Abstract Introduction: Exposure of non-transformed breast cells and breast microstructures to the medium-chain (MC) fatty acid (FA) octanoic acid (OA) induces a metabolic shift toward the serine, one-carbon, glycine and methionine pathways (SOG/methionine), enhancing epigenetic plasticity, increasing reactive oxygen species (ROS), promoting cell survival and disrupting cell-cell communication. Similarly, the aged mammary gland is characterized by disrupted cell-cell communication, epigenetic plasticity and increased ROS. We hypothesize that FA-induced metabolic reprogramming leads to biological aging of the mammary gland, contributing to pro-tumorigenic alterations observed during chronologic aging. Methods: MCF-10A cells were exposed to OA for proteomics. Breast microstructures exposed to ± OA were analyzed by scRNAseq. Breast microstructures and 3D mammary spheres derived from primary cells were embedded in Matrigel, exposed to ± OA for 7 days, stained for luminal and basal markers, F-actin, and nuclei, and imaged by confocal microscopy to assess migration/invasion. Migratory cell populations enriched in OA-containing media were identified with scRNAseq. Raman spectroscopy (RS) was used to characterize the lipid content in normal breast tissue. Results: OA treatment induced changes previously reported in aging and tumorigenic contexts, including: (1) upregulation (p < 0.01) of aging-related genes (GDF15, MDK, PLIN2), and downregulation (p < 0.01) of lineage markers and MMP7, a gene whose downregulation promotes mammary epithelial aging; (2) upregulation (p < 0.01) of Senescence-Associated Secretory Phenotype (SASP) genes, including AREG (reprogramming) and ANGPTL4 (migration); (3) increased secreted signaling via AREG, GDF15, and MDK; and (4) reduced extracelular matrix (ECM)-receptor and cell-cell interactions. Ex vivo, OA altered tissue architecture disrupting the basal barrier and promoting cellular migration. BMYO1, LASP1, and LHS1 epithelial subtypes were among the migratory cells in OA media and expressed SASP, cancer (MYC, EGFR, SREBF1), migration (S100A4, NCAM1), aging and SOG/methionine genes. FB1 fibroblasts dominated in vehicle media, but OA favored ECM-disassembling FB2 cells. RS analysis demonstrates the presence of both saturated and unsaturated FAs and revealed the presence of MCFAs, such as OA, with higher intensities observed in the postmenopausal tissue supporting the in vivo plausibility of our in vitro/ex vivo findings. Conclusions: Our data supports a model suggesting that chronological and biological aging processes increase the release of free FAs, due to elevated GDF15-induced lipolysis. The rise in FAs drives mammary gland remodeling and accelerates aging of the gland. Chronological and biological aging increase vulnerability to breast cancer. This model suggests potential preventive strategies such as targeting GDF15 and SOG/methionine. Citation Format: Mariana Bustamante Eduardo, Abul B.M.M.K. Islam, Curtis W. McCloskey, Maria Paula Zappia, Ashok Z. Samuel, Maxim V. Frolov, Rama Khokha, Rohit Bhargava, Elizaveta V. Benevolenskaya, Seema A. Khan, Susan E. Clare. Fatty acid exposure promotes age-related mammary tissue alterations with pro-tumorigenic potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6019.

The purpose of this project was to define gene expression changes associated with the acquisition and loss of resilience to diabetic retinopathy (RDR) in individual retinal cell types. A non-immune form of type 1 diabetes mellitus (DM) was induced by injecting male C57Bl6J mice with streptozotocin. Single-cell RNA sequencing was performed on retinas from mice that experienced DM for 5 or 15 days, along with retinas from age-matched, non-DM mice. The resulting data sets were analyzed to identify DM-associated differentially expressed genes and pathway enrichments after each duration of DM. We observed that acquisition of RDR, previously shown to arise after 5 days of DM was linked to altered expression of genes in a subset of retinal cells, mainly Müller cells. Pathway analysis indicated enhancement of numerous modes of protection, including reinforced neurovascular and structural homeostasis through phagocytosis, integrin signaling, and interferon-mediated defense. After 15 days of DM, when we previously showed that RDR is waning this pro-protection surge in gene expression subsided. We conclude that a duration of DM that is too short to cause diabetic retinopathy (DR) nonetheless evoked a profound change in the gene expression profile within a subset of retinal cell types. The nature and timing of this molecular shift indicated that it was not the preamble to DM-related damage that eventually develops. Rather, DM engaged numerous defense programs within Müller cells. The temporal alignment between RDR and activation of Müller cell-based defense provides a molecular foundation for the retina’s transient ability to remain healthy in the face of DM.

