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Matrix proteolysis mediated by MT1-MMP facilitates the invasive migration of tumor cells in dense tissues, which otherwise get trapped in the matrix because of limited nuclear deformability. A digest-on-demand response has been identified, which requires nucleus-microtubule linkage through the LINC complex and triggers MT1-MMP surface-exposure to facilitate nucleus movement.

Cancer cell dissemination is facilitated by actin-rich plasma membrane protrusions called invadopodia, which focally degrade matrix tissues. Zagryazhskaya-Masson et al. show that invadopodia formation and function depend on the interaction between the scaffolding protein, TKS5, and the CDC42 guanine exchange factor, FGD1., Tumor cells exposed to a physiological matrix of type I collagen fibers form elongated collagenolytic invadopodia, which differ from dotty-like invadopodia forming on the gelatin substratum model. The related scaffold proteins, TKS5 and TKS4, are key components of the mechanism of invadopodia assembly. The molecular events through which TKS proteins direct collagenolytic invadopodia formation are poorly defined. Using coimmunoprecipitation experiments, identification of bound proteins by mass spectrometry, and in vitro pull-down experiments, we found an interaction between TKS5 and FGD1, a guanine nucleotide exchange factor for the Rho-GTPase CDC42, which is known for its role in the assembly of invadopodial actin core structure. A novel cell polarity network is uncovered comprising TKS5, FGD1, and CDC42, directing invadopodia formation and the polarization of MT1-MMP recycling compartments, required for invadopodia activity and invasion in a 3D collagen matrix. Additionally, our data unveil distinct signaling pathways involved in collagenolytic invadopodia formation downstream of TKS4 or TKS5 in breast cancer cells.

Unraveling the mechanisms that govern the formation and function of invadopodia is essential towards the prevention of cancer spread. Here, we characterize the ultrastructural organization, dynamics and mechanical properties of collagenotytic invadopodia forming at the interface between breast cancer cells and a physiologic fibrillary type I collagen matrix. Our study highlights an uncovered role for MT1-MMP in directing invadopodia assembly independent of its proteolytic activity. Electron microscopy analysis reveals a polymerized Arp2/3 actin network at the concave side of the curved invadopodia in association with the collagen fibers. Actin polymerization is shown to produce pushing forces that repel the confining matrix fibers, and requires MT1-MMP matrix-degradative activity to widen the matrix pores and generate the invasive pathway. A theoretical model is proposed whereby pushing forces result from actin assembly and frictional forces in the actin meshwork due to the curved geometry of the matrix fibers that counterbalance resisting forces by the collagen fibers., The mechanism of force production by invadopodia is unclear. Here, the authors show that cell surface MT1-MMP when in contact with collagen, induces Arp2/3 branched actin polymerisation on the concave side of invadopodia, which generates a pushing force along with collagen cleavage by MT1-MMP to invade.

Summary Cellular stress responses serve as crucial decision points balancing persistence or culling of hematopoietic stem cells (HSCs) for lifelong blood production. Although strong stressors cull HSCs, the linkage between stress programs and self-renewal properties that underlie human HSC maintenance remains unknown, particularly at quiescence exit when HSCs must also dynamically shift metabolic state. Here, we demonstrate distinct wiring of the sphingolipidome across the human hematopoietic hierarchy and find that genetic or pharmacologic modulation of the sphingolipid enzyme DEGS1 regulates lineage differentiation. Inhibition of DEGS1 in hematopoietic stem and progenitor cells during the transition from quiescence to cellular activation with N-(4-hydroxyphenyl) retinamide activates coordinated stress pathways that coalesce on endoplasmic reticulum stress and autophagy programs to maintain immunophenotypic and functional HSCs. Thus, our work identifies a linkage between sphingolipid metabolism, proteostatic quality control systems, and HSC self-renewal and provides therapeutic targets for improving HSC-based cellular therapeutics., Graphical Abstract, Highlights • Sphingolipid composition is diverse across the human hematopoietic hierarchy • DEGS1 is a sphingolipid enzyme required for hematopoietic stem cell (HSC) function • Modulating DEGS1 function activates autophagy and the unfolded protein response • Variations in sphingolipid homeostasis serve to regulate HSC fate, Lipid metabolism is distinctly regulated in human hematopoietic stem cells (HSCs) versus progenitors. Xie et al. profiled the sphingolipidome in human cord blood and show that modulating sphingolipids during the transition from quiescence to cellular activation in ex vivo culture induced proteostatic cellular stress programs to maintain HSC self-renewal.

