The metabolic heterogeneity, and metabolic interplay between cells have been known as significant contributors to disease treatment resistance. However, with the lack of a mature high-throughput single cell metabolomics technology, we are yet to establish systematic understanding of the intra-tissue metabolic heterogeneity and cooperative mechanisms. To mitigate this knowledge gap, we developed a novel computational method, namely scFEA (single cell Flux Estimation Analysis), to infer cell-wise fluxome from single cell RNA-sequencing (scRNA-seq) data. scFEA is empowered by a systematically reconstructed human metabolic map as a factor graph, a novel probabilistic model to leverage the flux balance constraints on scRNA-seq data, and a novel graph neural network based optimization solver. The intricate information cascade from transcriptome to metabolome was captured using multi-layer neural networks to capitulate the non-linear dependency between enzymatic gene expressions and reaction rates. We experimentally validated scFEA by generating an scRNA-seq dataset with matched metabolomics data on cells of perturbed oxygen and genetic conditions. Application of scFEA on this dataset demonstrated the consistency between predicted flux and the observed variation of metabolite abundance in the matched metabolomics data. We also applied scFEA on five publicly available scRNA-seq and spatial transcriptomics datasets and identified context and cell group specific metabolic variations. The cell-wise fluxome predicted by scFEA empowers a series of downstream analysis including identification of metabolic modules or cell groups that share common metabolic variations, sensitivity evaluation of enzymes with regards to their impact on the whole metabolic flux, and inference of cell-tissue and cell-cell metabolic communications.

Most patients with pancreatic adenocarcinoma (PDAC) suffer cachexia; some do not. To model heterogeneity, we used patient-derived orthotopic xenografts. These phenocopied donor weight loss. Furthermore, muscle wasting correlated with mortality and murine IL-6, and human IL-6 associated with the greatest murine cachexia. In cell culture and mice, PDAC cells elicited adipocyte IL-6 expression and IL-6 plus IL-6 receptor (IL6R) in myocytes and blood. PDAC induced adipocyte lipolysis and muscle steatosis, dysmetabolism, and wasting. Depletion of IL-6 from malignant cells halved adipose wasting and abolished myosteatosis, dysmetabolism, and atrophy. In culture, adipocyte lipolysis required soluble (s)IL6R, while IL-6, sIL6R, or palmitate induced myotube atrophy. PDAC cells activated adipocytes to induce myotube wasting and activated myotubes to induce adipocyte lipolysis. Thus, PDAC cachexia results from tissue crosstalk via a feed-forward, IL-6 trans-signaling loop. Malignant cells signal via IL-6 to muscle and fat, muscle to fat via sIL6R, and fat to muscle via lipids and IL-6, all targetable mechanisms for treatment of cachexia.