In Drosophila, Arginine kinase 1 (Argk1) is involved in maintaining ATP homeostasis during bursts of activity in tissues with high and variable rates of energy turnover such as muscle. However, its role beyond stress conditions is less understood. Here, we show that Argk1 maintains energy homeostasis during flight muscle development and is required for animal viability and proper muscle function. The knockdown of Argk1 causes defects in both early and late stages of myogenesis. In the proliferating myoblasts associated with the wing disc, Argk1 depletion results in a reduction in cell size without changes in cell cycle progression. Single cell RNA-sequencing revealed that the overall composition of differentiating and undifferentiating myoblasts is not altered. Nonetheless, Argk1 knockdown causes broad alterations in the expression of genes involved in various metabolic pathways. This correlates with low levels in both ATP content and NAD+/NADH ratio. Later in muscle development, Argk1-depleted muscles completely lack spontaneous muscle contractions that are essential in myofibrillogenesis. Accordingly, Argk1 knockdown results in severe defects in sarcomere structure, while the mitochondrial network is highly fragmented. Furthermore, muscle growth is severely reduced. Thus, our data reveal an essential role for Argk1 in maintaining energy homeostasis throughout muscle development, which is required to meet the demand to support myofibrillogenesis, muscle growth and proper muscle function.

Introduction: We have investigated the breast microenvironment to identify factors that promote Estrogen Receptor Negative Breast Cancer (ERneg BC) and that may be disrupted for prevention. To that end, we have identified a lipid metabolism gene signature associated with the risk of ERneg BC. To better understand lipid metabolism in the breast, we studied the effect of fatty acids (FA) on non-transformed breast epithelial cells and tissues. FA exposure alters histone methylation, affecting gene expression and increase flux through serine, one-carbon, glycine (SOG) and methionine pathways. The association of the serine pathway and ERneg BC was first observed over a decade ago. A SOG pathway gene signature is significantly correlated with ERneg status. We hypothesized that the metabolism of FA results in a metabolic shift toward the de novo serine synthesis pathway (SSP), which ultimately increases S-adenosylmethionine (SAM), altering histone methylation, profoundly changing gene expression and fostering ERneg oncogenesis. Methods: Non-transformed MCF-10A cells were used for in vitro metabolic and epigenomic analyses. Cells exposed to the medium-chain FA octanoic acid (OA) were utilized for proteomics and U13C-glucose tracing. SAM, glutathione (GSH) and 2-hydroxyglutarate (2-HG) concentrations were measured following treatment with OA ± blockade of the serine pathway. Reactive Oxygen Species (ROS)-induced redox changes were monitored live cells. Comet assay was performed to detect DNA damage. CUT&RUN was performed for H3K4me3. Human breast tissue derived microstructures were utilized for genomic analysis. Single-cell RNA-seq (scRNAseq) was performed in microstructures exposed to ± OA. Metabolic flux analyses was performed using Compass. Results: 13C flux analysis revealed that OA led to increased flux to methylation. OA significantly increased the main methyl donor SAM, the antioxidant GSH via the transsulfuration pathway and the oncometabolite 2-HG after 15 min exposure. Blocking the first and rate limiting enzyme in the SSP, PHGDH, prevented these increases. Proteomics revealed the overexpression of PHGDH following OA exposure. Upon exposure to OA, scRNAseq analysis revealed increased expression of the SSP transcription factor (TF) ATF3 and the SSP genes PHGDH and PSAT1 in epithelial and non-epithelial clusters. Upon OA the proportion of three subtypes within the epithelial compartment increased: basal BSL1, Hormone sensing HS1 and luminal progenitor LP3. Compass, an algorithm to characterize cellular metabolic states, revealed flux greatly increased through the three enzymes of the SSP: PHGDH, PSAT1 and PSPH secondary to OA exposure in BSL1, LP3 and HS1 cells. H3K4me3 CUT&RUN revealed 661 differential peaks (FDR < 0.05) comparing OA to control. Motif analysis revealed an overrepresentation of binding sites for SSP TFs ATF3/4 (p < 0.05). After 5 min OA exposure, mitochondrial and nuclear ROS increased significantly (p < 0.01), peaking at 15 min. OA exposure triggered DNA damage likely due to ROS increase in the nucleus. Compass predicted an increase in GSH metabolism and ROS detoxification in BSL1. Conclusions: Protein levels of PHGDH are elevated in 70% of ERneg BCs. This cannot be explained by gene amplification alone as PHGDH gene amplification is observed in only approximately 6% of all breast cancers. This suggests that there are mechanisms other than gene amplification that contribute to PHGDH dysregulation. One of those mechanisms may be the lipid induced metabolic shift toward the SOG and methionine pathways that we have identified. The increased SAM and 2-HG foster epigenetic phenotypic plasticity via altered histone methylation. ROS increase shortly after OA exposure and are controlled by antioxidant defenses (e.g. GSH), which favors the survival of specific cell subtypes with acquired DNA damage which likely facilitates malignant transformation. Citation Format: Mariana Bustamante Eduardo, Curtis W. McCloskey, Gannon Cottone, Shiyu Liu, Flavio R. Palma, Maria Paula Zappia, Abul B.M.M.K. Islam, Jason Locasale, Marcelo G. Bonini, Maxim V. Frolov, Elizaveta V. Benevolenskaya, Rama Khokha, Navdeep S. Chandel, Seema A. Khan, Susan E. Clare. Medium chain fatty acids shift metabolism towards the de novo serine pathway fostering epigenetic plasticity and oxidative DNA damage [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-05-02.