The hematopoietic stem cells (HSC) field has long been perplexed by how the blood system d (~10e12 cells produced daily) - yet hematologic malignancies remain relatively rare. The risk of malignancy is mitigated in part by a complex hierarchy in which the quiescent long-term hematopoietic stem cells (LT-HSC) with high self-renewal capacity undergo a restricted number of cell divisions. Nonetheless, such a high production demand over a lifetime raises an inherent risk of malignancy due to DNA replication errors, misfolded proteins and metabolic stress that cause cellular damage in HSC. Previously, HSC dormancy, largely thought to be controlled by transcription factor networks, was held responsible for preventing mutation acquisition. However, recent studies suggest that LT-HSC contain critical cellular networks centered around the coordination of distinct HSC metabolic programs with proteostasis, which serve as crucial decision nodes to balance persistence or culling of HSC for lifelong blood production. While HSC culling mechanisms are known, the linkage between cellular stress programs and the self-renewal properties that underlie human HSC persistence remains unknown. Here, we ask how this HSC fate choice is influenced by lipid biosynthesis - an underexplored area of HSC metabolism. We observed a distinct sphingolipid transcriptional signature in human HSC and examined the consequences of sphingolipid perturbation in human cord blood (CB) stem cells during ex vivo activation. DEGS1 (Delta 4-Desaturase, Sphingolipid 1) is the final enzyme in de novo sphingolipid synthesis, converting dihydroceramide (dhCer) to ceramide (Cer); ablation of DEGS1 either genetically or by treatment with the synthetic retinoid fenretinide/N-(4-hydroxyphenyl) retinamide (4HPR) is sufficient to activate autophagy in mouse cells and human cell lines. DEGS1 gene expression was higher in HSC than in progenitors and was significantly increased in LT-HSC following 6 hours of cytokine stimulation, suggesting that it plays a role in cellular activation. Sphingolipid composition was altered in CB cultured with 4HPR for 8 days with an increase in dhCer levels and decrease in Cer levels shown by lipidomics. Remarkably, 4HPR treatment significantly increased in vitro colony forming efficiency from LT-HSC (50% over control), but not from short-term HSC or granulocyte-macrophage progenitors. Ex vivo 4HPR treatment of CB followed by serial xenotransplantation resulted in a 2.5-fold increase in long-term repopulation cell (LTRC) frequency over control-treated cells, suggesting that 4HPR treatment affects HSC self-renewal. RNA-seq analysis showed that 4HPR activates a set of proteostatic quality control (QC) programs that coalesce around the unfolded protein response (UPR) and autophagy, the latter confirmed by immunofluorescence and flow cytometry in CB stem cells. Ex vivo culture perturbs these programs and results in loss of chromatin accessibility at sites associated with uncultured LT-HSC as determined by ATAC-Seq. Addition of 4HPR to the culture activates these proteostatic programs to sustain immunophenotypic and functional HSC. These results suggest that ceramide, the central component to all sphingolipids, may act as a "lipid biostat" for measuring cellular stress and activating stress responses. We further asked if 4HPR could synergize with known compounds to enhance HSC self-renewal. Treatment of CB with a combination of 4HPR plus CD34+ agonists UM171 and StemRegenin-1 during ex vivo culture maintains a chromatin state more similar to uncultured LT-HSC as demonstrated by ATAC-seq, and led to a 4-fold increase in serial repopulating ability in xenotransplant assays over treatment with UM171 and SR1 alone. These results suggest that sphingolipid perturbation not only activates proteostatic mechanisms that protect HSC organelles from damage incurred during cellular activation, but also regulates the landscape of chromatin accessibility in cultured HSC when combined with CD34+ agonists. This work identifies a new linkage between sphingolipid metabolism, proteostatic QC systems and HSC self-renewal, and identifies novel strategies by which to expand HSC numbers and improve HSC quality for clinical applications. Disclosures Takayama: Megakaryon co. Ltd.: Research Funding.

