Hitosugi, Taro

Abstract Metabolic vulnerabilities in cancer have been targeted primarily to suppress tumor growth, but less is known about the metabolic requirements for tumor cell invasion. Here we report that lipid catabolism by cytosolic and lysosomal lipases supports pancreatic cancer cell invasion through both overlapping and distinct functional and metabolic mechanisms. Lysosomal acid lipase (LAL)-dependent lipid droplet catabolism promotes invadopodia formation and stabilization, enabling extracellular matrix degradation. In addition to modulating cellular energetics, lipidomics revealed that lipid droplet catabolism regulates cholesterol and membrane phospholipid levels. Using spatially resolved biosensors and cholesterol imaging, we found that lysosomal lipid catabolism occurs at invadopodia and sustains local ATP and membrane cholesterol. These findings identify spatially organized lipid catabolism as a mechanism that couples local energetics and membrane remodeling during the earliest steps of pancreatic cancer cell invasion.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy due in part to metabolic rewiring and a high rate of metastasis. Pancreatic tumors are particularly dependent upon glutamine (Gln) as a nutrient source for multiple metabolic pathways. To adapt to low levels of glutamine, PDAC cells upregulate the Rac1 dependent process of macropinocytosis, which internalizes extracellular macromolecules to scavenge them as a glutamine source. The proto oncogene Vav1 is an activator of the potent Rac/Cdc42 signaling cascades that regulate actin dynamics. While Vav1 is normally restricted to hematopoietic cells, Vav1 expression is significantly increased in PDAC, with high Vav1 correlating with worse overall survival. Here we uncover a novel Vav1 mediated regulation of glutamine metabolism in PDAC cells. While Vav1 is a potent activator of Rac1, surprisingly, Vav1 expression inhibited macropinocytosis in PDAC cells. Intriguingly, genomic and metabolomic analysis revealed that high Vav1 expression correlates with increased dependence on glutamine and glutamate (Glu) metabolism. Indeed, Vav1 dependent macropinocytosis was rescued by cell permeable glutamate, confirming disrupted Gln/Glu balance in Vav1 knockdown cells. RPPA screening indicated a Vav1 dependent effect on glutaminase 1 (GLS1, KGA isoform), which converts glutamine to glutamate. Mechanistically, we discovered that Vav1 modulates GLS1 localization to the mitochondria through regulation of GLS1 posttranslational modification. Disrupting GLS1 localization by deleting its mitochondrial targeting motif phenocopied loss of Vav1 in reducing the invasive potential of tumor cells. Investigation of Vav1 dependent metabolic consequences showed that knockdown of Vav1 alters 13C Gln derived metabolic flux into the TCA cycle, as well as a deficiency in the level of the antioxidant glutathione (GSH), which utilizes glutamate for its synthesis. Consequently, targeting Vav1 resulted in increased levels of cellular and mitochondrial reactive oxygen species (ROS) and increased sensitivity to lipid peroxidation. Notably, dual targeting of both glucose and glutamine pathways showed significant synergistic effects on cellular viability in Vav1 expressing cells. Here we established a new tumor promoting role for Vav1 via Gln/Glu regulation and uncover a novel subcellular regulation of mitochondrial glutamine metabolism. Our findings establish Vav1 as a critical regulator of metabolic rewiring in PDAC and orchestrates glutamine metabolism by controlling the KGA isoform of GLS1 mitochondrial localization. Importantly, Vav1 functions as a signaling adapter in glutamine metabolism, providing a potential therapeutic vulnerabilities for PDAC patients with high Vav1 expression. Citation Format: Mustafa Emre Gedik, Omar L. Gutierrez-Ruiz, Katherine M. Johnson, Ella Rose D. Chianis, Jing Chen, Alex Bittner, Adam Zahm, Ankit Chhoda, Taro Hitosugi, Mark A. McNiven, Gina Razidlo. Vav1 promotes pancreatic tumor cell invasion via GLS1-dependent glutamine metabolism [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 7322.

