle. Body weight was monitored weekly and sera and mammary tissues were collected at termination. The sera from each group was pooled and cytokine levels assessed using an antibody-pair-based assay. Results: Intriguingly, although the mice from the two study groups did not significantly differ in average body weight, the staggered LD exposure group had higher levels of inflammatory cytokines indicative of chronic, low-grade inflammation. Notably, the staggered LD group had 75% higher IL-6 levels, 157% higher IL-10 levels, and 49% higher TNF-alpha levels compared to the control group. To determine the impact of staggered light exposure on localized breast tissue gene expression, qPCR arrays of breast cancer risk and inflammatory genes will be done comparing expression levels between the control and staggered LD groups. IHC techniques will be used to determine Ki67 and macrophage subtype infiltration in the breast tissue of the two groups. Conclusion: Our data show that staggered LD exposure induces chronic, low-grade inflammation independent from body mass, which can be indicative of increased breast cancer risk. These data help elucidate the metabolic and immune responses induced by staggered light exposure, helping to define the mechanisms promoting health risks imposed by shift work, with the ultimate goal to develop preventive measures to limit breast cancer development. Citation Format: Lauren Palluth, Brittany Harlow, Bryan McCellan, Christopher Jolly, Molly Bray, Linda deGraffenried. Assessing the impact of staggered light exposure on breast cancer risk and metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 106.

Abstract BACKGROUND FASN expression is associated with a more aggressive breast cancer phenotype and is both transcriptionally and post-transcriptionally regulated downstream of receptor tyrosine kinase signaling pathways. Lipogenic transcripts, such as FASN, can be post-transcriptionally regulated through pre-mRNA splicing mediated through serine arginine rich protein kinases (SRPKs) and their respected substrates, serine-arginine rich splicing factors (SRSFs). Recent work has highlighted the mTORC1 signaling pathway as an upstream inducer of lipogenic pre-mRNA splicing, however, the role of extracellular environmental cues, such as growth factors and their respected receptors have not been explored. Here, an IGF-1-mTORC1-SRPK2 axis is demonstrated in the FASN regulation through SRSF-1 in breast cancer. METHODS: MDA-MB-231, MCF-7 breast cancer or MCF-10A non-transformed cells were exposed to IGF-1 followed by siRNA knockdown of IGF-1R, SRPK2, or SRSF-1. FASN expression was quantified by RT-qPCR or western blot analysis and de novo palmitate was measured by U-13C glucose incorporation followed by GS-MS. For mRNA stability, cells were pretreated with actinomycin-D with either vehicle or SRPK2 inhibitor for various timepoints followed by RT-qPCR for lipogenic and glycolytic mRNA abundance. eGFP-SRSF-1 was transfected in MDA-MB-231 and MCF-7 to visualize SRSF-1 localization in response to mTORC1 inhibition and/or SRPK2 knockdown and visualized by fluorescence microscopy. For intron retention, RT-PCR was performed with FASN intron and exon specific primers and resolved on a 2.5% agarose gel. RESULTS: Both IGF-1R and SRPK2 RNAi mediated knockdown significantly reduced FASN mRNA and protein and de novo synthesized palmitate levels. Similar results were obtained with mTORC1 inhibition. IGF-1 promoted the stabilization of FASN mRNA as well as reduced intron retention. This reduction of intron retention upon IGF-1 was abolished by SRPK2 knockdown. Additionally, IGF-1 contributed to a more diffuse localization in the nucleoplasm of SRSF-1, which become more retained in nuclear speckles upon both SRPK2 knockdown and mTORC1 inhibition. CONCLUSION These current findings establish a potential IGF-1-mTORC1-SRPK2 axis in breast cancer that contributes to metabolic programming through FASN. More specifically, SRSF-1 is the potential mediator of FASN expression through this pathway, which could be a potential therapeutic target for breast cancers that overexpress FASN and components of the IGF-1R signaling axis. Citation Format: Bryan Mcclellan, Paul Gries, Brittany Harlow, Andrew Brenner, Stefano Tiziani, Christopher Jolly, Linda deGraffenried. Regulation of FASN expression through a novel IGF-1R-mTORC1-SRPK2-SRSF1 pathway [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr CC02-01.