Background Pancreatic cancer is a complex disease with a desmoplastic stroma, extreme hypoxia, and inherent resistance to therapy. Understanding the signaling and adaptive response of such an aggressive cancer is key to making advances in therapeutic efficacy. Redox factor-1 (Ref-1), a redox signaling protein, regulates the conversion of several transcription factors (TFs), including HIF-1α, STAT3 and NFκB from an oxidized to reduced state leading to enhancement of their DNA binding. In our previously published work, knockdown of Ref-1 under normoxia resulted in altered gene expression patterns on pathways including EIF2, protein kinase A, and mTOR. In this study, single cell RNA sequencing (scRNA-seq) and proteomics were used to explore the effects of Ref-1 on metabolic pathways under hypoxia. Methods scRNA-seq comparing pancreatic cancer cells expressing less than 20% of the Ref-1 protein was analyzed using left truncated mixture Gaussian model and validated using proteomics and qRT-PCR. The identified Ref-1’s role in mitochondrial function was confirmed using mitochondrial function assays, qRT-PCR, western blotting and NADP assay. Further, the effect of Ref-1 redox function inhibition against pancreatic cancer metabolism was assayed using 3D co-culture in vitro and xenograft studies in vivo. Results Distinct transcriptional variation in central metabolism, cell cycle, apoptosis, immune response, and genes downstream of a series of signaling pathways and transcriptional regulatory factors were identified in Ref-1 knockdown vs Scrambled control from the scRNA-seq data. Mitochondrial DEG subsets downregulated with Ref-1 knockdown were significantly reduced following Ref-1 redox inhibition and more dramatically in combination with Devimistat in vitro. Mitochondrial function assays demonstrated that Ref-1 knockdown and Ref-1 redox signaling inhibition decreased utilization of TCA cycle substrates and slowed the growth of pancreatic cancer co-culture spheroids. In Ref-1 knockdown cells, a higher flux rate of NADP + consuming reactions was observed suggesting the less availability of NADP + and a higher level of oxidative stress in these cells. In vivo xenograft studies demonstrated that tumor reduction was potent with Ref-1 redox inhibitor similar to Devimistat. Conclusion Ref-1 redox signaling inhibition conclusively alters cancer cell metabolism by causing TCA cycle dysfunction while also reducing the pancreatic tumor growth in vitro as well as in vivo.

A key challenge in modeling single-cell RNA-seq data is to capture the diversity of gene expression states regulated by different transcriptional regulatory inputs across individual cells, which is further complicated by largely observed zero and low expressions. We developed a left truncated mixture Gaussian (LTMG) model, from the kinetic relationships of the transcriptional regulatory inputs, mRNA metabolism and abundance in single cells. LTMG infers the expression multi-modalities across single cells, meanwhile, the dropouts and low expressions are treated as left truncated. We demonstrated that LTMG has significantly better goodness of fitting on an extensive number of scRNA-seq data, comparing to three other state-of-the-art models. Our biological assumption of the low non-zero expressions, rationality of the multimodality setting, and the capability of LTMG in extracting expression states specific to cell types or functions, are validated on independent experimental data sets. A differential gene expression test and a co-regulation module identification method are further developed. We experimentally validated that our differential expression test has higher sensitivity and specificity, compared with other five popular methods. The co-regulation analysis is capable of retrieving gene co-regulation modules corresponding to perturbed transcriptional regulations. A user-friendly R package with all the analysis power is available at https://github.com/zy26/LTMGSCA.

We designed a novel haploidentical hematopoietic stem cell transplantation (haplo-HSCT) system using idarubicin (IDA) intensified conditioning regimens and combination of antithymocyte globulin and basiliximab for GvHD prophylaxis. The outcomes of 110 high-risk acute leukemia patients undergoing haplo-HSCT were compared with 69 contemporaneous high-risk patients receiving HLA-matched sibling transplantation using uniform IDA-intensified regimens. The relapse incidence of haplo-HSCT was 23.4%, and 3-year overall survival (OS) and disease-free survival (DFS) achieved 62.9%, 59.1%, respectively. The cumulative incidences of II-IV and III-IV aGvHD were 28.6 and 14.3%, while limited and extensive cGvHD were 19.4, 13.8%. All these results were equivalent to those of concurrent identical sibling transplantation. Three-year OS and DFS for patients in advance stage reached 48.5, 47.3%. Furthermore, the relapse, 3-year OS of positive minimal residual disease (MRD) patients did not differ from negative MRD patients (18.9% vs 11.5%, 63.6% vs 69.6%), indicating our intensified haplo-HSCT technique could circumvent the dismal prognosis of MRD. These data provide reinforcing evidence that our haplo-HSCT system could dramatically improve the survival of high-risk acute leukemia with low relapse and acceptable transplantation-related mortality, and might be a promising therapeutic option for high-risk patients.