The innate immune system plays a dual role in both mediating pathogenic processes following tissue damage and acting as a barrier to effective therapeutic delivery. Strategies that evade immune clearance while modulating host immune components offer promising solutions for treating complex chronic diseases, such as fibrosis. Here, an innate immune checkpoint material-based strategy is presented in which mesenchymal stromal cells, coated with a soft conformal microgel and functionalized with the CD47 self-marker agonist, effectively evade clearance by tissue resident macrophages. These engineered cells reverse persistent fibrotic damage in the lungs through a paracrine mechanism. Single-cell RNA sequencing identifies a transitional antigen-presenting macrophage subpopulation that mediates these reparative effects. By combining immune cloaking with the presentation of local signals encoded in the gel coatings, this strategy can be used to design secretory cells for long-term tissue remodeling, enabling a living pharmacy for chronic tissue damage.

A lipid metabolism gene signature is enriched in breast tissue at risk for estrogen receptor negative (ERneg) breast cancer (BC). Fatty acid (FA) exposure alters histone methylation, gene expression and increases metabolic flux through serine, one-carbon, glycine (SOG) and methionine pathways. We hypothesize that FA exposure induces a metabolic shift towards the SOG, increasing S-adenosylmethionine (SAM), altering histone methylation, gene expression, and promoting ERneg BC. Proteomics, metabolomics, Reactive Oxygen Species (ROS) measurement, comet assay and H3K4me3 CUT&RUN were performed in MCF-10A cells exposed to octanoic acid (OA). Single-cell RNA-seq (scRNAseq) was performed in breast tissue derived microstructures exposed to OA. Intracellular communication was analyzed using CellChat, and metabolic flux with Compass. OA increased SAM, glutathione (GSH) and 2-hydroxyglutarate (2-HG); blocking the serine pathway (SSP) prevented these increases. ScRNAseq revealed that OA increased expression of the SSP transcription factor ATF3 and genes PHGDH and PSAT1 in epithelial and stromal compartments. Metabolic flux analysis revealed a significant increase in flux through SSP in Basal BSL1, Luminal Progenitor LP3, and Hormone Sensing HS1 cells after OA exposure. Differential proteomics reveals PHGDH overexpression and downregulation of proteins involved in extracellular matrix (ECM)-receptor interaction and focal adhesion post-OA exposure, along with significant increase in mitochondrial and nuclear ROS (p < 0.01). OA exposure also induced DNA damage, likely due to elevated nuclear ROS. OA increased GSH metabolism and ROS detoxification in BSL1. CUT&RUN identified 661 peaks significantly enriched upon OA (FDR < 0.01) in regulatory regions of OA-induced genes involved in neural pathways and BC, including MDK, NGF, and NGFR. CellChat predicted a decrease in ECM-cell interactions, a reduction in cell-cell adhesions, and an increase of secreted signaling upon OA exposure. The strongest secreted signals in OA were AREG (linked to proliferation, growth, and invasiveness), GDF15 (involved in EMT, invasion, and aging), and MDK (linked to neurogenesis, and aging). We demonstrate an FA-induced shift towards the SOG and methionine pathways that promotes epigenetic plasticity, regulates ROS, and supports the survival of cells with 'inappropriate' phenotypes. These accumulate DNA damage, leading to age-related changes in the mammary gland (elevated ROS, disrupted junctions, altered ECM interactions, and increased MDK/GDF15 expression), all supporting carcinogenesis. Our findings also provide a metabolic explanation for the elevation of PHGDH in 70% of ERneg BCs, despite gene amplification in only 6%, and point to preventive strategies targeting the SSP. Mariana Bustamante Eduardo, Gannon Cottone, Curtis McCloskey, Flavio Palma, Shiyu Liu, Maria Paula Zappia, Abul B.M.M.K. Islam, Elizaveta Benevolenskaya, Maxim Frolov, Jason Locasale, Marcelo Bonini, Rama Khokha, Navdeep Chandel, Seema A. Khan, Susan E. Clare. Metabolic shift towards the de novo serine pathway in non-transformed breast cells drives epigenetic plasticity, oxidative DNA damage, and pro-tumorigenic cChanges associated with aging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5402.