Abstract Mitochondrial creatine kinase (MtCK) is a key enzyme in energy buffering and homeostasis in cells. It catalyzes transfer of phosphoryl group from ATP to creatine. Overexpression of MtCK occurs in many cancer cells to meet elevated energy demands, which is associated with poor prognosis. This suggests that MtCK may be a promising target for cancer therapeutics. We sought to discover first-in-class selective inhibitors of MtCK with diverse mechanisms of action using high-throughput screening, biochemical characterization and cryo-EM studies. Through these studies, we identified diverse types of compounds that modulate activity of MtCK in vitro , including fast-equilibrium and time-dependent orthosteric and allosteric inhibitors. Select hits were subjected to in vitro enzymatic and binding assays to assess MtCK inhibition and binding. A subset of inhibitors was advanced into structural studies using cryo-EM resulting in the molecular structure of MtCK with and without bound substrates in complex with an allosteric uncompetitive inhibitor that we discovered. These studies identified compound’s unique binding pocket on MtCK and established the molecular steps manifesting in the apparent uncompetitive mode of inhibition. We demonstrate that the compound stabilizes an active site loop in a closed conformation restricting access of the creatine substrate to and the release of phosphocreatine product from its binding pocket. These findings establish chemical tools suitable for validation of MtCK as a promising breast cancer target with high therapeutic potential and build a foundation for future structure-guided optimization of the hit compounds of MtCK we identified and de novo rational design of novel MtCK inhibitors. Abstract Figure

Abstract Background: Integrin αE (CD103) has been thought to mediate gut-homing Treg retention during GVHD by facilitating E-cadherin interaction on intestinal epithelial cells. However, only a small percentage of natural Tregs express CD103 in peripheral blood (PB). To leverage this mechanism and potentially enhance Treg therapeutic efficacy, we hypothesized that Tregs overexpressing CD103 (CD103high) would show improved gut-homing and GVHD mitigation. Methods: Murine CD4 Tregs were isolated from C57BL/6 (B6) mice via flow sorting and were retrovirally transduced with murine CD103 or truncated epidermal growth factor receptor as a control. Transduced Tregs were then tested in a murine acute GVHD model (B6 → BALB/c). Mouse organs, including small intestine (SI), colon, spleen, PB, axillary (aLN) and mesentric lymph nodes (mLN) were harvested on day 10 post-transplant and analyzed by flow cytometry, multiplex cytokine assay, histopathology, and single-cell RNA sequencing (scRNA-seq). Ex vivo expanded Tregs were subjected to 13C-glucose tracing assay to evaluate glycolytic flux, TCA cycle, and lipid biosynthesis. Results: Despite comparable FoxP3 expression at the end of ex vivo expansion, CD103high Tregs had significantly higher in vitro suppression compared to control Tregs (54% vs. 33% at Treg:Tcon ratio of 1:8, p=0.004). Upon re-stimulation, CD103high Tregs demonstrated increased proliferation compared to control Tregs (3.8 vs 2.8 fold expansion, p <0.0001). Bulk RNA seq on ex vivo expanded CD103high showed upregulation of immuneregulatory and metabolic programs including GTPase signaling and oxidative stress control. These transcriptomic changes suggest that CD103 intrinsically promotes a regulatory phenotype, stress tolerance and metabolic fitness. In vivo, CD103highTregs significantly decreased the severity and mortality of GVHD, compared to control Tregs, as measured by clinical scores, weight loss, and survival (50% vs 0% on day +60, p<0.0001 for three independent experiments combined; n = 24/group). Day 20 histopathology revealed significantly improved colitis scores in mice treated with CD103high Tregs vs controls (9.5 vs. 14.6, p=0.02). While these findings could be partially attributed to CD103 gut-homing function, we observed that CD103high Treg absolute number