Biology subjects

Awad, D.

Publications and source records attributed to Awad, D..

9 recordsLinked to original sources

bioRxiv2026

Synthetic Immunological Niche Reveals Early Immune Dysregulationand Stratifies Therapeutic Response in Type 1 Diabetes

In Type 1 Diabetes (T1D), disease onset and response to immunotherapy vary widely among individuals, reflecting heterogeneous stage-specific immune dysregulation that remains undefined. To investigate this heterogeneity, we engineered a microporous polycaprolactone scaffold that forms a synthetic immunological niche (IN) upon subcutaneous implantation, enabling in vivo capture of systemic immune dysregulation. In non-obese diabetic (NOD) mice, longitudinal transcriptomic profiling of IN-infiltrating cells identified early-stage genes relatively enriched for myeloid cells, followed by progressive increases in T cell-associated dysregulation at later stages that distinguished T1D progressors from non-progressors. We derived an early-stage IN-based T1D gene signature capturing immune alterations. The signature stratified NOD progressors from non-progressors as early as 6 weeks of age and was conserved across human T1D datasets, distinguishing T1D from non-diabetic individuals in spleen and pancreatic lymph node samples, but not peripheral blood. Signature-based stratification further revealed enrichment of macrophage-associated TNF- pathways in NOD progressors, validated in human T1D islets. Given heterogeneous response to anti-TNF- therapy, IN profiling identified resistance-associated mechanisms and enabled derivation of a pathway score that prospectively distinguished treatment-sensitive from resistant mice prior to therapy, establishing the IN as a minimally invasive platform for detecting stage-wise immune dysregulation and stratifying immunotherapy response in T1D.

bioengineering↗

bioRxiv2025

Aspartate transaminases are required for blood development

Red blood cells (RBCs) have a limited lifespan of approximately 120 days. This necessitates continuous RBC production, resulting in [~]200 billion new RBCs made per day to maintain oxygen delivery. Despite this enormous biosynthetic demand, the metabolic pathways supporting erythropoiesis are poorly understood. We profiled metabolites across four independent models of elevated erythropoiesis and a consistent increase in aspartate levels emerged when compared to controls. This suggested a potential role for aspartate metabolism in RBC production. To test this, we deleted the aspartate aminotransferases Got1 or Got2 globally or in an erythroid-specific manner. Loss of either enzyme resulted in anemia, with Got2 deficiency producing a more severe phenotype. Individual loss of either Got or dual Got deletion led to an erythroid defect, where early progenitors accumulated. In human and mouse models of erythropoiesis, GOT1 and GOT2 loss had opposing impacts on aspartate despite exhibiting similar anemic phenotypes, suggesting an aspartate-independent function for these enzymes. GOT1 and GOT2 are also components of the malate-aspartate shuttle (MAS), which regulates NAD(H) homeostasis. However, conditional deletion of another MAS enzyme, Mdh1, did not cause anemia, and alleviating NADH reductive stress in GOT2-deficient cells with cytoplasmic bacterial NADH oxidase (LbNOX) failed to restore erythropoiesis. Instead, transcriptomic and epigenetic analyses revealed dysregulation of chromatin histone modifications in GOT-deficient erythroid cells, implicating epigenetic dysfunction as a driver of defective erythropoiesis. Collectively, these findings identify a previously unrecognized role for GOT1 and GOT2 as a central link between metabolism and epigenetic regulation during erythroid development. These insights may inform the development of new therapeutic strategies for anemia.

biochemistry↗

bioRxiv2024

Isolation and characterization of microbiota from human pancreatic tumors and small intestine