A lipid metabolism gene signature is associated with the risk of estrogen negative breast cancer (ER-BC). In vitro, lipid exposure alters histone methylation affecting gene expression and increasing flux through various metabolic reactions; but little is known about the mechanism(s) linking lipids and epigenetic reprogramming with the genesis of ER-BC. Here we show that the metabolism of the medium-chain fatty acid Octanoic Acid (OA) in preference to glucose and glutamine results in a metabolic shift toward the serine pathway increasing the production of SAM, glutathione, and 2-HG, with implications for oncogenesis: SAM production results in epigenetic fostered plasticity leading to reprogramming/selecting cells that express Neural, EMT and BC related genes. 2-HG exposure results in appearance of DNA breaks, potentially consequent to the inhibition of essential demethylases for HR repair. ROS increases shortly after OA exposure and is mitigated by antioxidant defenses, which favors/enables the survival of specific cell subtypes.### Competing Interest StatementThe authors have declared no competing interest.

Abstract Introduction. Oncogenic factors that are local/in-breast are of great interest as they may be more specifically targetable for breast cancer prevention than systemic factors. We have identified a lipid metabolism gene signature that is enriched in breast tissue at risk for estrogen negative breast cancer (ER- BC). Utilizing the medium chain fatty acid Octanoic acid (OA) to probe lipid metabolism in non-transformed breast epithelial cells, we observed increased flux through several metabolic reactions and altered histone methylation with consequent changes in gene expression (e.g. neural genes). Neuronal signaling and regulatory circuits are observed in cancer cells of multiple origins, not just ones with ontological relationships to neurons. We hypothesize that the first and rate limiting step in the de novo serine pathway, which is catalyzed by Phosphoglycerate Dehydrogenase (PHGDH) is key to these observations. In the forward direction, PHGDH participates in the serine, one-carbon, glycine (SOG) and methionine pathways that produce the methyl donor S-adenosylmethionine (SAM) and in the reverse direction produces the oncometabolite 2-Hydroxyglycerate (2-HG). Methods. Non-transformed MCF-10A cells exposed to OA were utilized for U13C-glucose tracing. SAM and 2-HG concentrations following treatment with OA ± PHGDH inhibitor were measured by liquid chromatography. CUT&RUN for H3K4me3 was performed and genes affected by OA (PMID: 28263391) were compared with OA-responsive peaks. Single cell RNA-sequencing was carried out using breast microstructures derived from reduction mammoplasty tissue exposed to vehicle or OA. Microstructures were dissociated into single cells and sequenced using the 10x Genomics platform. The digital expression matrix file containing UMIs were analyzed with Seurat. Alkaline comet assay was performed to detect DNA breaks. Results. U13C-glucose tracing in presence of OA revealed that one-carbon-THF was redirected to the methionine cycle increasing flux to methylation. Concentrations of SAM and 2-HG increased after 15- and 30-min OA exposure, respectively; PHGDH inhibitor blocked these increases. H3K4me3 CUT&RUN revealed 661 differential peaks (FDR < 0.05) comparing OA to control. 73% of H3K4me3 OA-associated peaks were in regulatory regions of OA-induced genes (FDR < 0.01), these genes are involved in neural pathways, EMT and ER- BC. Motif analysis revealed an overrepresentation of binding sites for transcription factors ATF3/4 (p < 0.05), which are regulators of the serine pathway. Single cell RNA-sequencing revealed OA not only affected the distribution of cell subpopulations but also modulated the expression of many genes within each subcluster. The percentage of luminal progenitor subcluster 3 increased upon OA from less than 1% to about 13%. Within basal subcluster 3, OA drives the expression of ATF3, along with two of the enzymes in the de novo serine pathway: PHGDH and PSAT1. Alkaline comet assay showed DNA breaks in OA- and control 2-HG- treated cells. Conclusions. Metabolism of OA in preference to glucose and glutamine results in a metabolic shift toward the serine pathway increasing the production of SAM and 2-HG, with implications for oncogenesis: 1. SAM production results in epigenetic fostered plasticity leading to reprogramming/selecting cells that express genes consistent with a neural/neural crest-like state. These co-opted neuronal regulatory mechanisms can make critical contributions to the acquired functional capabilities that drive cancer development. 2. 2-HG exposure results in appearance of DNA breaks, which are likely consequent to the inhibition of the alpha-ketoglutarate-dependent dioxygenases KDM 4A/B by 2-HG. Their catalytic activity is required for homologous recombination repair; inhibition results in metabolic “BRCAness”. Citation Format: Mariana Bustamante Eduardo, Gannon Cottone, Shiyu Liu, Maria Paula Zappia, Elizaveta V. Benevolenskaya, Abul Bashar Mir Md. Khademul Islam, Maxim V. Frolov, Seema Khan, Susan Clare. Metabolic shift to serine pathway induced by lipids confers oncogenic properties in non-transformed breast cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-08-06.