Summary Regulated blood production is achieved through the hierarchical organization of dormant hematopoietic stem cell (HSC) subsets that differ in self-renewal potential and division frequency, with long-term (LT)-HSCs dividing the least. The molecular mechanisms underlying this variability in HSC division kinetics are unknown. We report here that quiescence exit kinetics are differentially regulated within human HSC subsets through the expression level of CDK6. LT-HSCs lack CDK6 protein. Short-term (ST)-HSCs are also quiescent but contain high CDK6 protein levels that permit rapid cell cycle entry upon mitogenic stimulation. Enforced CDK6 expression in LT-HSCs shortens quiescence exit and confers competitive advantage without impacting function. Computational modeling suggests that this independent control of quiescence exit kinetics inherently limits LT-HSC divisions and preserves the HSC pool to ensure lifelong hematopoiesis. Thus, differential expression of CDK6 underlies heterogeneity in stem cell quiescence states that functionally regulates this highly regenerative system., Graphical Abstract, Highlights • Human long-term (LT) and short-term (ST) HSCs are equally quiescent • LT- and ST-HSCs differ in division kinetics and expression of CDK6 • CDK6 expression regulates the timing of exit from quiescence • Differential regulation of quiescence helps maintain hematopoiesis, The hematopoietic stem cell (HSC) compartment is heterogeneous in terms of cell cycle properties. Laurenti et al. show that the timing of exit from quiescence in human HSC subsets is controlled by CDK6 expression levels. This differential control has an impact on the long-term preservation of the HSC pool.

Metabolic alterations are a cancer hallmark that are typically evaluated in bulk tissues. However most normal tissues and cancers are hierarchically organized and the metabolic requirements of both normal and cancer stem cells are poorly understood, particularly beyond energy metabolism. By analyzing a comprehensive transcriptional roadmap of human hematopoiesis, and comparing this to a leukemia stem cell (LSC) signature developed from 84 human acute myeloid leukemia (AML) samples, we found that: (1) several metabolic pathways, specifically in bioactive lipids, distinguish normal hematopoietic stem cells (HSC) from progenitors; and (2) while LSC are similar to HSC, specific metabolic pathways are more comparable to those of normal progenitors. We defined a lipid stem signature of 24 lipid genes, including sphingolipid genes, whose expression is higher in HSC than progenitors. Interestingly, sphingosine-1-phosphate (S1P) is known to play a role in HSC egress, and ceramide vs S1P levels serve as a rheostat to regulate cell growth and survival. To determine if sphingolipids play a functional role in the primitive hematopoietic compartment, we altered sphingolipid signaling by plating sorted populations of HSC or granulocyte-myeloid progenitors (GMP) in methylcellulose containing myriocin, which inhibits the first step of de novo sphingolipid synthesis, or FTY720, a S1P mimetic. Myriocin decreased GMP colony output but did not affect CFC derived from HSC. By contrast FTY720 affected HSC-derived CFC but not those from GMP, suggesting differential sensitivity to sphingolipid pathway inhibition between stem and progenitor cells. In vitro treatment of lineage depleted cord blood (Lin- CB) with myriocin for 8 days limited only myeloid differentiation compared to control treated cells. In contrast, FTY720 treatment reduced levels of immunophenotypic stem cells, erythroid and myeloid cells, as would be expected with inhibition of S1P proliferative signaling. However, in vitro FTY720-treated Lin- CB cells exhibit 16-week engraftment capacity comparable to that of controlled-treated cells, suggesting that any effects on HSC function are reversible after drug withdrawal. Furthermore, in vivo treatment with FTY720 in mice with established CB grafts did not decrease engraftment. Our lipid stem signature is enriched in LSC gene expression profiles from our 84 AML cohort by GSEA analysis, suggesting that differences in lipid metabolism may also exist in LSC vs. non-LSC. To determine if sphingolipids play a functional role in AML biology, we transplanted mice with peripheral blood cells from 13 AML patients, including those with therapy resistant and relapsed disease, and treated engrafted mice with myriocin or FTY720. We observed heterogeneous responses in our cohort, with reduction of leukemic burden in 3 and 5 samples following treatment with FTY720 and myriocin, respectively. Remarkably, serial transplantation of FTY720 responders into untreated secondary mice at limiting dilution demonstrated decreased LSC frequency in FTY720-treated primary mice compared to vehicle-treated controls, whereas myriocin responders showed no alteration of LSC frequency. These results suggest that FTY720 but not myriocin treatment affects LSC number and/or function. To stratify responders from nonresponders, we performed transcriptional analysis of untreated patient samples and compared these data to transcriptional signatures generated from the normal and AML hierarchy. Bioinformatic analysis demonstrated that FTY720 responders had an enriched LSC signature compared to nonresponders. In contrast, myriocin responders exhibited a strong GMP signature compared to nonresponders. Thus, normal human hematopoietic stem and progenitor cells display a variable dependence on sphingolipid biology that is also distinct between LSC and HSC, pointing to targeting of bioactive sphingolipids as a novel therapeutic strategy in AML to eradicate LSC while sparing HSC. Citation Format: Stephanie Z. Xie, Elisa Laurenti, Robin Ferrari, John E. Dick. Differential dependence on sphingolipid metabolism in the normal and leukemic human hematopoietic hierarchy. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A04.