Integration Analysis of Single-Cell Multi-Omics Reveals Prostate Cancer Heterogeneity.

Prostate cancer (PCa) is an extensive heterogeneous disease with a complex cellular ecosystem in the tumor microenvironment (TME). However, the manner in which heterogeneity is shaped by tumors and stromal cells, or vice versa, remains poorly understood. In this study, single-cell RNA sequencing, spatial transcriptomics, and bulk ATAC-sequence are integrated from a series of patients with PCa and healthy controls. A stemness subset of club cells marked with SOX9highARlow expression is identified, which is markedly enriched after neoadjuvant androgen-deprivation therapy (ADT). Furthermore, a subset of CD8+CXCR6+ T cells that function as effector T cells is markedly reduced in patients with malignant PCa. For spatial transcriptome analysis, machine learning and computational intelligence are comprehensively utilized to identify the cellular diversity of prostate cancer cells and cell-cell communication in situ. Macrophage and neutrophil state transitions along the trajectory of cancer progression are also examined. Finally, the immunosuppressive microenvironment in advanced PCa is found to be associated with the infiltration of regulatory T cells (Tregs), potentially induced by an FAP+ fibroblast subset. In summary, the cellular heterogeneity is delineated in the stage-specific PCa microenvironment at single-cell resolution, uncovering their reciprocal crosstalk with disease progression, which can be helpful in promoting PCa diagnosis and therapy.

PI3Kγ maintains the self-renewal of acute myeloid leukemia stem cells by regulating the pentose phosphate pathway.

ABSTRACT: Acute myeloid leukemia (AML) is an aggressive hematological malignancy originating from transformed hematopoietic stem or progenitor cells. AML prognosis remains poor owing to resistance and relapse driven by leukemia stem cells (LSCs). Targeting molecules essential for LSC function is a promising therapeutic approach. The phosphatidylinositol 3-kinase (PI3K)/AKT pathway is often dysregulated in AML. We found that although PI3Kγ is highly enriched in LSCs and critical for self-renewal, it was dispensable for normal hematopoietic stem cells. Mechanistically, PI3Kγ-AKT signaling promotes nuclear factor erythroid 2-related factor 2 (NRF2) nuclear accumulation, which induces 6-phosphogluconate dehydrogenase (PGD) and the pentose phosphate pathway, thereby maintaining LSC stemness. Importantly, genetic or pharmacological inhibition of PI3Kγ impaired expansion and stemness of murine and human AML cells in vitro and in vivo. Together, our findings reveal a key role for PI3Kγ in selectively maintaining LSC function by regulating AKT-NRF2-PGD metabolic pathway. Targeting the PI3Kγ pathway may, therefore, eliminate LSCs without damaging normal hematopoiesis, providing a promising therapeutic strategy for AML.

Lysine vitcylation is a novel vitamin C-derived protein modification that enhances STAT1-mediated immune response.

Vitamin C (vitC) is a vital nutrient for health and also used as a therapeutic agent in diseases such as cancer. However, the mechanisms underlying vitC's effects remain elusive. Here we report that vitC directly modifies lysine without enzymes to form vitcyl-lysine, termed "vitcylation", in a dose-, pH-, and sequence-dependent manner across diverse proteins in cells. We further discover that vitC vitcylates K298 site of STAT1, which impairs its interaction with the phosphatase PTPN2, preventing STAT1 Y701 dephosphorylation and leading to increased STAT1-mediated IFN pathway activation in tumor cells. As a result, these cells have increased MHC/HLA class-I expression and activate immune cells in co-cultures. Tumors collected from vitC-treated tumor-bearing mice have enhanced vitcylation, STAT1 phosphorylation and antigen presentation. The identification of vitcylation as a novel PTM and the characterization of its effect in tumor cells opens a new avenue for understanding vitC in cellular processes, disease mechanisms, and therapeutics.

STING agonism reprograms tumor-associated macrophages and overcomes resistance to PARP inhibition in BRCA1-deficient models of breast cancer.