Robust genetic systems to control the expression of transgenes in a spatial and temporal manner are a valuable asset for researchers. The GeneSwitch system induced by the drug RU486 has gained widespread use in the Drosophila community. However, some concerns were raised as negative effects were seen depending on the stock, transgene, stage and tissue under study. Here, we characterized the adverse effects triggered by activating the GeneSwitch system in adult muscles using the MHC-GS-GAL4 driver. When a control, mock UAS-RNAi transgene was induced by feeding adult flies with RU486, we found that the overall muscle structure, including myofibrils and mitochondrial shape, was significantly disrupted and led to a significant reduction in the lifespan. Remarkably, lifespan was even shorter when two copies of the driver were used even without the mock UAS-RNAi transgene. Thus, researchers should be cautious when interpreting the results given the adverse effects we found when inducing RU486-dependent MHC-GS-GAL4 in adult muscles. To counter the impact of these effects we recommend setting up additional control groups, such as a mock UAS-RNAi transgene, to validate the findings when using this inducible genetic system, as comparing the phenotypes between RU486-treated and untreated animals could be insufficient.

Supplementary Data from Transcriptional Repression by FoxM1 Suppresses Tumor Differentiation and Promotes Metastasis of Breast Cancer

IntroductionInflammatory epidermolysis bullosa acquisita (EBA) is characterized by a neutrophilic response to anti-type VII collagen (COL7) antibodies resulting in the development of skin inflammation and blistering. The antibody transfer model of EBA closely mirrors this EBA phenotype.MethodsTo better understand the changes induced in neutrophils upon recruitment from peripheral blood into lesional skin in EBA, we performed single-cell RNA-sequencing of whole blood and skin dissociate to capture minimally perturbed neutrophils and characterize their transcriptome.ResultsThrough this approach, we identified clear distinctions between circulating activated neutrophils and intradermal neutrophils. Most strikingly, the gene expression of multiple C-type lectin receptors, which have previously been reported to orchestrate host defense against fungi and select bacteria, were markedly dysregulated. After confirming the upregulation of Clec4n, Clec4d, and Clec4e in experimental EBA as well as in lesional skin from patients with inflammatory EBA, we performed functional studies in globally deficient Clec4e−/− and Clec4d−/− mice as well as in neutrophil-specific Clec4n−/− mice. Deficiency in these genes did not reduce disease in the EBA model.DiscussionCollectively, our results suggest that while the upregulation of Clec4n, Clec4d, and Clec4e is a hallmark of activated dermal neutrophil populations, their individual contribution to the pathogenesis of EBA is dispensable.

The canonical role of the transcription factor E2F is to control the expression of cell cycle genes by binding to the E2F sites in their promoters. However, the list of putative E2F target genes is extensive and includes many metabolic genes, yet the significance of E2F in controlling the expression of these genes remains largely unknown. Here, we used the CRISPR/Cas9 technology to introduce point mutations in the E2F sites upstream of five endogenous metabolic genes in Drosophila melanogaster . We found that the impact of these mutations on both the recruitment of E2F and the expression of the target genes varied, with the glycolytic gene, Phosphoglycerate kinase ( Pgk) , being mostly affected. The loss of E2F regulation on the Pgk gene led to a decrease in glycolytic flux, tricarboxylic acid cycle intermediates levels, adenosine triphosphate (ATP) content, and an abnormal mitochondrial morphology. Remarkably, chromatin accessibility was significantly reduced at multiple genomic regions in Pgk ΔE2F mutants. These regions contained hundreds of genes, including metabolic genes that were downregulated in Pgk ΔE2F mutants. Moreover, Pgk ΔE2F animals had shortened life span and exhibited defects in high-energy consuming organs, such as ovaries and muscles. Collectively, our results illustrate how the pleiotropic effects on metabolism, gene expression, and development in the Pgk ΔE2F animals underscore the importance of E2F regulation on a single E2F target, Pgk .