and frequency were higher not only in the the GI organs and mLN, but also in the spleen, PB, and aLN with similar distribution ratios across different organs (~5-fold higher). In vivo CFSE suppression assay, where CFSE labeled donor T cells were cotransferred with Tregs into B6 Rag1ko mice, demonstrated enhanced suppressive function of CD103high Tregs in the spleen (p=0.02), an effect that could not be accounted for by the conventional gut-homing mechanism attributed to CD103. Plasma cytokine analysis showed significantly elevated IL-10 levels in mice treated with CD103high Tregs (p<0.0001), consistent with increased Treg number and frequency. 13C-glucose tracing assay showed lower palmitate levels in CD103high Tregs, indicating lower de novo lipid synthesis, while glycolysis and TCA cycle tracing were similar. Day 10 scRNA-seq on colon samples revealed seven distinct immune clusters. The neutrophil cluster was absent in CD103high Treg group, suggesting reduced inflammation. Donor-derived dendritic cells (DCs) upregulated genes such as Cstb, and Slamf7, while pro-inflammatory genes including Cd52 and Cd74 were downregulated. STAT5 signaling was upregulated, indicating reprograming of myeloid cells toward an immunoregulatory state. Colon epithelial cells from CD103high Treg-treated mice displayed enhanced stress tolerance, with increased oxidative phosphorylation, fatty acid metabolism, and epithelial repair pathways. CellChat analysis revealed that CD103high Tregs reshaped the intestinal communication landscape, with DCs acting as key signaling hubs through interactions such as Ccl6–Ccr2 and Lgals3–Cd44. Enhanced DC-to-DC and DC-to-T cell communication suggest that CD103high Tregs promote a tolerogenic DC network that modulates downstream immune responses and supports epithelial recovery. Conclusion: Tregs overexpressing CD103 via retroviral transduction significantly improve overall survival and GVHD clinical scores in a murine transplant model. CD103High Tregs orchestrate broad remodeling of cell-cell communication across immune and stromal compartments, promoting an immunoregulatory microenvironment that underlies their protective effects in GVHD.

Abstract HER2+ breast cancer is an aggressive subtype of cancer that often develops resistance to the current first-line therapy, Trastuzumab. In our previous work, we demonstrated that HER2 signaling activates mitochondrial creatine kinase 1 (MtCK1), which enhances mitochondrial energy metabolism for rapid proliferation of breast cancer cells. However, pharmacological blockage of MtCK1 with a creatine analog, Cyclo-Creatine (CCr), requires millimolar concentration to inhibit breast cancer cell proliferation and is associated with brain toxicity, limiting its clinical application. This brain toxicity may result from the inhibition of the creatine transporter SLC6A8 by CCr, which decreases to transport creatine into brain. In this study, we took a prodrug development approach to identify and optimize protection groups for CCr, designed to bypass the SLC6A8 transporter, enhancing bioavailability and reducing toxicity. Liquid chromatography-mass spectrometry (LC-MS) was utilized to analyze drug uptake and creatine metabolites in cells treated with the prodrugs. Prodrug C, which is our lead prodrug candidate, demonstrated a seven-fold increase in cellular uptake and more than a hundred-fold improvement in cell-killing effects as compared to CCr in an MtCK1 specific manner in breast cancer cells. This approach offers a promising therapeutic strategy to inhibit HER2+ breast cancer cell by disrupting mitochondrial energy metabolism. Future investigations will focus on validating this prodrug’s pharmacokinetics (PK) and pharmacodynamics (PD) through further preclinical studies. Citation Format: Ya Li, Yetong Huang, Puja Dey, Sadae Hitosugi, Sarah A. Buhrow, Joel M. Reid, Taro Hitosugi. Prodrug development of Cyclo-Creatine: Enhancing permeability and reducing toxicity for HER2+ breast cancer treatment [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 4157.