PARP inhibitors (PARPi) have drastically changed the treatment landscape of advanced ovarian tumors with BRCA mutations. However, the impact of this class of inhibitors in patients with advanced BRCA-mutant breast cancer is relatively modest. Using a syngeneic genetically-engineered mouse model of breast tumor driven by Brca1 deficiency, we show that tumor-associated macrophages (TAMs) blunt PARPi efficacy both in vivo and in vitro. Mechanistically, BRCA1-deficient breast tumor cells induce pro-tumor polarization of TAMs, which in turn suppress PARPi-elicited DNA damage in tumor cells, leading to reduced production of dsDNA fragments and synthetic lethality, hence impairing STING-dependent anti-tumor immunity. STING agonists reprogram M2-like pro-tumor macrophages into an M1-like anti-tumor state in a macrophage STING-dependent manner. Systemic administration of a STING agonist breaches multiple layers of tumor cell-mediated suppression of immune cells, and synergizes with PARPi to suppress tumor growth. The therapeutic benefits of this combination require host STING and are mediated by a type I IFN response and CD8+ T cells, but do not rely on tumor cell-intrinsic STING. Our data illustrate the importance of targeting innate immune suppression to facilitate PARPi-mediated engagement of anti-tumor immunity in breast cancer.

Methylene-bridge tryptophan fatty acylation regulates PI3K-AKT signaling and glucose uptake.

Protein fatty acylation regulates numerous cell signaling pathways. Polyunsaturated fatty acids (PUFAs) exert a plethora of physiological effects, including cell signaling regulation, with underlying mechanisms to be fully understood. Herein, we report that docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) regulate PI3K-AKT signaling by modifying PDK1 and AKT2. DHA-administered mice exhibit altered phosphorylation of proteins in signaling pathways. Methylene bridge-containing DHA/EPA acylate δ1 carbon of tryptophan 448/543 in PDK1 and tryptophan 414 in AKT2 via free radical pathway, recruit both the proteins to the cytoplasmic membrane, and activate PI3K signaling and glucose uptake in a tryptophan acylation-dependent but insulin-independent manner in cultured cells and in mice. DHA/EPA deplete cytosolic PDK1 and AKT2 and induce insulin resistance. Akt2 knockout in mice abrogates DHA/EPA-induced PI3K-AKT signaling. Our results identify PUFA's methylene bridge tryptophan acylation, a protein fatty acylation that regulates cell signaling and may underlie multifaceted effects of methylene-bridge-containing PUFAs.

RIP3 targets pyruvate dehydrogenase complex to increase aerobic respiration in TNF-induced necroptosis.

Receptor-interacting protein kinase 3 (RIP3)-regulated production of reactive oxygen species (ROS) positively feeds back on tumour necrosis factor (TNF)-induced necroptosis, a type of programmed necrosis. Glutamine catabolism is known to contribute to RIP3-mediated ROS induction, but the major contributor is unknown. Here, we show that RIP3 activates the pyruvate dehydrogenase complex (PDC, also known as PDH), the rate-limiting enzyme linking glycolysis to aerobic respiration, by directly phosphorylating the PDC E3 subunit (PDC-E3) on T135. Upon activation, PDC enhances aerobic respiration and subsequent mitochondrial ROS production. Unexpectedly, mixed-lineage kinase domain-like (MLKL) is also required for the induction of aerobic respiration, and we further show that it is required for RIP3 translocation to meet mitochondria-localized PDC. Our data uncover a regulation mechanism of PDC activity, show that PDC activation by RIP3 is most likely the major mechanism activated by TNF to increase aerobic respiration and its by-product ROS, and suggest that RIP3-dependent induction of aerobic respiration contributes to pathologies related to oxidative stress.

Enoyl-CoA hydratase-1 regulates mTOR signaling and apoptosis by sensing nutrients.