Background. Treatment of newly diagnosed multiple myeloma (NDMM) consists of induction therapy followed by autologous stem cell transplant (ASCT) and indefinite maintenance. Continuous lenalidomide carries both significant clinical and financial toxicity. Minimal residual disease (MRD) has proven to be a powerful surrogate for PFS and OS but is not yet used to guide treatment. We hypothesize that MRD could be used to predict a subset of patients who can safely stop maintenance therapy and enter into a disease surveillance phase. Study Design and Methods. This prospective phase 2 study (NCT 05192122) is designed to stop post-ASCT maintenance therapy once sustained MRD negativity is achieved. MM patients are eligible if they have completed ≥2 years of maintenance therapy post-ASCT, and meet IMWG criteria for ≥ PR. Bone marrow (BM) MRD testing is performed twice, one year apart, using NGS. Sustained MRD-negativity is defined at a threshold of 10 -6, with negative MRD sustained between the Year 1 and Year 2 interval. Patients with sustained MRD negativity and absence of new bony lesions on PET then undergo maintenance therapy cessation. MRD-positive patients are removed from study. In patients who discontinue maintenance, BM testing to re-assess for MRD is repeated yearly for 3 years. At the time of MRD testing, peripheral blood (PB) samples are collected for MALDI-TOF mass spectrometry testing and circulating multiple myeloma cell (CMMC) enumeration. These are done in parallel with MRD assessment. Health-related quality of life (HRQoL) using the EORTC QLQ-MY20 questionnaire is measured before and after maintenance cessation. This study plans to enroll 50 patients to meet the primary objective of assessing sustained MRD-negativity 1 year after maintenance cessation. For all patients, 5-mL PB and BM samples are collected, processed, and kept frozen until analysis. Cellular indexing of transcriptomes and epitopes (CITE-seq) to simultaneously quantify cell surface protein and transcriptome data is used to characterize differences in the immune microenvironment between MRD-negative, MRD-positive, as well as between patients with biochemical and/or clinical progression and patients with MRD-resurgence but biochemically and clinically stable. Preliminary Results. FREEDMM enrollment began December 2021, and 31 patients have been enrolled, 29 of whom have undergone Year 1 BM for MRD assessment. Median follow up of our cohort is 414 (0-577) days. The median age is 69.5 (40-78) years, and the majority are either Black (n=9; 29%) or Hispanic (n=11; 35%). 18 (58%) patients had R-ISS 1 or 2, while 2 patients had high risk disease with presence of 17p deletion. The median number of years of maintenance therapy completed prior to Year 1 BM is 3.4 (2-7.9) years. 28 (96.6%) were in CR at time of Year 1 BM. Year 1 MRD results are pending in 13 patients. At Year 1, 11 (68.8 %) of 16 patients were found to be MRD-negative, while 5 (31.3%) patients were found to be MRD-positive with a median of 206 (1-9,748) cells per 10 6 nucleated cells detected. 24 (85%) of 28 patients had a negative MALDI-TOF result at the time of the Year 1 BM. MALDI-TOF results were false negative in 3 MRD-positive patients. PB CMMC samples were concordant with MRD results in all 5 (100%) patients who were tested. There was concordance between MALDI-TOF and CMMC results in 9 (75%) of 12 patients. At year 2, 4 patients continued to be MRD negative, while 1 patient who was previously MRD-negative became MRD positive. At most recent follow-up, 3 (42.9%) of 7 MRD positive patients at Year 1 experienced clinical progression and were removed from the study. 2 (50%) of 4 patients with sustained MRD-negativity have undergone maintenance cessation, with the remaining 2 awaiting confirmation of MRD-negative results. 59 paired PB (n=29) and BM (n=30) samples have been collected for CITE-seq analysis. Pilot CITE-seq experiments captured 10,000-15,000 cells with 33,000-56,000 reads/ cell on one MRD-positive BM and paired PB sample. CITE-seq results for remaining patients will be presented at the conference. Conclusion: This phase 2 trial applies a novel clinical trial design with an adaptive strategy to explore use of MRD testing as a guide to maintenance therapy cessation in MM patients and represents one of the first studies introducing transcriptome analysis of the tumor and immune microenvironment in the maintenance setting. Ongoing results will be reported at the conference.

