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.

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Graphical AbstractHighlights d EMT induces mitochondrial fusion through upregulation of MFN1 d MFN1 is required for PKCz-mediated NUMB phosphorylation for asymmetric cell division d MFN1-PKCz tethers fused mitochondria for segregation into stem cell progeny d Mitochondrial fusion enhances GSH synthesis to promote stem cell self-renewal In BriefWu et al. reveal that EMT, a key process in cancer progression, activates mitochondrial fusion protein, MFN1, which interacts with cell polarity protein complex to direct asymmetric cell division, allowing stem cell progeny to inherit fused mitochondria with enhanced reactive oxygen species scavenging capacity to sustain the stem cell pool.

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.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a poor response to current treatment regimens. The multifunctional DNA repair-redox signaling protein Ref-1 has a redox signaling function that activates several transcriptional factors (TFs) including NF-κB (RelA), STAT3, AP-1. These have been implicated in signaling in PDAC and associated with cancer progression and therapy resistance. Numerous studies have shown a role for RelA in PDAC inflammatory responses and therapy resistance, little is known as to how these inflammatory responses are modulated through Ref-1 redox signaling pathways during pancreatic pathogenesis. RelA and STAT3 are two major targets of Ref-1 and are important in PDAC pathogenesis. To decipher the mechanistic role of RelA in response to Ref-1 inhibition, we used PDAC cells (KC3590) from a genetically engineered KrasG12D-driven mouse model that also is functionally deficient for RelA (Parent/Vector) or KC3590 cells with fully functional RelA added back (clone 13; C13). We demonstrated that RelA deficient cells are more resistant to Ref-1 redox inhibitors APX3330, APX2009, and APX2014, and their sensitivity is restored in the RelA proficient cells. Knockdown of STAT3 did not change cellular sensitivity to Ref-1 redox inhibitors in either cell type. Gene expression analysis demonstrated that Ref-1 inhibitors significantly decreased IL-8, FOSB, and c-Jun when functional RelA is present. We also demonstrated that PRDX1, a known Ref-1 redox modulator, contributes to Ref-1 inhibitor cellular response. Knockdown of PRDX1 when functional RelA is present resulted in dramatically increased PDAC killing in response to Ref-1 inhibitors. The enhanced cell killing was not due to increased intracellular ROS production. Although Ref-1 inhibition decreased the NADP/NADPH ratio in the cells, the addition of PRDX1 knockdown did not further this redox imbalance. This data suggests that the mechanism of cell killing following Ref-1 inhibition is at least partially mediated through RelA and not STAT3. Further imbalancing of the redox signaling through disruption of the PRDX1-Ref-1 interaction may have therapeutic implications. Our data further support a pivotal role of RelA in mediating Ref-1 redox signaling in PDAC cells with the KrasG12D genotype and provide novel therapeutic strategies to combat PDAC drug resistance.

Background MPNST is a rare soft-tissue sarcoma that can arise from patients with NF1. Existing chemotherapeutic and targeted agents have been unsuccessful in MPNST treatment, and recent findings implicate STAT3 and HIF1-α in driving MPNST. The DNA-binding and transcriptional activity of both STAT3 and HIF1-α is regulated by Redox factor-1 (Ref-1) redox function. A first-generation Ref-1 inhibitor, APX3330, is being tested in cancer clinical trials and could be applied to MPNST. Methods We characterised Ref-1 and p-STAT3 expression in various MPNST models. Tumour growth, as well as biomarkers of apoptosis and signalling pathways, were measured by qPCR and western blot following treatment with inhibitors of Ref-1 or STAT3. Results MPNSTs from Nf1-Arfflox/floxPostnCre mice exhibit significantly increased positivity of p-STAT3 and Ref-1 expression when malignant transformation occurs. Inhibition of Ref-1 or STAT3 impairs MPNST growth in vitro and in vivo and induces apoptosis. Genes highly expressed in MPNST patients are downregulated following inhibition of Ref-1 or STAT3. Several biomarkers downstream of Ref-1 or STAT3 were also downregulated following Ref-1 or STAT3 inhibition. Conclusions Our findings implicate a unique therapeutic approach to target important MPNST signalling nodes in sarcomas using new first-in-class small molecules for potential translation to the clinic.

Targeting the hedgehog (HH) pathway to treat aggressive cancers of the brain, breast, pancreas, and prostate has been ongoing for decades. Gli gene amplifications have been long discovered within malignant glioma patients, and since then, inhibitors against HH pathway-associated molecules have successfully reached the clinical stage where several of them have been approved by the FDA. Albeit this success rate implies suitable progress, clinically used HH pathway inhibitors fail to treat patients with metastatic or recurrent disease. This is mainly due to heterogeneous tumor cells that have acquired resistance to the inhibitors along with the obstacle of effectively targeting the tumor microenvironment (TME). Severe side effects such as hyponatremia, diarrhea, fatigue, amenorrhea, nausea, hair loss, abnormal taste, and weight loss have also been reported. Furthermore, HH signaling is known to be involved in the regulation of immune cell maturation, angiogenesis, inflammation, and polarization of macrophages and myeloid-derived suppressor cells. It is critical to determine key mechanisms that can be targeted at different levels of tumor development and progression to address various clinical issues. Hence current research focus encompasses understanding how HH controls TME to develop TME altering and combinatorial targeting strategies. In this review, we aim to discuss the pros and cons of targeting HH signaling molecules, understand the mechanism involved in treatment resistance, reveal the role of the HH pathway in anti-tumor immune response, and explore the development of potential combination treatment of immune checkpoint inhibitors with HH pathway inhibitors to target HH-driven cancers.

Vismodegib, a Smoothened antagonist, is clinically approved for treatment of human basal cell carcinoma (BCC), in the clinical trials of medulloblastoma (MB) and other cancers. However, a significant proportion of these tumors fail to respond to Vismodegib after a period of treatment. Here, we find that AMPK agonists, A769662, and Metformin, can inhibit GLI1 activity and synergize with Vismodegib to suppress MB cell growth in vitro and in vivo. Furthermore, combination of AMPK agonists with Vismodegib is effective in overcoming Vismodegib‐resistant MB. This is the first report demonstrating that combining AMPK agonist (Metformin) and SHH pathway inhibitor (Vismodegib) confers synergy for MB treatment and provides an effective chemotherapeutic regimen that can be used to overcome resistance to Vismodegib in SHH‐driven cancers.