The oncogenic mechanisms of overnutrition, a confirmed independent cancer risk factor, remain poorly understood. Herein, we report that enoyl-CoA hydratase-1 (ECHS1), the enzyme involved in the oxidation of fatty acids (FAs) and branched-chain amino acids (BCAAs), senses nutrients and promotes mTOR activation and apoptotic resistance. Nutrients-promoted acetylation of lys101 of ECHS1 impedes ECHS1 activity by impairing enoyl-CoA binding, promoting ECHS1 degradation and blocking its mitochondrial translocation through inducing ubiquitination. As a result, nutrients induce the accumulation of BCAAs and FAs that activate mTOR signaling and stimulate apoptosis, respectively. The latter was overcome by selection of BCL-2 overexpressing cells under overnutrition conditions. The oncogenic effects of nutrients were reversed by SIRT3, which deacetylates lys101 acetylation. Severely decreased ECHS1, accumulation of BCAAs and FAs, activation of mTOR and overexpression of BCL-2 were observed in cancer tissues from metabolic organs. Our results identified ECHS1, a nutrients-sensing protein that transforms nutrient signals into oncogenic signals.Overnutrition has been linked to increased risk of cancer. Here, the authors show that exceeding nutrients suppress Enoyl-CoA hydratase-1 (ECHS1) activity by inducing its acetylation resulting in accumulation of fatty acids and branched-chain amino acids and oncogenic mTOR activation.

ULK1/2 Constitute a Bifurcate Node Controlling Glucose Metabolic Fluxes in Addition to Autophagy.

Metabolic reprogramming is fundamental to biological homeostasis, enabling cells to adjust metabolic routes after sensing altered availability of fuels and growth factors. ULK1 and ULK2 represent key integrators that relay metabolic stress signals to the autophagy machinery. Here, we demonstrate that, during deprivation of amino acid and growth factors, ULK1/2 directly phosphorylate key glycolytic enzymes including hexokinase (HK), phosphofructokinase 1 (PFK1), enolase 1 (ENO1), and the gluconeogenic enzyme fructose-1,6-bisphosphatase (FBP1). Phosphorylation of these enzymes leads to enhanced HK activity to sustain glucose uptake but reduced activity of FBP1 to block the gluconeogenic route and reduced activity of PFK1 and ENO1 to moderate drop of glucose-6-phosphate and to repartition more carbon flux to pentose phosphate pathway (PPP), maintaining cellular energy and redox homeostasis at cellular and organismal levels. These results identify ULK1/2 as a bifurcate-signaling node that sustains glucose metabolic fluxes besides initiation of autophagy in response to nutritional deprivation.

NADP(+)-IDH Mutations Promote Hypersuccinylation that Impairs Mitochondria Respiration and Induces Apoptosis Resistance.

Elucidating the tumorigenic mechanism of R-2-hydroxyglutarate (R-2HG) is critical for determining how NADP(+)-IDH mutations cause cancer. Here we report that R-2HG induces cancerous metabolism and apoptosis resistance through promoting hypersuccinylation. By competitive inhibition of the mitochondrial tricarboxylic acid cycle enzyme succinate dehydrogenase (SDH), R-2HG preferentially induced succinyl-CoA accumulation and hypersuccinylation in the mitochondria. IDH1 mutation-bearing glioma samples and cells were hypersuccinylated in the mitochondria. IDH1 mutation or SDH inactivation resulted in hypersuccinylation, causing respiration inhibition and inducing cancerous metabolism and mitochondrial depolarization. These mitochondrial dysfunctions induced BCL-2 accumulation at the mitochondrial membrane, leading to apoptosis resistance of hypersuccinylated cells. Relief of hypersuccinylation by overexpressing the desuccinylase SIRT5 or supplementing glycine rescued mitochondrial dysfunctions, reversed BCL-2 accumulation, and slowed the oncogenic growth of hypersuccinylated IDH1(R132C)-harboring HT1080 cells. Thus, R-2HG-induced hypersuccinylation contributes to the tumorigenicity of NADP(+)-IDH mutations, suggesting the potential of hypersuccinylation inhibition as an intervention for hypersuccinylation-related tumors.