The retinoblastoma (RB) and Hippo pathways interact to regulate cell proliferation and differentiation. How-ever, the mechanism of interaction is not fully understood. Drosophila photoreceptors with inactivated RB and Hippo pathways specify normally but fail to maintain their neuronal identity and dedifferentiate. We per-formed single-cell RNA sequencing to elucidate the cause of dedifferentiation and to determine the fate of these cells. We find that dedifferentiated cells adopt a progenitor-like fate due to inappropriate activation of the retinal differentiation suppressor homothorax (hth) by Yki/Sd. This results in the activation of a distinct Yki/Hth transcriptional program, driving photoreceptor dedifferentiation. We show that Rbf physically inter-acts with Yki and, together with the GAGA factor, inhibits the hth expression. Thus, RB and Hippo pathways cooperate to maintain photoreceptor differentiation by preventing inappropriate expression of hth in differ-entiating photoreceptors. Our work highlights the importance of both RB and Hippo pathway activities for maintaining the state of terminal differentiation.

The canonical role of the transcription factor E2F is to control the expression of cell cycle genes by binding to the E2F sites in their promoters. However, the list of putative E2F target genes is extensive and includes many metabolic genes, yet the significance of E2F in controlling expression of these genes remains largely unknown. Here, we used the CRISPR/Cas9 technology to introduce point mutations in the E2F sites upstream of five endogenous metabolic genes in Drosophila . We found that the impact of these mutations on both the recruitment of E2F and the expression of the target genes varied, with the glycolytic gene, Phosphoglycerate kinase ( Pgk) , being mostly affected. The loss of E2F regulation on Pgk gene led to a decrease in glycolytic flux, TCA cycle intermediates levels, ATP content and an abnormal mitochondrial morphology. Remarkably, chromatin accessibility was significantly reduced at multiple genomic regions in Pgk Δ E2F mutants. These regions contained hundreds of genes, including metabolic genes that were downregulated in Pgk Δ E2F mutants. Moreover, Pgk Δ E2F animals had shortened life span and exhibited defects in high-energy consuming organs, such as ovaries and muscles. Collectively, our results illustrate how the pleiotropic effects on metabolism, gene expression and development in the Pgk Δ E2F animals underscore the importance of E2F regulation on a single E2F target, Pgk .

BackgroundIndefinite maintenance in MM with continuous lenalidomide carries both significant clinical and financial toxicity. We hypothesize that MRD could be used to predict a subset of patients who can safely stop maintenance therapy and enter into a disease surveillance phase.Study Design and MethodsProspective phase 2 study (NCT 05192122) designed to stop post-ASCT maintenance therapy once sustained MRD negativity is achieved. MM patients are eligible if completed ≥2 years of maintenance therapy post-ASCT, and meet criteria for ≥ PR. Bone marrow (BM) MRD testing is performed twice, one year apart, using NGS. Sustained MRD-negativity is defined at a threshold of 10−6, with negative MRD sustained between the Year 1 and Year 2. Patients with sustained MRD negativity and absence of new bony lesions undergo maintenance cessation. MRD-positive patients are removed from study. In patients who discontinue maintenance, BM testing to re-assess for MRD is repeated yearly for 3 years. In parallel with MRD assessment, peripheral blood samples are collected for MALDI-TOF and circulating multiple myeloma cell (CMMC) enumeration. Cellular indexing of transcriptomes and epitopes (CITE-seq) to simultaneously quantify cell surface protein and transcriptome data is used to characterize differences in the immune microenvironmentPreliminary Results31 patients have been enrolled. Median follow up of our cohort is 414 days. The median age is 69.5 years, and the majority are either Black (n=9; 29%) or Hispanic (n=11; 35%). 18 patients had R-ISS 1 or 2, while 2 patients had 17p deletion. The median number of years of maintenance therapy prior to Year 1 BM is 3.4 years. At Year 1, 11 (68.8 %) of 16 patients were MRD-negative, while 5 (31.3%) patients were MRD-positive. MALDI-TOF results were false negative in 3 MRD-positive patients. PB CMMC samples were concordant with MRD results in all 5 (100%) patients who were tested. There was concordance between MALDI-TOF and CMMC results in 9 (75%) of 12 patients. At year 2, 4 patients continued to be MRD negative, while 1 patient who was previously MRD-negative became MRD positive. At most recent follow-up, 3 (42.9%) of 7 MRD positive patients at Year 1 experienced clinical progression and were removed from the study. All patients with sustained MRD-negativity have undergone maintenance cessation. Pilot CITE-seq captured 10,000-15,000 cells with 33,000-56,000 reads/cell on one MRD-positive BM and paired PB sample.ConclusionThis phase 2 trial applies a novel clinical trial design with an adaptive strategy to explore use of MRD testing as a guide to maintenance therapy cessation in MM patients and represents one of the first studies introducing transcriptome analysis of the tumor and immune microenvironment in the maintenance setting. CITE-seq results for remaining patients will be presented at the conference.