Introduction The tumor ecosystem in multiple myeloma (MM) involves complex interactions among various cell types, including clonal plasma cells (PCs) and immune cells. Previous research has indicated that the bone marrow (BM) microenvironment's metabolite and lipid profiles differ between MM patients and those with precursor conditions. This study aims to explore if differences in cell composition, metabolic phenotype of clonal PCs, and the spatial organization of various cell types are linked to the early versus late progression of high-risk smoldering MM (SMM) to MM. Methods We identified high-risk SMM patients using the IMWG 20-2-20 criteria, focusing on early progressors (EP), who progressed to MM within one year, and later progressors (LP), who progressed after more than four years. Using a high-dimensional single-cell spatial imaging platform, CODEX (CO-Detection by indEXing), we identified clonal PCs and immune cell types as well as their metabolic phenotypes at the single-cell level, in paired FFPE BM samples at SMM diagnosis and at the time of progression to MM. Our validated panel included markers such as CD3e, CD4, CD8, CD14, CD20, CD31, CD38, CD45, CD66a, CD68, CD138, HLA-DR, ASCT2, pNRF2, GLUT1, LDHA, ATPA5, CPT1A, and Citrate synthase. We employed single-cell segmentation, artifact cleanup, signal thresholding, and spatial analysis. Repeated measure models accommodated multiple regions of interest (ROIs) per patient and accounted for potential intrapatient correlation, while Poisson models estimated incidence rate ratios (RR) of cell types. Spatial statistical models evaluated cell type segregation and integration at the time of SMM diagnosis. Results Single cell events were acquired from 6 unique SMM patients who had paired FFPE BM samples from the time of their SMM diagnosis and at the time of their progression to MM. There were 3 EP SMM patients (Time to Progression (TTP):8, 8, 10 mos) and 3 LP SMM patients (TTP: 51, 58, 117 mos). The median number of single cell events identified per patient slide sample was 104,390 (Range: 24,357 - 314,877). We identified 8 main cell types: PCs, CD4+ T-cells, CD8+ T-cells, B-cells, granulocytes, Monocyte/Macrophages and two groups of other immune cells. The PCs and the remainder of the non-PCs were further classified based on the expression of the following metabolic markers: ASCT2 (Glutamine importer), CPT1A (Fatty acid metabolism), GLUT1 (Glucose importer), LDHA (Glycolysis), Citrate synthase and ATPA5 (Oxidative Phosphorylation). There was a higher RR of granulocytes (RR 2.1, CI 1.6-2.8, p<0.001) in EP compared to LP samples at the time of SMM diagnosis, but a lower RR of CD4 T cells (RR 0.40, CI 0.3-0.6, p< 0.001). Focusing on metabolic phenotypes of the PCs, we observed higher incidence RR of most metabolic markers: ASCT2 (RR=1.77; p=0.032), ATPA5 (RR=1.25; p<0.001), CPT1a (RR=2.42; p=0.015), and LDHA (RR 2.13; p<0.001) in EP compared to LP samples at the time of SMM diagnosis. However, GLUT1 had a lower incidence RR in EP patients (RR=0.79; p<0.001). The same modeling for non-PCs at the time of SMM diagnosis yielded higher expression of LDHA (RR=1.98; p<0.001) and ASCT2 (RR=3.3; p=0.002). Using the x and y coordinates and the corresponding phenotype classifications, we performed spatial statistical modeling based on spatial point processes to generate aggregated measures of how different types of cells of interest interacted. CD4 T cells were more isolated in LPs compared to EPs (p =0.046). Segregation indices for PCs and CD4 T cells indicated that they were more integrated/collocated in EPs. Granulocytes were more isolated in EPs although the difference was not as pronounced (p= 0.07). PCs were more integrated with granulocytes (p=0.06) and B cells (p=0.07) in LP patients. Further analyses of paired samples at the time of progression to MM will be reported at the time of the presentation. Conclusion This study is the first to provide whole-slide, single-cell, spatial proteomic characterization of clonal PCs and immune cells in the FFPE BMs of high-risk SMM patients with varying progression times to MM. Distinct metabolic pathway markers in clonal PCs and their spatial relationships with immune cell subsets are closely associated with the risk of progression to MM. These findings illuminate the complex interactions between the metabolic states of clonal PCs and their spatial orientation relative to immune cells in the pathogenesis of MM.