Pancreatic ductal adenocarcinoma has a unique tumor microbiome and the systemic depletion of bacteria or fungi using antibiotic/antifungal cocktails leads to a decrease in pancreatic tumor burden in mice. However, functional studies remain rare due to the limited availability of clinically relevant microbiota. Here, we describe in detail the isolation of bacteria and fungi from the small intestine and tumor of pancreatic cancer patients at the Rogel Cancer Center. We then further characterized the impact of a newly isolated Klebsiella oxytoca strain (UMKO1) on the pancreatic tumor microenvironment using bacterial genome sequencing, untargeted and targeted metabolomics, as well as an ex vivo tumor transplant system. We found that UMKO1 possesses a gene for the long form of cytidine deaminase, which can inactivate the standard PDAC chemotherapeutic agent gemcitabine. In addition, we found that UMKO1 can produce several indoles when grown in tumor-like conditions, metabolites that can lead to an immune suppressive environment and interfere with therapy outcome. To test this in detail, we assessed changes in immune populations in pancreatic tumor explants upon exposure to the supernatant of UMKO1 and other isolated bacteria grown in tumor Interstitial fluid media (TIFM). We found that while none of the bacterial supernatants changed the abundance of CD8 T cells, granzyme B positive CD8 T cells were the lowest in tumor explants exposed to UMKO1, and not other isolated Klebsiella species or the non-pathogenic laboratory strain E. coli K12. In summary, the isolated collection of bacteria and fungi from this study are a valuable toolbox to study the impact of microbiota on pancreatic cancer.

cancer biology↗

bioRxiv2024

A metabolic atlas of mouse aging

Humans are living longer, but this is accompanied by an increased incidence of age-related chronic diseases. Many of these diseases are influenced by age-associated metabolic dysregulation, but how metabolism changes in multiple organs during aging in males and females is not known. Answering this could reveal new mechanisms of aging and age-targeted therapeutics. In this study, we describe how metabolism changes in 12 organs in male and female mice at 5 different ages. Organs show distinct patterns of metabolic aging that are affected by sex differently. Hydroxyproline shows the most consistent change across the dataset, decreasing with age in 11 out of 12 organs investigated. We also developed a metabolic aging clock that predicts biological age and identified alpha-ketoglutarate, previously shown to extend lifespan in mice, as a key predictor of age. Our results reveal fundamental insights into the aging process and identify new therapeutic targets to maintain organ health.

molecular biology↗

bioRxiv2024

Targeting PIKfyve-driven lipid homeostasis as a metabolic vulnerability in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) subsists in a nutrient-deregulated microenvironment, making it particularly susceptible to treatments that interfere with cancer metabolism1 2. For example, PDAC utilizes and is dependent on high levels of autophagy and other lysosomal processes3-5. Although targeting these pathways has shown potential in preclinical studies, progress has been hampered by the challenge of identifying and characterizing favorable targets for drug development6. Here, we characterize PIKfyve, a lipid kinase integral to lysosomal functioning7, as a novel and targetable vulnerability in PDAC. In human patient and murine PDAC samples, we discovered that PIKFYVE is overexpressed in PDAC cells compared to adjacent normal cells. Employing a genetically engineered mouse model, we established the essential role of PIKfyve in PDAC progression. Further, through comprehensive metabolic analyses, we found that PIKfyve inhibition obligated PDAC to upregulate de novo lipid synthesis, a relationship previously undescribed. PIKfyve inhibition triggered a distinct lipogenic gene expression and metabolic program, creating a dependency on de novo lipid metabolism pathways, by upregulating genes such as FASN and ACACA. In PDAC, the KRAS-MAPK signaling pathway is a primary driver of de novo lipid synthesis, specifically enhancing FASN and ACACA levels. Accordingly, the simultaneous targeting of PIKfyve and KRAS-MAPK resulted in the elimination of tumor burden in a syngeneic orthotopic model and tumor regression in a xenograft model of PDAC. Taken together, these studies suggest that disrupting lipid metabolism through PIKfyve inhibition induces synthetic lethality in conjunction with KRAS-MAPK-directed therapies for PDAC.

cancer biology↗

bioRxiv2024

Type I interferon governs immunometabolic checkpoints that coordinate inflammation during Staphylococcal infection

Fine-tuned inflammation during infection enables pathogen clearance while minimizing host damage. Inflammation regulation depends on macrophage metabolic plasticity, yet coordination of metabolic and inflammatory programs is underappreciated. Here we show that type I interferon (IFN) temporally guides metabolic control of inflammation during methicillin-resistant Staphylococcus aureus (MRSA) infection. In macrophages, staggered Toll-like receptor and type I IFN signaling permitted a transient respiratory burst followed by inducible nitric oxide synthase (iNOS)-mediated OXPHOS disruption. OXPHOS disruption promoted type I IFN, suppressing other pro-inflammatory cytokines, notably IL-{beta}. iNOS expression peaked at 24h post-infection, followed by lactate-driven iNOS repression via histone lactylation. In contrast, type I IFN pre-conditioning sustained infection-induced iNOS expression and amplified type I IFN. Cutaneous MRSA infection in mice constitutively expressing epidermal type I IFN led to elevated iNOS levels, impaired wound healing, vasculopathy, and lung infection. Thus, kinetically regulated type I IFN signaling coordinates immunometabolic checkpoints that control infection-induced inflammation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/575104v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@977b1eorg.highwire.dtl.DTLVardef@1142c6corg.highwire.dtl.DTLVardef@170776borg.highwire.dtl.DTLVardef@b02c2f_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2022