Hexokinase 2 (HK2), which catalyzes the first committed step in glucose metabolism, is induced in cancer cells. HK2’s role in tumorigenesis has been attributed to its glucose kinase activity. Here, we describe a kinase independent HK2 activity, which contributes to metastasis. HK2 binds and sequesters glycogen synthase kinase 3 (GSK3) and acts as a scaffold forming a ternary complex with the regulatory subunit of protein kinase A (PRKAR1a) and GSK3β to facilitate GSK3β phosphorylation and inhibition by PKA. Thus, HK2 functions as an A-kinase anchoring protein (AKAP). Phosphorylation by GSK3β targets proteins for degradation. Consistently, HK2 increases the level and stability of GSK3 targets, MCL1, NRF2, and particularly SNAIL. In addition to GSK3 inhibition, HK2 kinase activity mediates SNAIL glycosylation, which prohibits its phosphorylation by GSK3. Finally, in mouse models of breast cancer metastasis, HK2 deficiency decreases SNAIL protein levels and inhibits SNAIL-mediated epithelial mesenchymal transition and metastasis.

AbstractThe transcription factor Forkhead box M1 (FoxM1) is overexpressed in breast cancers and correlates with poor prognosis. Mechanistically, FoxM1 associates with CBP to activate transcription and with Rb to repress transcription. Although the activating function of FoxM1 in breast cancer has been well documented, the significance of its repressive activity is poorly understood. Using CRISPR–Cas9 engineering, we generated a mouse model that expresses FoxM1-harboring point mutations that block binding to Rb while retaining its ability to bind CBP. Unlike FoxM1-null mice, mice harboring Rb-binding mutant FoxM1 did not exhibit significant developmental defects. The mutant mouse line developed PyMT-driven mammary tumors that were deficient in lung metastasis, which was tumor cell-intrinsic. Single-cell RNA-seq of the tumors revealed a deficiency in prometastatic tumor cells and an expansion of differentiated alveolar type tumor cells, and further investigation identified that loss of the FoxM1/Rb interaction caused enhancement of the mammary alveolar differentiation program. The FoxM1 mutant tumors also showed increased Pten expression, and FoxM1/Rb was found to activate Akt signaling by repressing Pten. In human breast cancers, expression of FoxM1 negatively correlated with Pten mRNA. Furthermore, the lack of tumor-infiltrating cells in FoxM1 mutant tumors appeared related to decreases in pro-metastatic tumor cells that express factors required for infiltration. These observations demonstrate that the FoxM1/Rb-regulated transcriptome is critical for the plasticity of breast cancer cells that drive metastasis, identifying a prometastatic role of Rb when bound to FoxM1.Significance:This work provides new insights into how the interaction between FoxM1 and Rb facilitates the evolution of metastatic breast cancer cells by altering the transcriptome.

The E2F transcription factors play a critical role in controlling cell fate. In Drosophila, the inactivation of E2F in either muscle or fat body results in lethality, suggesting an essential function for E2F in these tissues. However, the cellular and organismal consequences of inactivating E2F in these tissues are not fully understood. Here, we show that the E2F loss exerts both tissue-intrinsic and systemic effects. The proteomic profiling of E2F-deficient muscle and fat body revealed that E2F regulates carbohydrate metabolism, a conclusion further supported by metabolomic profiling. Intriguingly, animals with E2F-deficient fat body had a lower level of circulating trehalose and reduced storage of fat. Strikingly, a sugar supplement was sufficient to restore both trehalose and fat levels, and subsequently rescued animal lethality. Collectively, our data highlight the unexpected complexity of E2F mutant phenotype, which is a result of combining both tissue-specific and systemic changes that contribute to animal development.