Abstract Senescent cells drive tissue dysfunction through the senescence-associated secretory phenotype (SASP). We uncovered a central role for mitochondria in the epigenetic regulation of the SASP, where mitochondrial-derived metabolites, specifically citrate and acetyl-CoA, fuel histone acetylation at SASP gene loci, promoting their expression. We identified the mitochondrial citrate carrier (SLC25A1) and ATP-citrate lyase (ACLY) as critical for this process. Inhibiting these pathways selectively suppresses SASP without affecting cell cycle arrest, highlighting their potential as therapeutic targets for age-related inflammation. Notably, SLC25A1 inhibition reduces systemic inflammation and extends healthspan in aged mice, establishing mitochondrial metabolism as pivotal to the epigenetic control of aging.

Abstract B cell activation is accompanied by dynamic metabolic reprogramming, supported by a multitude of nutrients that include glucose, amino acids, and fatty acids. Although several studies have indicated that fatty acid mitochondrial oxidation is critical for immune cell functions, contradictory findings have been reported. Carnitine palmitoyltransferase II (CPT2) is a critical enzyme for long-chain fatty acid oxidation in mitochondria. In this study, we test the requirement of CPT2 for humoral immunity using a mouse model with a lymphocyte-specific deletion of CPT2. Stable [13C] isotope tracing reveals highly reduced fatty acid–derived citrate production in CPT2-deficient B cells. Yet, CPT2 deficiency has no significant impact on B cell development, B cell activation, germinal center formation, and Ab production upon either thymus-dependent or -independent Ag challenges. Together, our findings indicate that CPT2-mediated fatty acid oxidation is dispensable for humoral immunity, highlighting the metabolic flexibility of lymphocytes.

Background: It has been recently demonstrated that altered lipid metabolism involving increased free fatty acids (FFAs) uptake supports the growth and proliferation of MM cells (Panaroni et al. Blood 2022; Farell et al. Elife 2023). Whether the availability of FFAs in the bone marrow (BM) microenvironment is associated with the pathogenesis of MM from MGUS has not been previously evaluated. Thus, we evaluated if individual species of FAs or FA-containing lipids such as triacylglycerols (TAGs), diacylglycerols (DAGs), and monoacylglycerols (MAGs) are differentially present between the BM microenvironment of MGUS and MM patients. Methods: Lipid profiles of BM plasma samples (reflective of the BM microenvironment) from patients with a spectrum of plasma cell disorders (22 MGUS and 24 MM (15 newly diagnosed and 9 relapsed/refractory)) were acquired by liquid chromatography (LC)/tandem mass spectrometry (MS), and individual lipid species were quantified based on single-point quantification using 55 isotopically labeled internal standards. Concentration-based lipid species data was analyzed by the web-based tool MetaboAnalyst 5.0. Data was normalized by quantile normalization, log transformed and scaled by Pareto scaling. p values <0.05 were considered significant. Results: A total of 993 distinct lipid species were quantified by the MS-based targeted lipidomics assessment. Of these lipid species, the levels of individual mid-chain (≤50 carbon chain length) and long-chain (≥52 carbon chain length) polyunsaturated TAGs were the most differentially present in the BM plasma of MM compared to MGUS patients (Figure 1A). Even though a principal component analysis (PCA) involving all lipid species showed moderate separation of MM from MGUS patients, five MGUS patients (categorized as MGUS-other (MGO)) who clustered more towards MM patients compared to the remaining MGUS patients had a distinct pattern of individual mid and long-chain polyunsaturated TAG levels that were comparable to MM patients (Figure 1B). Furthermore, the proliferation rate of clonal plasma cells in the BM of these MM and MGUS as measured by the S-phase% was found to be directly correlated with the level of long-chain polyunsaturated TAGs. Conclusions: Mid and long-chain polyunsaturated TAGs are present in lower and higher levels, respectively, in the BM microenvironment of MM compared to MGUS patients. These findings continue to support the importance of FA-containing TAGs and other lipids in the pathogenesis of MM. Future confirmatory studies evaluating the biological significance of these differences are required to determine if they can be exploited for diagnostic and therapeutic purposes.