Adipose triglyceride lipase is regulated by CAMKK2-AMPK signaling and drives advanced prostate cancer

Lipid metabolism plays a central role in prostate cancer. To date, the major focus on prostate cancer lipid metabolism has centered on de novo lipogenesis and lipid uptake with little consideration for how cancer cells access these lipids once they are created or taken up and stored. Patient-derived phosphoproteomics identified adipose triglyceride lipase (ATGL), a previously suspected tumor suppressor, as a CAMKK2-AMPK signaling target that, conversely, promotes castration-resistant prostate cancer (CRPC) progression. Phosphorylation of ATGL increased its lipase activity, cancer cell proliferation, migration, and invasion. Shotgun lipidomics and mass spectrometry imaging demonstrated ATGLs profound regulation of lipid metabolism in vitro and in vivo, remodeling membrane composition. Inhibition of ATGL induced metabolic plasticity, causing a glycolytic shift that could be exploited therapeutically by co-targeting both metabolic pathways. Together, these data nominate ATGL and intracellular lipolysis as potential therapeutic targets for the treatment of CRPC and provide insights for future combination therapies.

cancer biology↗

bioRxiv2022

SGC-CAMKK2-1: A chemical probe for CAMKK2

The serine/threonine protein kinase calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) plays critical roles in a range of biological processes. Despite its importance, only a handful of inhibitors of CAMKK2 have been disclosed. Having a selective small molecule tool to interrogate this kinase will help demonstrate that CAMKK2 inhibition can be therapeutically beneficial. Herein, we disclose SGC-CAMKK2-1, a selective chemical probe that targets CAMKK2.

cancer biology↗

bioRxiv2022

Systemic ablation of Camkk2 impairs metastatic colonization and improves insulin sensitivity in TRAMP mice: Evidence for cancer cell-extrinsic CAMKK2 functions in prostate cancer

Despite early studies linking calcium-calmodulin protein kinase kinase 2 (CAMKK2) to prostate cancer cell migration and invasion, the role of CAMKK2 in metastasis in vivo remains unclear. Moreover, while CAMKK2 is known to regulate systemic metabolism, whether CAMKK2s effects on whole body metabolism would impact prostate cancer progression and/or related comorbidities is not known. Here, we demonstrate that germline ablation of Camkk2 slows, but does not stop, primary prostate tumorigenesis in the TRansgenic Adenocarcinoma Mouse Prostate (TRAMP) genetic mouse model. Consistent with prior epidemiological reports supporting a link between obesity and prostate cancer aggressiveness, TRAMP mice fed a high-fat diet exhibited a pronounced increase in the colonization of lung metastases. We demonstrated that this effect on metastatic spread was dependent on CAMKK2. Notably, diet-induced lung metastases exhibited a highly aggressive neuroendocrine phenotype. Concurrently, Camkk2 deletion improved insulin sensitivity in the same mice. Histological analyses revealed that cancer cells were smaller in the TRAMP;Camkk2-/- mice compared to TRAMP;Camkk2+/+ controls. Given the differences in circulating insulin levels, a known regulator of cell growth, we hypothesized that systemic CAMKK2 could promote prostate cancer cell growth and disease progression in part through cancer cell-extrinsic mechanisms. Accordingly, host deletion of Camkk2 impaired the growth of syngeneic murine prostate tumors in vivo, confirming nonautonomous roles for CAMKK2 in prostate cancer. Cancer cell size and mTOR signaling was diminished in tumors propagated in Camkk2-null mice. Together, these data indicate that, in addition to cancer cell-intrinsic roles, CAMKK2 promotes prostate cancer progression via tumor-extrinsic mechanisms. Further, we propose that CAMKK2 inhibition may also help combat common metabolic comorbidities in men with advanced prostate cancer.

cancer biology↗