Sestrin 3 (Sesn3) belongs to the three‐member sestrin protein family. Sestrins have been implicated in antioxidative stress, adenosine monophosphate–activated protein kinase and mammalian target of rapamycin signal transduction, and metabolic homeostasis. However, the role of Sesn3 in the development of nonalcoholic steatohepatitis (NASH) has not been previously studied. In this work, we generated Sesn3 whole‐body knockout and liver‐specific transgenic mice to investigate the hepatic function of Sesn3 in diet‐induced NASH. With only 4 weeks of dietary treatment, Sesn3 knockout mice developed severe NASH phenotype as characterized by hepatic steatosis, inflammation, and fibrosis. Strikingly, after 8‐week feeding with a NASH‐inducing diet, Sesn3 transgenic mice were largely protected against NASH development. Transcriptomic analysis revealed that multiple extracellular matrix–related processes were up‐regulated, including transforming growth factor β (TGF‐β) signaling and collagen production. Further biochemical and cell biological analyses have illustrated a critical control of the TGF‐β–mothers against decapentaplegic homolog (Smad) pathway by Sesn3 at the TGF‐β receptor and Smad3 levels. First, Sesn3 inhibits the TGF‐β receptor through an interaction with Smad7; second, Sesn3 directly inhibits the Smad3 function through protein–protein interaction and cytosolic retention. Conclusion: Sesn3 is a critical regulator of the extracellular matrix and hepatic fibrosis by suppression of TGF‐β–Smad3 signaling.

Activation of the epithelial-to-mesenchymal transition (EMT) program is a critical mechanism for initiating cancer progression and migration. Colorectal cancers contain many genetic and epigenetic alterations that can contribute to EMT. Mutations activating the PI3K/AKT signaling pathway are observed in >40% of patients with colorectal cancer contributing to increased invasion and metastasis. Little is known about how oncogenic signaling pathways such as PI3K/AKT synergize with chromatin modifiers to activate the EMT program. Lysine-specific demethylase 1 (LSD1) is a chromatin-modifying enzyme that is overexpressed in colorectal cancer and enhances cell migration. In this study, we determine that LSD1 expression is significantly elevated in patients with colorectal cancer with mutation of the catalytic subunit of PI3K, PIK3CA, compared with patients with colorectal cancer with WT PIK3CA. LSD1 enhances activation of the AKT kinase in colorectal cancer cells through a noncatalytic mechanism, acting as a scaffolding protein for the transcription-repressing CoREST complex. In addition, growth of PIK3CA-mutant colorectal cancer cells is uniquely dependent on LSD1. Knockdown or CRISPR knockout of LSD1 blocks AKT-mediated stabilization of the EMT-promoting transcription factor Snail and effectively blocks AKT-mediated EMT and migration. Overall, we uniquely demonstrate that LSD1 mediates AKT activation in response to growth factors and oxidative stress, and LSD1-regulated AKT activity promotes EMT-like characteristics in a subset of PIK3CA-mutant cells.Implications: Our data support the hypothesis that inhibitors targeting the CoREST complex may be clinically effective in patients with colorectal cancer harboring PIK3CA mutations.

Ribonucleotides (rNMPs) mis-incorporated during DNA replication are removed by RNase H2 dependent excision repair or by Topoisomerase I – catalyzed cleavage. Top1 cleavage of rNMPs produces 3’ ends harboring terminal adducts, such as 2’, 3’ cyclic phosphate or Top1 cleavage complex (Top1cc), and leads to frequent mutagenesis and DNA damage checkpoint induction. We surveyed a range of candidate enzymes from Saccharomyces cerevisiae for potential roles in Top1 dependent genomic rNMP removal. Genetic and biochemical analyses reveal that Apn2 resolves phosphotyrosine-DNA conjugates, terminal 2’, 3’ cyclic phosphates and their hydrolyzed products. APN2 also suppresses 2-bp slippage mutagenesis in RNH201 -deficient cells. Our results define additional activities of Apn2 in resolving a wide range of 3’- end blocks and identify a role of Apn2 in maintaining genome integrity during rNMP repair.