Introduction Multiple myeloma (MM) patients often become unresponsive to immunomodulatory drugs (IMiDs) over time. Research has indicated that the cellular ability to counteract oxidative stress plays a crucial role in determining the sensitivity to IMiDs in MM. This study aims to comprehend the metabolic pathways responsible for MM's antioxidative capability and develop new treatments to augment the effectiveness of IMiDs. Methods We experimented using MM cells to investigate how different nutrient conditions affect cell proliferation and drug response. We utilized stable isotope-labeled amino acids and mass spectrometry to analyze the metabolites in these cells. We evaluated cell viability through the MTT assay, the CellTiter-Glo® Luminescent Assay, and direct cell counting. Furthermore, we conducted Western blots to measure the levels of various proteins expressed in MM cells grown under different nutrient conditions. To create CRBN knockout cells, we employed the CRBN CRISPR/Cas9 KO plasmid, and for the overexpression of CRBN in MM cells, we used a lentivirus system. Results Our research findings reveal that the production of cellular cysteine enhances the cell's ability to counteract oxidative damage, leading to resistance to IMiDs even with the presence of the wild-type CRBN protein. However, MM cells not producing enough cysteine rely on consuming extracellular cystine, which increases sensitivity to lenalidomide and reduces antioxidative capacity. Lenalidomide-sensitive cells acquire more cystine from the extracellular environment than resistant cells. The analysis of cysteine uptake in various MM cell lines using C13-labeled cysteine revealed that the lenalidomide-sensitive cell line, OCIMY5-CRBN, consumed more cysteine from the media, and the presence of L-glutamine further increased this uptake. Lenalidomide treatment increased the catabolism of cysteine into taurine in sensitive cell lines, confirming the link between lenalidomide's molecular action and cysteine catabolism. Our study also identified that lenalidomide treatment led to an accumulation of CDO protein, which adds molecular oxygen to cysteine, transforming it into cysteine sulfinic acid. Our study suggests that CRBN may play a role in maintaining intracellular CDO protein levels, and the accumulation of CDO protein induced by lenalidomide is the molecular factor responsible for cystine catabolism and sensitivity to IMiDs in multiple myeloma. We found that by inhibiting intracellular cysteine biosynthesis, we could improve the effectiveness of IMiD against MM. One drug that can achieve this is 5-aza-2-deoxycytidine (DAC), which acts as a DNA methylation inhibitor. Additionally, combining DAC with IMiDs improves their effectiveness, even in cell lines that are resistant to treatment. Furthermore, we attempted to inhibit cysteine biosynthesis by promoting the synthesis of methionine from homocysteine with the supplementation of 5-methyltetrahydrofolate. We discovered that treatment with 5-methyltetrahydrofolate further enhanced the sensitivity of IMiDs in resistant MM cell lines. Our results confirm that interfering with intracellular cysteine biosynthesis is a promising therapeutic approach for MM and can improve the effectiveness of IMiDs. Conclusions The biosynthesis of L-Cysteine is vital for the cellular ability to resist oxidation. Cancer cells that create high levels of cysteine within the cell from methionine generate a more significant amount of reduced cysteine, which can act as antioxidants and make MM cells resistant to IMiDs. On the other hand, cells that cannot produce enough cysteine increase their consumption of extracellular cystine and are more susceptible to IMiDs. Cells that consume an oxidized form of cystine from outside the cell require a reduction process inside the cell, which further reduces their ability to resist oxidation. Additionally, the accumulation of CDO protein induced by lenalidomide can enhance the breakdown of intracellular cysteine and cystine uptake in vulnerable cells. Overall, MM cells that rely on extracellular cysteine are sensitive to IMiDs. Therefore, interfering with intracellular cysteine biosynthesis is a better option for myeloma treatment and enhancing the sensitivity of IMiDs.